JP2010243373A - Analysis method and analysis apparatus in analysis device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analysis method and an analysis device with improved analytical precision, regardless of the fluctuations in the working accuracy, at measurement spots. <P>SOLUTION: The analysis method includes steps of feeding a sample liquid to a first reaction chamber (43), containing a first reagent and analyzing a first reaction liquid (62) of the first reagent and the sample liquid by transmitting light through the first reaction chamber (43); feeding and reacting the first reaction liquid (62) to a second reaction chamber (44a), containing a second reagent from the first reaction chamber (43); and feeding a second reaction liquid (63) of the reaction product of the second reagent from the second chamber (44a) to the first chamber (43) and the first reaction liquid (62) and analyzing, by transmitting light through the second reaction liquid (63) of the first reaction chamber (43). <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an analytical device used for analyzing a liquid collected from a living organism, an analytical apparatus and an analytical method using the analytical device, and more specifically, a solution mixed in the analytical device is described below. The present invention relates to a technique for transferring to a process.

Patent Document 1 describes an analysis method for analyzing a plurality of items of a sample solution using the analysis device 1.
As shown in FIGS. 4 and 5, the analysis apparatus used for this analysis is configured to rotate the analysis device holding member 103 and optically measure the solution in the analysis device 1. The optical measurement means 109, the control means 108 for controlling the rotation speed and direction of the analysis device holding member 103, the measurement timing of the optical measurement means, the signal obtained by the optical measurement means 109, and the measurement result It is comprised with the calculating part 110 for calculating, and the display part 111 for displaying the result obtained by the calculating part 110. FIG.

  The rotation driving means 107 not only rotates the analysis device 1 around the axis 102 in a predetermined direction at a predetermined rotation speed via the analysis device holding member 103 but also rotates the axis 102 at a predetermined stop position. The analyzing device 1 can be swung by reciprocating left and right with a predetermined amplitude range and period at the center.

  The optical measuring unit 109 includes a light source 105 for irradiating light to the measurement unit of the analyzing device 1 and a photo detector for detecting the amount of transmitted light that has passed through the analyzing device 1 out of the light emitted from the light source 105. 106. When the analysis device holding member 103 is made of a material having poor translucency or a material that is not translucent, holes 51 and 52 are formed at the mounting position of the analysis device 1 on the analysis device holding member 103. Yes.

  28 to 30 show measurements in a denaturation reaction chamber 38, an immunoassay chamber 43, and an agglutination reaction chamber 46, which are three measurement spots among a plurality of measurement spots having a microchannel structure formed inside the analysis device 1. FIG. Shows the process.

  By rotating the analytical device holding member 103 from FIG. 28A, a prescribed amount of the diluted solution held in the holding cavity 35 is transferred to the denaturation reaction chamber 38 via the connecting channel 37, and the denaturation reaction chamber 38 The denaturing reagent previously supported on 38 is dissolved.

Thereafter, at the position shown in FIG. 28B, the motor 104 is controlled so as to give the analytical device 1 a swing of about ± 1 mm, and the denaturing solution 61 in the denaturing reaction chamber 38 is stirred.
Next, after the analysis device 1 is stopped and the denaturing solution 61 is subjected to a denaturation reaction, the analysis device holding member 103 is rotated to perform the first measurement.

  In the first measurement, the denaturing solution 61 subjected to the denaturation reaction in the denaturation reaction chamber 38 of the analytical device 1 is read at a timing interposed between the light source 105 and the photodetector 106. The calculation unit 110 displays the modified hemoglobin concentration obtained by processing the measurement value obtained by the first measurement based on the reference value obtained by reading the diluent overflow chamber 29 in advance on the display unit 111.

  Next, the analysis device 1 is set to the position shown in FIG. 29A, and the motor 104 is controlled at a frequency of 1500 rpm so as to give the analysis device 1 a swing of about ± 1 mm. The denaturing solution 61 held in the tube is transferred to the denaturing solution quantification chamber 39, which is constituted by a capillary cavity, and a predetermined amount of the denaturing solution 61 is held in the denaturing solution quantification chamber 39.

  Next, by rotating the analysis device holding member 103, the denaturing solution 61 flows from the denaturing solution quantification chamber 39 into the immunoassay chamber 43 via the connection channel 41, and is previously carried in the immunoassay chamber 43. Dissolve the latex reagent.

  Thereafter, at the position shown in FIG. 29 (b), the motor 104 is controlled at a frequency of 1000 rpm so as to give a swing of about ± 1 mm to the analyzing device 1, and the immunoassay solution 62 in the immunoassay chamber 43 is stirred. .

Next, after the analysis device 1 is stopped and the second reaction liquid 62 is subjected to an antigen-antibody reaction, the analysis device holding member 103 is rotated to perform the second measurement.
In the second measurement, reading is performed at a timing when the second reaction liquid 62 that has reacted with the antigen-antibody in the immunoassay chamber 43 is interposed between the light source 105 and the photodetector 106 in a light emission state in which the wavelength of the light source 105 is switched to 625 nm.

  Next, the analysis device 1 is set to the position shown in FIG. 30A, and the motor 104 is controlled at a frequency of 1500 rpm so as to give the analysis device 1 a swing of about ± 1 mm. Capillary transfer to an immunoassay quantification chamber 44 consisting of capillary cavities.

  Thereafter, the analysis device holding member 103 is rotated (left rotation indicated by C 1, 2000 rpm), whereby a prescribed amount of the second reaction liquid 62 held in the immunoassay quantification chamber 44 is aggregated via the connection channel 45. It flows into the reaction chamber 46 and dissolves the agglutinating reagent carried in the agglutination reaction chamber 46.

  Thereafter, the motor 104 is controlled at a frequency of 1000 rpm so as to give the analytical device 1 a swing of about ± 1 mm at a position near 180 ° shown in FIG. 46 aggregation solution 63 is stirred.

  Next, after the analysis device 1 is stopped and the aggregation solution 63 is agglutinated, the analysis device holding member 103 is rotated (left rotation indicated by C1 · 1500 rpm) to perform the third measurement.

  In the third measurement, reading is performed at the timing when the aggregation solution 63 that has undergone the aggregation reaction in the aggregation reaction chamber 46 is interposed between the light source 105 and the photodetector 106. The calculation unit 110 digitizes the measurement values obtained by the second measurement and the third measurement by processing the dilution liquid overflow chamber 29 based on a reference value read in advance with the wavelength of the light source 105 set to 625 nm. The display unit 111 displays the HbA1c concentration and the HbA1c% value calculated based on the modified hemoglobin concentration.

JP 2009-31116 A

  In the analysis method using the analysis device and the analysis apparatus, in order to increase the accuracy of the analysis, processing is required to make the thickness of the immunoassay chamber 43 and the agglutination reaction chamber 46 uniform. It is.

  An object of the present invention is to provide an analysis method and an analysis apparatus that can improve the accuracy of analysis even if the processing accuracy of measurement spots varies.

  The analysis method for an analysis device according to claim 1 of the present invention is performed by transmitting light to the reaction solution in the measurement spot of the analysis device having a microchannel structure that transports the solution toward the measurement spot by centrifugal force. In doing so, the step of supplying the sample solution to the first reaction chamber having the first reagent and transmitting the light to the first reaction chamber to perform analysis of the first reaction solution of the first reagent and the sample solution Supplying the first reaction liquid from the first reaction chamber to the second reaction chamber having the second reagent and reacting the second reaction liquid, and reacting the second reagent and the first reaction liquid with each other. Supplying the first reaction chamber from the second reaction chamber and transmitting light to the second reaction solution in the first reaction chamber to perform analysis.

  The analysis apparatus according to claim 2 of the present invention is an analysis apparatus that analyzes light by transmitting light through a reaction solution in the measurement spot of an analytical device having a microchannel structure that transports a solution toward the measurement spot by centrifugal force. A rotation driving means for rotating the set analysis device to generate the centrifugal force, and a mode for controlling the rotation drive means to swing the set analysis device and the centrifugal force. And a control means for performing an analysis by transmitting light to the measurement spot of the set analysis device and providing the first reagent with the first reagent. A sample solution is supplied to the first reaction chamber of the analytical device and the light is transmitted through the first reaction chamber so that the first reagent and the The first reaction solution is analyzed with the liquid material, the first reaction solution is supplied from the first reaction chamber to the second reaction chamber of the analytical device having the second reagent, and the second reaction chamber is reacted. A second reaction solution obtained by reacting a reagent and the first reaction solution is supplied from the second reaction chamber to the first reaction chamber, and light is transmitted through the second reaction solution in the first reaction chamber to perform analysis. It is characterized by having comprised.

  According to this configuration, the sample solution is supplied to the first reaction chamber of the analytical device having the first reagent, and light is transmitted through the first reaction chamber to analyze the first reaction solution of the first reagent and the sample solution. The first reagent is supplied from the first reaction chamber to the second reaction chamber of the analytical device having the second reagent, and the second reagent and the first reaction liquid are reacted. The second reaction liquid in which the reaction has occurred is returned from the second reaction chamber to the first reaction chamber, light is transmitted again to the same first reaction chamber, the analysis is performed, and the first reaction is performed using the same first reaction chamber. Since the measurement of the liquid and the measurement of the second reaction liquid are performed, it is possible to improve the accuracy of the analysis.

