JP6805637B2 - Hematocrit value measuring method, hematocrit value measuring device, amount of substance to be measured, and amount of substance to be measured - Google Patents

Description

The present invention relates to a measuring method and a measuring device for measuring a hematocrit value of a sample containing blood, and a measuring method and a measuring device for measuring the amount of a substance to be measured in the sample.

If a small amount of a substance to be measured in a sample, such as protein or DNA, can be detected with high sensitivity and quantitatively in a clinical test, it is possible to quickly grasp the patient's condition and perform treatment. For example, when measuring an antigen (substance to be measured) in blood, whole blood collected from a patient or plasma or serum in which a blood cell component is separated from the whole blood can be used as a sample. In addition, in order to grasp the patient's condition, it is necessary to measure the amount (concentration) of the substance to be measured with respect to plasma or serum. However, since the ratio of plasma or serum to whole blood varies from patient to patient, when whole blood is used as a sample and a measured value indicating the amount of the substance to be measured is obtained, the ratio of plasma or serum to whole blood is used. It is necessary to correct the measured value accordingly. At this time, the hematocrit value can be used to correct the measured value.

The microhematocrit method is the most classic and highly accurate method for measuring hematocrit values. However, the microhematocrit method has a problem that the centrifuge used is large and it is difficult to automate the measurement process. Therefore, various methods for measuring the hematocrit value have been developed (see, for example, Patent Documents 1 to 3).

Patent Document 1 describes a method of measuring a hematocrit value based on the bulk conductivity of a sample. Further, Patent Document 2 describes a method of measuring a hematocrit value based on the absorbance of a sample. Further, Patent Document 3 describes a method of measuring a hematocrit value based on the viscosity of a sample. In the method described in Patent Document 3, when a sample is supplied to the capillary space in a state where a voltage is applied, the hematocrit value of the sample is determined based on the time until a current through the sample is detected. be able to.

Japanese Unexamined Patent Publication No. 2011-002468 Japanese Unexamined Patent Publication No. 2009-236487 Japanese Unexamined Patent Publication No. 2011-137816

In the method for measuring a hematocrit value described in Patent Document 1 and Patent Document 3, a measuring system for measuring a current value (conductivity) is required when measuring a hematocrit value. Further, in the method for measuring a hematocrit value described in Patent Document 2, a measuring system for measuring the absorbance is required when measuring the hematocrit value. In addition, when correcting the measured value indicating the amount of the substance to be measured in the sample containing blood with the hematocrit value, if the measuring device does not have a measuring system for measuring the hematocrit value, a separate measuring system is provided. Need to be added. However, if a measuring system is added separately, there is a problem that the cost of the measuring device becomes high and the measuring device becomes large.

On the other hand, a measuring device that handles a liquid usually has a pipette tip and a pressure sensor for detecting the pressure in the pipette tip.

The present invention has been made in view of this point, and a measuring method and a measuring device for measuring a hematocrit value of a sample containing blood by using a pressure sensor for detecting the pressure in the pipette tip. The purpose is to provide. Another object of the present invention is to provide a measuring method and a measuring device capable of measuring the hematocrit value of a sample and measuring the amount of a substance to be measured in the sample with high accuracy without adding a new measuring system separately. And.

In order to solve the above problem, the method for measuring the hematocrit value according to the embodiment of the present invention is to measure the hematocrit value for measuring the hematocrit value of a sample containing blood by using a measuring chip having a flow path. The method, wherein the sample is injected from the pipette tip into the flow path while detecting the pressure in the pipette tip, or the sample is sucked into the pipette tip from the flow path. It includes a step of acquiring information on a time change of pressure in a pipette tip and a step of determining a hematocrit value of the sample based on the information.

In order to solve the above problem, the method for measuring the amount of the substance to be measured according to the embodiment of the present invention is to measure the amount of the substance to be measured in a sample containing blood. According to the method for measuring the hematocrit value according to the present invention, the step of measuring the hematocrit value of the sample and the substance to be measured contained in the sample are immobilized in the flow path. In addition, the step of acquiring a measured value indicating the amount of the substance to be measured in the sample in the absence of the sample, and the step of correcting the measured value based on the hematocrit value are included.

In order to solve the above problem, the hematocrit value measuring device according to the embodiment of the present invention is a hematocrit value measuring device for measuring the hematocrit value of a sample containing blood, and is a measurement having a flow path. The pipette tip has a tip holder for holding the tip, a pipette tip, and a pressure sensor for detecting the pressure in the pipette tip, and the pipette tip is detected by the pressure sensor while detecting the pressure in the pipette tip. The time change of the pressure in the pipette tip acquired by the pressure sensor and the liquid handling unit for injecting the sample into the flow path or sucking the sample from the flow path into the pipette tip. It has a processing unit for determining a hematoclit value of the sample based on the information regarding the sample.

In order to solve the above problems, the device for measuring the amount of the substance to be measured according to the embodiment of the present invention is for measuring the amount of the substance to be measured in the sample containing blood. The substance to be measured contained in the sample is immobilized in the measuring device for measuring the hematocrit value according to the present invention and the flow path of the measuring chip held in the chip holder. And, in the absence of the sample, it has a measurement value acquisition unit for acquiring a measurement value indicating the amount of the substance to be measured in the sample, and the processing unit further obtains the hematocrit value. The measured value is corrected based on the above.

According to the present invention, the hematocrit value can be measured by using the pipette tip of the device that handles the liquid and the pressure sensor for detecting the pressure in the pipette tip, so that the height of the measuring device is high. It is possible to suppress the increase in cost and size.

FIG. 1 is a schematic view showing an example of the configuration of the SPFS apparatus according to the embodiment. FIG. 2 is a cross-sectional view showing an example of the configuration of the measuring chip. FIG. 3 is a flowchart showing an example of the operation procedure of the SPFS device according to the embodiment. FIG. 4 is a flowchart showing a process in the measurement value acquisition process shown in FIG. FIG. 5 is a flowchart showing a process in the process of measuring the hematocrit value shown in FIG. 6A and 6B are graphs for explaining a method of determining a hematocrit value. FIG. 7 is a graph showing an example of a calibration curve for determining the hematocrit value from the time until the equilibrium state is reached when the sample is injected into the flow path. FIG. 8 is a graph showing the accuracy when the amount of the substance to be measured in plasma is determined by the viscosity method.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Here, as a typical example of the measuring device according to the present invention, a measuring device (SPFS device) using surface plasmon-field enhanced Fluorescence Spectroscopy (hereinafter abbreviated as “SPFS”) will be described. ..

FIG. 1 is a schematic view showing an example of the configuration of the SPFS device 100 according to the present embodiment. As shown in FIG. 1, the SPFS device 100 includes an excitation light emitting unit 110, a signal detecting unit 120, a liquid handling unit 130, a transport unit 140, and a control processing unit (processing unit) 150. In the present embodiment, the excitation light emitting unit 110 and the signal detecting unit 120 both form a measured value acquisition unit for acquiring a measured value indicating the amount of the substance to be measured in the sample.

The SPFS device 100 is used with the measuring chip 10 mounted on the chip holder 142 of the transport unit 140. Therefore, the measuring chip 10 will be described first, and then the SPFS device 100 will be described. The chip holder 142, the liquid handling unit 130, and the control processing unit 150 all constitute a hematocrit value measuring device for measuring the hematocrit value of the sample.

