JP5354947B2 - Bioanalytical device and sample quantitative stirring method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a living body analyzing device capable of realizing the quantitation of a sample liquid, a reagent reaction and inspection at the same portion. <P>SOLUTION: A liquid in a sample quantifying capillary (109) is transferred to a sample photometry part (106) placed at more outer circumferential side of rotary drive than the sample quantifying capillary (109) by a centrifugal force generated with rotary drive, the liquid in the sample photometry part (106) is arranged to be sucked by the capillary force of the sample quantifying capillary (109) by reducing the centrifugal force, and a flow velocity V1 for sucking a liquid along one sidewall being close to a connection port (116) is arranged to be lower than a flow velocity V2 for sucking a liquid along the other sidewall being distant from the connection port (116). Accordingly, the quantitation of a sample liquid, sample agitation and measurement can be realized at the same portion by using both a capillary force and a centrifugal force. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to an analysis device used for analyzing a sample collected from a living organism and the like, and an analysis method using the same, and more specifically, quantification of a sample solution and a sample solution and a reagent in the analysis device. This relates to the stirring technology.

  2. Description of the Related Art Conventionally, as a method for optically analyzing a biological fluid, a method for analyzing using a microdevice having a liquid flow path is known. Microdevices can control fluids using a rotational analyzer, and can use centrifugal force to quantitate samples, separate cytoplasmic materials, transfer and distribute separated fluids, etc. Various biochemical analyzes can be performed.

Patent Document 1 can be cited as an analysis method for performing quantification of a sample, reaction with a reagent, and detection using centrifugal force and capillary force.
FIG. 7 shows the technique of Patent Document 1.

  In this analytical device, a whole blood separation chamber 31 into which whole blood (blood) 45 as a specimen is injected from the injection port 31a in the direction of the arrow 15 from the side closest to the axis serving as the centrifugal force source toward the outer periphery. , A separation chamber 32, a quantitative chamber 33, and a reaction / detection chamber 34 are provided.

  The whole blood separation chamber 31 and the separation chamber 32 are connected to the separation chamber 32 via a flow path 35, a reagent chamber 36, a flow path 37, a stirring unit 38, and a flow path 39. The separation chamber 32 and the quantitative chamber 33 are connected via a flow path 40. The quantitative chamber 33 and the reaction / detection chamber 34 are connected via a flow path 41.

  In the conventional analysis method, the sample liquid generated in the whole blood separation chamber 31 and the separation chamber 32 is transferred to the quantitative chamber 33 by applying a centrifugal force to the analysis device. And about the sample liquid which flowed into the fixed_quantity | quantitative_assay chamber 33, the sample liquid which flowed in excess is discharged | emitted from the discharge port 33c, and the flowed-in sample liquid is quantified according to the volume of the fixed_quantity | quantitative_assay chamber 33.

Next, when the centrifugal force is stopped, the sample solution is filled up to the outlet 41c by the capillary force and stopped by the surface tension. When the centrifugal force is applied again to the analysis device, all of the sample liquid in the quantitative chamber 33 is transferred to the reaction / detection chamber 34. The sample liquid transferred to the reaction / detection chamber 34 reacts by touching the reaction reagent 43, and the sample liquid can be analyzed by detecting the absorbance of the reacted sample liquid by an optical method.
JP 2006-214955 A

  However, in Patent Document 1, two chamber configurations, a quantification chamber 33 and a reaction / detection chamber 34, are required as a minimum configuration for quantifying a sample solution and performing a reagent reaction and detection. Reaction / detection cannot be performed at the same site.

  Therefore, when the quantification chamber 33 is set to a depth at which the capillary force acts and the quantification / reaction / detection of the sample liquid is configured at the same site, the sample is sucked up by the capillary force acting on the quantification chamber 33. At this time, the sample solution is discharged from the discharge port 33c, and there is a problem that the quantitative property of the sample solution cannot be maintained.

  An object of the present invention is to solve the above-mentioned conventional problems, and to provide a bioanalytical device capable of realizing the quantification of the sample solution and the reaction with the reagent at the same site and a quantitative stirring method using the same.