The principal part perspective view of the state which set the device for analysis in the analyzer of the embodiment of the present invention Exploded perspective view of the analysis device of the same embodiment External view of the analyzer of the same embodiment Configuration of the same embodiment and a conventional analyzer Sectional view of the same embodiment and a conventional analyzer The figure which shows the rotation stop position of the device for analysis in the embodiment A plan view and a sectional view of the dilution unit opening part in the same embodiment An enlarged perspective view around the injection port and its front view in the same embodiment A plan view showing a microchannel configuration of the analysis device according to the embodiment The top view which shows the cross-sectional position of the device for analysis in the embodiment Sectional drawing of each part of the analytical device in the same embodiment The top view which shows the hydrophilic treatment position of the device for analysis in the embodiment Configuration diagram of analysis device in the same embodiment Explanatory drawing of injection process and separation / weighing process in the same embodiment Action explanatory drawing of separation cavity 18 which has capillary cavity 19 in the embodiment Action explanatory diagram of the separation cavity 18 not having the capillary cavity 19 Explanatory drawing of the weighing process and mixing process of the analytical device in the same embodiment Explanatory drawing of the mixing process of the analytical device in the same embodiment Explanatory diagram of dilute solution transfer process and metering process in the same embodiment Explanatory drawing of transfer process and reagent reaction / measurement process in the same embodiment Explanatory drawing of transfer process and reagent reaction / measurement process in the same embodiment Explanatory drawing of transfer process and reagent reaction / measurement process in the same embodiment Detailed view of FIG. Measurement results of hemoglobin concentration-absorbance of the mixture Measurement result of glycated hemoglobin concentration-absorbance change Measurement result diagram of glycated hemoglobin concentration by conventional analysis method Measurement result diagram of glycated hemoglobin concentration in the case of the analysis method of the present embodiment Illustration of conventional transfer process and reagent reaction / measurement process Illustration of conventional transfer process and reagent reaction / measurement process Illustration of conventional transfer process and reagent reaction / measurement process

  FIG. 1 shows a state in which the analysis device 1 according to the embodiment of the present invention is set on the analysis device holding member 103 of the analyzer, and FIG. 2 is in contact with the analysis device holding member 103 of the analysis device 1. The disassembled state is shown with the surface facing upward.

  The analysis device 1 includes a protective cap 2 for preventing scattering of a sample liquid, a base substrate 3 on which a microchannel structure having fine irregularities on its surface is formed, a cover substrate 4 that covers the surface of the base substrate 3, and a dilution. Opening button for discharging the dilution liquid in the dilution unit 5 that is set in one of the plurality of recesses formed on the upper surface of the base substrate 3 and the dilution unit 5 that holds the liquid. 6 is composed of 5 parts.

  The analysis device may have a fan shape, a cube shape, or other shapes, and a plurality of these analysis devices 1 may be mounted on the analysis device holding member 103 simultaneously. Absent.

  The base substrate 3 and the cover substrate 4 are joined with the dilution unit 5 and the like set therein, and the protective cap 2 is attached to the joined substrate. The opening button 6 is joined around the position of the opening hole 7 formed in the cover substrate 4.

  By covering the openings of several concave portions formed on the upper surface of the base substrate 3 with the cover substrate 4, a flow path connecting between a plurality of storage areas described later (same as measurement spots described later) and the storage areas. Etc. are formed (see FIG. 2). Necessary ones of the storage areas are loaded with reagents necessary for various analyses.

  This analytical device 1 can collect a sample liquid, for example, a solution such as blood, from the inlet 11, and closes the protective cap 2 and sets the sample liquid on the analytical device holding member 103 of the analytical apparatus. The component analysis can be performed. Reference numeral 102 denotes an axis of the analysis device holding member 103 during rotation.

  The analysis device 1 includes a centrifugal force generated by rotating the analysis device 1 around the axial center 102 located on the inner periphery of the sample solution taken in from the injection port 11 and the analysis solution. The capillary cap force of the capillary channel provided in the device 1 is used to transfer the solution inside the analytical device 1, and the protective cap 2 is a sample solution adhering to the vicinity of the inlet 11. Is attached to prevent scattering due to centrifugal force during analysis.

  As a material of the parts constituting the analytical device 1 of the present invention, a resin material having a low material cost and excellent mass productivity is desirable. Since the analysis apparatus analyzes the sample liquid by an optical measurement method that measures light transmitted through the analysis device 1, the material of the base substrate 3 and the cover substrate 4 may be PC, PMMA, AS, MS, or the like. A highly transparent resin is desirable.

  Moreover, as the material of the dilution unit 5, since it is necessary to enclose the dilution liquid inside the dilution unit 5 for a long period of time, a crystalline resin having a low moisture permeability such as PP or PE is desirable. Since the opening button 6 is deformed and used when the dilution unit 5 is opened, a crystalline resin such as PP having a high elastic modulus is desirable. As a material of the protective cap 2, there is no particular problem as long as it is a material with good moldability, and an inexpensive resin such as PP or PE is desirable.

  The base substrate 3 and the cover substrate 4 are preferably joined by a method that hardly affects the reaction activity of the reagent carried in the storage area, such as ultrasonic welding or laser welding, in which reactive gas or solvent is less likely to be emitted during joining. Is desirable.

  Further, a hydrophilic treatment for increasing the capillary force is performed on the portion where the solution is transferred by the capillary force due to the minute gap between the substrates 3 and 4 by joining the base substrate 3 and the cover substrate 4. This reduces the viscous resistance and facilitates fluid movement. Specifically, a hydrophilic treatment is performed using a hydrophilic material, or using a hydrophilic polymer or a surfactant. Further, a hydrophilic agent such as a silica powder such as silica gel may be added 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, hydrophilic means that the contact angle with water is less than 90 °, more preferably less than 40 °.

3 to 6 show an analysis apparatus in which the analysis device 1 is set.
In FIG. 3, the analysis device 1 is placed on the analysis device holding member 103 that rotates about the axis 102 of the analysis apparatus 100, with the cover substrate 4 side of the base substrate 3 and the cover substrate 4 down. The analysis is performed with the lid 101 closed.

  As shown in FIGS. 4 and 5, the analyzer 100 includes a rotation driving means 107 for rotating the analysis device holding member 103 and an optical measurement for optically measuring the solution in the analysis device 1. Means 109, a control means 108 for controlling the rotational speed and direction of the analysis device holding member 103, the measurement timing of the optical measurement means, and the like, processing the signals obtained by the optical measurement means 109 and calculating the measurement results And a display unit 111 for displaying the result obtained by the calculation unit 110.

  The rotation driving means 107 not only rotates the analysis device 1 around the axis 102 in a predetermined direction at a predetermined rotation speed via the analysis device holding member 103 but also rotates the axis 102 at a predetermined stop position. The analyzing device 1 can be swung by reciprocating left and right with a predetermined amplitude range and period at the center. Here, the motor 104 is used as the rotation driving means 107 to rotate the analysis device holding member 103 around the axis 102. The shaft center 102 is attached so as to be rotatable at an inclination angle θ ° around a predetermined position on the shaft center 102.

  Here, the rotational operation and the swinging operation of the analysis device 1 are performed by the single rotational driving means 107. However, in order to reduce the load on the rotational driving means 107, the driving means for the swinging operation is separately provided. It does not matter if it is provided. Specifically, by directly or indirectly contacting the analysis device 1 set on the analysis device holding member 103 with a vibration means such as a vibration motor prepared separately from the motor 104. The analytical device 1 is swung to apply an inertial force to the solution in the analytical device 1.

  The optical measuring means 109 detects the light source 105 for irradiating the measuring unit of the analyzing device 1 with laser light, and the amount of transmitted light that has passed through the analyzing device 1 out of the light emitted from the light source 105. And a photodetector 106. When the analysis device holding member 103 is made of a material having poor translucency or a material that is not translucent, holes 51 and 52 are formed at the mounting position of the analysis device 1 on the analysis device holding member 103. Yes.

Here, a light source 105 that can switch the wavelength of the emitted light is used, and a photodetector 106 that can detect light of any wavelength of the emitted light from the light source 105 is used.
Note that a plurality of pairs of the light source 105 and the photodetector 106 may be provided according to the type of wavelength necessary for measurement.

  Further, the analysis apparatus 100 has an opening means for automatically opening the dilution unit 5 in the analysis device 1, specifically, an opening button 6 of the analysis device 1 set on the analysis device holding member 103. May be provided with an arm that can move up and down on the analysis device holding member 103 and a mechanism for pushing up the opening button 6 by the arm.

  The analysis device holding member 103 is attached to an inclined axis 102 as shown in FIG. 5 and is inclined by an inclination angle θ ° with respect to the horizontal line, and the analysis device 1 is analyzed according to the rotation stop position of the analysis device 1. The direction of gravity applied to the solution in the device 1 can be controlled.

  Specifically, when the analysis device 1 is stopped at the position shown in FIG. 6A (a position near 180 ° when expressed as 0 ° (360 °) directly above), the analysis device 1 Since the lower side 53 of the lower side 53 faces downward as viewed from the front, the solution in the analytical device 1 receives gravity toward the outer peripheral direction (lower side 53).

