JP4645211B2 - HDL-cholesterol analysis disk and HDL-cholesterol analysis device - Google Patents

Description

  The present invention relates to a disk for HDL-cholesterol analysis and an apparatus for HDL-cholesterol analysis.

 A method for measuring HDL-cholesterol in blood as a specimen will be described below. In the conventional method for measuring HDL-cholesterol, as shown in FIG. 11, first, whole blood (blood) 1 is injected into a container 2, and then centrifugal force is applied to the container 2. Then, the whole blood 1 is separated into blood cells 3 having a high specific gravity and plasma 4 having a low specific gravity, as shown in FIG. At this time, the plasma 4 having a light specific gravity is separated above the container 2.

 Next, as shown in FIG. 13, only this plasma 4 is transferred to another container 5. Then, a reagent 6 for aggregating cholesterol components other than HDL-cholesterol is injected into the container 5. Then, as shown in FIG. 14, centrifugal force is applied to the container 5 to precipitate the aggregates.

 Next, a reaction reagent containing an enzyme such as cholesterol esterase and cholesterol oxidase or a dye precursor is added to the reagent 6 in the container 5 and the melted plasma 7, and the color change caused by the reaction is measured with a spectrophotometer or the like. To measure HDL cholesterol in the blood. Here, in the step of separating the blood cell 3 and the plasma 4 by applying a centrifugal force to the whole blood 1 or the step of melting the plasma 4 and the reagent 6 by applying a centrifugal force, a disk is used as the centrifugal force generating means. There was also.

As prior art document information related to the invention of this application, for example, Patent Document 1 is known.
JP-A-3-225276

 However, in such a conventional method for measuring HDL-cholesterol in blood, of the whole blood 1 separated into plasma 4 and blood cell 3, only the supernatant plasma 4 is transferred to another container 5, and the reagent is transferred to this container 5. After adding 6 and melting, cholesterol components other than HDL-cholesterol are aggregated and precipitated by centrifugal force. The plasma 7 in the container 5 is again taken out and mixed with a reagent containing an enzyme and a chromogen to produce a color reaction. After that, the measurement must be performed with a spectrophotometer or the like, and manual operations such as transfer of the containers 2 and 5 and mixing with a plurality of reaction reagents are complicated.

  Therefore, the present invention has been made to solve this problem, and an object of the present invention is to provide a sample measuring means capable of automatically measuring a sample.

 In order to achieve this object, the HDL-cholesterol analysis disk of the present invention agglomerates cholesterol components other than HDL-cholesterol in order from the axial center toward the outer peripheral direction of the first chamber having the specimen inlet. A second chamber holding a reagent to be sampled, a third chamber for collecting a predetermined volume of a sample, a reagent group that reacts with HDL-cholesterol and changes color, and detects discoloration due to the reaction. A first chamber having a capillary force between the first and second chambers, between the second and third chambers, and between the third and fourth chambers. To the third flow path, and the first to third flow paths are each bent in an inverted “U” shape, and the top of the inverted “U” is A HDL- Cholesterol Analysis disc, characterized in that located on the axis side of the upper end of the sample entering the flow side of the chamber.

 The HDL-cholesterol analysis apparatus of the present invention is equipped with the above-mentioned HDL-cholesterol analysis disk, and a centrifugal force generating means for generating a centrifugal force around the axis of the HDL-cholesterol analysis disk; Control means for controlling the strength of the centrifugal force generated from the force generating means, a light emitting part for emitting laser light toward at least the fourth chamber, and light receiving for receiving the laser light emitted from the light emitting part And a portion. Thereby, the initial purpose can be achieved.

 As described above, according to the present invention, the first chamber and the second chamber have a capillary force and are connected by the reverse “U-shaped” first flow path. By applying a centrifugal force, the blood sample in the first chamber can be separated into blood cells and plasma by the specific gravity difference.

 Thereafter, the supply of centrifugal force is stopped or reduced by the control means, so that only the plasma in the first chamber is advanced by capillary force to the inlet to the second chamber through the top of the first flow path. The inflow into the second chamber is stopped by the surface tension at the outlet.

 Then, a reagent that overcomes this surface tension by the centrifugal force applied next, automatically transfers the specimen to the second chamber, and agglutinates cholesterol components other than HDL-cholesterol held in the second chamber And plasma. By precipitating the aggregation caused by the centrifugal force and stopping or reducing the supply of the centrifugal force, the plasma containing the cholesterol component of only HDL-cholesterol in the second chamber is caused to flow through the top of the second flow path by capillary force. Then, the flow advances to the inlet to the third chamber, and the inflow into the third chamber is stopped by the surface tension at the outlet.

 Then, the surface tension is overcome by the centrifugal force applied next, the specimen is automatically transferred to the third chamber, and is advanced to the specimen discharge port provided in the third chamber. The surplus specimen that has reached the axial center side relative to the attachment position of the discharge port is discharged from the third chamber through the discharge port, and a predetermined amount of the sample can be held in the third chamber.

 Thereafter, by stopping or reducing the supply of centrifugal force, plasma containing a predetermined amount of HDL-cholesterol-only cholesterol component in the third chamber is caused by capillary force through the top of the third flow path. The flow advances to the inlet to the fourth chamber, and the inflow into the fourth chamber is stopped by the surface tension at the outlet.

 Then, the surface tension is overcome by the next applied centrifugal force, the specimen is automatically transferred to the fourth chamber, reacts with cholesterol held in the fourth chamber, and corresponds to the amount of cholesterol. The plasma is mixed with a reagent that produces an amount of dye. And HDL-cholesterol can be quantified automatically by detecting the amount of pigment | dyes produced. That is, all processes can be performed automatically by controlling the centrifugal force using the centrifugal force and the capillary force.

 Therefore, if blood is used as a specimen, HDL-cholesterol in the blood can be easily measured.

 In addition, since it is sufficient that the amount of the sample is sufficient to fill only the first chamber, the amount of the sample may be small compared to the conventional case using a container, and the burden on the human body is reduced.

 Furthermore, since all the processes can be automatically performed using centrifugal force and capillary force, the specimen can be measured quickly.