(Measuring chip)
FIG. 2 is a cross-sectional view showing an example of the configuration of the measuring chip 10. FIG. 2 is a cross-sectional view in a plane perpendicular to the paper surface of FIG. 1 and along the flow direction of the liquid in the flow path 41. The measuring chip 10 has a prism 20, a metal film 30, and a flow path lid 40. In the present embodiment, the flow path lid 40 of the measuring chip 10 is integrated with the liquid chip 50 described later.

The prism 20 has an incident surface 21, a film forming surface 22, and an emitting surface 23. The incident surface 21 causes the excitation light α from the excitation light emitting unit 110 to enter the inside of the prism 20. A metal film 30 is arranged on the film-forming surface 22. The excitation light α incident on the inside of the prism 20 is reflected at the interface (deposition surface 22) between the prism 20 and the metal film 30 to become reflected light. The exit surface 23 emits the reflected light to the outside of the prism 20.

The shape of the prism 20 is not particularly limited. In the present embodiment, the shape of the prism 20 is a pillar body having a trapezoidal bottom surface. The surface corresponding to one base of the trapezoid is the film forming surface 22, the surface corresponding to one leg is the incident surface 21, and the surface corresponding to the other leg is the exit surface 23. The trapezoid to be the bottom surface is preferably an isosceles trapezoid. As a result, the incident surface 21 and the exit surface 23 become symmetrical, and the S wave component of the excitation light α is less likely to stay in the prism 20.

The incident surface 21 is formed so that the excitation light α from the excitation light emitting unit 110 is not reflected by the incident surface 21 and returned to the excitation light emitting unit 110. When the light source of the excitation light α is a laser diode (hereinafter, also referred to as “LD”), when the excitation light α returns to the LD, the excited state of the LD is disturbed and the wavelength and output of the excitation light α fluctuate. .. Therefore, the angle of the incident surface 21 is set so that the excitation light α is not incident perpendicularly to the incident surface 21 in the scanning range centered on the ideal resonance angle or enhancement angle.

Here, the "resonance angle" means the incident angle when the amount of reflected light emitted from the emitting surface 23 is minimized when the incident angle of the excitation light α with respect to the metal film 30 is scanned. Further, the "enhanced angle" is scattered light having the same wavelength as the excitation light α emitted above the measurement chip 10 when the incident angle of the excitation light α with respect to the metal film 30 is scanned (hereinafter, “plasmon scattered light””. It means the incident angle when the amount of light of γ is maximized. In the present embodiment, the angle between the incident surface 21 and the film forming surface 22 and the angle between the film forming surface 22 and the emitting surface 23 are both about 80 °.

The resonance angle (and the augmentation angle in the immediate vicinity thereof) is roughly determined by the design of the measurement chip 10. The design elements include the refractive index of the prism 20, the refractive index of the metal film 30, the thickness of the metal film 30, the extinction coefficient of the metal film 30, the wavelength of the excitation light α, and the like. The substance to be measured captured on the metal film 30 shifts the resonance angle and the enhancement angle, but the amount is less than a few degrees.

The prism 20 is made of a dielectric material that is transparent to the excitation light α. The prism 20 has not a little birefringence characteristic. Examples of materials for the prism 20 include resin and glass. Examples of the resins constituting the prism 20 include polymethyl methacrylate (PMMA), polycarbonate (PC), and cycloolefin-based polymers. The material of the prism 20 is preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence.

The metal film 30 is arranged on the film forming surface 22 of the prism 20. As a result, surface plasmon resonance (hereinafter abbreviated as "SPR") occurs between the photons of the excitation light α incident on the film-forming surface 22 under total reflection conditions and the free electrons in the metal film 30. , Localized field light (generally also referred to as "evanescent light" or "proximity field light") can be generated on the surface of the metal film 30. The localized field light extends from the surface of the metal film 30 to a distance of about the wavelength of the excitation light α. The metal film 30 may be formed on the entire surface of the film-forming surface 22, or may be formed on a part of the film-forming surface 22. In the present embodiment, the metal film 30 is formed on the entire surface of the film-forming surface 22.

A trapping body for capturing the substance to be measured is immobilized on the metal film 30. The region on the metal film 30 where the trap is immobilized is particularly referred to as a "reaction field". In the present embodiment, the trap is fixed substantially uniformly over the metal film 30. Therefore, the reaction field is formed over the metal film 30. In addition, the trapping body specifically binds to the substance to be measured. Therefore, the substance to be measured can be immobilized on the metal film 30 via the trap.

The type of capture body is not particularly limited as long as it can capture the substance to be measured. For example, the trap is an antibody (primary antibody) or a fragment thereof that can specifically bind to the substance to be measured, an enzyme that can specifically bind to the substance to be measured, or the like.

The material of the metal film 30 is not particularly limited as long as it is a metal capable of causing surface plasmon resonance. Examples of materials for the metal film 30 include gold, silver, copper, aluminum, and alloys thereof. In the present embodiment, the metal film 30 is a gold thin film. The thickness of the metal film 30 is not particularly limited, but is preferably in the range of 20 to 60 nm from the viewpoint of efficiently generating SPR. The method for forming the metal film 30 is not particularly limited. Examples of methods for forming the metal film 30 include sputtering, vapor deposition, and plating.

The flow path lid 40 is arranged on the metal film 30. When the metal film 30 is formed only on a part of the film forming surface 22 of the prism 20, the flow path lid 40 may be arranged on the film forming surface 22. In the present embodiment, the flow path lid 40 is arranged on the metal film 30. By arranging the flow path lid 40 on the metal film 30, the flow path 41, which is a cavity for accommodating the liquid, is formed. In the present embodiment, the flow path 41 has a bottom surface, a top surface, and a pair of side surfaces connecting the bottom surface and the top surface. In the present specification, the surface of the flow path 41 on the prism 20 side is referred to as "the bottom surface of the flow path 41", and the surface of the flow path 41 facing the bottom surface of the flow path 41 is referred to as the "top surface of the flow path 41". The details will be described later, but from the viewpoint of obtaining the desired flow path resistance, the length between the pair of side surfaces of the flow path 41 is preferably 0.1 mm or more and 5 mm or less, and is equal to the top surface of the flow path 41. The length between the flow path 41 and the bottom surface is preferably 0.1 mm or more and 1 mm or less, and the length of the flow path 41 in the sample flow direction (longitudinal direction of the flow path 41) is 10 mm or more and 50 mm. The following is preferable.

As shown in FIG. 2, the flow path lid 40 has a frame body 42, a liquid injection portion coating film 43, and a liquid storage portion coating film 44. Two through holes are formed in the frame body 42. A recess (flow path groove) is formed on the back surface of the frame body 42. The flow path lid 40 (frame body 42) is arranged on the metal film 30 (and the prism 20), and the opening of the recess is closed by the metal film 30 to form the flow path 41. Further, the opening of one through hole is closed by the liquid injection portion coating film 43 to form the liquid injection portion 45, and the opening of the other through hole is closed by the liquid storage portion coating film 44. Then, the liquid storage unit 46 is formed. The liquid storage portion coating film 44 is provided with ventilation holes 47.