  When the analysis device 1 is stopped at a position near 60 ° shown in FIG. 6B, the upper left side 54 of the analysis device 1 faces downward as viewed from the front. The solution is subjected to gravity in the upper left direction. Similarly, at the position near 300 ° shown in FIG. 6C, the upper right side 55 of the analytical device 1 faces downward as viewed from the front, so that the solution in the analytical device 1 is gravitational toward the upper right direction. Receive.

  In this manner, by providing the axis 102 with an inclination and stopping the analysis device 1 at an arbitrary position, it can be used as one of driving forces for transferring the solution in the analysis device 1 in a predetermined direction. .

  The magnitude of gravity applied to the solution in the analysis device 1 can be set by adjusting the inclination angle θ of the axis 102, and the amount of liquid to be transferred and the force attached to the wall surface in the analysis device 1. It is desirable to set according to the relationship.

  The inclination angle θ is preferably in the range of 10 ° to 45 °. If the inclination angle θ is smaller than 10 °, the gravity applied to the solution may be too small to obtain the driving force necessary for the transfer. If the angle exceeds 45 °, the load on the shaft center 102 may increase, or the solution transferred by centrifugal force may move freely by its own weight and cannot be controlled.

  In the analyzer 100 of this embodiment, the tilt angle θ is fixed at an arbitrary angle in the range of 10 ° to 45 °, and the motor 104, the light source 105, and the photodetector 106, which are the rotation drive means 107, also have a tilt. Although it is attached in parallel with the axis 102, the inclination angle θ can be adjusted to an arbitrary angle, and the motor 104, the light source 105, and the photodetector 106 are also configured to follow and change the angle. The optimum inclination angle can be set according to the specifications of 1 and the transfer process in the analysis device 1. Here, in the case of a configuration in which the inclination angle θ can be adjusted to an arbitrary angle, the range of the inclination angle θ is preferably 0 ° to 45 °, and the inclination angle is 0 ° when analysis is not desired to be affected by gravity, that is, analysis. The device holding member 103 can be rotated horizontally.

7 to 13 show details of the analysis device 1.
FIG. 7 shows a dilution unit opening part of the analytical device 1.
Fig.7 (a) is a top view which shows the attachment position of the opening button 6, FIG.7 (b) shows AA sectional drawing of Fig.7 (a).

  As shown in FIG. 7B, the dilution unit 5 is opened and discharged by pushing up the center of the opening button 6 joined to the cover substrate 4 from below so that the pin 8 is stuck on the surface of the dilution unit 5. The aluminum seal 10 is broken and the dilution unit 5 is opened. Further, when the analysis device 1 is rotated in a state where the dilution unit 5 is opened, the diluted solution in the dilution unit 5 becomes a space formed between the opening hole 7 and the discharge hole 9 (the base substrate 3 and the cover substrate). 4 and a space formed between the cover substrate 4 and the unsealing button 6) and then discharged to the holding cavity 14.

FIG. 8A is an enlarged perspective view of the vicinity of the injection port of the analysis device 1, and FIG. 8B is a front view thereof. FIG. 9 is a plan view of a joint surface of the base substrate 3 shown in FIG. 2 with the cover substrate 4.
Since the analysis device 1 attaches the sample solution to the injection port 11 so that the sample solution can be sucked by the capillary force of the capillary cavity 17 formed inside, the blood can be directly collected from the fingertip or the like. it can. Here, since the injection port 11 has a shape that protrudes in the direction of the axial center 102 from one side surface of the analysis device 1 main body, a finger or the like comes into contact with a place other than the injection port 11, and blood adheres to it. It has an effect of preventing blood adhering at the time of analysis from being scattered outside.

  Also, cavities 12 and 13 having a cross-sectional dimension in the thickness direction larger than that of the capillary cavity 17 and communicating with the atmosphere are provided on the side surfaces of the capillary cavity 17. By providing the cavities 12 and 13, the sample liquid flowing in the capillary cavity 17 is filled not as a capillary flow in which the side portion precedes, but as a capillary flow in which the central portion precedes, so that the sample liquid flows a plurality of times. Even in the case where the sample liquid is divided and filled, the sample liquid held in the capillary cavity 17 and the central part of the sample liquid collected later flow so as to come into contact with each other first, and the air in the capillary cavity 17 is allowed to flow into the side cavity. 12 and 13 are filled while being discharged. For this reason, even when the amount of the sample liquid to be attached to the injection port 11 is insufficient during collection or when a fingertip or the like is separated from the injection port 11 during collection, the collection into the capillary cavity 17 is completed. It can be collected any number of times. Here, the cross-sectional dimension of the capillary cavity 17 in the thickness direction is 50 to 300 μm, and the cross-sectional dimension of the cavities 12 and 13 in the thickness direction is 1000 to 3000 μm. However, the capillary cavity 17 can collect the sample liquid by capillary force. The dimensions and cavities 12 and 13 are not particularly limited as long as the sample liquid is not transferred by capillary force.

  In addition, the enlarged view of the cross section of each position of AA-AA, BB, CC, DD, and EE shown in FIG. 10 is shown to Fig.11 (a)-(e). 20a, 20b1, 20b2, 20c, 20d, 20e, 20f, 20g and 20h are air holes. Moreover, the position where the hydrophilic treatment is performed is shown by hatching in FIG.

Next, the microchannel configuration of the analytical device and the solution transfer process in Embodiment 1 of the present invention will be described in detail.
FIG. 13 is a block diagram showing the structure of the analytical device 1. The analytical device 1 has a sample liquid collecting unit 150 for collecting the sample liquid and a diluting liquid for diluting the sample liquid. In order to collect the sample liquid containing a predetermined amount of the solid component after holding the sample liquid transferred from the diluent holding section 151 and the sample liquid collecting section 150 and centrifuging the solution into a solid component Separating unit 152, a diluent measuring unit 153 for measuring the diluent transferred from the diluent holding unit 151, a sample solution transferred from the separating unit 152, and a diluent transferred from the diluent measuring unit 153 The mixing unit 154 for measuring the diluted solution in an amount necessary for analysis after the mixing, and the measurement unit for measuring the diluted solution transferred from the mixing unit 154 by reacting with the analysis reagent 155 is formed .

  As shown in FIG. 9, the sample liquid collecting unit 150 has an inlet 11 for collecting a sample liquid, a capillary cavity 17 that collects the sample liquid through the inlet 11 with a capillary force and holds a predetermined amount, and a sample liquid collecting It is sometimes composed of cavities 12 and 13 for discharging air in the capillary cavity 17.

As shown in FIG. 9, the diluent holding unit 151 holds the diluent in the dilution unit 5, and the diluent is developed by the opening operation described with reference to FIG.
As shown in FIG. 9, the separation unit 152 is formed so as to communicate with the capillary cavity 17 through the cavity 12 on the lower side of the sample liquid collecting unit 150 and transfers the sample liquid transferred from the capillary cavity 17 by centrifugal force. A separation cavity 18 that holds and separates the sample liquid into a solution component and a solid component by centrifugal force, and one of the solid components that is formed between the separation cavity 18 and the diluent measurement unit 153 and separated by the separation cavity 18. A measuring channel 23 in which the part is transferred and held; a connecting channel 21 for connecting the measuring channel 23 and the separation cavity 18 to transfer the sample liquid in the separation cavity 18; An overflow that preferentially holds the solution component of the sample solution formed between the measuring unit 153 and separated in the connecting channel 21 and transfers only the solid component to the measuring channel 23. A flow path, a capillary cavity 19 formed in the separation cavity 18 for suppressing the solution component in the separated separation cavity 18 from being transferred to the measurement flow path 23, and a metering flow with the separation cavity 18 as a boundary. A separation channel 18, a connection channel 21, and a connection channel 24 for discharging a sample liquid that is not required for analysis in the overflow channel 22 are formed on the side opposite to the channel 23, and are transferred via the connection channel 24. And overflow cavities 25 and 26 for holding unnecessary sample liquid.

  Here, the connecting channel 21, overflow channel 22, metering channel 23, connecting channel 24, capillary cavity 19, and overflow cavity 26 have a thickness dimension of 50 to 300 μm. There is no particular limitation as long as the sample liquid can be transferred by force. Moreover, although the cross-sectional dimension of the thickness direction of the separation cavity 18 and the overflow cavity 25 is 1000-3000 micrometers, it can adjust according to the quantity of a required sample liquid.

  As shown in FIG. 9, the dilution liquid measuring unit 153 is formed on the lower side of the dilution liquid holding unit 151 and has a holding cavity 14 for holding a predetermined amount of dilution liquid transferred from the dilution unit 5 by centrifugal force. , A siphon-shaped connection flow path 15 formed between the holding cavity 14 and the separation part 152 for transferring the diluent measured in the holding cavity 14 to the mixing part 154, and the holding cavity 14 as a boundary. It is held by the holding cavity 14 and the overflow channel 16 for overflowing the outside of the holding cavity 14 when the dilution liquid formed on the side opposite to the channel 15 and transferred to the holding cavity 14 exceeds a predetermined amount. The overflow cavity 27 in which the diluent is overflowed via the overflow channel 16 with the liquid surface height being defined, and the overflowed diluent is held for reference measurement of the optical measuring means 109. And the diluent overflow chamber 29 to be use, and a capillary portion 28 for preventing the diluent held in the diluent overflow chamber 29 flows out to another area to reflux.