 Hereinafter, elemental technologies for realizing the embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a side view of a sample (liquid) measuring apparatus. In FIG. 1A, reference numeral 11 denotes a first chamber (container), and reference numeral 12 denotes a specimen injection port provided at the upper end of the first chamber 11. Reference numeral 13 denotes an air vent provided at the upper end of the first chamber 11.

 Reference numeral 14 denotes a second chamber, and the second chamber 14 is located on the downstream side with respect to the direction of the centrifugal force indicated by the arrow 15. Reference numeral 16 denotes a flow path that connects the first chamber 11 and the second chamber 14, and is connected to an upper end of the second chamber 14 from an inlet 17 provided on a side surface of the first chamber 11. . The flow path 16 is bent in an inverted “U” shape. The inverted “U-shaped” top 16 a is located on the side of the centrifugal force source (existing in the direction opposite to the direction of the centrifugal force) from the upper end of the specimen entering the first chamber 11. Reference numeral 18 denotes an air vent provided at the upper end of the second chamber 14.

 Here, as shown in FIG. 1B, the flow path 16 has a thickness 19 of 100 μm. This is a dimension for generating a capillary force (force that causes the liquid to travel through the flow path by capillary action) in the flow path 16. The first chamber 11 and the second chamber 14 both have a thickness dimension 20 of 200 to 600 μm. The dimension 20 is a dimension for preventing capillary force from being generated (non-generated) in the first chamber 11 and the second chamber 14. Further, the wall surface of the flow path 16 is subjected to hydrophilic treatment. On the other hand, the wall surfaces of the first chamber 11 and the second chamber 14 are subjected to a hydrophilic process that is weaker than the flow path 16. This is preferable in improving the capillary force.

 The operation of the sample measuring apparatus using the flow channel 16 configured as described above will be described below. First, the sample 25 is filled in the first chamber 11 as shown in FIG. At this time, if the centrifugal force is applied in the direction of the arrow 15, the liquid level of the test liquid 25 is the same as the liquid level 25 a in the first chamber 11 and the liquid level 25 b in the flow path 16. It has become.

 Here, when the centrifugal force is stopped (or the centrifugal force is made smaller than the capillary force; the same applies hereinafter), the liquid 25 in the first chamber 11 fills the flow path 16 with the capillary force as shown in FIG. To do. At this time, as shown in FIG. 3B, the specimen 25 is not transferred to the second chamber 14 due to the surface tension of the specimen 25 on the joint surface 26 between the flow path 16 and the second chamber 14.

 Next, as shown in FIG. 4, when the centrifugal force is again applied in the direction indicated by the arrow 15, the specimen 25 in the flow channel 16 overcomes the surface tension applied to the joint surface 26 and is transferred to the second chamber 14. Is done. Due to this centrifugal force, the transfer of the specimen 25 in the flow path 16 to the second chamber 14 is continued, and this transfer stops at the position of the inlet 17 of the first chamber 11. That is, when the specimen 25 flows out to the inlet 17 of the first chamber 11, no further specimen 25 can flow into the flow path 16 due to the action of the attachment position of the inlet 17, and the transfer of the specimen 25 is stopped. The

 In this way, the test solution 25 in the first chamber 11 is automatically transferred to the second chamber 14 by centrifugal force and capillary force. However, the specimen 25 from the bottom surface 11 a of the first chamber 11 to the inlet 17 remains in the first chamber 11.

 As described above, the first chamber 11 and the second chamber 14 have a capillary force and are connected by the flow path 16 having an inverted “U-shape”, so that a centrifugal force is applied by the control means. The specimen 25 in the chambers 11 and 14 can be separated by specific gravity. Further, by stopping the centrifugal force, the specimen 25 in the first chamber 11 is advanced by capillary force to the outlet to the second chamber 14 via the top portion 16a of the flow path 16, and the surface at the outlet. Outflow can be stopped by tension. Then, this surface tension can be overcome by the centrifugal force applied next and transferred to the second chamber 14. That is, all processes can be performed automatically by controlling the centrifugal force.

Further, since the sample 25 only needs to have an amount sufficient to fill only the first chamber 11, the amount of the sample 25 may be small compared to the conventional one using the containers 2 and 5, and the burden on the human body is reduced.
Furthermore, since the entire process can be automatically performed using centrifugal force and capillary force, the sample 25 can be measured speedily.

 The centrifugal force in the above description is obtained by forming a sample measuring device in a disk and rotating the disk. The details of the generating means for generating the centrifugal force and the control means for controlling the stop of the centrifugal force will be described in the third embodiment.

(Embodiment 1)
FIG. 5 is a side view of the sample measuring apparatus according to the first embodiment. In the present embodiment, blood is used as the specimen. In FIG. 5, reference numeral 31 denotes a whole blood separation chamber (the first chamber) provided on the side closest to the axis serving as a centrifugal force source, and hereinafter, the separation chamber 32 sequentially toward the downstream (arrow 15). (Second chamber), quantitative chamber 33 (third chamber), and reaction / detection chamber 34 (fourth chamber) are provided.

 Reference numeral 35 denotes a flow path connected to the side surface of the whole blood separation chamber 31, and the flow path 35 is bent in an inverted “U-shape”. The top 35a of the reverse “U” is positioned on the centrifugal force source side from the upper end of the blood entering the whole blood separation chamber 31.

  Further, the downstream side of the top portion 35 a of the flow path 35 is connected to the upper end of the reagent chamber 36, and the lower end of the reagent chamber 36 is connected to the inlet of the stirring unit 38 through the flow path 37. The outlet of the stirring unit 38 is connected to the upper end of the separation chamber 32 by a flow path 39.

 From the side surface of the separation chamber 32, the flow path 40 is connected to the upper end of the quantitative chamber 33. The flow path 40 is bent in an inverted “U” shape. The top portion 40a of the reverse “U” is located on the centrifugal force source side from the upper end of the plasma entering the separation chamber 32.

 In addition, a channel 41 is connected to the upper end of the reaction / detection chamber 34 from the bottom surface 33 b of the quantitative chamber 33. The flow path 41 is bent in an inverted “U” shape. The top 41a of the reverse “U” is located on the centrifugal force source side from the upper end of the plasma entering the quantitative chamber 33. Here, in order to stably measure a certain amount of plasma, it is important that the flow path 41 is led out from the bottom surface 33 b of the quantitative chamber 33. In addition, a discharge port 33 c is provided on the upper surface of the quantitative chamber 33. That is, a certain amount of plasma can be measured with the volume from the bottom surface 33b to the discharge port 33c.

