JP2006052950A - Blood analyzer and blood analyzing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a blood analyzer capable of performing blood corpuscle/blood plasma separation by centrifugal operation and the feed of blood plasma and a calibration liquid, capable of performing an analysis of high precision by certainly discharging the calibration liquid from a sensor and capable of preventing the damage of the sensor caused by centrifugal force at the time of the blood corpuscle/blood plasma separation, and also provide a blood analyzing method. <P>SOLUTION: A blood corpuscle/blood plasma separation substrate and a sensor substrate for analyzing blood plasma are made attachable and detachable as separate bodies and blood corpuscle/blood plasma separation is performed by centrifugalizing only a blood corpuscle separation substrate. The sensor groove of the sensor substrate is arranged in a first centrifugal force applying direction with respect to the sensor substrate and a waste calibration liquid sump is arranged in a second centrifugal force applying direction. The calibration liquid in the calibration liquid sump is fed to the sensor groove by the centrifugation in a first centrifugal direction and centrifugalized in a second centrifugal direction after sensor calibration to discharge the calibration liquid from the sensor groove. After the discharge of the calibration liquid, the sensor substrate is connected to the blood corpuscle separation substrate and the calibration liquid is again centrifugalized in the first centrifugal direction to feed blood plasma to the sensor groove from the blood corpuscle separation substrate. The sensor part can be used so as to apply only weak centrifugal force to the sensor part and the damage of the sensor part is prevented to enable a highly precise analysis. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a blood analyzer constituted by an ultra-small groove channel fabricated on an insulating material substrate such as a quartz plate or a polymer resin plate. In particular, when a small amount (several μL or less) of blood is introduced into the groove channel on the substrate and centrifuged to separate blood cell components and plasma components and then measure various chemical substance concentrations in the plasma components Further, the present invention relates to a flow path and a substrate structure for carrying a liquid such as a calibration liquid or blood of an analysis sensor by centrifugal force.

  Conventional health examinations and diagnosis of disease states have been carried out by taking a large amount of blood of several cc from a patient and analyzing it from measurements obtained with a large-scale automatic blood analyzer. Usually, such an automatic analyzer is installed in a medical institution such as a hospital, has a large scale, and its operation is limited to those having specialized qualifications.

  However, in recent years, by applying the microfabrication technology used for the production of semiconductor devices that have advanced extremely recently, analyzers such as various sensors are arranged on a substrate of several millimeters to several centimeters square, and the blood of the subject is placed there. There is a growing interest in the development and practical application of new devices that can guide body fluids and instantly grasp the health status of subjects. With the advent of such inexpensive devices, health insurance benefits, which continue to increase, can be reduced by enabling daily health management of elderly people at home in the coming aging society. Also, in the field of emergency medicine, various social effects are expected, such as being able to take appropriate measures if this device can be used to quickly determine the presence or absence of infection (such as hepatitis or acquired immune deficiency). This is a technical field that is attracting a great deal of attention. As described above, instead of the conventional automatic analyzer, a small and simple blood analysis method and a blood analyzer aiming to perform blood analysis by one's own hands in each home have been developed (for example, see Patent Document 1). ).

JP 2001-258868 A

  FIG. 1 shows an example of a micro-module blood analyzer described in Patent Document 1. Reference numeral 101 denotes a lower substrate of the blood analyzer, and a fine groove channel (microcapillary) 102 formed by etching on the lower substrate is provided. On the lower substrate 101, an upper substrate (not shown) having substantially the same size is attached to seal the groove channel 102 from the outside.

  In the flow channel 102, a blood collecting means 103, a plasma separating means 104, an analyzing means 105, and a moving means 106 are sequentially provided from the most upstream part to the most downstream part. A hollow blood collection needle 103a is attached to the blood collection means 103 at the forefront of the flow path, and this needle 103a is pierced into the body to serve as an inlet for blood into the substrate. Separating means 104 is formed by curving the middle of the flow path 102 and is made of, for example, a U-shaped microcapillary. After the collected blood is guided to this U-shaped microcapillary, the substrate is accelerated in a certain direction by a centrifuge to precipitate blood cell components at the bottom of the U-shaped part and separate plasma as a supernatant. To do. The analysis means 105 is a sensor for measuring each concentration of blood such as pH value, oxygen, carbon dioxide, sodium, potassium, calcium, glucose, lactic acid.

  The moving means 106 located at the most downstream part of the flow path moves blood in the microcapillary by electroosmotic flow, and includes electrodes 107 and 108 and a flow path portion 109 connecting between the electrodes 107 and 108. The buffer solution previously filled in the flow path is moved to the downstream side of the flow path by the electroosmotic flow generated by applying a voltage between the electrodes, and the suction force that is generated moves the sampling means 103 at the forefront of the flow path 102 into the substrate. Take in blood. In addition, the plasma obtained by centrifugation is guided to the analysis means 105.

  Reference numeral 110 denotes an output means for taking out information from the analysis means, and is composed of electrodes and the like. Reference numeral 111 denotes a control means for controlling the collection means, plasma separation means, analysis means, movement means, and output means as necessary.

  The blood collected from the collection means 103 is separated into plasma and blood cell components by the separation means 104, and this plasma is guided to the analysis means 105, where the pH value in the plasma, oxygen, carbon dioxide, sodium, potassium, calcium, Each concentration of glucose, urea nitrogen, creatinine, lactic acid, etc. is measured. The movement of blood between each means is performed by the moving means 106 having a pumping capability such as those using phenomena such as electrophoresis and electroosmosis. In FIG. 1, the downstream area of the flow path 102 is divided into five, and an analysis means 105 and a movement means 106 are provided for each of them.