  Here, the cross-sectional dimension in the thickness direction of the connecting channel 15, the overflow channel 16, and the capillary portion 28 is configured to be 50 to 300 μm, but there is no particular limitation as long as the capillary force works. The holding cavity 14, the overflow cavity 27, and the dilution liquid overflow chamber 29 have a cross-sectional dimension in the thickness direction of 1000 to 3000 μm. Conditions for measuring the required amount of sample liquid and absorbance (optical path) Length, measurement wavelength, etc.).

  As shown in FIG. 9, the mixing unit 154 is formed so as to communicate with the measurement channel 23 and the connection channel 15 on the lower side of the separation unit 152 and the diluent measurement unit 153, and is transferred from the measurement channel 23. A mixing chamber 30 serving as a third holding unit that holds the sample solution and the diluted solution transferred from the holding cavity 14 and mixes the sample solution therein, and an air hole 20c provided in the mixing chamber 30 during mixing. The rib 31 formed so as to prevent the liquid from flowing out and the dilute solution held in the mixing chamber 30 are formed on the inner side of the liquid surface height with respect to the direction of the axis 102 and mixed and transferred from the mixing chamber 30. A holding cavity 32 that holds the diluted solution, a holding cavity 35 that is formed on the lower side of the holding cavity 32 and holds a predetermined amount of the diluted solution transferred from the holding cavity 32 by centrifugal force, and a holding key A capillary section 33 for suppressing the dilute solution formed between the bitty 32 and the overflow cavity 27 and transferred to the holding cavity 32 from flowing out into the overflow cavity 27, the holding cavity 32, and the holding cavity 35; And a measuring channel 155 located on the lower side of the holding cavity 35 and the holding cavity 35. Between the holding cavity 35 and the overflow cavity 27 and to the holding cavity 35. The connecting channel 37 is used for transferring the diluted solution measured between the holding cavity 35 and the measuring cavity 155. The overflow flow path 36 is configured to overflow the holding cavity 35 when the diluted solution to be transferred exceeds a predetermined amount.

  Here, the capillary section 33, the connecting channel 34, the overflow channel 36, and the connecting channel 37 have a cross-sectional dimension in the thickness direction of 50 to 300 μm. Absent. Moreover, although the cross-sectional dimension of the holding | maintenance cavity 32 and the holding | maintenance cavity 35 is 1000-3000 micrometers in thickness direction, it can adjust according to the quantity of a required dilution solution.

  As shown in FIG. 9, the measuring unit 155 is formed to communicate with the holding cavity 35 via the connection channel 37 on the lower side of the mixing unit 154 and is connected from the holding cavity 35 with the reagent carried inside. It is an operation cavity when viewed from the denaturation reaction chamber 38 which is a measurement spot for reacting and holding the diluted solution transferred through the flow path 37 and performing the first measurement, and the immunoassay chamber 43 which is the measurement spot. Denaturing solution quantification, which is formed inside the liquid surface height of the denaturing solution 61 held in the denaturing reaction chamber 38 with respect to the direction of the axis 102, and collects the denaturing solution 61 in the denaturing reaction chamber 38 after measuring the denaturing solution 61. A capillary cavity 40 for stabilizing the amount of the denaturing solution 61 formed between the chamber 39, the denaturing reaction chamber 38 and the denaturing solution quantitative chamber 39 and returning to the denaturing reaction chamber 38; A connection channel 41 that prevents the denaturing solution 61 formed on the lower side of the volume chamber 39 and collected in the denaturing solution quantification chamber 39 from flowing into the immunoassay chamber 43 as a measurement spot; A rib 42 that is located at a connection portion with the capillary cavity 40 and breaks the denaturing solution 61 in the denaturing solution quantification chamber 39 by centrifugal force to return a predetermined amount of the diluted solution to the denaturation reaction chamber 38; On the lower side, a reagent which is formed so as to communicate with the denaturing solution quantification chamber 39 via the connecting channel 41 and the denaturing solution 61 transferred from the denaturing solution quantification chamber 39 via the connecting channel 41 are supported. The immunoassay chamber 43, which is a measurement spot for performing the second measurement by reacting the liquid, and the height of the liquid surface relative to the axial center 102 direction of the immunoassay solution 62 held in the immunoassay chamber 43 Is formed inside, the immunoassay solution 62 of the immunoassay chamber 43 taken after the measurement of the immunoassay solution 62, and a coagulation reaction chamber 44a to react with a reagent carried on the inside.

  The agglutination reaction chamber 44a has the same configuration as the immunoassay quantification chamber 44 configured by a capillary cavity, and no reagent is provided in the immunoassay quantification chamber 44. In this embodiment, a conventional example is used. The agglutination reagent provided in the agglutination reaction chamber 46 shown in FIG. 29B is different from the conventional one.

  Here, the cross-sectional dimensions in the thickness direction of the denaturing solution quantification chamber 39, the capillary cavity 40, the connection channel 41, and the aggregation reaction chamber 44a are configured to be 50 to 500 μm, but there is no particular limitation as long as the capillary force acts. . The denaturation reaction chamber 38 and immunoassay chamber 43 have a cross-sectional dimension in the thickness direction of 1000 to 3000 μm. Conditions for measuring the required amount of mixed solution and absorbance (optical path length, measurement wavelength, sample It can be adjusted according to the reaction concentration of the solution, the type of reagent, and the like.

Next, the sample solution analysis step of the analysis device 1 will be described in detail by taking the concentration measurement of hemoglobin and HbA1c contained in blood cells in blood as an example.
14 to 22 show the analysis device 1 set on the analysis device holding member 103 as viewed from the surface side of the analysis device holding member 103, and the rotation direction with respect to the axis 102. C1 indicates the left rotation in FIG. 1, and the rotation direction C2 with respect to the axis 102 indicates the right rotation in FIG. FIG. 14 shows the injection process and separation / metering process of the analytical device.

− Step 1 −
In FIG. 14A, blood as a sample solution is collected from the punctured fingertip or the like through the inlet 11 of the analytical device 1 until the inside of the capillary cavity 17 is filled by the capillary force of the capillary cavity 17. The Here, the sample liquid, for example, about 10 μL of blood can be measured by the volume determined by the gap and the facing area of the capillary cavity 17, but the shape dimension of the capillary cavity 17 is defined according to the amount necessary for analysis, and collected. You can adjust the capacity. The analysis device 1 that has collected a necessary amount of blood is mounted on the analysis device holding member 103 of the analysis apparatus 100, and an unsealing operation is performed by the unsealing means of the dilution unit 5.

− Step 2 −
After the opening of the dilution unit 5 is finished, the analysis device holding member 103 is rotated (right rotation indicated by C2 and 3000 rpm), whereby the blood and the diluted solution in the capillary cavity 17 are as shown in FIG. The diluting liquid in the dilution unit 5 is transferred to the holding cavity 14. Here, when diluting the blood and taking out the measurement component in the blood cell, in order to reduce the variation in dilution due to the influence of the hematocrit value (the ratio of the blood cell component contained in the blood) having individual differences, the separation cavity 18 The blood transported to is separated into a plasma component and a blood cell component by centrifugal force, and high-hematocrit blood on the outer periphery is collected and diluted to reduce variation in dilution.

  In addition, the diluent that has been transferred to the holding cavity 14 during this rotation and has exceeded the specified amount flows into the diluent overflow chamber 29 via the overflow channel 16, the overflow cavity 27, and the capillary portion 28. Held in.

FIG. 15 shows the centrifugal separation operation in the separation cavity 18 having the capillary cavity 19 and the transfer flow to the mixing chamber 30 via the measuring channel 23.
As shown in FIG. 15A, blood 57 collected at the bottom of the separation cavity 18 is separated into a plasma component 57a and a blood cell component 57b by centrifugal force as shown in FIG. 15B. When the rotation is stopped and the centrifugal force is lost, as shown in FIG. 15C, the plasma component 57a in the separation cavity 18 is capillary-transferred to the capillary cavity 19, and the plasma component 57a and the blood cell component 57b in the connection channel 21 are Then, it is capillary-transferred toward the overflow channel 22 connected to the cavity 58 having the air hole 20a communicating with the atmosphere. The plasma component 57a and the blood cell component 57b in the connection channel 24 are capillary-transferred toward the overflow cavity 26 having the air hole 20d communicating with the atmosphere. Here, the end of the measurement flow path 23 is connected to the connection flow path 21 at the position where the blood cell component 57b reaches, and as shown in FIG. Only the blood cell component 57b is transferred by the capillary force of the measuring channel 23.