 Here, the reagent chamber 36 holds a reagent 42 excluding cholesterol components other than HDL-cholesterol (hereinafter referred to as HDL-C), and the reaction / detection chamber 34 holds a reaction reagent 43. . The stirring unit 38 is formed by bending the flow path 37 into a “U” shape and connecting a plurality of these “U” shapes. When the plasma passes through the stirring portion 38 formed in this way, the flow is disturbed by the “U-shape”, and thus the plasma is stirred.

 In FIG. 5, the reagent chamber 36 and the stirring unit 38 are provided, but these are not formed, and the whole blood separation chamber 31 and the separation chamber 32 are directly connected to each other through a flow path, and cholesterol components other than HDL-C. It is also possible to hold the reagent 42 except for in the separation chamber 32. This makes it easier to create an analysis disk.

 A valve 35 d is provided at the upper end of each chamber 31, 32, 33, 34 and in the vicinity of the inlet 35 b of the flow path 35. The chambers 31, 32, 33, 34, and 36 have a thickness of 400 μm so that no capillary force is generated. On the other hand, the thickness of the flow paths 35, 37, 39, 40, 41 and the stirring unit 38 is set to 100 μm, and is set to a thickness at which capillary force is generated.

 The operation of the sample measuring apparatus configured as described above will be described below. First, whole blood (blood) 45 is injected from the inlet 31 a of the whole blood separation chamber 31. At this time, it is assumed that the centrifugal force is stopped (or the centrifugal force is made smaller than the capillary force. The same shall apply hereinafter).

 Whole blood 45 flows into whole blood separation chamber 45. When this whole blood 45 reaches the inlet 35b of the flow path 35, it flows into the flow path 35 by capillary force. The blood sample 45 that has flowed in is stopped by a valve 35d that is thicker than the flow path 35 provided in the vicinity of the inflow port 35b. That is, when the whole blood 45 reaches the valve 35d of the flow path 35, the whole blood 45 is prohibited from flowing into the valve 35d due to the surface tension of the whole blood 45 on the inlet surface of the valve 35d. In this state, the whole blood 45 is filled into the whole blood separation chamber 45.

 Next, when a centrifugal force is applied, the whole blood 45 is pressed in the direction of the arrow 15. As a result, the whole blood 45 overcomes the surface tension of the inlet surface of the valve 35 d and flows into the valve 35 d in the flow path 35, and further proceeds in the flow path 35. Then, the liquid level 45 b is the same as the liquid level 45 a of the whole blood separation chamber 31 and is stopped. The whole blood 45 flowing into the whole blood separation chamber 31 and the flow path 35 is separated into plasma and blood cells by centrifugal force, and the blood cells move to the region of the whole blood separation chamber 31 in the direction of the arrow 15 from the inlet 35b. To do.

 Next, when the centrifugal force is stopped, only the plasma separated in the whole blood separation chamber 31 and the flow path 35 flows in from the inlet 35b of the flow path 35 provided on the side surface. Together with the plasma flowing into the surface 45b, the reagent 42 is stirred with the reagent 42 in the stirring part 38 while dissolving the reagent 42 in the flow path 36 over the top 35a by capillary force. Thereafter, it reaches the outlet 39c of the flow path 39, and the outflow to the reagent chamber 32 is prohibited by the surface tension at the outlet 39c.

 In this state, when a centrifugal force is applied, the plasma that fills the flow channel on the centrifugal force direction side of the whole blood separation chamber 31, that is, the flow channel 37, the stirring unit 38, and the flow channel 39 is pushed down, As a result, the specimen 45 in the whole blood separation chamber 31 is sucked out to the position of the inlet 35b of the flow path 35. Only the plasma separated in the whole blood separation chamber 31 by this negative pressure flows into the separation chamber 32 through the flow paths 35, 37 and 39. In the separation chamber 32, cholesterol components other than HDL-C are separated as aggregated precipitates from plasma cholesterol components by centrifugal force.

 The separated HDL-C also flows into the flow path 40 and stops at the same level as the plasma level in the separation chamber 32 before the top 40a. At this time, the plasma in the whole blood separation chamber 31 stops flowing at the position of the inlet 35b. This is an effect due to the position from the inlet 35 b and the bottom surface 31 b of the whole blood separation chamber 31. Of the whole blood 45, the separated blood cells accumulate between the bottom surface 31b and the inflow port 35b. Even if the centrifugal force is stopped here, the blood cells on the bottom surface 31b side from the inlet 35b no longer flow into the flow path 35 due to capillary action.

 In this way, the plasma in the whole blood separation chamber 31 can be transferred to the separation chamber 32 without manual intervention by centrifugal force and capillary force. Hereinafter, since the mechanism for transferring the specimen to the chamber provided downstream is the same, the description of this transfer mechanism is simplified.

 That is, when the centrifugal force is stopped next, the plasma containing only HDL-C in the cholesterol component of the specimen exceeds the top 40 a of the flow path 40 by the capillary force. And it stops by the surface tension at the outflow port 40c of the flow path 40. Then, when centrifugal force is applied next, plasma containing only HDL-C in the cholesterol component of the specimen in the separation chamber 32 flows into the quantitative chamber 33. The HDL-C flows into the flow path 41, but stops at the same position as the liquid level of the metering chamber 33 (without exceeding the top 41a). The extra HDL-C that has flowed in is discharged from the discharge port 33c. Here, in the separation chamber 32, cholesterol components other than the separated HDL-C remain between the bottom surface 32b and the inflow port 40b as a coagulated precipitate.

 Next, when the centrifugal force is stopped, the HDL-C is stopped by the surface tension at the outlet 41c of the flow path 41 beyond the top 41a due to the capillary force.

 When a centrifugal force is applied next, all of the HDL-C in the quantitative chamber 33 is transferred to the reaction / detection chamber 34. Here, since the outflow port 41 b is provided on the bottom surface 33 b of the quantitative chamber 33, plasma does not remain in the quantitative chamber 33. That is, the metering according to the volume of the quantitative chamber 33 is performed.