  A glass material such as quartz is often used for the substrate of such a blood analyzer, but it is more suitable for manufacturing the device in large quantities and at low cost, and in recent years considering the disposal at the time of disposable. Materials are starting to be used.

  In the conventional blood analyzer shown in FIG. 1, a moving means such as an electroosmotic pump 106 is required when a blood sample is introduced into the apparatus. After the introduced blood is centrifuged together with the substrate to obtain plasma, it is necessary to operate the electroosmotic pump 106 again in order to move the plasma to the analysis means 105. In addition, when the analyzing means is a sensor configured based on an electrochemical principle, it is necessary to calibrate the sensor using a calibration solution in advance. That is, before the plasma is guided to the sensor, the sensor must be immersed in the calibration solution to calibrate the sensor, and the calibration solution after calibration must be discharged from the analysis means. A moving means such as a pump is also required for transferring the calibration liquid.

  As the moving means, it is conceivable to use an electroosmotic pump provided in the same substrate as shown in FIG. 1 or a negative pressure pump installed outside the substrate. By these moving means, blood, plasma, calibration liquid, etc. Is moved by being pumped or sucked. At this time, in order to move the desired liquid to a desired position in the blood analyzer, it is necessary to accurately control the suction force of the moving means. For this purpose, there has been a problem that a liquid position sensor must be newly installed in or outside the blood analyzer, and the apparatus becomes expensive because of the addition of these control mechanisms and position sensors.

  When the analysis means is a sensor constructed on the basis of electrochemical principles, after calibrating the sensor with a calibration solution (standard solution) containing a known concentration of the test component, the calibration solution is discharged from the analysis means. There must be. However, even if the calibration liquid is discharged, some calibration liquid remains on the surfaces of the analysis means and the flow path means according to the wettability of the surface. As described above, the current blood analyzer is designed to analyze the concentration of various chemical substances present in a minute amount of blood of several microliters. ing. In general, when the size of an object is reduced, the ratio S / V of the surface area (S) to the volume (V) is increased, which means that the effect of the surface appears remarkably. Therefore, even if the amount of calibration solution remaining on the surface of the flow path means or the analysis means is small, an analyzer that introduces a small amount of plasma has a problem that the concentration of the measured chemical substance varies. For this purpose, it is necessary to discharge the calibration solution after the calibration from the analysis means before introducing the plasma into the analysis means.

  In view of such circumstances, the present inventors are blood analyzers that perform plasma separation in a flow path by centrifugal operation, and transport blood, plasma, and calibration fluid in the apparatus without using a pump or the like. In addition, an attempt was made to develop a blood analyzer that enables highly accurate analysis by reliably discharging the calibration solution from the sensor portion (see, for example, Patent Document 2).

Japanese Patent Application No. 2003-040481

  FIG. 2 shows an example of a blood analyzer described in Patent Document 2 (unpublished). Reference numeral 201 denotes an upper substrate on which a flow path is formed, and 202 denotes a lower substrate on which an electrode terminal 204 for taking out sensor electrodes 203 and sensor signals to the outside is formed. A blood collection needle 205 is attached to the upper substrate 201, and the collected blood is moved to the blood reservoir 207 through the guide channel 206 by a negative pressure of an external pump (not shown) from the suction / pressure feed port 208. The flow path 209 and the flow path 210 are connected to the opening holes 211 and 212 provided on the side wall of the upper substrate 201, respectively, but when sucking blood, the opening holes are provided by a holder (not shown) to which the blood analysis substrate is attached. 211 and 212 are closed. Similarly, the calibration liquid reservoir 213 stores the calibration liquid injected from the suction / pressure feeding port 208.

  An example of the operation of this blood analyzer substrate will be described next. First, when the blood analyzer substrate is centrifuged around the first centrifugal force center 214, the calibration liquid in the calibration liquid reservoir 213 passes through the guide channels 215 and 216, and a plurality of sensors 203 are accommodated. It is carried into the sensor groove 217. After calibration of the sensor 203, the blood analyzer substrate is rotated 90 degrees clockwise and placed on the centrifuge. That is, when the substrate is centrifuged around the second centrifugal force center 218 located on the left side in FIG. 2, the calibration liquid filling the sensor groove 217 passes through the guide flow paths 216 and 219 and the calibration liquid waste liquid reservoir 220. Is housed in.

  Thereafter, the blood analyzer substrate is turned back 90 degrees counterclockwise and placed on the centrifuge. That is, when the substrate is centrifuged around the first centrifugal force center 214, the blood is conveyed from the blood reservoir 207 to the sensor groove 217 via the guide channel 221. If centrifugal force is continuously applied in this state, blood cell components in the blood are fractionated in the direction in which gravity is applied, that is, below the sensor groove 217, and plasma components are separated as a supernatant above the sensor groove 217. Since the sensor group 203 is arranged in that region, the pH value in the blood, oxygen, carbon dioxide, sodium, potassium, calcium, glucose, lactic acid, and other concentrations are connected via a plurality of electrode terminals 204 connected to each sensor. Measured by an external measuring instrument.

  This hematology analyzer is capable of centrifugal operation in two different directions, and the calibration liquid in the calibration liquid reservoir is conveyed to the sensor unit by the centrifugal operation in the first centrifugal direction. By centrifuging in the centrifugal direction 2, the calibration liquid can be reliably discharged from the sensor unit. After discharging the calibration liquid, the blood in the blood reservoir can be transported to the sensor unit and separated into blood cells and plasma by centrifuging in the first centrifugal direction.