From the viewpoint of sufficiently securing the region covered by the localized field light, the height of the flow path 41 (depth of the flow path groove) is preferably large to some extent. From the viewpoint of reducing the amount of impurities mixed in the flow path 41, the height of the flow path 41 (depth of the flow path groove) is preferably small. From such a viewpoint, the height of the flow path 41 is preferably in the range of 0.05 to 0.15 mm. Both ends of the flow path 41 are connected to a liquid injection section 45 and a liquid storage section 46 formed in the flow path lid 40 so as to communicate the inside and the outside of the flow path 41, respectively.

The frame body 42 is preferably made of a material that is transparent to the light (fluorescent β and plasmon scattered light γ) emitted from the metal film 30. Examples of materials for the flow path lid 40 include glass and resin. Examples of such resins include polymethyl methacrylate resin (PMMA). Further, as long as it is transparent to the above light, the other portion of the frame body 42 may be formed of an opaque material. The frame body 42 is joined to the metal film 30 or the prism 20 by, for example, bonding with double-sided tape or an adhesive, laser welding, ultrasonic welding, crimping using a clamp member, or the like.

The liquid injection portion coating film 43 is a film into which the pipette tip 134 can be inserted, and when the pipette tip 134 is inserted, the film can be brought into close contact with the outer periphery of the pipette tip 134 without a gap. For example, the liquid injection portion coating film 43 is a two-layer film of an elastic film and an adhesive film. The liquid injection portion covering film 43 may be provided with a fine through hole for inserting the pipette tip 134. In the present embodiment, the liquid injection portion covering film 43 is provided with a through hole 48 for inserting a pipette tip having an outer diameter of 1.2 mm. Since the liquid injection portion coating film 43 is configured to be in close contact with the outer periphery of the pipette tip 134 without a gap, the inside of the flow path 41 on the liquid injection portion 42 side can be sealed at the time of liquid feeding.

As described above, the liquid storage portion covering film 44 has ventilation holes 47. The structure of the liquid storage portion covering film 44 is not particularly limited. For example, the liquid storage portion covering film 44 may be a two-layer film similar to the liquid injection portion covering film 43 described above.

As described above, in the present embodiment, the measuring chip 10 and the liquid chip 50 are integrated (see FIG. 1). More specifically, the frame body 42 and the liquid chip 50 are integrated. The liquid chip 50 has a well for containing the liquid. The opening of the well may be closed with a film or the like while containing the liquid. The film that closes the well openings may be removed by the user prior to use of the liquid chip 50. If the pipette tip 134 can penetrate the film, the liquid tip 50 may be used with the well openings closed by the film.

Examples of the liquid contained in the liquid chip 50 include a sample containing blood, a labeling solution containing a catcher labeled with a fluorescent substance, a washing solution (buffer solution), and a diluted solution thereof.

The measuring tip 10 and the liquid tip 50 are usually replaced at each measurement. Further, the measuring chip 10 is preferably a structure having a length of several mm to several cm, but is a smaller structure or a larger structure that is not included in the category of “chip”. May be good.

(SPFS device)
Next, each component of the SPFS apparatus 100 will be described. As described above, the SPFS device 100 includes an excitation light emitting unit 110, a signal detecting unit 120, a liquid handling unit 130, a transport unit 140, and a control processing unit (processing unit) 150.

The excitation light emitting unit 110 emits the excitation light α. When the fluorescence β is detected, the excitation light emitting unit 110 emits a P wave with respect to the metal film 30 toward the incident surface 21 so that surface plasmon resonance occurs in the metal film 30. Here, the "excitation light" is light that directly or indirectly excites a fluorescent substance. For example, the excitation light α is light that generates localized field light that excites a fluorescent substance on the surface of the metal film 30 when the metal film 30 is irradiated through the prism 20 at an angle at which surface plasmon resonance occurs. is there. The excitation light emitting unit 110 includes a light source unit 111, an angle adjusting mechanism 112, and a light source control unit 113.

The light source unit 111 emits light that is collimated and has a constant wavelength and light amount so that the shape of the irradiation spot on the back surface of the metal film 30 is substantially circular. The light source unit 111 includes, for example, a light source, a beam shaping optical system, an APC mechanism, and a temperature adjusting mechanism (all not shown).

The type of light source is not particularly limited, and is, for example, a laser diode (LD). Other examples of light sources include laser light sources such as light emitting diodes and mercury lamps. The wavelength of the excitation light α emitted from the light source is, for example, 400 nm or more and 1000 nm or less. When the excitation light α emitted from the light source is not a beam, the excitation light α is converted into a beam by a lens, a mirror, a slit, or the like. When the excitation light α emitted from the light source is not monochromatic light, the excitation light α is converted into monochromatic light by a diffraction grating or the like. Further, when the excitation light α emitted from the light source is not linearly polarized light, the excitation light α is converted into linearly polarized light by a polarizer or the like.

The beam shaping optical system includes, for example, a collimator, a bandpass filter, a linear polarizing filter, a half-wave plate, a slit, a zoom means, and the like. The beam shaping optical system may include all of these, or may include some of them.

The collimator collimates the excitation light α emitted from the light source.

The bandpass filter converts the excitation light α emitted from the light source into narrow-band light having only a central wavelength. This is because the excitation light α emitted from the light source has a slight wavelength distribution width.

The linearly polarized light filter converts the excitation light α emitted from the light source into linearly polarized light.

The half-wave plate adjusts the polarization direction of light so that the P wave component is incident on the metal film 30.

The slit and zoom means adjust the beam diameter and contour shape of the excitation light α emitted from the light source so that the shape of the irradiation spot on the back surface of the metal film 30 becomes a circle of a predetermined size.

The APC mechanism controls the light source so that the output of the light source is constant. More specifically, the APC mechanism detects the amount of light branched from the excitation light α with a photodiode (not shown) or the like. Then, the APC mechanism controls the output of the light source to be constant by controlling the input energy with the regression circuit.

The temperature adjusting mechanism is, for example, a heater or a Peltier element. The wavelength and energy of the excitation light α emitted from the light source may vary depending on the temperature. Therefore, by keeping the temperature of the light source constant by the temperature adjusting mechanism, the wavelength and energy of the excitation light α emitted from the light source are controlled to be constant.

The angle adjusting mechanism 112 adjusts the incident angle of the excitation light α with respect to the metal film 30 (the interface between the prism 20 and the metal film 30 (deposition surface 22)). The angle adjusting mechanism 112 connects the optical axis of the excitation light α emitted from the light source and the chip holder 142 in order to irradiate the metal film 30 with light at a predetermined angle of incidence toward a predetermined position via the prism 20. Rotate relatively. For example, the angle adjusting mechanism 112 rotates the light source unit 111 on the metal film 30 about an axis orthogonal to the optical axis of the excitation light α (the axis perpendicular to the paper surface of FIG. 1). At this time, the position of the rotation axis is set so that the position of the irradiation spot on the metal film 30 hardly changes even if the incident angle is scanned. In particular, the position of the center of rotation is set near the intersection of the optical axes of the excitation light α emitted from the two light sources at both ends of the scanning range of the incident angle (between the irradiation position on the film forming surface 22 and the incident surface 21). By setting, the deviation of the irradiation position can be minimized.