 The HDL-C transferred to the reaction / detection chamber 34 undergoes an enzyme reaction with the reaction reagent 43. The reaction / detection chamber 34 is irradiated with laser light, and HDL-C is measured from the absorbance of the dye produced by the reaction. In this way, the concentration of HDL-C in the specimen can be automatically measured without using human hands by utilizing centrifugal force and capillary force.

 The centrifugal force in the present embodiment is obtained by forming a specimen measuring device in a disk and rotating the disk.

 Here, the reagent 42 excluding cholesterol components other than HDL-C is not limited to be disposed in the flow path 36, but may be disposed in the stirring unit 38 and the separation chamber 32.

 The functions of the separation chamber 32 and the quantitative chamber 33 may be combined into one chamber. For example, the separation chamber 32 is provided with a discharge port similar to the discharge port 33c of the quantitative chamber 33. 41 may be omitted.

 Further, the reaction / detection chamber 34 is not limited to one chamber. For example, in consideration of the stability of each reagent constituting the reaction reagent 43, a chamber is further provided on the upstream side of the reaction / detection chamber 34. In addition, an arbitrary reagent constituting the reaction reagent 43 may be arranged separately from the reaction reagent 43 in the chamber. In addition, the reaction reagent 43 is not limited to the reaction / detection chamber 34 although it is the same, and an arbitrary reagent constituting the reaction reagent 43 is separated from the reaction reagent 43 in the quantitative chamber 33. May be.

 That is, the three functional elements of the whole blood separation chamber 31 and the reagent 42 and the separation chamber 32 are the minimum necessary in terms of structure. The structure to be realized can be arbitrarily selected within the range allowed by the space on the disk and within the range to which the above method can be applied, and the detection means is not limited to the laser beam, but electrochemically. It may be detected.

 The details of the generating means for generating the centrifugal force and the control means for controlling the stop of the centrifugal force will be described in the third embodiment.

(Embodiment 2)
In the second embodiment, a process of detecting HDL-C from whole blood 45 will be described. Note that the same reference numerals are assigned to the same components as those in the first embodiment to simplify the description.

 In FIG. 6, reference numeral 51 denotes a sample injection process in which the centrifugal force is stopped and the whole blood 45 is injected from the injection port 31 a of the whole blood separation chamber 31.

 Reference numeral 52 denotes a whole blood separation step. In this whole blood separation step 52, centrifugal force is applied to the whole blood separation chamber 31 (since the centrifugal force is obtained by rotating the disk, it is simply expressed as “rotation” hereinafter. The whole blood 45 in the whole blood separation chamber 31 is separated into plasma and blood cells.

  53 is a plasma separation / transfer process. In this plasma separation / transfer process 53, the centrifugal force is stopped and only the plasma and blood cells separated in the whole blood separation chamber 31 are transferred by capillary force. . Then, the reagent 42 excluding cholesterol components other than HDL-C mounted in the reagent chamber 36 is dissolved in plasma. The dissolved plasma is stirred in the process of passing through the stirring unit 38. That is, a precipitation reagent dissolving / stirring step 53a and an agglutination reaction step 53b are also included.

  As described above, when the reagent chamber 36 and the stirring unit 38 are not formed, the precipitation reagent dissolving / stirring step 53 a and the aggregation reaction step 53 b are performed in the separation chamber 32.

  54 is a sample quantitative fractionation step, and in this sample quantitative fractionation step 54, a centrifugal force is applied by rotating the disk. First, cholesterol components other than HDL-C in the plasma are precipitated in the separation chamber 32. A fixed volume of the volume of plasma containing only HDL-C among the cholesterol components in the sample is measured in the quantitative chamber 33. That is, the sample quantitative fractionation step 45 includes an HDL-C centrifugation step 45a and a sample quantitative fractionation step 45b.

Reference numeral 55 denotes a sample transfer process. In this sample transfer process 55, the rotation of the disk is stopped. Then, the HDL-C measured by a certain amount in the flow path 41 is transferred to the outlet 41c over the top 41a by capillary force.
Reference numeral 56 denotes an HDL-C detection step. When a centrifugal force is applied, the disk is rotated and a certain amount of HDL-C measured in the quantitative chamber 33 is taken into the reaction / detection chamber 34 from the flow path 41. Then, the reaction reagent 43 mounted in the reaction / detection chamber 34 is dissolved. That is, the reaction reagent 43 is dissolved in the reaction reagent dissolving step 56a and reacted with the enzyme in the enzyme reaction step 56b. And HDL-C is detected by the HDL-C detection process 56c.
That is, a laser beam is irradiated to the dye produced by the reaction between HDL-C and the enzyme, and HDL-C is measured from the absorbance. In this way, it is possible to automatically measure the amount of HDL-C in a certain amount in the whole blood 45 without using human hands by utilizing centrifugal force and capillary force.

 As shown in FIG. 15, the flow branches from the flow path 35 connected to the whole blood separation chamber 31, or the flow paths 61 and 62 are directly connected to the whole blood separation chamber 31. Then, quantitative chambers 63 and 64 (the fifth and seventh chambers) are connected to the flow paths 61 and 62, respectively. These quantitative chambers 63 and 64 are connected to flow paths 65 and 66, respectively, and these flow paths 65 and 66 are connected to reaction / detection chambers 67 and 68 (the sixth and eighth chambers), respectively.

  That is, the flow path 61, the quantitative chamber 63, the flow path 65, and the reaction / detection chamber 67 are connected in series in this order to form a first serial connection body, and the flow path 62, the quantitative chamber 64, and the flow path 66. Then, the reaction / detection chamber 68 is connected in series in this order to form a second series connection body. Then, by connecting the first series connection body and the second series connection body in parallel, it is possible to form two similar sample measuring apparatuses.

 If the reagent for detecting total cholesterol is held in the reaction / detection chamber 67 and the reagent for detecting triglyceride in the reaction / detection chamber 68, the plasma separated in the whole blood separation chamber 31 is used. In the reaction / detection chamber 67, total cholesterol (TC) can be detected, and at the same time, the reaction / detection chamber 68 can detect triglyceride (TG). That is, HDL-C, total cholesterol, and triglyceride can be detected automatically and simultaneously from the whole blood 45 without human intervention.