    However, while having these advantages, a problem that cannot be ignored in order to perform blood analysis in a short time due to the use of centrifugal force.

  Needless to say, it is important that the measurement time by the blood analyzer chip is as short as possible. In this blood analyzer, the distance from the centrifugal center axis to the chip center is 5 cm, and the time required for injection and discharge of the calibration liquid is usually about 1 second even under a low centrifugal force of 3000 rpm or less. However, in order to separate blood cells and plasma in blood in a few seconds to several minutes, a centrifugal force of at least 4000 rpm is required in the blood cell separation region. FIG. 15 shows the relationship between the rotational speed (rpm) and the acceleration (G) at this time, which corresponds to the application of 500 G gravity acceleration at 3000 rpm and 1000 G gravity acceleration at 4000 rpm.

  As a result of the examination, it was found that the output of the sensor decreases when the centrifugal operation for separating blood cells and plasma by such a large gravitational acceleration is performed. For example, looking at the output voltage when the calibration solution (containing 137 mM sodium ions) was measured with a sodium ion sensor, it was affected by the centrifugal rotation speed (rpm) as shown in FIG. Up to about 3000 rpm, the sensor output shows a stable value of about 200 mV. However, at higher speeds, the sensor output shows a decreasing tendency and the dispersion of values increases. In this measurement, a sensor showing a stable value of about 200 mV up to 1000 rpm was prepared and used for each rotation experiment. Although not particularly shown, the same tendency was found in potassium ion measurement.

  The sodium ion concentration measurement sensor uses Bis (12-crown-4), an ion-sensitive membrane that captures sodium ions, and an anion that plays a role in preventing plasma anions (anions) from entering the sensitive membrane. An excluding agent is mixed with PVC (polyvinyl chloride), and this is immobilized on a carbon electrode to form a sensor. At that time, a large amount of plasticizer is mixed with the PVC in order to facilitate the incorporation of sodium ions into the sensitive membrane. If the centrifugal force at 7000 rpm is estimated from the weight per sensor, the force applied to the sensor is about piconewton. However, the cause of the decrease in the sensor output at this high rotational speed is that the PVC film containing the plasticizer containing the ion sensitive film is deformed on the carbon electrode by a strong centrifugal force, and a part of the PVC film is a carbon electrode. It is assumed that it was because of water intrusion. Although it is conceivable to change the composition of the film to cure the film and enhance the fixation of the film to the carbon electrode, curing the film loses the characteristics of the original electrochemical sensor.

  The present invention has been made in view of such circumstances, and is a blood analyzer that separates plasma by a centrifugal operation, and can transport plasma and calibration fluid in the apparatus without using a pump or the like. A first object of the present invention is to provide a blood analyzer that can discharge a calibration solution from a sensor part reliably and that can be analyzed more accurately without damaging the sensor due to centrifugation during plasma separation. And

  In addition, the present invention can transport plasma and calibration liquid in the apparatus only by centrifugal operation, and the calibration liquid can be surely discharged from the sensor portion, and the sensor may be damaged by the centrifugal operation during plasma separation. A second object is to provide a blood analysis method that enables analysis with higher accuracy.

  According to the present invention, a first object is a sensor substrate having a blood cell / plasma separation substrate for separating blood cells and plasma, and a sensor for analyzing a test component of a blood liquid component, and the blood cell / plasma separation substrate And a blood analysis device comprising a detachable sensor substrate.

  Here, the blood cell / plasma separation substrate includes a U-shaped channel for separating blood cells and plasma when a centrifugal force is applied; a blood cell reservoir provided at a curved portion (lowermost part) of the U-shaped channel; A plasma outlet for discharging plasma to the sensor substrate. The sensor substrate includes a sensor groove for storing the sensor; a calibration liquid reservoir for storing a calibration liquid for calibrating the sensor; a calibration liquid waste liquid reservoir for storing the calibration liquid after sensor calibration; a calibration liquid reservoir and a sensor groove. A calibration fluid introduction channel that communicates; a calibration fluid discharge channel that communicates the sensor groove with the calibration fluid waste reservoir; and a sensor groove that communicates with the plasma outlet when the blood cell / plasma separation substrate is assembled to the sensor substrate. And a plasma introduction channel for introducing plasma into the plasma.

  The sensor groove is a position on the side where the calibration liquid in the calibration liquid reservoir can be stored when a centrifugal force is applied to the sensor board in the first centrifugal direction, and the blood cell / plasma separation board is used as the sensor board. When assembled and applied with a centrifugal force in the first centrifugal direction, the blood cell / plasma separation substrate is disposed at a position where the plasma can be accommodated through the plasma introduction flow path. The calibration liquid waste reservoir is positioned on the side where the calibration liquid in the sensor groove can be accommodated in the calibration liquid waste reservoir by applying a centrifugal force in the second centrifugal direction.

  That is, the blood analyzer of the present invention is configured such that a substrate for blood cell plasma separation and a sensor substrate for analyzing a test component in the separated plasma are separated and removable. The blood cell / plasma separation is performed by centrifuging only the blood cell / plasma separation substrate at a high rotation, and the strong centrifugal force at this time prevents the sensor in the sensor substrate from being damaged. The sensor substrate can be independently centrifuged in two different directions, and the calibration liquid in the calibration liquid reservoir is conveyed to the sensor groove by the centrifugal operation in the first centrifugal direction, and the second centrifugal direction after the sensor calibration. The calibration solution can be reliably discharged from the sensor groove by centrifugation. Centrifugation at this time can be performed at a low rotation, and the sensor can be prevented from being damaged due to excessive centrifugal force.