  In this embodiment, since the capillary cavity 19 is formed in the separation cavity 18, most of the plasma component 57 a remaining in the separation cavity 18 can be held by the capillary cavity 19, and a necessary amount of the measurement channel 23 is provided in the measurement channel 23. Only the blood cell component 57b is useful for capillary transfer. Specifically, as shown in FIG. 16A, in the comparative example in which the capillary cavity 19 is not formed in the separation cavity 18, the plasma component 57a is accumulated at the bottom of the separation cavity 18, and When the capillary is transferred by the capillary force, as shown in FIG. 16 (b), the plasma component 57a accumulated at the bottom of the separation cavity 18 is mixed from the connection channel 21 toward the measurement channel 23, and the necessary amount is obtained. Cannot be obtained.

  On the other hand, the diluted liquid transferred to the holding cavity 14 overflows via the overflow channel 16 when the level of the retained liquid exceeds the connecting position of the overflow channel 16 and the overflow cavity 27. Since it is discharged into the cavity 27, it is held in the holding cavity 14 by a specified amount. Here, since the connecting channel 15 has a siphon shape including a curved pipe disposed radially inward from the connecting position of the overflow channel 16 and the overflow cavity 27, the analysis device 1 is rotating. The diluted solution can be held in the holding cavity 14.

  In addition, since the overflow channel 16 that connects the holding cavity 14 and the overflow cavity 27 is a capillary tube, the diluent is transferred from the holding cavity 14 to the overflow cavity 27 by inertia force or surface tension when the analysis device 1 is decelerated and stopped. Can be prevented by capillary force, and the diluent can be accurately measured.

− Step 3 −
After the rotation (right rotation indicated by C2: 3000 rpm) of the analysis device holding member 103 is stopped and stopped, the analysis device holding member 103 is rotated (right rotation indicated by C2: 2000 rpm) from FIG. As a result, the necessary amount of blood cell component 57b held in the measurement flow channel 23 and the diluted solution in the holding cavity 14 flow into the mixing chamber 30 and are mixed and diluted. The excess blood cell component 57b is shown in FIG. As shown, it is retained in the overflow cavity 26. The optical measurement unit 109 performs reference measurement in which the diluted solution in the diluted solution overflow chamber 29 of the analytical device 1 is read at a timing interposed between the light source 105 and the photodetector 106. At this time, reference measurement is performed by switching the wavelength of the light source 105 between 535 nm and 625 nm.

− Step 4 −
Next, the analysis device 1 is set to a position near 60 ° shown in FIG. 18A, and the dilution liquid is controlled by controlling the motor 104 at a frequency of 1000 rpm so as to give the analysis device 1 a swing of about ± 1 mm. Stir.

− Step 5 −
Thereafter, the analysis device 1 is set at a position near 180 ° shown in FIG. 18B, and the motor 104 is controlled at a frequency of 1000 rpm so as to give the analysis device 1 a swing of about ± 1 mm. Stir.

  Here, the mixing chamber 30 and the holding cavity 32 are communicated with each other by a connecting portion 59, and the position of the connecting portion 59 at the time of stirring is held in the mixing chamber 30 with respect to the rotation axis 102 that generates centrifugal force. Since the liquid is positioned on the inner peripheral side of the liquid surface of the diluted solution, the diluted liquid during the stirring and mixing does not flow out to the holding cavity 32.

-Step 6-
Next, the analysis device 1 is set at a position near 300 ° shown in FIG. 19A, and the motor 104 is controlled at a frequency of 1000 rpm so as to give the analysis device 1 a swing of about ± 1 mm. The diluted blood cell component 57 b (diluted solution) in the chamber 30 is oscillated and transferred to the holding cavity 32 via the connecting portion 59.

  Here, the diluted solution held in the mixing chamber 30 is held by the surface tension acting on the wall surface of the mixing chamber 30 even if the analytical device 1 is moved to a position near 300 ° shown in FIG. (Because the surface tension is greater than the gravity acting on the diluted solution), the analytical device 1 is swung to give the diluted solution an inertial force, thereby breaking the surface tension acting on the wall surface of the mixing chamber 30 and The diluted solution can be transferred to the holding cavity 32 by the working inertia force and gravity.

-Step 7-
Next, the analysis device 1 is held from the holding cavity 32 through the connection channel 34 as shown in FIG. 19B by rotating the analysis device holding member 103 (right rotation indicated by C2 / 2000 rpm). A prescribed amount of diluted solution is transferred to the cavity 35. A portion exceeding the predetermined amount of the diluted solution transferred to the holding cavity 35 overflows to the overflow cavity 27 via the overflow channel 36, and only the prescribed amount of the diluted solution 60 is held in the holding cavity 35.

− Step 8 −
By stopping the rotation of the analysis device holding member 103 (right rotation indicated by C2 and 2000 rpm) and allowing it to stand still, the diluted solution in the holding cavity 35 is drawn into the connection channel 37 as shown in FIG. Further, by rotating the analysis device holding member 103 (left rotation indicated by C1 · 2000 rpm) from FIG. 20A, a predetermined amount of the diluted solution held in the holding cavity 35 is passed through the connection channel 37. Transferred to the denaturation reaction chamber 38, the denaturing reagent previously supported in the denaturation reaction chamber 38 is dissolved.

-Step 9-
Thereafter, at a position near 180 ° shown in FIG. 20B, the motor 104 is controlled at a frequency of 1000 rpm so as to give the analytical device 1 a swing of about ± 1 mm, and the denaturation reaction chamber of the analytical device 1 38 denaturing solution 61 is stirred.

  Here, the denaturation reaction chamber 38 and the immunoassay chamber 43 are in communication with each other via a capillary cavity 40 and a denaturing solution quantification chamber 39. The position of the capillary cavity 40 at the time of stirring is located on the inner peripheral side of the surface of the diluted solution held in the denaturation reaction chamber 38 with respect to the rotation axis 102 that generates centrifugal force. The solution does not flow out into the denaturing solution quantification chamber 39 on the side of the immunoassay chamber 43.

-Step 10-
Next, after the analysis device 1 is stopped and the denaturing solution 61 is subjected to a denaturation reaction, the analysis device holding member 103 is rotated (left rotation indicated by C1 · 1500 rpm) to perform the first measurement.

  In the first measurement, in the light emission state in which the wavelength of the light source 105 is switched to 535 nm, the denaturing solution 61 subjected to the denaturation reaction in the denaturation reaction chamber 38 of the analytical device 1 is read at a timing interposed between the light source 105 and the photodetector 106. I do. The calculation unit 110 displays the denatured hemoglobin concentration obtained by processing the measurement value obtained by the first measurement and digitizing the dilution overflow chamber 29 based on a reference value read in advance with the wavelength of the light source 105 set to 535 nm. Displayed on the unit 111.

  After the reaction between the mixed solution and the denaturing reagent held in the denaturing reaction chamber 38, the concentration of the hemoglobin derivative is measured in the measurement region formed in the denaturing reaction chamber, so that the total amount in the mixed solution or the denaturing solution is measured. Make hemoglobin determination.

  In addition, before modification | denaturation, in the mixing chamber 30, you may measure the hemoglobin by the colorimetric degree by the light source of LED with a wavelength of 410 nm using absorption of (gamma) peak (sole band) of hemoglobin.

  As used herein, “denaturation” means that a specific portion is exposed (exposed) from within the structure of the protein, and the antigen-antibody reaction described later is a portion exposed from within the structure of the protein. This is done with a latex reagent that reacts specifically with “denatured sites”.

− Step 11 −
Next, by controlling the motor 104 at a frequency of 1500 rpm so that the analyzing device 1 is placed at a position near 60 ° shown in FIG. The denaturing solution 61 held in the denaturing reaction chamber 38 is capillary-transferred to the denaturing solution quantification chamber 39, and a predetermined amount of denaturing solution 61 is held in the denaturing solution quantification chamber 39.

− Step 12 −
Next, the denaturing solution 61 flows into the immunoassay chamber 43 from the denaturing solution quantitation chamber 39 through the connection channel 41 by rotating the analysis device holding member 103 (left rotation indicated by C1 · 2000 rpm), and immunization The latex reagent previously supported in the assay chamber 43 is dissolved.

-Step 13-
Thereafter, at a position near 180 ° shown in FIG. 21B, the motor 104 is controlled at a frequency of 1000 rpm so as to give the analytical device 1 a swing of about ± 1 mm, and the immunoassay chamber of the analytical device 1 43 second reaction liquid 62 is stirred. In the immunoassay chamber 43, the immunoassay solution undergoes an antigen-antibody reaction with a reagent (for example, a latex reagent) containing an antibody specific to the denatured portion of the hemoglobin derivative.

  Here, between the immunoassay chamber 43 and the agglutination reaction chamber 44a side, the position of the connecting portion 44b connecting the immunoassay chamber 43 and the agglutination reaction chamber 44a during stirring is the axis of rotation that generates centrifugal force. Since 102 is positioned on the inner peripheral side of the liquid surface of the mixed solution held in the immunoassay chamber 43, the mixed solution being stirred and mixed does not flow out to the agglutination reaction chamber 44a.

− Step 14 −
Next, after the analysis device 1 is stopped and the immunoassay solution 62 is reacted with the antigen and antibody, the analysis device holding member 103 is rotated (left rotation indicated by C1 · 1500 rpm) to perform the second measurement.