In the biological analysis device according to claim 1 of the present invention, a microchannel is formed from the vicinity of the rotation center toward the outer circumferential direction, and the liquid is transferred around the rotation channel by being driven to rotate around the rotation center. The microchannel is a device for biological analysis, in which the sample holding portion into which the sample solution is injected performs sample solution quantification and reagent reaction via a capillary siphon formed toward the center of rotation. The sample liquid capillary is connected to a connection port on one side wall on the inner peripheral side as viewed from the rotation center of the sample quantification capillary to be measured, and the sample liquid reacted with the reagent on the outer peripheral side as viewed from the rotation center of the sample quantification capillary is measured. Before the sample photometry unit is sucked up by the capillary force by the sample metering unit and the liquid transferred from the sample metering capillary to the sample photometry unit by the centrifugal force generated by the rotation drive A first recess is provided so as to surround the sample quantification capillary, and the sample quantification capillary has a second recess formed along a side wall close to the connection port, and a side wall away from the connection port. The second concave portion is deeper than the sample quantitative capillary.

The bioanalytical device according to claim 2 of the present invention is characterized in that, in claim 1, the sample quantitative capillary carries a reagent that reacts with the sample solution.
According to a third aspect of the present invention, there is provided the bioanalytical device according to the first aspect, wherein an outer peripheral end of the rotational drive of the sample quantitative capillary is formed so as to protrude into the sample photometric unit. .

The bioanalytical device according to claim 4 of the present invention is characterized in that, in claim 3 , the sample quantitative capillary is connected to a side wall in the outer peripheral direction of the sample photometry unit.
The bioanalytical device according to claim 5 of the present invention is characterized in that, in claim 1, the depth of the sample fixed capillary is the same as the depth of the capillary siphon or shallower than the depth of the capillary siphon.

According to a sixth aspect of the present invention, the bioanalytical device according to the first aspect is characterized in that an outer peripheral end of the rotational drive of the capillary siphon protrudes into the sample holding portion.

According to a seventh aspect of the present invention, in the bioanalytical device according to the sixth aspect , the outer peripheral end of the rotational drive of the capillary siphon is separated from the bottom in the outer peripheral direction of the sample holder. .

The bioanalytical device according to an eighth aspect of the present invention is characterized in that, in the first aspect, a hydrophilic treatment is applied to a part or the whole of the inner wall of the sample fixed capillary and the capillary siphon.

In the sample quantitative stirring method according to claim 9 of the present invention, the sample solution to be analyzed is injected into the sample holder of the bioanalytical device according to claim 1, and the rotation and stop of the bioanalytical device are repeated. The sample solution and the reagent are mixed and stirred inside the sample quantification capillary by the centrifugal force generated by rotating the bioanalysis device and the capillary force in the sample quantification capillary of the bioanalysis device.

In the sample fixed quantity stirring method according to claim 10 of the present invention, in claim 9 , the rotational speed for generating the centrifugal force is such that the centrifugal force applied to the sample liquid transferred to the sample fixed capillary is stronger than the capillary force. It is the number of revolutions.

  According to the configuration of the present invention, it is possible to perform the determination of the sample solution and the reaction with the reagent at the same site.

DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments of a bioanalytical device and a quantitative stirring method using the same according to the present invention will be described in detail with reference to the drawings.
FIG. 1 shows a bioanalytical device 101 according to an embodiment of the present invention.

  The bioanalytical device 101 is configured by bonding a circular base substrate 102 and a circular cover substrate 103 together. FIG. 2 shows a bonding surface of the base substrate 102 to the cover substrate 103, and FIG. 3 shows a cross-sectional view taken along the line A-AA in FIG.

  On the bonding surface of the base substrate 102 to the cover substrate 103, a plurality of recesses having different depths are formed, and a microchannel 114 formed from a capillary channel, a storage unit, an inspection unit, and the like is formed. The base substrate 102 and the cover substrate 103 are both made of a synthetic resin material, and the microchannel 114 is formed at the time of injection molding of the base substrate 102 or by cutting the base substrate 102. The cover substrate 103 is bonded to the base substrate 102 so as to cover the concave portion formed in the base substrate 102 to form a microchannel 114.