  After the calibration liquid is discharged, the blood cell / plasma separation substrate is coupled to the sensor substrate, and the plasma separated in the plasma / separation substrate is conveyed to the sensor groove in the sensor substrate by centrifugation in the first centrifugal direction. I can do it. Since the centrifugal force at this time does not need to be an excessive centrifugal force required for blood cell / plasma separation, the sensor is not damaged. If the blood cell reservoir provided on the blood cell / plasma separation substrate is curved and recessed so as to be directed to the first centrifugal direction side from the U-shaped flow path, the blood cell fraction is transferred to the sensor substrate during plasma transfer by centrifugation. It can be prevented from flowing out.

  It is preferable that the first centrifugal direction in which the calibration liquid and plasma are transported to the sensor groove and the second centrifugal direction in which the calibration liquid is discharged from the sensor groove are substantially orthogonal. For example, when a sensor groove is provided on the lower side of a quadrangular sensor substrate, a calibration solution waste reservoir is provided on the right side (or left side) substantially orthogonal to the lower side. The plasma introduction channel and the calibration solution reservoir are located at the center, right side, or upper side of the substrate. When the blood cell / plasma separation substrate is combined with the sensor substrate, the U-shaped flow path in the blood cell / plasma separation substrate and its plasma outlet are located above the sensor substrate. That is, the blood cell / plasma separation substrate and the sensor substrate are such that the first centrifugal force pressurization direction (plasma transport direction) of the sensor substrate and the U-shaped flow path extending direction of the blood cell / plasma separation substrate are substantially orthogonal to each other. Preferably it is arranged. However, the first and second centrifugal directions do not necessarily need to be substantially orthogonal. When the plasma is introduced into the sensor groove by centrifuging in the first centrifugal direction, the calibration liquid waste reservoir and the calibration liquid waste flow path may be provided so that the calibration liquid does not flow back into the sensor groove.

  A plurality of sensor grooves may be provided, and a plurality of sensors for analyzing different test components may be accommodated in each sensor groove. In this case, the calibration liquid introduction flow path branches and communicates with each of the plurality of sensor grooves on the first centrifugal force pressurizing direction side (substrate lower side). In that case, it is preferable that the plurality of sensor grooves be arranged substantially in parallel with the first centrifugal force pressing direction.

  If the blood collection needle can be attached to the blood introduction port of the U-shaped flow path of the blood cell / plasma separation substrate, the whole blood collected from the blood collection needle can be introduced into the U-shaped flow path. Alternatively, a blood collection mechanism that stores already collected blood may be attached to the blood inlet. A blood sample can be smoothly introduced by hydrophilizing the U-shaped channel. When the plasma introduction channel, the calibration solution introduction channel, the calibration solution reservoir, and the sensor groove of the sensor substrate are respectively hydrophilicized, the calibration solution conveyance and the plasma conveyance become smoother.

A second object of the present invention is a blood analysis method comprising the following steps:
(1) A U-shaped channel that separates blood cells and plasma when centrifugal force is applied, a blood cell reservoir provided in a curved portion of the U-shaped channel, and a U-shaped channel Providing a blood cell / plasma separation substrate having a plasma outlet for discharging plasma;
(2) A blood sample is introduced into the U-shaped channel, the blood cell / plasma separation substrate is centrifuged so that the blood reservoir is in the direction of centrifugal force pressurization, and blood cell / plasma separation is performed in the U-shaped channel. Let the blood cell fraction settle in the blood cell reservoir;
(3) A sensor groove for storing the sensor, a calibration liquid reservoir for storing a calibration liquid for calibrating the sensor; a calibration liquid waste liquid reservoir for storing the calibration liquid after sensor calibration; and a communication between the calibration liquid reservoir and the sensor groove. A calibration liquid introduction flow path; a calibration liquid discharge flow path that communicates the sensor groove and the calibration liquid waste reservoir; and a blood cell / plasma separation substrate that is communicated with the discharge port when the blood cell / plasma separation substrate is assembled to the sensor groove. Providing a sensor substrate with a plasma introduction channel to be introduced;
(4) introducing the calibration liquid in the calibration liquid reservoir into the sensor groove by centrifuging the sensor substrate in the first centrifugal direction so that the sensor groove is in the direction of centrifugal force pressurization;
(5) Calibrate the sensor;
(6) Disposing the calibration liquid in the sensor groove to the calibration liquid reservoir by arranging and centrifuging the sensor substrate in the second centrifugal direction so that the calibration liquid reservoir is in the centrifugal force pressurizing direction;
(7) Combine the two substrates so that the plasma outlet of the blood cell / plasma separation substrate and the plasma introduction channel of the sensor substrate communicate with each other;
(8) By placing and centrifuging the combined substrates in the first centrifugal direction so that the sensor groove is in the direction of centrifugal force pressurization, the plasma fractionated in the U-shaped flow path can be obtained. Introduced into the sensor groove;
(9) The liquid component in the plasma in the sensor groove is analyzed by the sensor.
Is achieved.

  4 and 5 are plan views of a blood cell / plasma separation substrate of the blood analyzer according to one embodiment of the present invention, FIG. 6 is a plan view of a sensor substrate combined with the blood cell / plasma separation substrate, and FIG. 7 is a blood cell / plasma separation substrate. It is a top view which shows the state which couple | bonded the board | substrate and the sensor board | substrate. FIG. 4 shows a blood cell separation substrate when a blood sample is introduced, and FIG. 5 shows a blood cell / plasma separation substrate in a state where blood cells / plasma are separated by centrifugation.