  In the second measurement, the concentration of the hemoglobin derivative is measured in a light emission state in which the wavelength of the light source 105 is switched to 625 nm. The immunoassay solution 62 that has reacted with an antigen in the immunoassay chamber 43 of the analytical device 1 is used as the light source. A blank measurement is performed by reading at a timing interposed between the photo detector 105 and the photodetector 106.

− Step 15 −
Next, the assay device 1 is set at a position near 60 ° shown in FIG. 22A, and the motor 104 is controlled at a frequency of 1500 rpm so as to give the analysis device 1 a swing of about ± 1 mm. The solution 62 is capillary-transferred to the agglutination reaction chamber 44a, and the agglutination reagent carried in the agglutination reaction chamber 44a is dissolved and stirred.

− Step 16 −
Thereafter, the analysis device holding member 103 is rotated (left rotation indicated by C1 · 2000 rpm), so that the aggregation solution 63 held in the aggregation reaction chamber 44a is converted into the immunoassay chamber 43 as shown in FIG. The immunoassay chamber 43 holds the aggregation solution 63 again.

− Step 17 −
Next, when the analysis device 1 is stopped at a position near 60 ° shown in FIG. 22B, the aggregation solution 63 transferred to the immunoassay chamber 43 is again sucked into the aggregation reaction chamber 46 by the action of capillary force. It is done. Here, since the surface of the wall connecting the immunoassay chamber 43 and the agglutination reaction chamber 46 was wetted by the transfer of the immunoassay solution 62 in step 15, it does not move into the agglutination reaction chamber 46 even if it is not shaken as in step 15. The solution can be transferred.

  Aggregation solution 63 can promote agitation by repeating the operations of steps 16 and 17, and in immunoassay chamber 43 and agglutination reaction chamber 46, the aggregating agent binds the antibody to the hemoglobin derivative. Aggregation occurs due to reactive bonding with unreacted unreacted material.

− Step 18 −
Next, after the analysis device 1 is stopped and the aggregation solution 63 is agglutinated, the analysis device holding member 103 is rotated (left rotation indicated by C1 · 1500 rpm) to perform the third measurement.

  In the third measurement, in the light emission state in which the wavelength of the light source 105 is switched to 625 nm, reading is performed at a timing when the agglutination solution 63 in the immunoassay chamber 43 of the analytical device 1 is interposed between the light source 105 and the photodetector 106. And measure the degree of turbidity of the aggregation solution 63.

  The calculation unit 110 digitizes the measurement values obtained by the second measurement and the third measurement by processing the dilution liquid overflow chamber 29 based on a reference value read in advance with the wavelength of the light source 105 set to 625 nm. The display unit 111 displays the HbA1c concentration and the HbA1c% value calculated based on the modified hemoglobin concentration.

  In the present embodiment, a light emitting diode is used as the light source 105, but the present invention is not limited to this, and the light source wavelength may be in a wavelength region where hemoglobin absorbs light in colorimetric measurement. What is necessary is just to use the gamma peak (sole absorption band) which has a light absorption effect in the region of 400 to 450 nm, and the light absorption effect in the visible region of hemoglobin and hemoglobin derivatives having an absorption effect in the region of 500 to 580 nm. . About turbidity, 500-800 nm is suitable although there is absorption in the visible light region.

FIG. 23 shows FIG. 13 in more detail with respect to steps 1 to 18 described above.
The case of measuring a hemoglobin derivative for the reagent held in the analysis device 1 will be specifically described.

The method for measuring a hemoglobin derivative, which is an embodiment of the present invention, includes a step of denaturing the hemoglobin derivative in the sample with a denaturing reagent (denaturing agent).
Hemoglobin (hereinafter sometimes referred to as Hb) is based on a tetrameric structure formed by binding and association of α-chain and non-α-chain (β, γ, δ chain) globin with heme. To do.

  In particular, HbA1 to which sugar is bound, acetaldehyde-modified Hb by drinking, carbamylated Hb found in dialysis patients and the like are various. Thus, the hemoglobin derivative means a part of hemoglobin modified as described above and having a different structure. HbA (α2β2), which accounts for about 90% of the hemoglobin derivative, and about 3% of HbA2 ( α2δ2) and about 1% HbF (α2γ2). In HbA, there are HbA0 in which no sugar is bound to the β-chain amino acid terminal and HbA1 in which a sugar is bound. Further, among the HbA1, there are HbA1a, HbA1b, and HbA1c, which are also hemoglobin derivatives. Among them, hemoglobin A1c in which sugar in blood is bound to the β chain N-terminal of hemoglobin is widely known as an index reflecting the blood sugar concentration in the past 2 to 3 months.

  The point that determines the hemoglobin derivative is whether there is an amino acid sequence in a region specific to the peptide structure, or whether there is a region in which amino acid residues or peptide ends are phosphorylated or glycated. For example, HbA1a is phosphorylated at the β-chain N-terminus, HbA1b is aldehyded at the β-chain N-terminus, and HbA1c is fructosylated at the β-chain N-terminus. Moreover, subunits of HbF are different between β and γ. In the amino acid unit difference, the 9th and 21st residues from the N-terminal of the β subunit of HbA2 are different. The hemoglobin derivative of the present invention refers to those having different partial regions. In addition to these, there are various types of hemoglobin derivatives, such as the above-mentioned acetaldehyde-hemoglobin adducts due to alcohol abuse, urea-hemoglobin adducts present in the blood of uremic patients, for example, Aspirin-hemoglobin complex, carboxymethylated hemoglobin, etc. Examples of the hemoglobin derivative include glycated hemoglobin produced by a non-enzymatic reaction between a reactive amine group on hemoglobin protein and glucose, but are not limited thereto.

  In measuring the hemoglobin derivative shown in the present invention, it is necessary to distinguish and recognize slightly different regions of the hemoglobin derivative and to identify and quantify each hemoglobin derivative. In the present invention, denaturing the different portion, that is, a specific portion of the hemoglobin derivative from the structure of the protein to the outside of the structure is referred to as denaturation.

  The degree of modification is such that the subunit structure constituting the quaternary structure is dissociated, the hydrophobic bond constituting the tertiary structure, the hydrogen bond, the van der Waals force, and the ionic bond are dissociated. Α constituting the secondary structure -It may be a degree to change the structure of the helix or β sheet, or a linear structure. Generally, proteins exist as functional substances in vivo, because proteins maintain a precise three-dimensional structure formed from these structures. Therefore, changing the structure can be said to have changed the function and properties of the protein. This includes both reduced functionality and improved functionality. Examples of the modifying agent for achieving these include a method of modifying hemoglobin with an anion in the form of a lithium salt (Japanese Patent Laid-Open No. 3-51759), a method including a nonionic surfactant (WO 2006/112339), and the like. However, it is not limited to these.

  The reagent for immunoassay in the present embodiment is a reagent containing an antibody specific for the denatured portion of the hemoglobin derivative, and if it is a measurement principle based on the immunoassay antigen-antibody reaction of the present invention. Well known, immunoturbidimetric method, immunonephelometric method, latex immunoagglutination method, immunoaggregation inhibition method, latex immunoaggregation inhibition method, fluorescence immunoassay, chemiluminescence immunoassay, electrochemical immunization Any of a measurement method, a fluorescence polarization immunoassay method, and an immunochromatography method may be used.

  Of particular importance in the method for measuring a hemoglobin derivative of the present invention is an immunoassay using an antibody specific to the glycation site of the hemoglobin derivative. The glycated hemoglobin is HbA1a, HbA1b, and HbA1c as described above. Among them, HbA1c is an index for managing diabetic patients, which has recently become a problem as one of the three major adult diseases, and is a measure for long-term blood glucose control for 1 to 3 months. It is. Specifically, it includes a reagent containing an antibody against a glycosylated β-chain N-terminal amino acid unique to HbA1c after HbA1c denaturation treatment.

  In the present embodiment, the aggregating agent for aggregation is an aggregated polyvalent antigen that specifically reacts with an antibody by an antigen-antibody reaction, and immunity is measured using an antibody against a site specific to a hemoglobin derivative. The measurement reaction is not limited to the above.

The method for retaining the reagent of the present invention is not limited to a liquid state, a dried product state, a lyophilized product state, and the like, and can be retained by a method that is meaningful in practice.
Next, with respect to hemoglobin A1c, which is a representative test item of a hemoglobin derivative, in FIGS. 24 to 27 and (Table 1) to (Table 4) in the following Examples 1 to 8, more specific examples will be given. Explained.

Example 1
As an example of the practice of the present invention, the reagent composition when examined by the HbA1c latex aggregation inhibition method will be described. In this example, the antibody is used in a state labeled with latex beads.
(A) Preparation of monoclonal antibody
Preparation of immunogen Preparation of an immunogen of HbA1c was performed as follows. First, in order to produce a structure equivalent to the β-chain N-terminal site of HbA1c, a fructose is added to a valine of a polypeptide composed of amino acids linked to a valine-histidine-leucine-threonine-cysteine (hereinafter abbreviated as VHLTC) sequence. To make fructosyl VHLTC. Next, in order to enhance immunogenicity, the amino group of chicken γ globulin (hereinafter abbreviated as CGG) is labeled using the condensation reagent N- (6-Maleimidocaproyloxy) succinimide through the cysteine residue of fructosyl VHLTC. The resulting fructosyl VHLTC-labeled CGG was used as an immunogen.