The microchannel 114 will be described in detail.
As shown in FIG. 1, the microchannel 114 is formed from the vicinity of the rotation center 104 toward the outer peripheral direction of the base substrate 102.

  Specifically, the sample holding unit 105 arranged at a position away from the rotation center 104, the sample quantification capillary 109 for performing the quantification of the sample liquid 118 and the reaction with the reagent 112, and the sample liquid reacted with the reagent 112 A sample photometry unit 106 for optically measuring 118, and a sample holding unit 105 and a sample quantitative capillary 109 are connected by a capillary siphon 107 having a siphon apex 108 at a position closest to the rotation center 104. Has been.

  A first recess 110 deeper than the depth of the sample quantitative capillary 109 is formed around the sample quantitative capillary 109. This is to prevent air bubbles from being mixed during the centrifugal transfer or capillary transfer of the sample liquid 118 by forming a passage through which air flows around the capillary quantification unit 109. The depth relationship between the sample fixed capillary 109 and the capillary siphon 107 is formed at the same depth, but the sample fixed capillary 109 may be formed shallower than the capillary siphon 107.

  The sample fixed capillary 109 is formed so as to protrude into the sample holder 105 and the sample photometer 106. Furthermore, the sample fixed capillary 109 protruding into the sample holding part 105 is formed so as not to be connected to the outer peripheral side wall of the sample holding part 105.

  The sample quantitative capillary 109 protruding into the sample photometry unit 106 is formed so as to be connected to the outer peripheral side wall of the sample photometry unit 106. This is because in the sample holding unit 105, the liquid level of the sample liquid 118 transferred into the sample holding unit 105 is transferred to the sample quantitative capillary 109 only when it reaches the sample quantitative capillary 109. This is to prevent unnecessary sample liquid 118 after quantification from being mixed into the sample quantification capillary 109. Further, the sample photometry unit 106 is for transferring all the quantified sample solution 118 to the sample quantification capillary 109 in order to improve the reactivity between the sample solution 118 and the reagent 112.

  Further, in the sample fixed amount capillary 109, a second concave portion 117 is formed on the capillary siphon 107 side, and a convex portion 111 is formed at a position away from the capillary siphon 107. Specifically, a second recess 117 is formed along the side wall (109s) close to the connection port 116 and in contact with the side wall (109s). As shown in FIG. 3, the second concave portion 117 is deeper than the sample quantitative capillary 109, and the convex portion 111 is formed in contact with the cover substrate 103.

  With such a configuration, when the sample liquid 118 is sucked up by capillary force, the flow velocity V1 is slower than the flow velocity V2 on the convex portion 111 side on the concave portion 110 side, and the sample liquid from the side far from the capillary siphon 107 is obtained. 118, the liquid surface tends to shrink due to the surface tension acting on the sample liquid 118 as the sample liquid 118 approaches the connection port 116, so that the sample liquid 118 is finally stopped at the connection port 116. Thus, backflow to the capillary siphon 107 can be prevented.

  Conventionally, when there is no such configuration of the second concave portion 117 and the convex portion 111, when the sample liquid 118 is sucked up by a capillary tube, the liquid surface speed V1 is faster than V2, so the sample liquid Before 118 fills the sample fixed capillary 109, it flows into the capillary siphon 107, and the quantitative property of the sample liquid 118 cannot be maintained.

A reagent 112 for measuring the characteristics of the sample liquid 118 is applied to the surface of the sample fixed capillary 109, and the type of the reagent 112 can be changed depending on the measurement contents.
Next, the configuration of the bioanalytical device 101 will be specifically described.

  The bioanalytical device 101 according to the present invention includes a base substrate 102 and a cover substrate 103, and each substrate includes an injection molded or cut substrate. The thicknesses of the base substrate 102 and the cover substrate 103 are 1 mm to 5 mm, but are not particularly limited as long as the microchannel 114 can be formed. As for the shapes of the base substrate 102 and the cover substrate 103, a circular shape is preferable when the bioanalytical device 101 is rotated alone, but the bioanalytical device 101 is configured to be mounted on an external attachment. In the case of making it, there is no particular limitation, and a shape according to the purpose of use, for example, a shape such as a quadrangular shape, a triangular shape, a sector shape, or other complicated shapes is possible.