  In these drawings, reference numeral 10 denotes a blood cell / plasma separation substrate (hereinafter also referred to as a blood cell separation substrate), and an upper substrate is laminated and bonded to a lower substrate in which a flow path is formed. The upper and lower substrates are made of a resin such as polyethylene terephthalate (PET) or polycarbonate (PC). Inside the substrate 10, there are provided a U-shaped channel 12 extending in the vertical direction and bent at the lower part, and a blood reservoir 14 provided at the lower end thereof. A blood inlet 16 is provided upstream of the U-shaped channel, and a plasma outlet 18 is provided downstream for discharging separated plasma out of the substrate.

  A blood collection mechanism 20 that contains already collected blood is inserted into the blood introduction port 16. The blood collection mechanism 20 is integrally formed with a stainless painless needle 22 having an outer diameter of 100 μm, a reinforcing stainless tube 24, and a primary blood reservoir 26 for storing the blood after blood collection. It is sterilized and stored, and after blood collection, it is inserted into the blood inlet 16 of the substrate 10. By sucking with negative pressure from the opening hole 18, the blood is conveyed to the entire area inside the U-shaped flow path 12. If the blood collection mechanism 20 is inserted into the blood introduction port 16 of the substrate 10 in advance, a blood sample can be introduced into the U-shaped channel 12 simultaneously with blood collection by suction from the opening hole 18.

  When the blood cell separation substrate 10 is fixed to a centrifuge (not shown) and centrifuged about the centrifugal force center C1, the whole blood 28 in FIG. 4 is fractionated into plasma 30 and blood cell fraction 32 as shown in FIG. Is done. That is, the blood cell fraction 32 is precipitated in the blood cell reservoir 14, and the plasma fraction 30 of the centrifugal supernatant is separated above the U-shaped channel 12. If this centrifugation operation is performed while the blood collection mechanism 20 is inserted into the blood introduction port 16 of the substrate 10, introduction of a blood sample into the U-shaped channel 12 and blood cell plasma separation can be performed simultaneously. .

  In addition, a connecting pipe (not shown) for liquid-tight coupling with a plasma introduction path opening of a sensor substrate 40 described later is provided in the opening hole 18 serving as a plasma discharge port on the back side of the substrate 10. Further, connection protrusions 34 a and 36 a and connection locking holes 34 b and 36 b are provided on the back surface or the upper surface of the substrate 10, and can be coupled to the sensor substrate 40.

  The sensor substrate 40 shown in FIG. 6 is obtained by stacking upper and lower substrates made of a resin such as polyethylene terephthalate (PET) or polycarbonate (PC), and a flow path structure is provided between the substrates. That is, a calibration solution reservoir 42 is provided at the upper left of the sensor substrate 40 at the center of the substrate, and an opening hole 44 that opens to the substrate surface side and is coupled to the plasma outlet 18 of the blood cell separation substrate 10 is provided. A plurality of sensor grooves 46 are provided below, and a calibration liquid waste reservoir 48 is provided on the right side of the substrate.

  Reference numeral 50 denotes a calibration liquid introduction flow path for introducing the calibration liquid from the calibration liquid reservoir 42 into the sensor groove 46, and branches below the sensor grooves 46 and communicates with the respective sensor grooves 46. The calibration liquid introduction flow path 50 communicates with the calibration liquid discharge flow path 52, whereby the sensor groove 46 communicates with the calibration liquid reservoir 48. Reference numeral 52 a denotes a backflow prevention weir for preventing the backflow of the calibration liquid from the calibration liquid waste liquid reservoir 48 to the sensor groove 46. Reference numeral 54 denotes an air escape passage.

  Reference numeral 56 denotes a plasma introduction channel, which communicates with the plasma outlet 18 of the blood cell separation substrate 10 through the opening hole 44 and moves the plasma 30 moving from the U-shaped channel 12 of the blood cell separation substrate 10 by centrifugation to the sensor groove 46. To introduce.

  Each sensor groove 46 accommodates a sensor 58, and the output of each sensor is guided to each electrode terminal 62 exposed to the outside of the substrate through each wiring 60. The sensor 58 includes, for example, an electrode such as silver / silver chloride or carbon, and a silver / silver chloride reference electrode. The wiring 60 is made of, for example, silver-containing carbon, and the external electrode 62 is made of, for example, silver. These silver / silver chloride, carbon electrode, silver / silver chloride reference electrode, silver-containing carbon wiring, silver electrode and the like are formed by screen printing, for example.

  Protrusions 64 a and 66 a and through holes 64 b and 66 b are provided on the upper surface of the sensor substrate 40. When the blood cell separation substrate 10 of FIG. 5 is rotated 90 degrees counterclockwise and superimposed on the sensor substrate 40 as shown in FIG. 7, the protrusions 64a and 66a are engaged with the locking holes 34b and 36b of the blood cell separation substrate. Then, the protrusions 34a and 36a of the blood cell separation substrate are inserted into the through holes 64b and 66b. At this time, the connection tube of the plasma outlet 18 of the blood cell separation substrate 10 is inserted into the opening hole 44 of the sensor substrate 40.

  A method of using this blood analyzer will be described with reference to FIGS. First, the sensor is calibrated before blood analysis.