As described above, among the incident angles of the excitation light α emitted from the light source to the metal film 30, the angle at which the amount of plasmon scattered light γ is maximized is the enhancement angle. By setting the incident angle of the excitation light α emitted from the light source to an angle at or near the enhancement angle, it becomes possible to detect high-intensity fluorescent β and plasmon scattered light γ. The basic incident conditions of the excitation light α emitted from the light source are determined by the material and shape of the prism 20, the thickness of the metal film 30, the refractive index of the liquid in the flow path 41, and the like. The optimum incident conditions vary slightly depending on the type and amount of light sources, the shape error of the prism 20, and the like. Therefore, it is preferable to obtain the optimum enhancement angle for each measurement.

The light source control unit 113 controls various devices included in the light source unit 111 to control the emission of the excitation light α from the light source unit 111. The light source control unit 113 is composed of, for example, a known computer or microcomputer including an arithmetic unit, a control device, a storage device, an input device, and an output device.

In the signal detection unit 120, the excitation light emitting unit 110 is transmitted to the metal film 30 via the prism 20 in a state where the substance to be measured contained in the sample is immobilized on the metal film 30 and the sample does not exist. When the excitation light α is emitted at an incident angle where surface plasmon resonance occurs, a signal (for example, fluorescence β, reflected light or plasmon scattered light γ) generated by the measuring chip 10 is detected. The signal detection unit 120 outputs a signal indicating the detected signal amount (for example, the light amount of fluorescent β, the light amount of reflected light, or the light amount of plasmon scattered light γ) to the control processing unit 150. The signal detection unit 120 includes a light receiving optical system unit 121, a position switching mechanism 122, and a sensor control unit 127.

The light receiving optical system unit 121 is arranged on the normal line of the metal film 30 of the measuring chip 10. The light receiving optical system unit 121 includes a first lens 123, an optical filter 124, a second lens 125, and a light receiving sensor 126.

The position switching mechanism 122 switches the position of the optical filter 124 on or out of the optical path of the light receiving optical system unit 121. Specifically, when the first light receiving sensor 126 detects the fluorescence β, the optical filter 124 is arranged on the optical path of the light receiving optical system unit 121, and when the first light receiving sensor 126 detects the plasmon scattered light γ. , The optical filter 124 is arranged outside the optical path of the light receiving optical system unit 121.

The first lens 123 is, for example, a condensing lens, which condenses light (signal) emitted from the metal film 30. The second lens 125 is, for example, an imaging lens, and the light collected by the first lens 123 is imaged on the light receiving surface of the first light receiving sensor 126. The light is a substantially parallel luminous flux between the two lenses.

The optical filter 124 is arranged between the first lens 123 and the second lens 125. At the time of fluorescence detection, the optical filter 124 transmits only the fluorescence component among the light incident on the optical filter 124, and removes the excitation light component (plasmon scattered light γ). As a result, only the fluorescent component can be guided to the first light receiving sensor 126, and the fluorescent β can be detected with a high S / N ratio. Examples of the types of optical filters 124 include excitation light reflection filters, short wavelength cut filters and bandpass filters. Examples of the optical filter 124 include a filter including a multilayer film that reflects a predetermined light component, and a colored glass filter that absorbs a predetermined light component.

The light receiving sensor 126 detects fluorescent β and plasmon scattered light γ. The light receiving sensor 126 has high sensitivity capable of detecting weak fluorescence β from a trace amount of the substance to be measured. The light receiving sensor 126 is, for example, a photomultiplier tube (PMT), an avalanche photodiode (APD), a silicon photodiode (SiPD), or the like.

The sensor control unit 127 controls detection of the output value of the light receiving sensor 126, management of the sensitivity of the light receiving sensor 126 based on the output value, change of the sensitivity of the light receiving sensor 126 in order to obtain an appropriate output value, and the like. The sensor control unit 127 is composed of, for example, a known computer or microcomputer including an arithmetic unit, a control device, a storage device, an input device, and an output device.

The liquid handling unit 130 supplies a liquid such as a sample into the flow path 41 of the measuring chip 10 held by the chip holder 142. Further, the liquid handling unit 130 removes the liquid from the flow path 41 of the measuring chip 10. The liquid handling unit 130 includes a pipette 131 and a pipette control unit 136.

The pipette 131 is a pressure connected between the syringe pump 132, the nozzle unit 133 connected to the syringe pump 132, the pipette tip 134 attached to the tip of the nozzle unit 133, and the syringe pump 132 and the nozzle unit 133. It has a sensor 135.

The reciprocating motion of the plunger in the syringe pump 132 quantitatively sucks and drains the liquid in the pipette tip 134. The opening area of the opening of the pipette tip 134 is larger than the cross-sectional area of the flow path 41 perpendicular to the liquid flow direction. The flow path resistance of the flow path 41 is larger than the resistance when the pipette tip 134 is injected into the flow path 41. As a result, the flow path resistance can be adjusted according to the cross-sectional area of the flow path 41.

The pressure sensor 135 detects the pressure in the pipette tip 134. The type of the pressure sensor 135 is not particularly limited as long as the pressure in the pipette tip 134 can be detected by the pressure sensor 135. Examples of the types of pressure sensors 135 include strain gauge resistance pressure sensors, semiconductor piezoresistive pressure sensors, capacitive pressure sensors and silicon resonant pressure sensors.

The pipette control unit 136 includes a driving device for the syringe pump 132 and a moving device for the nozzle unit 133. The driving device of the syringe pump 132 is a device for reciprocating the plunger of the syringe pump 132, and includes, for example, a stepping motor. The moving device of the nozzle unit 133, for example, freely moves the nozzle unit 133 in the vertical direction. The moving device of the nozzle unit 133 is composed of, for example, a robot arm, a two-axis stage, or a vertically movable turntable.

The pipette control unit 136 drives the syringe pump 132 to suck various liquids from the liquid tip 50 into the pipette tip 134. Then, the pipette control unit 136 moves the nozzle unit 133 to insert the pipette tip 134 into the flow path 41 of the measuring tip 10, and drives the syringe pump 132 to flow the liquid in the pipette tip 134. It is injected into 41. After introducing the liquid, the pipette control unit 136 drives the syringe pump 132 to suck the liquid in the flow path 41 into the pipette tip 134. By sequentially exchanging the liquid in the flow path 41 in this way, the trapped substance and the substance to be measured are reacted in the reaction field (primary reaction), or the substance to be measured is reacted with the trapped substance labeled with the fluorescent substance. (Secondary reaction). The pipette control unit 136 injects the liquid into the flow path 41 or sucks the liquid from the flow path 41 while detecting the pressure in the pipette tip 134 by the pressure sensor 135.

The transport unit 140 transports and fixes the measuring chip 10 to the installation position, the measuring position, or the liquid feeding position. Here, the "installation position" is a position for installing the measuring chip 10 in the SPFS device 100. The "measurement position" is a position for acquiring a measured value indicating the amount of the substance to be measured in the sample, and when the excitation light emitting unit 110 emits the excitation light α toward the measurement chip 10. This is the position where the signal detection unit 120 detects the signal emitted from the measurement chip 10. Further, the “liquid feeding position” is a position where the liquid handling unit 130 supplies the liquid into the flow path 41 of the measuring chip 10 or removes the liquid in the flow path 41 of the measuring chip 10.

The transport unit 140 includes a transport stage 141 and a tip holder 142.

The transport stage 141 moves the tip holder 142 in one direction and vice versa. The transport stage 141 also has a shape that does not obstruct the optical path of the excitation light α, the reflected light of the excitation light α, the fluorescence β, the plasmon scattered light γ, and the like. The transport stage 141 is driven by, for example, a stepping motor or the like.