  Synthetic resin is used as the material of the base substrate 102 and the cover substrate 103 from the viewpoint of easy moldability, high productivity, and low cost, but there is a limit as long as it is a material that can be joined, such as glass, silicon wafer, metal, and ceramic There is no.

  The base substrate 102 and the cover substrate 103 are subjected to hydrophilic treatment on part or all of the wall surfaces in order to reduce the viscous resistance in the microchannels 114 formed on the respective substrates and promote fluid movement. It is also possible to use a hydrophilic material such as glass, or to add a hydrophilic agent such as a surfactant, a hydrophilic polymer, or a silica powder such as silica gel during molding to impart hydrophilicity to the material surface. . Examples of the hydrophilic treatment method include a surface treatment method using an active gas such as plasma, corona, ozone, and fluorine, and a surface treatment with a surfactant. Here, the hydrophilic property means that the contact angle with water is less than 90 °, and more preferably the contact angle is less than 40 °. It is sufficient that at least a part or the entire inner wall of the sample fixed capillary 109 and the capillary siphon 107 is subjected to a hydrophilic treatment.

  In this embodiment, the base substrate 102 and the cover substrate 103 are bonded by ultrasonic welding, but may be bonded by a bonding method such as an adhesive bonding sheet, anodic bonding, or laser bonding depending on the material to be used. Absent.

Next, a process from injection of the sample liquid 118 to transfer and measurement of the components of the sample liquid 118 will be described in detail.
5 (a), 5 (b), and 5 (c) are schematic diagrams for explaining from the injection of the sample liquid 118 to the quantitative distribution, and FIG. 6 (a), FIG. 6 (b), FIG. FIGS. 6C and 6D are schematic views showing a state in which the sample liquid 118 is transferred into the capillary.

  As shown in FIG. 5A, the sample liquid 118 is injected into the sample holding unit 105 from the injection port 115 with a pipette 501. The injected sample liquid 118 fills the sample holding unit 105 as shown in FIG. 5 (b), is sucked up by the capillary force 502 working in the capillary siphon 107, and is transferred to the sample fixed capillary 109. Further, when the injection is continued, the sample fixed capillary 109 is filled with the sample liquid 118. At that time, the sample liquid 118 is not transferred to the first and second recesses 110 and 117.

  This is because the depth of the first and second concave portions 110 and 117 is formed deeper than the depth of the sample quantitative capillary 109, so that the sample quantitative capillary 109 and the first and second concave portions 110 and 117 have a depth. This is because the capillary force 502 is cut off at the connecting portion and the interface of the sample solution is maintained by the surface tension, thereby preventing the sample solution from entering the first and second recesses 110 and 117. Further, when the sample liquid 118 comes into contact with the reagent 112 applied to the sample fixed capillary 109, the reagent 112 is dissolved by the sample liquid 118 and the reaction is started.

  Here, it is said that the capillary force generally has a large influence when the inner diameter of the capillary is 2.5 mm or less, and the surface acting between the contact angle between the wall surface and the liquid and the gas-liquid interface. This is the force by which the liquid inside the capillary moves due to the force that tries to maintain the balance between the tensions.

Next, in order to quantify the sample liquid 118, the centrifugal force 503 is generated by rotating the bioanalytical device 101 around the rotation center 104.
As a result, the sample liquid 118 is transferred to the sample holder 105 and the sample photometer 106 arranged in the outer circumferential direction by the centrifugal force 503 as shown in FIG. At that time, the sample liquid 118 is quantified by being divided at the siphon apex 108, and the sample liquid 118 that has started to dissolve the reagent 112 is transferred to the sample photometric unit 106 together with the dissolved reagent 112, and unnecessary sample liquid is obtained. 118 is transferred to the sample holder 105.