(Embodiment 3)
FIG. 7 is a side view of the HDL-cholesterol analysis device described in the first and second embodiments. In FIG. 7, reference numeral 70 denotes a disk motor as drive means provided connected to the axis 93 of the disk 71, and the disk 71 is rotated by the disk motor 70. The disk motor 70 is controlled and stopped by a control circuit 73 as control means.

 Reference numeral 74 denotes a laser light output drive circuit. A laser diode forming the light emitting unit 75 emits light based on the output of the laser light output drive circuit 74, and the laser light output at this time is used as a measurement chamber 113 (measurement chamber) of the disk 71. 114). Two laser beams are output, but only one is shown in FIG.

 Of this laser light, the laser light used for controlling the rotation of the disk 71 is reflected by the disk 71 and the information on the disk 71 is detected by the photodetector 76. The output is input to the control circuit 73, and the rotation of the disk 71 is controlled. That is, the rotation speed is controlled and stopped.

 Further, laser light passing through the measurement chamber 113 of the disk 71 in the laser light is detected by a photodetector that forms the light receiving portion 77, and its output is processed by the video signal processing circuit 78. In this way, the absorbance of the test solution (liquid for component inspection) 85 in the measurement chamber 113 is measured.

 The disk 71 has a disk shape, a diameter of 12 cm, and a thickness 149 of 2 mm. The rotational speed of the disk 71 that generates centrifugal force is 500 to 6000 revolutions per minute. The disk 71 also stores thickness information and calibration information of the measurement chamber 113, and the video signal processing circuit 78 measures the absorbance of the test solution 85 based on this information.

 FIG. 8 is a cross-sectional view of the main part in the vicinity of the measurement chamber 113. In FIG. 8, reference numeral 81 denotes a base substrate. The base substrate 81 is made of polycarbonate (PC), glass, acrylic, plastic, or the like, and is made of a transparent material.

 An upper cover 83 is adhered to the upper surface of the base substrate 81 via an adhesive (two-component mixed adhesive, thermosetting adhesive, glue, etc.) 82. The upper cover 83 is also made of polycarbonate (PC), glass, acrylic, plastic, etc., and is made of a transparent material. In this way, the upper cover 83 is firmly fixed to the base substrate 81 with the adhesive 82.

 Reference numeral 84 denotes a recess provided in the upper cover 83, and the recess 84 opens toward the base substrate 81. That is, the measurement chamber 113 is formed by the recess 84 and the base substrate 81. The measurement chamber 113 formed in this way is filled with the test solution 85.

 A light emitting unit 75 is provided below the measurement chamber 113 and is formed of a laser diode. A light receiving unit 76 is provided above the measurement chamber 113 and is formed of a photodetector. The laser beam emitted from the light emitting unit 75 passes through the test solution 85 in the measurement chamber 113 and is received by the light receiving unit 77. The property and the like of the test solution 85 are measured from the attenuation (absorbance) of the laser beam caused by the laser light passing through the test solution 85.

 FIG. 9 is a partially fragmented plan view of the disk 71 constituting the sample measuring apparatus. The disk 71 is composed of a plurality (six in this embodiment) of independent sample analyzers 91. In FIG. 9, reference numeral 92 denotes an inlet for the test solution 85 provided in the vicinity of the shaft 93. The injection port 92 for the test solution 85 is connected to the test solution analyzing unit 91. In addition, the sample analysis unit 91 causes the sample analysis unit 91 to generate a centrifugal force in the outer circumferential direction by rotating the disk 71 in the 94 direction. In the present embodiment, since each of the six independent sample analyzers 91 has six independent sample analyzers 91, each of the sample analyzers 91 can independently analyze different types of samples 85 at a time. .

 FIG. 10 is an enlarged view of a main part of the test liquid analysis unit 91. In FIG. 10, the injection port 92 for the test solution 85 is connected to the injection chamber 95. Reference numeral 96 denotes a flow path derived from the vertical side surface of the injection chamber 95, and a valve 96 b is provided in the vicinity of the inlet 96 c of the flow path 96. The flow path 96 is bent in an inverted “U” shape. The inverted “U-shaped” top portion 96 a is located on the axial center 93 side from the upper end of the injection chamber 95. Further, the flow path 96 is branched in the middle, and one of them is connected to the upper end of the reagent chamber 97. The other branched portion is connected to a flow path 98, and this flow path 98 is connected to an outer peripheral chamber 99 provided near the outer periphery of the disk 71.

 The lower end of the reagent chamber 97 is connected to the upper end of the quantitative chamber 101 through the flow channel 100. The flow path 100 is formed with a stirring portion 102 in which “U” shapes are connected in opposite directions. The stirring portion 102 is formed of a plurality of “U” shapes that open toward the axial center 93 side (the direction opposite to the arrow 103).

 Note that the reagent chamber 97 and the stirring unit 102 may not be specially formed. In this case, the reagent to be held in the reagent chamber 97 can be held in the quantitative chamber 101.

 From the lower end of the quantitative chamber 101, a flow path 104 is connected to the upper end of the first reaction chamber 105. The connecting channel 104 is bent in an inverted “U” shape. The inverted “U-shaped” top portion 104 a is located on the axial center 93 side from the upper end of the metering chamber 101. Reference numeral 106 denotes a flow path that connects the upper end of the metering chamber 101 and the upper end of the injection chamber 95 and branches from the middle to connect to the outer peripheral chamber 99. An air vent is provided at the upper end 106 a of the channel 106. Reference numeral 107 denotes a flow path that connects the upper end of the quantitative chamber 101 and the outer peripheral chamber 99.

 From the lower end of the first reaction chamber 105, it is connected to the upper end of the second reaction chamber 109 by a flow path 108. The connected channel 108 is bent in an inverted “U” shape. The inverted “U-shaped” top portion 108 a is located closer to the axial center 93 than the upper end of the first reaction chamber 105. Reference numeral 105 a denotes an air vent provided at the upper end of the first reaction chamber 105.