The tip holder 142 is fixed to the transport stage 141 and holds the measuring tip 10 detachably. The shape of the chip holder 142 is a shape that can hold the measurement chip 10 and does not obstruct the optical path of light such as excitation light α, reflected light of excitation light α, fluorescence β, and plasmon scattered light γ. For example, the chip holder 142 is provided with an opening through which the above light passes.

The control processing unit 150 controls the angle adjusting mechanism 112, the light source control unit 113, the position switching mechanism 122, the sensor control unit 127, the pipette control unit 136, and the transfer stage 141. The control processing unit 150 also functions as a processing unit that processes the detection results of the signal detection unit 120 (light receiving sensor 126) and the liquid handling unit 130 (pressure sensor 135). In the present embodiment, the control processing unit 150 determines a measured value indicating the amount of the substance to be measured in the sample based on the detection result of the fluorescence β by the signal detection unit 120. Further, the control processing unit 150 determines the hematocrit value of the sample based on the information regarding the time change of the pressure in the pipette tip 134 by the liquid handling unit 130. At the same time, the control processing unit 150 corrects the measured value based on the hematocrit value. Thereby, the control processing unit 150 determines the amount of the substance to be measured in plasma or serum.

In addition, predetermined information (for example, data related to various conversion coefficients and calibration curves) used when processing the above detection result may be recorded in advance in the control processing unit 150. In the present embodiment, the control processing unit 150 previously records data on a calibration curve for determining the hematocrit value based on information on the time change of the pressure in the pipette tip 134. The control processing unit 150 is composed of, for example, a known computer or microcomputer including an arithmetic unit, a control device, a storage device, an input device, and an output device.

(Optical path in SPFS device)
As shown in FIG. 1, the excitation light α is incident on the prism 20 from the incident surface 21. The excitation light α incident on the prism 20 is incident on the metal film 30 at a total reflection angle (angle at which SPR occurs). In this way, by irradiating the metal film 30 with the excitation light α at an angle at which SPR is generated, localized field light can be generated on the metal film 30. The localized field light excites the fluorescent substance that labels the substance to be measured existing on the metal film 30, and emits the fluorescent β. The SPFS device 100 detects the amount (intensity) of the fluorescent β emitted from the fluorescent substance. Although not particularly shown, the reflected light of the excitation light α on the metal film 30 is emitted to the outside of the prism 20 on the exit surface 23.

(Operation procedure of SPFS device)
Next, the operation procedure of the SPFS device 100 according to the present embodiment (measurement method according to the present embodiment) will be described. FIG. 3 is a flowchart showing an example of the operation procedure of the SPFS device 100. In the present embodiment, the fluorescence value, which is the amount of light of fluorescence β, is measured as a measurement value indicating the amount of the substance to be measured in the sample. FIG. 4 is a flowchart showing a process in the process (process S120) for acquiring the measured value shown in FIG. FIG. 5 is a flowchart showing a step in the step (step S130) for determining the hematocrit value shown in FIG.

The measuring methods according to the present embodiment are 1) a step of preparing for measurement (step S110), 2) a step of acquiring a measured value (step S120), and 3) a step of measuring a hematocrit value (step S130). And 4) a step of correcting the measured value (step S140).

1) Preparation for measurement First, preparation for measurement is performed (step S110). Specifically, the measuring chip 10 is installed in the chip holder 142 arranged at the installation position of the SPFS device 100. When the storage reagent is present on the metal film 30 of the measurement chip 10, the metal film 30 is washed to remove the storage reagent so that the trapping body can properly capture the substance to be measured. The sample is housed in the liquid chip 50.

2) Acquisition of measured value Next, a measured value indicating the amount of the substance to be measured in the sample is acquired (step S120). The step of acquiring the measured value (step S120) includes a step of setting the incident angle to the augmented angle (step S121), a step of measuring the optical blank value (step S122), and a step of performing a primary reaction (step S123). A step of performing the secondary reaction (step S124) and a step of measuring the fluorescence value (step S125) are included.

First, the incident angle of the excitation light α with respect to the metal film 30 (deposition surface 22) is set to the enhancement angle (step S121). Specifically, the control processing unit 150 controls the transfer stage 141 to move the measuring chip 10 from the installation position to the liquid feeding position. The control processing unit 150 controls the pipette control unit 136 to provide a buffer solution for measurement in the liquid chip 50 into the flow path 41. The control processing unit 150 controls the transfer stage 141 to move the measuring chip 10 from the liquid feeding position to the measuring position. The control processing unit 150 controls the position switching mechanism 122 to move the optical filter 124 out of the optical path of the light receiving optical system unit 121. The control processing unit 150 controls the light source control unit 113, the angle adjustment unit 112, and the sensor control unit 127 to send the excitation light α from the light source unit 111 to a predetermined position of the metal film 30 with respect to the metal film 30. While scanning the incident angle of the light source, the light receiving sensor 126 detects the plasmon scattered light γ. As a result, the control processing unit 150 obtains data including the relationship between the incident angle of the excitation light α and the amount of the plasmon scattered light γ. The obtained data is stored in the control processing unit 150. Then, the control processing unit 150 analyzes the data to determine the augmentation angle, which is the incident angle at which the amount of plasmon scattered light γ is maximized. Finally, the control processing unit 150 controls the angle adjusting unit 112 to set the incident angle of the excitation light α with respect to the metal film 30 (deposition surface 22) to a determined enhancement angle.

The augmentation angle is determined by the material and shape of the prism 20, the thickness of the metal film 30, the refractive index of the liquid in the flow path 41, and the like, but the type and amount of the trapped body in the flow path 41 and the shape error of the prism 20. It fluctuates slightly due to various factors such as. Therefore, it is preferable to determine the enhancement angle each time the measurement is performed. The augmentation angle is determined on the order of about 0.1 °.

Next, the optical blank value is measured (step S122). Here, the "optical blank value" means the amount of light having the same wavelength as the fluorescence β emitted above the measuring chip 10.

The control processing unit 150 controls the position switching mechanism 122 to move the optical filter 124 on the optical path of the light receiving optical system unit 121. Next, the control processing unit 150 controls the light source control unit 113 to emit the excitation light α from the light source unit 111 toward the metal film 30 (film-forming surface 22). At the same time, the control processing unit 150 controls the sensor control unit 127, and the light receiving sensor 126 detects the amount of light having the same wavelength as the fluorescence β. As a result, the light receiving sensor 126 can measure the amount of light (optical blank value) that becomes noise in the measurement of the fluorescence value (step S125). The optical blank value is transmitted to the control processing unit 150 and recorded.

Next, the substance to be measured in the sample is reacted with the trapped material on the metal film 30 (primary reaction; step S123). Specifically, the control processing unit 150 controls the transfer stage 141 to move the measuring chip 10 from the measuring position to the liquid feeding position. After that, the control processing unit 150 controls the pipette control unit 136 to discharge the buffer solution in the flow path 41, collects the sample in the liquid tip 50 on the pipette tip 134, and collects the collected sample in the flow path. Inject into 41. As a result, when the substance to be measured is present in the sample, at least a part of the substance to be measured is captured by the trap on the metal film 30. After that, the inside of the flow path 41 is washed with a buffer solution or the like to remove substances that have not been captured by the trap.