  The calibration liquid is introduced from the opening hole 42 a of the sensor substrate 40 in FIG. 6 to fill the calibration liquid reservoir 42. By filling the calibration liquid reservoir 42, a calibration liquid having a volume of approximately 1 μL is weighed. This calibration solution may be introduced immediately before blood analysis, or may be placed in the calibration solution reservoir 42 in advance. After introducing the calibration liquid into the sensor substrate 40, the substrate is attached to the centrifuge and the centrifugal operation is performed about the centrifugal axis C1. By centrifuging the sensor groove 46 on the pressure direction side of the centrifugal force F <b> 1, the calibration liquid passes through the calibration liquid introduction channel 50 and is conveyed to each sensor groove 46 and covers the sensor 58. At that time, the air in the sensor groove 46 is expelled from the opening hole 44. In this state, each sensor is calibrated using the wiring 60 and the external electrode terminal 62.

  After the sensor is calibrated, the calibration solution in the sensor groove is discharged. The sensor substrate 40 is removed from the centrifuge, rotated 90 degrees clockwise, and set in the centrifuge. That is, the sensor substrate 40 is centrifuged around the second centrifugal center axis C2, and a centrifugal force is applied in the direction F2. The calibration liquid moves to the calibration liquid waste liquid reservoir 48 via the calibration liquid discharge channel 52 by centrifugal force, and the calibration liquid discharge is completed. The centrifugal force can remove the calibration liquid covering the sensor, and the error of the analysis value due to the residual calibration liquid is eliminated.

  It should be noted that the centrifugal force used for conveying and discharging the calibration liquid is preferably a gravitational acceleration that does not damage the sensor 58, and the gravitational acceleration applied to the sensor 58 is preferably 500 G or less.

  Blood cell / plasma separation is performed by centrifuging only the blood cell separation substrate 10. When the whole blood sample 28 is introduced into the blood cell separation substrate 10 shown in FIG. 4 and then centrifuged around the centrifugal axis C1, plasma and blood cells are separated, and the blood cell fraction 32 is precipitated in the blood cell reservoir 28, and the plasma 30 is fractionated as a supernatant in the upper part of the U-shaped channel 12. The centrifugal operation at this time is for completely separating blood cells, and it is preferable to apply a gravitational acceleration of 1000 G or more to the lowermost part of the U-shaped flow path.

  The blood cell separation substrate 10 from which the plasma has been separated is overlapped and bonded to the sensor substrate 40 that has already been subjected to sensor calibration. That is, the blood cell separation substrate 10 of FIG. 5 is rotated 90 degrees counterclockwise, overlapped with the sensor substrate of FIG. 6, and fixed so that the locking protrusions and the locking holes of both substrates are engaged with each other. As a result, the protruding tube of the plasma discharge port 18 protruding from the back surface of the blood cell separation substrate 10 and the opening hole 44 of the sensor substrate 40 are connected. Both integrated substrates are as shown in FIG.

  Then, both the substrates 10 and 40 are centrifuged around the centrifugal axis C1, and a centrifugal force is applied in the first centrifugal direction F1. The plasma 30 is conveyed to the entire area of each sensor groove 46 through the plasma discharge port 18, the opening hole 44, and the plasma introduction channel 56. At that time, the air in the sensor groove 46 is expelled from the opening hole 42a of the calibration liquid reservoir 42 that is already empty. FIG. 7 shows the state of both substrates after plasma transfer.

  Finally, both substrates 10 and 40 are removed from the centrifuge, and the test components in the plasma stored in the sensor grooves 46 are analyzed by the sensor electrodes 58.

  In the above-described embodiment, the blood cell separation substrate 10 is overlapped on the upper surface of the sensor substrate 40 and bonded three-dimensionally. However, as shown in FIG. 8, both the substrates 10A and 40A may be bonded in a plane. In this second embodiment, the plasma outlet 18A of the U-shaped flow path 12 of the blood cell separation substrate 10A opens to the side surface of the substrate 12A, and the insertion tube 18B is provided at the tip thereof. The sensor board 40A is provided with a receiving port 68 that is fluid-tightly engaged with the insertion tube 18B, and a plasma introduction channel 56A communicates therewith. The opening hole 44 shown in FIG. 6 is omitted. In addition, the protrusions for locking and the engaging holes are omitted from both substrates. Other configurations are the same as those of the first embodiment. The operation method other than the method of joining both the substrates 10A and 40A is the same as that of the first embodiment.

  9A to 9J show an example in which a series of operations of sensor calibration, blood cell plasma separation, plasma transport, and test component are automated using the blood analyzer of the first embodiment. Here, reference numeral 70 denotes a centrifuge rotating table that applies a centrifugal force, and a rotatable first table 72 and a rotatable second table 74 are arranged on opposite sides of the rotating table. A substrate feeding mechanism, a substrate fixing / demounting mechanism to / from the turntable 70, a calibration liquid introducing mechanism, a calibration and measurement instrument for a test component, etc. are not shown.

  First, the sensor substrate 40 is placed on the first table 72 with the external electrode 62 facing outward (FIG. 9A). In this state, the sensor substrate 40 is fixed to the first area 76 indicated by the dotted line on the turntable 70, and the calibration liquid is injected into the calibration liquid reservoir 42 (FIG. 9B). The rotating table 70 is rotated to introduce the calibration liquid into the sensor groove 46 by centrifugal force, the rotation of the sensor substrate 40 is stopped so as to come to the position on the first table 72 side, and the sensor substrate 40 is moved to the first table 74. Let the sensor calibrate here.

  After the sensor calibration, the first table is rotated to rotate the sensor substrate 40 by 90 degrees in the clockwise direction, and then again moves to the first region 76 of the turntable 70 and is fixed here (FIG. 9 (c)). The rotary table 70 is rotated and the calibration liquid is conveyed to the calibration liquid waste reservoir 48 by centrifugal force. The rotation is stopped so that the sensor substrate 40 is located in the second region 78 located in front of the second table 74, and the sensor substrate 40 that has been calibrated is transported and retracted from the second region to the second table (FIG. 9 ( d)).