 From the lower end of the second reaction chamber 109, two flow paths 111 and 112 are connected to the lower ends of the measurement chambers 113 and 114, respectively. The connecting channels 111 and 112 are bent in an inverted “U” shape. The tops 111 a and 112 a of the inverted “U” shape are located on the axial center 93 side from the upper end of the second reaction chamber 109. 109 a is an air vent provided at the upper end of the second reaction chamber 109. The upper ends of the measurement chambers 113 and 114 and the outer peripheral chamber 99 are connected by a flow path 115.

 Note that the flow path 98 may be branched to form another determination system. That is, a plurality of configurations from the reagent chamber 97 or the quantitative chamber 101 of the present embodiment to the measurement chambers 113 and 114 can be provided in parallel. Then, the test solution 85 injected into the injection chamber 95 can be used for another independent determination.

 Here, the injection chamber 95, the reagent chamber 97, the quantitative chamber 101, the first reaction chamber 105, the second reaction chamber 109, and the measurement chambers 113 and 114 have a thickness of 200 to 600 μm, and the flow paths 96, 100, 104 , 108, 111, 112 have a thickness of 100 μm. Therefore, a capillary force is generated in these flow paths 96, 100, 104, 108, 111, 112.

 The operation of the measuring apparatus configured as described above will be described below. This specimen measuring apparatus alternately generates centrifugal force and capillary force, and sequentially transfers the test solution 85 in each chamber in the direction of centrifugal force (direction of arrow 103). Each time, each step of the sample analysis is performed.

 First, the rotation of the disk 71 is stopped. Then, the test solution 85 is injected from the injection port 92. The test solution 85 is temporarily stored up to the inlet 96c of the flow path 96 provided in the injection chamber 95. When the test solution 85 reaches the inflow port 96c, it proceeds to the valve 96b provided in the flow path 96 by capillary force. Further, further infiltration is stopped by the surface tension of the valve 96b. In this stopped state, the test solution 85 is further injected.

 Then, centrifugal force is applied by rotating the disk 71 at 4000 to 6000 rpm. Then, the test solution 85 in the injection chamber 95 is pushed down in the direction of the centrifugal force (the direction of the arrow 103). As a result, the test solution 85 in the flow path 96 passes through the position of the valve 96b by this centrifugal force, stops at the same position as the liquid level of the injection chamber 95. At this time, the test solution 85 does not reach the top 96a. At this time, if the test solution 85 in the injection chamber 95 is whole blood, the blood cell component is centrifuged and moved in the centrifugal force direction from the inlet 96c, and the centrifugal force source direction is filled with the plasma component from the interface. It is.

 Next, the disk 71 is stopped (or decelerated until the centrifugal force is weaker than the capillary force. The same applies hereinafter). As a result, the test solution 85 consisting only of plasma components in the injection chamber 95 passes through the top 96a of the flow path 96, the reagent chamber 97, and the stirring unit 102 by capillary force, and proceeds to the inlet to the quantitative chamber 101. This is stopped by surface tension at the inlet. At this time, the test solution 85 proceeds while dissolving the reagent composed of sodium phosphotungstate and magnesium chloride held in the reagent chamber 97, and passes through the agitation unit 102 to mix the reagent and the test solution 85. This reagent composed of sodium phosphotungstate and magnesium chloride is prepared so as to aggregate and cause precipitation of cholesterol components other than HDL-C among plasma cholesterol components, and this technique is publicly known.

 This reagent is also known in the art as other polyanion compounds (phosphomolybdic acid, tungstic acid, inorganic salts of molybdic acid, or sulfated polysaccharides such as dextran sulfate, heparin, amylose sulfate, amylopectin sulfate) and water-soluble metal ions. Compound (compound having a divalent metal ion selected from calcium, manganese, cobalt, nickel, strontium, zinc, barium and copper, or a compound having a metal ion other than divalent selected from aluminum, iron and chromium) Alternatively, a combination of ammonium ion compounds may be used.

 Next, when a centrifugal force is applied by the rotation of the disk 71, the test solution 85 overcomes the surface tension and is guided to the quantitative chamber 101. Here, the test solution 85 filled in the stirring unit 102 and the flow path 100 is pushed down by the centrifugal force, and the test solution 85 in the injection chamber 95 is sucked out to the injection port 96 c and flows into the quantitative chamber 101. Further, a part of the sucked test solution 85 is discharged to the outer peripheral chamber 99 through the flow path 98.

 Of the test solution 85 that has flowed into the quantitative chamber 101, the overflowed test solution 85 is discharged to the outer peripheral chamber 99 through the channel 107. Therefore, the test solution 85 in the quantitative chamber 101 becomes constant at the liquid level at the outlet of the flow channel 107. In addition, the test solution 85 that has flowed into the flow path 104 is below the top 104 a of the flow path 104 and is at the liquid level at the outlet of the flow path 107. As a result, a fixed amount of the test solution 85 is measured in the fixed quantity chamber 101. Further, aggregation of cholesterol components other than HDL-C produced in the reagent chamber 97 and the stirring unit 102 is separated from the test solution 85 and precipitated by the centrifugal force on the centrifugal force direction side of the quantitative chamber 101.

Next, the rotation of the disk 71 is stopped. As a result, the test solution 85 in which the cholesterol component in the quantitative chamber 101 is only HDL-C passes through the top 104a of the flow path 104 by capillary force, and due to surface tension at the inlet to the first reaction chamber 105. Stop.
Next, when a centrifugal force is applied by the rotation of the disk 71, the test solution 85 in which the cholesterol component is only HDL-C overcomes the surface tension and flows into the first reaction chamber 105. The test solution 85 reacts by dissolving the first reagent in the first reaction chamber 105. The first reagent consists of cholesterol esterase, β-diphosphopyridine nucleotide disodium (NAD +), diaphorase, sodium cholate, n-octyl-β-D-thioglucoside and buffer components. Further, while the centrifugal force is applied, the test solution 85 is stopped below the top portion 108a of the flow path 108.

 Here, by stopping the disk 71, the test solution 85 in the first reaction chamber 105 passes through the top part 108 a of the flow path 108 by capillary force, and due to surface tension at the inlet to the second reaction chamber 109. Stop.

 Next, when a centrifugal force is applied by the rotation of the disk 71, the test solution 85 overcomes the surface tension and flows into the second reaction chamber 109. Then, the test solution 85 dissolves the second reagent in the second reaction chamber 109 by centrifugal force. The second reagent consists of cholesterol dehydrogenase. In addition, while the centrifugal force is applied, the test solution 85 is stopped below the high points 111a and 112a of the channels 111 and 112.