Next, the substance to be measured captured by the trap on the metal film 30 is labeled with a fluorescent substance (secondary reaction; step S124). Specifically, the control processing unit 150 controls the pipette control unit 136 to inject the fluorescent labeling liquid in the liquid chip 50 into the flow path 41. As a result, the substance to be measured can be labeled with a fluorescent substance. The fluorescent labeling solution is, for example, a buffer solution containing an antibody (secondary antibody) labeled with a fluorescent substance. After that, the inside of the flow path 41 is washed with a buffer solution or the like to remove free fluorescent substances or the like.

Next, the fluorescence β emitted from the fluorescent substance labeling the substance to be measured in the reaction field is detected, and the fluorescence value is measured (step S125). Specifically, the control processing unit 150 controls the transfer stage 141 to move the measuring chip 10 from the liquid feeding position to the measuring position. After that, the control processing unit 150 controls the light source control unit 113, and the light source unit 111 of the excitation light emitting unit 110 is in a state where the substance to be measured contained in the sample is immobilized and the sample does not exist. The excitation light α is irradiated to the back surface of the metal film 30 corresponding to the region where the capture body is fixed via the prism 20 at an incident angle at which surface plasmon resonance occurs. At the same time, the control processing unit 150 controls the sensor control unit 127 to detect the fluorescence β (signal) generated in the measuring chip 10 with the light receiving sensor 126. As a result, the light receiving sensor 126 acquires the fluorescence value (measured value) which is the amount of light of the fluorescence β. The fluorescence value is transmitted to the control processing unit 150 and recorded. In the present specification, the "state in which the sample does not exist" means a state in which the operation of removing the sample from the flow path 41 is performed. That is, it suffices that the sample does not substantially exist in the flow path 41, and a small amount of the sample that cannot be completely removed may remain in the flow path 41.

3) Measurement of hematocrit value Next, the hematocrit value is measured (step S130). The step of determining the hematocrit value (step S130) is a step of detecting a time change of the pressure in the pipette tip 134 when the sample is injected into the flow path (step S131) and a step of determining the hematocrit value (step S131). S132) and.

First, the sample is injected into the flow path 41 and the time change of the pressure in the pipette tip 134 is detected (step S131). Specifically, the control processing unit 150 controls the transfer stage 141 to move the measuring chip 10 from the measuring position to the liquid feeding position. The control processing unit 150 controls the pipette control unit 136 to collect the sample in the liquid tip 50 on the pipette tip 134. In the present embodiment, the control processing unit 150 collects a sample having a liquid volume of 100 μL on the pipette tip 134. Next, the control processing unit 150 controls the pipette control unit 136 to insert the pipette tip 134 into the liquid injection unit 45 so that the outer periphery of the tip end portion of the pipette tip 134 is in close contact with the liquid injection unit coating film 43. Next, the collected sample is injected into the flow path 41 while detecting the pressure in the pipette tip 134 from the pipette tip 134 into the flow path 41. At this time, since the pipette tip 134 and the liquid injection portion film 43 are in close contact with each other, the flow path 41 on the liquid injection portion 45 side is sealed. As a result, the sample can enter the flow path 41. The pressure sensor 135 acquires information on the time change of the pressure in the pipette tip 134 when the sample is injected into the flow path 41. The information is transmitted to the control processing unit 150 and recorded.

From the viewpoint of the measurement range and resolution of the pressure sensor 135, the flow rate of the sample when the liquid handling unit 130 injects the sample into the flow path 41 is preferably 10 μL / min or more and 15000 μL / min or less, preferably 500 μL. More preferably, it is 1/minute or more and 10000 μL / min or less. Further, from the viewpoint of the measurement range of the pressure sensor 135 and the volume of the pipette tip 134, the liquid volume of the sample when the liquid handling device 130 injects the sample into the flow path 41 is preferably 50 μL or more and 500 μL or less. More preferably, it is 100 μL or more and 200 μL or less.

Next, the hematocrit value is determined (step S132). The control processing unit 150 determines the hematocrit value of the sample based on the information obtained by the pressure sensor 135 regarding the time change of the pressure in the pipette tip 134 when the sample is injected into the flow path 41.

6A and 6B are graphs for explaining a method of determining a hematocrit value. In FIGS. 6A and 6B, the horizontal axis represents time (seconds) and the vertical axis represents pressure (kPa) in the pipette tip 134. 6A and 6B show the time of pressure in the pipette tip 134 when a sample (liquid volume 100 μL, hematocrit value 68%) is injected into a flow path 41 having a size of 20 mm × 4.6 mm × 0.1 mm. It is a graph which shows the change. In FIGS. 6A and 6B, the horizontal axis is time and the vertical axis is the pressure in the pipette tip 134. FIG. 6A is a graph when the sample is injected into the flow path 41 at a high flow rate (10000 μL / min), and FIG. 6B is a graph when the sample is injected into the flow path 41 at a low flow rate (500 μL / min). It is a graph.

When the sample is injected into the flow path 41 from the pipette tip 134, the sample receives resistance (flow path resistance) from the inner wall surface of the flow path 41. Therefore, as shown in FIGS. 6A and 6B, the pressure in the pipette tip 134 becomes high. Further, as described above, the opening area of the opening of the pipette tip 134 is larger than the cross-sectional area of the flow path 41 perpendicular to the liquid flow direction. Therefore, the magnitude of the flow path resistance is determined according to the size of the cross-sectional area of the flow path 41 in the cross section perpendicular to the flow direction of the sample. That is, the size of the cross-sectional area of the flow path 41 can be determined according to the flow path resistance of the desired flow path 41.

As time passes after the sample is injected into the flow path 41, the pressure in the pipette tip 134 decreases and reaches an equilibrium state. The time t from the peak pressure in the pipette tip 134 to the equilibrium state becomes longer as the viscosity of the sample increases. The viscosity of the sample increases as the proportion of blood cell components in the sample increases (the higher the hematocrit value). That is, there is a correlation between the time t and the hematocrit value of the sample. Therefore, the hematocrit value of the sample can be determined based on the time t until the equilibrium state is reached. Criteria for determining whether or not equilibrium has been reached can be appropriately determined as needed. For example, the time t to reach the equilibrium state may be the time from when the pressure in the pipette tip 134 reaches the peak value to 1% or less, or per unit time of the pressure in the pipette tip 134. It may be the time until the amount of change of is 2% or less with respect to the peak value.

FIG. 7 is a graph showing an example of a calibration curve for determining the hematocrit value Hct from the time t until the equilibrium state is reached when the sample is injected into the flow path 41. In FIG. 7, the horizontal axis represents the time t (seconds) until the equilibrium state is reached, and the vertical axis represents the hematocrit value. The calibration curve shown in FIG. 7 is until an equilibrium state is reached when a sample (liquid volume 100 μL) having a known hematocrit value Hct is injected into a flow path 41 having a size of 20 mm × 4.6 mm × 0.1 mm. It was created by measuring each time t and plotting the time t and the hematocrit value Hct. In FIG. 7, black circles (●) indicate the results when the sample was injected into the flow path 41 at a high flow rate (10000 μL / min), and white circles (◯) indicate the results when the sample was injected into the flow path 41 at a low flow rate (500 μL / min). The result when the sample was injected into the inside is shown. As shown in FIG. 7, the hematocrit value Hct tends to increase as the time t to reach the equilibrium state becomes longer. That is, by measuring the time t until the measured equilibrium state is reached, the hematocrit value Hct of the sample can be determined based on the measured time t and the calibration curve prepared in advance.