  More specifically, the rotational speed at this time is set so that the force applied to the sample liquid 118 transferred to the sample fixed capillary 109 is 500 G or more, and the sample liquid 118 is securely held between the sample photometry unit 106 and the sample. It can be transferred to the unit 105. In this embodiment, the rpm is 2500 rpm, which is the rotational speed at which the centrifugal force applied to the sample liquid transferred to the sample fixed capillary is stronger than the capillary force.

After the sample liquid 118 is reliably transferred to the sample holding unit 105 and the sample photometry unit 106, the capillary force of the sample quantitative capillary 109 becomes dominant by stopping the rotation, and the sample photometry is performed as shown in FIG. The sample liquid 118 quantified in the unit 106 is transferred again to the sample quantification capillary 109. At that time, since the capillary force on the side wall side where the second concave portion 117 is formed becomes weaker than the capillary force on the side wall side where the convex portion 111 is formed, the sample liquid 118 reaches the convex surface 111. Between the velocity V2 of the liquid surface and the velocity V1 of the liquid level reaching the second recess 117,
V1 <V2
A speed difference is generated. In this state, as shown in FIG. 6B, the liquid surface at the speed V2 approaches the vicinity of the connection port 116 first. Further, as shown in FIG. 6 (c), when the liquid surface at the speed V1 and the liquid surface at the speed V2 approach each other, a force for reducing the area of the liquid surface is generated by the surface tension St of the liquid surface of the sample liquid 118. Since it becomes dominant, the liquid level finally stops at the connection port 116 as shown in FIG.

  By utilizing such a flow mechanism of the sample liquid 118, it is possible to control the liquid level to reach the connection port 116 when the sample liquid 118 is all sucked into the sample fixed capillary 109. It is possible to prevent 118 from being transferred to the capillary siphon 107 beyond the siphon apex 108, and the quantitative property of the sample liquid 118 can be maintained.

  On the other hand, the unnecessary sample liquid 118 transferred to the sample holder 105 is disposed so that the liquid surface does not reach the capillary, that is, the outer peripheral end of the rotational drive of the capillary siphon 107 is in the outer peripheral direction of the sample holder 105. Since it is separated from the bottom, it is held by the sample holder 105.

After all the sample liquid 118 is transferred to the sample quantitative capillary 109, the sample liquid 118 sucked into the sample quantitative capillary 109 by centrifugal force is transferred again to the sample photometric unit 106.
In this way, by generating or stopping the centrifugal force, the sample liquid 118 is alternately transferred between the sample photometry unit 106 and the sample quantitative capillary 109, and the interface of the sample liquid 118 contacts the reagent 112 many times. Thus, dissolution and stirring of the reagent 112 are promoted, and the reagent 112 can be surely dissolved.

After the sample liquid 118 and the reagent 112 are mixed and stirred by this method, a centrifugal force is finally generated and transferred to the sample photometry unit 106.
The mixed liquid of the transferred sample liquid 118 and the reagent 112 can be analyzed by measuring by an optical method.

  In this bioanalytical device 101, by configuring such a microchannel 114, it is possible to realize the determination of the sample liquid 118 and the reaction with the reagent 112 at the same site.

FIG. 4 shows the analyzer 401.
The analysis apparatus 401 includes a rotation driving unit 407 for rotating the biological analysis device 101, an optical measurement unit 402 for optically measuring a solution in the biological analysis device 101, and a rotation speed of the biological analysis device 101. Control unit 408 for controlling the rotation direction and the measurement timing of the optical measurement unit, a calculation unit 404 for processing a signal obtained by the optical measurement unit 402 and calculating a measurement result, and a calculation unit 404 And a display unit 405 for displaying the result. The optical measurement unit 402 includes a laser light source 406 and a photodetector 403.

  After the sample liquid 118 to be inspected and the reagent 112 are reacted, the sample photometric unit 106 is irradiated with transmitted light from the laser light source 406, the reaction state is received by the photodetector 403, analyzed by the arithmetic unit 404, and displayed. At 405, the measurement result is displayed.

  At the time of this measurement, since the reaction solution filled in the sample photometry unit 106 changes the absorbance at the rate of reaction, the sample photometry unit 106 is irradiated with transmitted light from the laser light source 406, and the amount of the transmitted light is detected by the photodetector 403. By measuring the above, it is possible to analyze the components of the sample liquid 118 by measuring the change in the amount of light transmitted through the reaction liquid.