 Next, by stopping the disk 71, the test solution 85 in the second reaction chamber 109 passes through the top portions 111a and 112a of the flow paths 111 and 112 by capillary force, and flows to the measurement chambers 113 and 114. Stop by surface tension at the entrance.

 Next, when a centrifugal force is applied by the rotation of the disk 71, the test solution 85 overcomes the surface tension and flows into the measurement chambers 113 and 114. Therefore, the test solution 85 in the second reaction chamber 109 is divided by half and flows into the measurement chambers 113 and 114. Then, the test solution 85 dissolves the third reagent held only in the measurement chamber 114, the main reaction is started in the measurement chamber 114, and a dye that absorbs an amount of laser light corresponding to the concentration of HDL-C. Is generated. The third reagent comprises the dye precursor WST-9 (manufactured by Dojindo Laboratories).

 The properties of the test solution 85 are measured by comparing the absorbances of the measurement chambers 113 and 114. That is, laser light is emitted from one of the measurement chambers 113 and 114 in the vertical direction by the laser diode of the light emitting unit 75 described with reference to FIG. 7, and received by the photodetector of the light receiving unit 77 provided on the other side. The absorbance of the test solution 85 is measured based on the intensity of light reception (transmittance).

 In the measurement chambers 113 and 114, the test solution 85 can be further stirred to make the concentration uniform by the following method. As a result, the absorbance can be measured with little error.

 That is, by alternately rotating and stopping the disk 71, centrifugal force and capillary force are alternately generated. Then, by this action, the test solution 85 reciprocates between the measurement chambers 113 and 114 and the flow paths 111 and 112. Thus, further stirring of the test solution 85 can be promoted. In this case, it is desirable to increase the volume of the flow paths 111 and 112, and it is preferable to set the volume of the measurement chambers 113 and 114 to 25% to 100%.

 Further, in order to obtain the stirring action by alternately generating the centrifugal force and the capillary force, it is important to connect the two flow paths 111 and 112 to the lower ends of the measurement chambers 113 and 114, respectively. This is because the flow path 111 (112) is always connected to the measurement chamber 113 (114) by the test solution 85.

  Since the HDL-cholesterol analysis disk and the HDL-cholesterol analysis device according to the present invention can automatically measure the properties of the specimen, they are useful for measuring total cholesterol, triglyceride, etc. in addition to HDL-cholesterol. It is.

(A) is a top view of the sample measuring apparatus for demonstrating the elemental technology used as the premise of this invention. (B) is the principal part sectional drawing. Same as above, a plan view showing the first state (A) is a plan view showing the second state, (b) is a cross-sectional view of the relevant part. The top view which shows a 3rd state same as the above The top view of the disk for HDL-cholesterol analysis in Embodiment 1 of this invention Process drawing which shows the analysis procedure in Embodiment 2 of this invention The top view of the apparatus for HDL-cholesterol analysis in Embodiment 3 of this invention Cross-sectional view of the main part of a disk for HDL-cholesterol analysis used in the apparatus Same as above, partially broken plan view of the disk Same part enlarged view First process diagram of conventional specimen measurement method Same as above, second process diagram Third process diagram 4th process drawing The top view of the disk for other HDL-cholesterol analysis in Embodiment 2 of this invention

Explanation of symbols

11 First chamber 12 Inlet 14 Second chamber 15 Arrow 16 First flow channel 16a Top 25 Sample

Claims (14)