1. Immunization of mice 10 mice (Balb / c) about 8 weeks after birth were made into an emulsion of the immunogen (fructosyl VHLTC-labeled CGG) prepared as described above, and 10 μg per mouse was injected intraperitoneally. This immunization work was performed every two weeks.

2. Antibody production check For mice immunized 5 times, 50-100 μL of blood was collected into a centrifuge tube from the ocular vein. The serum was centrifuged and the antibody titer was evaluated by ELISA. As a result, production of anti-HbA1c antibody was confirmed in all mice.

3. Mouse Boost In the above-described evaluation of antibody titer, a boost (injection of weak immunogen) was performed to enlarge the spleen of the mouse, which had a particularly high titer. As the immunogen, a solution obtained by diluting 10 μg of fructosyl VHLTC-labeled CGG with PBS was used.

4). Cell Fusion Spleen cells of mice that had passed 3 days after the boost were removed and fused with mouse myeloma-derived cells by a conventional method using polyethylene glycol. The fused cells were cultured in a HAT medium containing 15% fetal calf serum (hereinafter referred to as FCS) on a 96-well plate. After one week, the medium was replaced with HT medium containing 15% FCS.

5). Cloning The antibody titer was measured by ELISA, and wells with high titers were selected.
The solution was diluted to a concentration containing 1 cell per well (limit dilution) and dispensed into a 96-well microplate. The culture was advanced while increasing the size of the plate, and the antibody titer measurement by the ELISA method was repeated with respect to the timely supernatant, and the cell group showing high titer against HbA1c and showing good proliferation was finally selected.

6). Cryopreservation of cells The finally selected cells were frozen at −80 ° C. at a concentration of 3 × 10 ⁶ cells / ml, and then transferred to liquid nitrogen for long-term storage.

7). Antibody Evaluation Ascites was prepared by a general technique of injecting the obtained cells into a mouse abdominal cavity, and the antibody was purified by a column packed with protein A Sepharose gel. Regarding the antibody thus obtained, the half value of fructosyl VHLTC-labeled CGG (inhibition half value) that inhibits the binding of each antibody to human hemoglobin A1c coated on an ELISA plate is the National Institute of Advanced Industrial Science and Technology. Patent biological deposit center accession number (international deposit) The monoclonal antibody derived from FERM BP-10895 strain is 2 × 10 ⁻¹⁰ M.
(B) Latex reagent The purified antibody was labeled by a physical adsorption method by stirring for 2 hours at room temperature using a 0.15 μm polystyrene latex manufactured by Sekisui Chemical Co., Ltd. Thereafter, blocking was performed in a 0.5% BSA (manufactured by Sigma-Aldrich) / PBS suspension, and then the unlabeled antibody was separated and washed by centrifugation, and again 0.5% BSA (Sigma The antibody latex reagent was obtained by resuspension in an Aldrich) / PBS suspension.
(C) Aggregation reagent The aggregation reagent used was an aggregation reagent (synthetic multivalent HbA1c antigen) of Cobas reagent HbA1c (manufactured by Roche Diagnostics).
(D) Denaturing reagent A method comprising a nonionic surfactant, and the following composition was determined with reference to (WO 2006/112339).

1. Sucrose monocaprate (manufactured by Wako Pure Chemical Industries) / Concentration at reaction 0.25%
2. Potassium ferricyanide (Wako Pure Chemical Industries, Ltd.) / Reaction concentration 0.25%
(E) Diluent The pure water adjusted by the general ion exchange method was used for the diluent.

(Example 2)
Below, the Example which examined the hemoglobin density | concentration measurement is described in detail.
(A) Method for producing analytical device The analytical device is the analytical device of the first embodiment, and the latex reagent, agglutinating reagent, and denaturing reagent shown in Example 1 are respectively added to the immunoassay chamber 43 and the agglutination reaction chamber 46. It was prepared by carrying it in the denaturation reaction chamber 38 by lyophilization.
(B) Preparation of mixed solution The sample solution (blood) was prepared by diluting blood collected from a person with pure water.

The Hb concentration of the liquid for mixing was determined using “Hemoglobin B-Test Wako” sold by Wako Pure Chemical Industries, Ltd. This is a method for detecting hemoglobin by the SLS-hemoglobin method.
(C) Hb measurement by analytical device Using an analytical device carrying a reagent, an injection hole was formed in the upper part of the mixing chamber 30 by a drill, and after directly injecting the mixed solution, the injector was sealed. Thereafter, the analysis device 1 is set in the analyzer main body 100, transferred from the mixing chamber 30 to the denaturation reaction chamber 38 as described in Example 1, and the absorbance value at 535 nm when reacted with the denaturing reagent for 1 minute is obtained. Detected.

  In FIG. 24, the horizontal axis represents the hemoglobin concentration of the mixed solution, and the vertical axis represents the absorbance. As a result, the Hb concentration and the absorbance value are proportional to each other in the range of 0 to 50.0 mg / dL by this method, which suggests that Hb measurement according to the Hb concentration is sufficiently possible. It was done.

Example 3
Below, the Example which examined glycated hemoglobin (HbA1c) measurement is described in detail.
(A) Method for producing analytical device: as described in Example 2 (b) Preparation of mixed solution 24.6 μM saccharification enclosed with Cobas reagent saccharified hemoglobin commercially available from Roche Diagnostics Inc. A hemoglobin standard solution was diluted with pure water to prepare a mixed solution having a known glycated hemoglobin concentration.
(C) Glycated hemoglobin measurement using an analytical device Using an analytical device carrying a reagent, an injection hole was formed in the upper part of the mixing chamber 30 by a drill, and the mixed solution was directly injected, and then the injection port was sealed. Thereafter, the analysis device 1 is set in the analyzer main body 100 and transferred from the mixing chamber 30 to the denaturation reaction chamber 38 as described in Example 1. In each step of the transfer, the denaturation reagent is used in the denaturation reaction chamber 38. In the immunoassay chamber 43, the latex blank (turbidity) measured at 625 nm was measured at 625 nm, and the aggregation turbidity (turbidity) when the final agglutination reaction was performed. Was measured at 625 nm. The amount of change in cohesive turbidity from the latex blank was calculated.

FIG. 25 is a graph in which the glycated hemoglobin concentration is plotted on the horizontal axis and the absorbance change is plotted on the vertical axis.
In order to change the glycated hemoglobin concentration from mol to mg / dL, the calculation was performed with the hemoglobin molecular weight of 64,500. As a result, according to this method, the HbA1c concentration and the absorbance value are almost proportional in the range of the HbA1c concentration of 0.1 to 10.0 mg / dL (more preferably 0.2 to 4.0 mg / dL). Therefore, it was suggested that measurement of glycated hemoglobin is sufficiently possible.

Example 4
Below, the Example which examined the method of measuring the abundance ratio of glycated hemoglobin / Hb is described in detail.
(A) Method for producing analytical device: as described in Example 2 (b) Preparation of sample solution Three types of whole blood were used as the sample solution. In Table 1, glycated hemoglobin (%) was measured with an automated glycated hemoglobin analyzer (HLC-723GHbV) manufactured by Tosoh Corporation based on the HPLC method widely used for measuring glycated hemoglobin. It is a result.

（ｃ）分析用デバイスによる測定
分析用デバイス１の試料収容室に前記３種類の血液試料と希釈液をそれぞれ注入し、分析装置本体１００にセットし、液体を移送し、５３５ｎｍでのヘモグロビン誘導体の吸光度と、６２５ｎｍでの濁度を測定することにより、ラテックスブランク及び、凝集度合いを吸光度により測定した。ヘモグロビン濃度の決定は、測定吸光度により実施例２のヘモグロビン濃度プロットより図２４から変性溶液３８に含まれるヘモグロビン濃度を算出した。また糖化ヘモグロビン濃度の決定は、濁度の変化量から、図２５から凝集溶液６３に含まれる糖化ヘモグロビン濃度を算出した。その結果を（表２）に示す。

(C) Measurement by analytical device The three kinds of blood samples and diluent are respectively injected into the sample storage chamber of the analytical device 1, set in the analyzer main body 100, the liquid is transferred, and the hemoglobin derivative at 535 nm By measuring the absorbance and turbidity at 625 nm, the latex blank and the degree of aggregation were measured by absorbance. The hemoglobin concentration was determined by calculating the hemoglobin concentration contained in the denaturing solution 38 from the hemoglobin concentration plot of Example 2 based on the measured absorbance. The glycated hemoglobin concentration was determined by calculating the glycated hemoglobin concentration contained in the aggregation solution 63 from FIG. 25 from the amount of change in turbidity. The results are shown in (Table 2).

（表２）によれば、本分析用デバイス１により糖化ヘモグロビンの占める割合を測定した結果は、あらかじめ東ソー社の自動糖化ヘモグロビン分析計（ＨＬＣ−７３２ＧＨｂＶ）にて、糖化ヘモグロビンの占める割合を測定した結果（（表１）参照）と非常に近く、本分析用デバイス１によって正確な糖化ヘモグロビン濃度の測定が可能であることが確認された。

According to (Table 2), as a result of measuring the proportion of glycated hemoglobin by the device for analysis 1, the proportion of glycated hemoglobin was measured in advance with an automatic glycated hemoglobin analyzer (HLC-732GHbV) manufactured by Tosoh Corporation. It was confirmed that it was very close to the results (see (Table 1)) and that the glycated hemoglobin concentration could be accurately measured by the analytical device 1.