  During this centrifugation operation, the blood cell separation substrate 10 into which the blood collection mechanism 20 (see FIG. 4) in which blood is collected and stored with a painless needle is inserted on the first table 72 in advance. When the sensor substrate 40 is retracted to the second table, the blood cell separation substrate 10 is moved to the first region 76 on the turntable 70 and fixed. During this time, the second table 74 is rotated to rotate the sensor substrate 40 counterclockwise by 90 degrees (FIG. 9 (e)). The whole blood sample accommodated in the blood collection mechanism in the blood cell separation substrate 10 is rotated by rotating the turntable 70 and separated into blood cells and plasma by centrifugal force, and the blood cell separation substrate 10 is in the first table 72. The rotation of the turntable 70 is stopped so as to come to the side (FIG. 9 (f)). After the blood cell separation, the blood cell separation substrate 10 is moved to the first table 72, rotated 90 degrees counterclockwise, and then again moved to the first region 76 on the turntable 70 and fixed (FIG. 9 (g)). . The turntable 70 is rotated 180 degrees, and the blood cell separation substrate 10 is moved to the second region 78 facing the second table 74 (FIG. 9 (h)). Thereafter, the sensor substrate 40 that has been retracted on the second table is moved onto the blood cell separation substrate 10, and the two substrates are coupled (FIG. 9 (i)). In this state, the turntable 70 is rotated to transport the plasma separated in the blood cell separation substrate 10 by centrifugal force to the sensor substrate 40, and the rotation is stopped at the same position as in FIG. 9 (i). The sensor substrate 40 to which the plasma has been transferred is moved to the second table 74, where the test component is analyzed.

  As described above, the blood analyzer of the present invention is capable of carrying in and discharging the calibration solution into the sensor groove, separating the blood cells, and introducing the blood plasma into the sensor unit by centrifugal force without using any pump. is there. There is no need to use a conventional negative pressure pump, and an inexpensive and simple blood analyzer can be realized. Further, since the blood cell / plasma separation substrate and the sensor substrate can be detached, the blood cell separation can be reliably performed by applying a strong centrifugal force to the blood cell / plasma separation substrate. Carrying in and discharging the calibration liquid into the sensor groove can be performed by centrifuging only the sensor substrate with a weak centrifugal force. The plasma separated by the blood cell / plasma separation substrate can be introduced into the sensor groove with a weak centrifugal force after the two substrates are combined. Therefore, it can be used so that only weak centrifugal force is applied to the sensor. The multi-layered sensor composed of different components is not damaged by the strong centrifugal force at the time of blood cell separation, and more accurate analysis is possible.

It is a conceptual diagram which shows an example of the conventional blood analyzer made into the micromodule. It is a whole perspective view of the blood analyzer (unknown) which inventors proposed. It is a figure which shows that an output voltage when a calibration liquid (137 mM sodium ion containing) is measured with a sodium ion sensor changes with centrifugal rotation speed (rpm). FIG. 3 is a plan view of a blood cell / plasma separation substrate of the blood analyzer according to one embodiment of the present invention, showing the time of blood introduction. The blood cell / plasma separation substrate of FIG. 4 shows a state where blood cell / plasma separation is performed by centrifugation. It is a top view of a sensor substrate combined with a blood cell / plasma separation substrate. FIG. 3 is a plan view of the blood analyzer of the first embodiment of the present invention in which a blood cell / plasma separation substrate after separation of blood cells and a sensor substrate are combined and integrated, and plasma is conveyed into a sensor groove by centrifugation. Indicates. It is a top view of the blood analyzer by 2nd embodiment of this invention. It is a figure which shows a series of operation | movement which measures a to-be-tested component using the blood analyzer of this invention. It is a figure which shows the relationship between the gravity acceleration (G) produced when a rotary body with a radius of 50 mm rotates, and rotation speed (rpm).

Explanation of symbols

10, 10A Blood cell / plasma separation substrate (blood cell separation substrate)
12 U-shaped channel (blood cell / plasma separation means)
14 Blood cell reservoir 16 Blood inlet 18, 18A Plasma outlet (opening hole)
18B Insertion tube 20 Blood collection mechanism 22 Blood collection needle 26 Primary blood reservoir 26
28 Whole blood 30 Plasma fraction 32 Blood cell fractions 34a, 36a, 66a, 68a Connection projections 34b, 36b, 66b, 68b Connection locking holes 40, 40A Sensor substrate 42 Calibration liquid reservoir 44 Opening hole 46 Sensor groove 48 Calibration Liquid waste reservoir 50 Calibration liquid introduction flow path 52 Calibration liquid discharge flow path 54 Air escape flow path 56, 56A Plasma introduction flow path 58 Sensor 60 Wiring 62 External electrode terminal 68 Insertion tube receiving port 70 Turntable 72 A rotatable first table 74 A rotatable second table 76 A first area 78 on the turntable A second area C1 on the turntable C1 First centrifugal force center C2 Second centrifugal force center

Claims (13)