A first chamber having a specimen injection port and a second chamber holding a reagent that agglutinates cholesterol components other than HDL-cholesterol in order from the axial center toward the outer periphery thereof, and the first and first chambers. The two chambers are connected by a first flow path having a capillary force, the first flow path is bent in an inverted “U-shape”, and the top of the inverted “U-shape” is formed in the first flow path. The HDL-cholesterol analysis disk, which is located on the axial side from the upper end of the specimen entering the chamber. A first chamber having an inlet for a specimen, a second chamber holding a reagent for aggregating a cholesterol component other than HDL-cholesterol, and a specimen having a predetermined volume in order from the axial center toward the outer circumference. A third chamber that separates the first and second chambers, and the second and third chambers are connected by first and second flow paths having a capillary force, respectively. In addition, each of the first and second flow paths is bent in an inverted “U-shape”, and the top of the inverted “U-shape” is located on the axial center side from the upper end of the specimen entering the upstream chamber. A disc for HDL-cholesterol analysis, characterized by A first chamber having a specimen injection port, a second chamber holding a reagent for agglutinating cholesterol components other than HDL-cholesterol, and a specimen having a predetermined volume in order from the axial center toward the outer periphery. And a fourth chamber for holding a reagent that changes color by reacting with HDL-cholesterol and for detecting a color change caused by the reaction, the first and second chambers And the second and third chambers and the third and fourth chambers are respectively connected by first to third flow paths having capillary force, and the first to third chambers are connected to each other. Each of the flow paths is bent in an inverted “U-shape”, and the top of the inverted “U-shape” is located on the axial center side from the upper end of the sample entering the upstream chamber. To HDL- cholesterol analysis for the disk. Instead of holding a reagent for agglutinating cholesterol components other than HDL-cholesterol in the second chamber, it is connected between the first chamber and the second chamber by a flow path, respectively. The HDL-cholesterol analysis disk according to any one of claims 1 to 3, wherein the reagent is held in a chamber provided in the disk. The reagent for aggregating cholesterol components other than HDL-cholesterol is a reagent comprising a combination of sodium phosphotungstate and magnesium chloride, or inorganic salts such as phosphomolybdic acid, tungstic acid, molybdic acid, or dextran sulfate, heparin, A polyanion compound selected from the group consisting of sulfate polysaccharides such as amylose sulfate and amylopectin sulfate, a compound having a divalent metal ion selected from calcium, manganese, cobalt, nickel, strontium, zinc, barium, copper, and aluminum And a reagent comprising any combination of a compound having a metal ion other than divalent selected from iron and chromium and a water-soluble compound selected from the group consisting of compounds having ammonium ions, Do HDL- Cholesterol Analysis disc according to any one of Motomeko 1 4. The HDL-cholesterol analysis disk according to any one of claims 1 to 3, wherein the reagent that reacts with HDL-cholesterol contains a tetrazolium salt. A fifth chamber that is connected to the outer circumference side of the first chamber by a fourth flow path so that the sample from the first chamber flows, and that collects a predetermined volume of the sample; A sixth chamber for holding a reagent that changes color by reacting with cholesterol and detecting a color change caused by the reaction, between the first and fifth chambers and between the fifth and sixth chambers; The fourth and fifth flow paths are connected by capillary forces, respectively, and the fourth and fifth flow paths are bent in a reverse “U” shape, and the top of the reverse “U” shape. The HDL-cholesterol analysis disk according to claim 3, wherein is located on the axial center side from the upper end of the specimen entering the upstream chamber. A seventh chamber which is connected to the outer periphery of the first chamber by a sixth flow path so that the sample from the first chamber flows, and which dispenses a predetermined volume of the sample, and triglyceride And an eighth chamber for detecting a color change caused by the reaction, and a space between the first and seventh chambers and a space between the seventh and eighth chambers, respectively. The sixth and seventh flow paths having capillary force are connected to each other, and the sixth and seventh flow paths are bent in a reverse “U” shape, and the top of the reverse “U” is 4. The HDL-cholesterol analysis disk according to claim 3, wherein the HDL-cholesterol analysis disk is positioned on the axial center side from the upper end of the specimen entering the upstream chamber. The HDL-cholesterol analysis disk according to claim 1 is mounted, a centrifugal force generating means for generating a centrifugal force around an axis of the HDL-cholesterol analysis disk, and a centrifugal force generated from the centrifugal force generating means Control means for controlling the strength, and after injecting whole blood as a specimen into the first chamber, the centrifugal force is applied by the centrifugal force generating means to separate blood cell components in the first chamber. Then, after the centrifugal force is stopped or weakened by the centrifugal force control means, only the plasma component is moved to the second chamber by applying the centrifugal force again, and in the second chamber, other than HDL-cholesterol After aggregating the cholesterol component, the aggregate is separated by centrifugal force, and then the centrifugal force is stopped or weakened, and the centrifugal force is applied again to regenerate HDL-core. Terol only features that HDL- cholesterol analytical device to so as to obtain a plasma containing cholesterol components. The HDL-cholesterol analysis disk according to claim 2 is mounted, a centrifugal force generating means for generating a centrifugal force around an axis of the HDL-cholesterol analysis disk, and a centrifugal force generated from the centrifugal force generating means Control means for controlling the strength,
After injecting whole blood into the first chamber as a specimen, centrifugal force is applied by the centrifugal force generating means to separate blood cell components in the first chamber, and then centrifugal force is controlled by the centrifugal force control means. After stopping or weakening, only the plasma component is moved to the second chamber by applying centrifugal force again. After the cholesterol component other than HDL-cholesterol is aggregated in the second chamber, the aggregate is centrifuged. Then, the centrifugal force is stopped or weakened, and the centrifugal force is applied again to move the plasma containing the cholesterol component of only HDL-cholesterol to the third chamber, and a predetermined volume in the third chamber. After fractionating the plasma, the centrifugal force is stopped or weakened, and the centrifugal force is applied again. Terol only features that HDL- cholesterol analytical device to so as to obtain a plasma containing cholesterol components. 9. A centrifugal force generating means for generating a centrifugal force around an axis of the HDL-cholesterol analysis disk, wherein the HDL-cholesterol analysis disk according to claim 3, 7 or 8 is mounted, and generated from the centrifugal force generating means Provided with a control means for controlling the strength of the centrifugal force, a light emitting part for emitting laser light toward at least the fourth chamber, and a light receiving part for receiving the laser light emitted from the light emitting part. HDL-cholesterol analyzer. After injecting whole blood into the first chamber as a specimen, centrifugal force is applied by the centrifugal force generating means to separate blood cell components in the first chamber. Thereafter, the centrifugal force is controlled by the centrifugal force control means. After stopping or weakening, only the plasma component is moved to the second chamber by applying centrifugal force again, and after the cholesterol component other than HDL-cholesterol is aggregated in the second chamber, the aggregate is centrifuged. Then, the centrifugal force is stopped or weakened, and the centrifugal force is applied again to move the plasma containing the cholesterol component of only HDL-cholesterol to the third chamber, and a predetermined volume in the third chamber. Then, the centrifugal force is further stopped or weakened, and the centrifugal force is applied again to obtain a predetermined volume of plasma. The HDL-cholesterol is quantified by detecting the color change according to the amount of HDL-cholesterol in the fourth chamber through the light emitting part and the light receiving part. For HDL-cholesterol analysis. After injecting whole blood into the first chamber as a specimen, centrifugal force is applied by the centrifugal force generating means to separate blood cell components in the first chamber. Thereafter, the centrifugal force is controlled by the centrifugal force control means. After stopping or weakening, only a plasma component is moved to the second chamber and the fifth chamber by applying centrifugal force again, and a predetermined volume of plasma is fractionated in the fifth chamber. Thereafter, the centrifugal force is further stopped or weakened, and a predetermined volume of plasma is moved to the sixth chamber by applying the centrifugal force again, and a color change corresponding to the total cholesterol amount in the sixth chamber is performed. The apparatus for analyzing HDL-cholesterol according to claim 12, wherein total cholesterol is quantified by detection through a light receiving part. After injecting whole blood into the first chamber as a specimen, centrifugal force is applied by the centrifugal force generating means to separate blood cell components in the first chamber. Thereafter, the centrifugal force is controlled by the centrifugal force control means. After stopping or weakening, only a plasma component is moved to the second chamber and the seventh chamber by applying centrifugal force again, and a predetermined volume of plasma is fractionated in the seventh chamber. Thereafter, the centrifugal force is further stopped or weakened, and a predetermined volume of plasma is moved to the eighth chamber by applying the centrifugal force again, and the discoloration according to the triglyceride amount in the eighth chamber is changed to the light emitting unit and The apparatus for HDL-cholesterol analysis according to claim 12, wherein triglyceride is detected through a light receiving unit and the triglyceride is quantified.

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