  The present invention can realize the quantification of the sample solution and the reagent reaction at the same site, and thus can contribute to the spread of a small-sized bioanalyzer used in a clinic or the like.

1 is an exploded perspective view of a bioanalytical device according to an embodiment of the present invention. Plan view of a bonding surface of base substrate 102 to cover substrate 103 in the same embodiment A-AA sectional view of FIG. 2 in the same embodiment Configuration diagram of analyzer in the same embodiment Process diagram of injection process from sample liquid injection in the same embodiment Process diagram of capillary transfer state of sample liquid in the same embodiment Plan view of a conventional bioanalytical device

Explanation of symbols

101 Bioanalytical device 102 Base substrate 103 Cover substrate 104 Center of rotation 105 Sample holding unit 106 Sample photometric unit 107 Capillary siphon 108 Siphon apex 109 Sample quantitative capillary 110 First concave portion 111 Convex portion 112 Reagent 113 Air hole 114 Micro channel 115 Note Inlet 116 Connection port 117 Second recess 118 Sample liquid 401 Analyzer 402 Optical measurement unit 403 Photo detector 404 Calculation unit 405 Display unit 406 Laser light source 407 Rotation drive unit 408 Control unit 502 Capillary force 503 Centrifugal force V1, V2 Flow rate St Surface tension

Claims (10)

A device for bioanalysis in which a microchannel is formed from the vicinity of a rotation center toward an outer peripheral direction, and is driven to rotate around the rotation center to control liquid transfer inside the microchannel.
The microchannel is
A sample holding part into which the sample liquid is injected is connected to one of the inner peripheral sides as viewed from the rotation center in the sample quantification capillary that performs the quantification of the sample liquid and the reagent reaction via the capillary siphon formed toward the rotation center. Connected to the side wall side connection port ,
A sample photometry unit for measuring the sample solution that has reacted with the reagent on the outer peripheral side when viewed from the rotation center of the sample fixed capillary,
And a first recess arranged to surround the sample quantification capillary that sucks up the liquid transferred from the sample quantification capillary to the sample photometry unit by the capillary force generated by the rotational drive by the capillary force;
In the sample fixed capillary,
A second recess formed along the side wall proximate to the connection port;
Providing a convex portion formed along the side wall away from the connection port,
The second recess is a bioanalytical device deeper than the sample quantitative capillary. The bioanalytical device according to claim 1, wherein the sample quantification capillary carries a reagent that reacts with the sample solution. The bioanalytical device according to claim 1, wherein an outer peripheral end of the rotational drive of the sample fixed capillary is formed so as to protrude into the sample photometry unit. The bioanalytical device according to claim 3, wherein the sample quantification capillary is connected to a side wall in an outer peripheral direction of the sample photometry unit. The bioanalytical device according to claim 1, wherein a depth of the sample fixed capillary is the same as a depth of the capillary siphon or shallower than a depth of the capillary siphon. The bioanalytical device according to claim 1, wherein an outer peripheral end of the rotational drive of the capillary siphon is formed so as to protrude into the sample holding portion. The bioanalytical device according to claim 6, wherein an outer peripheral end of the rotational drive of the capillary siphon is separated from a bottom portion in an outer peripheral direction of the sample holding unit. The bioanalytical device according to claim 1, wherein a hydrophilic treatment is applied to a part of or the entire inner wall of the sample fixed capillary and the capillary siphon. The sample liquid to be analyzed is injected into the sample holder of the bioanalytical device according to claim 1,
Repeat the rotation and stop of the bioanalytical device,
A sample quantitative stirring method in which a sample liquid and a reagent are mixed and stirred inside a sample quantitative capillary by a centrifugal force generated by rotating the biological analytical device and a capillary force in the sample quantitative capillary of the biological analytical device. The sample quantitative stirring method according to claim 9, wherein the rotational speed for generating the centrifugal force is a rotational speed at which the centrifugal force applied to the sample liquid transferred to the sample quantitative capillary is stronger than the capillary force.

Images