(Example 5)
The measurement accuracy when measuring the abundance ratio of glycated hemoglobin / Hb using the analytical device of the present embodiment will be described.

  First, the measurement accuracy in the case of using the analysis device having the conventional configuration shown in FIGS. The analysis device having a conventional configuration is configured to measure latex blank and glycated hemoglobin at different measurement spots. After measuring the latex blank in the immunoassay chamber 43, the immunoassay solution 62 is immunoassayed. The glycated hemoglobin concentration is calculated by measuring the turbidity of the agglutination solution 63 in the agglutination reaction chamber 46 by reacting with the agglutination reagent by transferring to the agglutination reaction chamber 46 by centrifugal force. ing.

FIG. 26 shows the measurement results of the glycated hemoglobin concentration when the conventional analytical device shown in FIGS. 28 to 30 is used.
The horizontal axis is the result of measuring glycated hemoglobin (%) with an automated glycated hemoglobin analyzer (HLC-723GHbV) manufactured by Tosoh Corporation based on the principle of the HPLC method, and the vertical axis is glycated hemoglobin with an analysis device having a conventional configuration. It is the result of measuring (%). In addition, Table 3 shows the measurement CV when measured with a conventional analysis device.

図２６によれば、糖化ヘモグロビンの含有量が異なる４つの生物学的試料について測定した結果、ＨＰＬＣ法で測定した値に対して乖離しており、（表３）に示すように測定ＣＶが４．９％〜１８．４％と、ばらつきが大きいことが確認できる。

According to FIG. 26, as a result of measuring four biological samples having different glycated hemoglobin contents, there was a deviation from the value measured by the HPLC method, and the measured CV was 4 as shown in (Table 3). It can be confirmed that the variation is as large as .9% to 18.4%.

Next, the measurement accuracy when the analysis device of the present embodiment is used will be described.
The analysis device of the present embodiment is configured to perform measurement of latex blank and measurement of glycated hemoglobin at the same measurement spot. After measuring the latex blank in the immunoassay chamber 43, the immunoassay solution 62 is agglutinated. The aggregation solution 63 reacted with the aggregation reagent by being transferred to the chamber 46 and further reacted with the aggregation reagent by centrifugal force is returned to the immunoassay chamber 43, and the turbidity of the aggregation solution 63 in the immunoassay chamber 43 is measured. Is used to calculate the concentration of glycated hemoglobin.

  FIG. 27 shows the measurement result of the glycated hemoglobin concentration when the analytical device of the present embodiment is used. The horizontal axis is the result of measuring glycated hemoglobin (%) with an automated glycated hemoglobin analyzer (HLC-723GHbV) manufactured by Tosoh Corporation based on the HPLC method, and the vertical axis is the analytical device of the present embodiment. It is the result of measuring glycated hemoglobin (%). In addition, Table 4 shows the measurement CV when measured with the analytical device of the present embodiment.

図２７によれば、糖化ヘモグロビンの含有量が異なる４つの生物学的試料について測定した結果、ＨＰＬＣ法で測定した値に対して乖離が少なく、（表４）に示すように測定ＣＶが３．５％〜５．３％と、従来構成の分析用デバイスを用いた場合の結果に対して、ばらつきが小さくなっていることが確認できる。

According to FIG. 27, as a result of measuring four biological samples having different glycated hemoglobin contents, there was little difference from the value measured by the HPLC method, and the measured CV was 3. From 5% to 5.3%, it can be confirmed that the variation is small with respect to the result when the analysis device having the conventional configuration is used.

  Thus, it can be seen that the measurement accuracy is further improved over the analytical device of the conventional configuration by performing the measurement of the latex blank and the measurement of glycated hemoglobin at the same measurement spot according to the present embodiment.

  Factors that can be improved include measurement at the same measurement spot, variation in optical path length of measurement spots due to molding accuracy and bonding accuracy between measurement spots, and variation in transmittance due to differences in materials and surface conditions. This is because it can be ignored.

  Further, in the analysis device having the conventional configuration, when the immunoassay solution 62 is transferred to the agglutination reaction chamber 46 through the immunoassay quantification chamber 44 after measurement in the immunoassay chamber 43, it adheres to the immunoassay chamber 43. Since the reaction concentration with the agglutination reagent in the agglutination reaction chamber 46 is considered to vary due to the variation in the amount of the remaining immunoassay solution 62, the measurement with the latex blank and the glycated hemoglobin It can be inferred that the variation in reaction concentration could be suppressed by reacting the measurement with the reagent in the same liquid amount.

  In the present invention, since all the solutions transferred at different timings can be held at the same place and transferred to the next step at the necessary timing, the solution can be transferred to the next step in a completely mixed state. It is possible to improve the analysis accuracy, and is useful as a transfer control means for an analysis device used for component analysis of a liquid collected from a living organism.

DESCRIPTION OF SYMBOLS 1 Analytical device 2 Protective cap 3 Base board 4 Cover board 5 Dilution unit 6 Opening button 7 Opening hole 8 Pin 9 Discharge hole 10 Aluminum seal 11 Inlet 12, 13 Cavity 14 Holding cavity 15 Connection flow path 16 Overflow flow path 17 Capillary cavity 18 Separation cavity 19 Capillary cavities 20a, 20b1, 20b2, 20c-20h Air holes 21, 24 Connection flow path 22 Overflow flow path 23 Metering flow paths 25, 26, 27 Overflow cavity 28 Capillary section 29 Diluent overflow Chamber 30 Mixing chamber 31 Rib 32 Holding cavity 33 Capillary part 34 Connection channel 35 Holding cavity 36 Overflow channel 37 Connection channel 38 Denaturation reaction chamber (measurement spot)
39 Denaturing solution quantification chamber 40 Capillary cavity 41 Connection channel 42 Rib 43 Immunoassay chamber 43 (measurement spot, first reaction chamber)
44a Immunoassay quantification chamber (second reaction chamber)
44b Connection 46 Aggregation reaction chamber (measurement spot)
56 Hydrophilic treatment position 57 Blood 57a Plasma component 57b Blood cell component 58 Cavity 59 Connection part 61 Denatured solution 62 Immunoassay solution (first reaction solution)
63 Aggregation solution (second reaction solution)
DESCRIPTION OF SYMBOLS 100 Analyzing apparatus 101 Cover 102 Center 103 Analysis device holding member 104 Motor 105 Light source 106 Photo detector 107 Rotation drive means 108 Control means 109 Optical measurement means 110 Calculation part 111 Display part 150 Sample liquid collection part 151 Diluent holding part 152 Separation part 153 Diluent measuring unit 154 Mixing unit 155 Measuring unit C1 Left rotation C2 Right rotation θ Inclination angle

Claims (3)

When analyzing by transmitting light to the reaction solution in the measurement spot of the analytical device having a microchannel structure that transports the solution toward the measurement spot by centrifugal force,
Supplying a sample solution to a first reaction chamber having a first reagent and transmitting light to the first reaction chamber to analyze the first reaction solution of the first reagent and the sample solution;
Supplying the first reaction solution from the first reaction chamber to the second reaction chamber having a second reagent and reacting the second reaction chamber;
A second reaction solution obtained by reacting the second reagent and the first reaction solution is supplied from the second reaction chamber to the first reaction chamber, and light is transmitted through the second reaction solution in the first reaction chamber. An analysis method in an analysis device having a step of performing analysis. An analysis apparatus for analyzing light by transmitting light through a reaction solution in the measurement spot of an analytical device having a microchannel structure for transferring a solution toward the measurement spot by centrifugal force,
Rotation driving means for generating the centrifugal force by rotating the set analysis device is provided,
According to the program, the measurement spot of the set analysis device is switched between a mode for controlling the rotation driving means to swing the set analysis device and a mode for rotating the analysis device to generate centrifugal force. A control means for transmitting the light to perform the analysis,
The control means;
The sample solution is supplied to the first reaction chamber of the analytical device having the first reagent, and light is transmitted through the first reaction chamber to analyze the first reaction solution of the first reagent and the sample solution. Then, the first reaction solution is supplied from the first reaction chamber to the second reaction chamber of the analytical device having the second reagent and reacted, and the second reagent and the first reaction solution react with each other. An analyzer configured to perform analysis by supplying a reaction solution from the second reaction chamber to the first reaction chamber and transmitting light to the second reaction solution in the first reaction chamber. The sample solution is a solution obtained by mixing a diluted solution obtained by diluting the blood to be examined and a denaturing reagent,
The first reagent is a latex reagent,
A latex solution in which the first reaction solution is an antigen-antibody reaction of glycated hemoglobin and a latex reagent in the sample solution;
The second reagent is an agglutination reagent,
2. The analysis method for an analytical device according to claim 1, wherein the second reaction solution is a latex agglutination solution obtained by subjecting a latex solution and an agglutination reagent to an antigen-antibody reaction.

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