The viscosity of the sample may vary depending on factors other than the proportion of blood cell components in the sample, but the influence of the other factors can be reduced by adjusting the dilution rate of the sample.

Further, it can be seen that when the sample is injected from the pipette tip 134 into the flow path 41, the time until the pressure in the pipette tip 134 reaches its peak becomes shorter as the flow rate of the sample increases (FIGS. 6A and 6B). See comparison). On the other hand, the time t to reach the equilibrium state is almost the same even if the flow rate of the sample is different. That is, the hematocrit value Hct of the sample can be determined without depending on the flow rate when the sample is injected into the flow path 41 from the pipette tip 134.

［上記式（１）において、Ｈｃｔはヘマトクリット値（０〜１００％）であり、ｄｆは検体の希釈倍率である。］ Finally, the measured value is corrected based on the hematocrit value (step S140). The fluorescence value includes a fluorescence component (signal component) derived from the fluorescence substance that labels the substance to be measured, and a noise component (optical blank value) caused by a factor other than the fluorescence substance. Therefore, the control processing unit 150 calculates a measured value (signal component) indicating the amount of the substance to be measured in the sample by subtracting the optical blank value obtained in the step S122 from the fluorescence value obtained in the step S125. can do. Further, the control processing unit 150 converts the calculated measured value into the amount of the substance to be measured in plasma by multiplying the calculated measured value by the conversion coefficient c represented by the following formula (1).

[In the above formula (1), Hct is a hematocrit value (0 to 100%), and df is a dilution ratio of the sample. ]

By the above procedure, the amount (concentration) of the substance to be measured in plasma can be determined.

In the above embodiment, the step of setting the incident angle to the augmented angle (step S121), the step of measuring the optical blank value (step S122), and the step of performing the primary reaction (step S123) are performed in this order. Was explained. However, the measuring method and the measuring device according to the present invention are not limited to this order. For example, the incident angle may be set to the augmentation angle after the primary reaction is performed, or the primary reaction may be performed after the optical blank value is measured.

Further, in the above embodiment, a secondary reaction (step S124) was carried out after the primary reaction (step S123) (two-step method). However, the timing of labeling the substance to be measured with the fluorescent substance is not particularly limited. For example, before introducing the sample into the flow path 41 of the measurement chip 10, a labeling solution may be added to the sample to label the substance to be measured with a fluorescent substance in advance. Further, the sample and the labeling solution may be simultaneously injected into the flow path 41 of the measuring chip 10. In the former case, by injecting the sample into the flow path 41 of the measurement chip 10, the substance to be measured labeled with the fluorescent substance is captured by the capture body. In the latter case, the substance to be measured is labeled with a fluorescent substance, and the substance to be measured is captured by the trap. In either case, both the primary reaction and the secondary reaction can be completed by introducing the sample into the flow path 41 of the measurement chip 10 (one-step method).

(effect)
In the present embodiment, the hematocrit value can be measured based on the viscosity of the sample that correlates with the hematocrit value by using the pressure sensor 135 for detecting the pressure in the pipette tip 134. Measuring devices that handle liquids usually have a pressure sensor. Therefore, according to the measuring method according to the present embodiment, when the measured value indicating the amount of the substance to be measured in the sample containing blood is corrected by the hematocrit value, the measuring device measures the hematocrit value. Even if it does not have a system, it is not necessary to add a measurement system separately. Therefore, it is possible to suppress the increase in cost and size of the measuring device.

In the above embodiment, the mode in which the hematocrit value of the sample is determined based on the information on the time change of the pressure in the pipette tip 134 when the sample is injected into the flow path 41 from the pipette tip 134 has been described. , The present invention is not limited to this aspect. For example, the hematocrit value of the sample may be determined based on the information on the time change of the pressure in the pipette tip 134 when the sample is sucked into the pipette tip 134 from the flow path 41.

Further, in the above-described embodiment, the embodiment in which the hematocrit value measurement step (step S130) is performed after the measurement value acquisition step (step S120) has been described, but the present invention is not limited to this embodiment. For example, in the step of performing the primary reaction (step S123), when the sample is injected into the flow path 41, the time change of the pressure in the pipette tip 134 may be detected. The measurement time can be shortened by simultaneously detecting the time change of the pressure in the pipette tip 134 and performing the primary reaction.

Further, in the above embodiment, an embodiment in which the fluorescence value of fluorescence β from a fluorescent substance is measured as a measurement value by using the SPFS method has been described, but the present invention is not limited to this embodiment. For example, the SPR method may be used to measure the amount of reflected light of the excitation light α as a measured value. Alternatively, in the present invention, the measured value may be acquired by using an ELISA method, an RIfS method, a QCM method, or the like.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

In this embodiment, the hematocrit value Hct is measured by using the micro hematocrit method or the viscosity method (the method for measuring the hematocrit value according to the above embodiment), and the amount of the substance to be measured obtained in advance is shown. The measured values were corrected by the hematocrit value Hct, and the measurement results were compared.

1. 1. Acquisition of measured values As a sample, 24 kinds of whole blood containing myocardial troponin (cTn) I to which heparin was added were used. The concentration of cTnI in the sample was measured using a high-sensitivity automatic immunoassay device (SPFS device according to the above embodiment).

2. 2. Microhematocrit method The above sample was placed in a glass capillary and centrifuged at 12000 rpm for 5 minutes using a known hematocrit centrifuge (Centec 3220; manufactured by Kubota Seisakusho Co., Ltd., “Centec” is a registered trademark of the same company). The hematocrit value of the sample after centrifugation was measured using the hematocrit measuring instrument attached to the hematocrit centrifuge. Then, based on the measured hematocrit value, the concentration of cTnI in the sample was corrected to the concentration of cTnI in plasma.

3. 3. Viscosity method Using a high-sensitivity automatic immunoassay device, from a pipette tip having an opening with an opening diameter of φ1 mm in the flow path of a measuring tip having a flow rate of 20 mm × 4.6 mm × 0.1 mm in size. The above sample (flow rate 10000 μL / min, liquid volume 100 μL) was injected. At this time, based on the time change of the pressure in the pipette tip, the time when the pressure in the pipette tip becomes 1% or less from the peak when the sample is injected into the flow path is measured, and based on the time. The hematocrit value of the sample was determined. Then, based on the measured hematocrit value, the concentration of cTnI in the sample was corrected to the concentration of cTnI in plasma.

Specimen No. And the cTnI concentration in the sample WB-cTnI, the hematocrit value Hct obtained by the microhematocrit method, and the cTnI concentration in plasma (correction value A (reference value)) until the equilibrium state obtained by the viscosity method is reached. Table 1 shows the time t, the hematocrit value Hct, the cTnI concentration in plasma (correction value B), and the deviation amount BiasB of the correction value B with respect to the reference value. In Table 1, BiasB is a value calculated by the following formula (2).