In a blood analyzer that separates whole blood samples by centrifugation and analyzes the test components in the blood fluid components:
(a) a blood cell / plasma separation substrate for separating blood cells and plasma;
(b) a sensor for analyzing a test component of a blood liquid component, and a blood cell / plasma separation substrate and a removable sensor substrate;
The blood cell / plasma separation substrate is:
(a-1) a U-shaped channel that separates blood cells and plasma when a centrifugal force is applied;
(a-2) a blood cell reservoir provided in the curved portion of the U-shaped flow path;
(a-3) a plasma discharge port for discharging plasma to the sensor substrate;
The sensor substrate is:
(b-1) a sensor groove for accommodating the sensor;
(b-2) a calibration fluid reservoir containing calibration fluid for calibrating the sensor;
(b-3) a calibration liquid waste reservoir containing calibration liquid after sensor calibration;
(b-4) a calibration fluid introduction flow path that communicates the calibration fluid reservoir and the sensor groove;
(b-5) a calibration liquid discharge channel that communicates the sensor groove with the calibration liquid waste reservoir;
(b-6) provided with a plasma introduction flow path for introducing plasma into the sensor groove in communication with the plasma outlet when the blood cell / plasma separation substrate is assembled to the sensor substrate;
The sensor groove is a position on the side where the calibration liquid in the calibration liquid reservoir can be accommodated when a centrifugal force is applied to the sensor board in the first centrifugal direction, and the blood cell / plasma separation board is detected by the sensor groove. Disposed at a position where plasma can be accommodated through the plasma introduction channel from the plasma outlet of the blood cell / plasma separation substrate when the centrifugal force is applied to the substrate in the first centrifugal direction;
The calibration liquid waste reservoir is disposed at a position where the calibration liquid in the sensor groove can be accommodated in the calibration liquid waste reservoir by applying a centrifugal force in the second centrifugal direction. Analysis equipment.   In the blood cell reservoir, a blood cell / plasma separation substrate is assembled to a sensor substrate, and a centrifugal force is applied to both substrates in a first centrifugal direction to discharge a plasma fraction in the U-shaped channel to the sensor substrate. 2. The blood analyzer according to claim 1, wherein the blood analyzer has a volume enough to prevent the blood cell fraction in the blood cell reservoir from flowing out, and is recessed toward the first centrifugal direction.   3. The blood analyzer according to claim 1 or 2, wherein the sensor groove is a plurality of sensor grooves in which sensors for analyzing different test components are accommodated.   4. The blood analyzer according to claim 3, wherein the calibration solution introduction channel branches and communicates with each of the plurality of sensor grooves on the first centrifugal force pressurizing direction side.   The blood analyzer according to claim 1, wherein the sensor is an electrochemical sensor.   The blood analyzer according to claim 1, wherein a blood collection needle or a blood collection mechanism storing already collected blood can be attached to the blood introduction port of the U-shaped channel.   The U-shaped flow path of the blood cell / plasma separation substrate, the plasma introduction flow path, the calibration liquid introduction flow path, the calibration liquid reservoir, and the sensor groove of the sensor board are respectively subjected to a hydrophilic treatment. Item 7. The blood analyzer according to items 1 to 6.   The blood analyzer according to claim 1, wherein the first centrifugal force pressurizing direction and the second centrifugal force pressurizing direction are substantially orthogonal to each other.   The blood cell / plasma separation substrate and the sensor substrate can be coupled to each other so that the first centrifugal force pressurization direction of the sensor substrate and the U-shaped flow path extending direction of the blood cell / plasma separation substrate are substantially orthogonal to each other. The blood analyzer according to any one of claims 1 to 8, wherein: Blood analysis method consisting of the following steps:
(1) A U-shaped channel that separates blood cells and plasma when centrifugal force is applied, a blood cell reservoir provided in a curved portion of the U-shaped channel, and a U-shaped channel Providing a blood cell / plasma separation substrate having a plasma outlet for discharging plasma;
(2) A blood sample is introduced into the U-shaped channel, the blood cell / plasma separation substrate is centrifuged so that the blood reservoir is in the direction of centrifugal force pressurization, and blood cell / plasma separation is performed in the U-shaped channel. Let the blood cell fraction settle in the blood cell reservoir;
(3) A sensor groove for storing the sensor, a calibration liquid reservoir for storing a calibration liquid for calibrating the sensor; a calibration liquid waste liquid reservoir for storing the calibration liquid after sensor calibration; and a communication between the calibration liquid reservoir and the sensor groove. A calibration liquid introduction flow path; a calibration liquid discharge flow path communicating with the sensor groove and the calibration liquid waste reservoir; Providing a sensor substrate with a plasma introduction channel to be introduced;
(4) introducing the calibration liquid in the calibration liquid reservoir into the sensor groove by centrifuging the sensor substrate in the first centrifugal direction so that the sensor groove is in the direction of centrifugal force pressurization;
(5) Calibrate the sensor;
(6) Disposing the calibration liquid in the sensor groove to the calibration liquid reservoir by arranging and centrifuging the sensor substrate in the second centrifugal direction so that the calibration liquid reservoir is in the centrifugal force pressurizing direction;
(7) Combine the two substrates so that the plasma outlet of the blood cell / plasma separation substrate and the plasma introduction channel of the sensor substrate communicate with each other;
(8) By placing and centrifuging the combined substrates in the first centrifugal direction so that the sensor groove is in the direction of centrifugal force pressurization, the plasma fractionated in the U-shaped flow path is sensored. Introduced into the groove;
(9) The liquid component in the plasma in the sensor groove is analyzed by the sensor.   The blood analysis method according to claim 10, wherein the first centrifugal force pressurizing direction and the second centrifugal force pressurizing direction are substantially orthogonal to each other.   The blood analysis method according to any one of claims 10 and 11, wherein a gravitational acceleration applied to the sensor by centrifugation performed in steps (4), (6), and (8) is 500 G or less.   The blood analysis method according to claim 10, 11, or 12, wherein the gravitational acceleration applied to the U-shaped flow path performed by centrifugation in step (2) is 1000 G or more.