FIG. 8 is a graph showing the accuracy when the amount of the substance to be measured in plasma is determined by the viscosity method. In FIG. 8, the horizontal axis represents the average cTnI concentration, and the vertical axis represents BiasB, which is the amount of deviation of the correction value B with respect to the correction value A (reference value). As shown in FIG. 8 and Table 1, BiasB was within 5% in all the samples used in this example. As described above, it can be seen that the viscosity method can determine the amount of the substance to be measured in plasma with high accuracy in the same manner as the microhematocrit method. Compared to the microhematocrit method, the viscosity method does not require a separate device such as a centrifuge. According to the present invention, it is possible to easily and accurately measure a hematocrit value in the same device without separately preparing a device for measuring the hematocrit value.

According to the present invention, the hematocrit value and the amount of the substance to be measured can be detected with high reliability, which is useful for, for example, a disease test.

10 Measuring chip 20 Prism 21 Incident surface 22 Film formation surface 23 Exit surface 30 Metal film 40 Flow path lid 41 Flow path 42 Frame 43 Liquid injection section coating film 44 Liquid storage section coating film 45 Liquid injection section 46 Liquid storage section 47 Pore 48 Through hole for inserting pipette tip 50 Liquid tip 100 SPFS device 110 Excitation light emission unit 111 Light source unit 112 Angle adjustment mechanism 113 Light source control unit 120 Signal detection unit 121 Light receiving optical system unit 122 Position switching mechanism 123 First lens 124 Optical Filter 125 Second lens 126 Light receiving sensor 127 Sensor control unit 130 Liquid handling unit 131 Pipette 132 Syringe pump 133 Nozzle unit 134 Pipette tip 135 Pressure sensor 136 Pipette control unit 140 Transfer unit 141 Transfer stage 142 Chip holder 150 Control processing unit α excitation Light β Fluorescent γ Plasmon scattered light

Claims (14)

A measuring tip with a flow path held in the tip holder,
It has a pipette tip and a pressure sensor for detecting the pressure in the pipette tip, and while detecting the pressure in the pipette tip by the pressure sensor, a sample is injected from the pipette tip into the flow path. Or, a liquid handling unit for sucking a sample from the flow path into the pipette tip,
Is a method for measuring a hematocrit value for measuring a hematocrit value of a sample containing blood using the above.
In the liquid handling unit, the sample is placed in the flow path from the pipette tip while detecting the pressure in the pipette tip that changes according to the resistance that the sample receives from the inner wall surface of the flow path of the measurement tip. The pressure in the pipette tip that changes according to the resistance that the sample receives from the inner wall surface of the flow path of the measurement tip when injecting or sucking the sample into the pipette tip from the flow path. The process of acquiring information on time changes and
Based on the above information, a step of determining the hematocrit value of the sample from the relationship between the hematocrit value and the information on the time change of the pressure in the pipette tip acquired in advance ,
How to measure the hematocrit value, including. The flow path has a top surface, a bottom surface, and a pair of side surfaces connecting the top surface and the bottom surface.
The length between the pair of side surfaces is 0.1 mm or more and 5 mm or less.
The length between the top surface and the bottom surface is 0.1 mm or more and 1 mm or less.
The length of the flow path in the flow direction of the sample is 10 mm or more and 50 mm or less.
The method for measuring a hematocrit value according to claim 1. The first or second aspect, wherein the flow rate of the sample when the sample is injected into the flow path or the sample is sucked from the flow path is 10 μL / min or more and 15,000 μL / min or less. How to measure the hematocrit value of. The liquid amount of the sample when the sample is injected into the flow path or the sample is sucked from the flow path is 50 μL or more and 500 μL or less, according to any one of claims 1 to 3. How to measure the hematocrit value of. A method for measuring the amount of a substance to be measured, which is used to measure the amount of the substance to be measured in a sample containing blood.
A step of measuring the hematocrit value of the sample by the method for measuring the hematocrit value according to any one of claims 1 to 4.
A step of acquiring a measured value indicating the amount of the substance to be measured in the sample in a state where the substance to be measured contained in the sample is immobilized in the flow path and the sample is not present. When,
A step of correcting the measured value based on the hematocrit value, and
A method for measuring the amount of a substance to be measured, including. The measuring chip has a prism, a metal film arranged on the prism, and a flow path arranged on the metal film.
In the step of acquiring the measured value, the substance to be measured contained in the sample is immobilized on the metal film, and the sample is not present on the metal film via the prism. , The measured value is obtained by detecting a signal indicating the amount of the substance to be measured, which is generated when light is irradiated at an incident angle at which surface plasmon resonance occurs.
The method for measuring the amount of a substance to be measured according to claim 5. The method for measuring the amount of a substance to be measured according to claim 6, wherein the signal is fluorescence emitted from a fluorescent substance that labels the substance to be measured. A hematocrit measuring device for measuring the hematocrit value of a sample containing blood.
A tip holder for holding a measuring tip having a flow path,
The pipette has a pipette tip and a pressure sensor for detecting the pressure in the pipette tip, and the pressure sensor changes according to the resistance that the sample receives from the inner wall surface of the flow path of the measurement tip. A liquid handling unit for injecting a sample from the pipette tip into the flow path or sucking the sample from the flow path into the pipette tip while detecting the pressure in the tip.
Based on the information on the time change of the pressure in the pipette tip acquired by the pressure sensor, the sample is based on the relationship between the information on the time change of the pressure in the pipette tip and the hematocrit value obtained in advance . The processing unit that determines the hematocrit value,
A device for measuring hematocrit values. The flow path has a top surface, a bottom surface, and a pair of side surfaces connecting the top surface and the bottom surface.
The length between the pair of side surfaces is 0.1 mm or more and 5 mm or less.
The length between the top surface and the bottom surface is 0.1 mm or more and 1 mm or less.
The length of the flow path in the flow direction of the sample is 10 mm or more and 50 mm or less.
The hematocrit value measuring device according to claim 8. 8. The flow rate of the sample when the liquid handling unit injects the sample into the flow path or sucks the sample from the flow path is 10 μL / min or more and 15000 μL / min or less. The hematocrit value measuring device according to claim 9. Any of claims 8 to 10, wherein the liquid volume of the sample is 50 μL or more and 500 μL or less when the liquid handling unit injects the sample into the flow path or sucks the sample from the flow path. The hematocrit value measuring device according to item 1. A device for measuring the amount of a substance to be measured for measuring the amount of the substance to be measured in a sample containing blood.
The hematocrit value measuring device according to any one of claims 8 to 11.
The substance to be measured contained in the sample is immobilized in the flow path of the measurement chip held in the chip holder, and the sample to be measured in the sample does not exist. A measurement value acquisition unit for acquiring a measurement value indicating the amount of a substance,
Have,
The processing unit further corrects the measured value based on the hematocrit value.
A device for measuring the amount of a substance to be measured. The measuring chip has a prism, a metal film arranged on the prism, and a flow path arranged on the metal film.
The measured value acquisition unit
A light emitting part for emitting light and
In a state where the substance to be measured contained in the sample is immobilized on the metal film and the sample does not exist, the light emitting portion causes surface plasmon resonance on the metal film via the prism. A signal detection unit for detecting a signal indicating the amount of the substance to be measured, which is generated when light is irradiated at the incident angle generated.
Have,
The processing unit acquires the measured value based on the detection result of the signal detection unit.
The device for measuring the amount of a substance to be measured according to claim 12. The device for measuring the amount of a substance to be measured according to claim 13, wherein the signal is fluorescence emitted from a fluorescent substance that labels the substance to be measured.

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