Blood test device and blood test method

Abstract

A blood test device (10) comprises: a bottom plate (100); a top plate (200); a lower electrode (300); an upper electrode (400); a voltage application part (500); and at least one detection part (600) provided on the lower electrode (300) and capable of detecting a substance to be measured in the blood. The lower electrode (300) includes a reservoir electrode (310) and a plurality of driver electrodes (320). The voltage application part (500) can apply a low frequency voltage between the reservoir electrode (310) and the upper electrode (400) and can selectively apply a low frequency voltage or a high frequency voltage between each driver electrode (320) and the upper electrode (400). At least one detection part (600) is disposed on the driver electrode (320).

Description

[Technical Field]

[0001] The present invention relates to a blood test apparatus and a blood test method. [Background technology]

[0002] Conventionally, techniques for separating plasma droplets from a blood sample have been known. For example, Non-Patent Document 1 below discloses a technique for separating plasma droplets from a blood sample using a digital microfluidics (DMF) device. The digital microfluidics device includes a bottom plate, a top plate positioned opposite the bottom plate, a lower electrode provided on the upper surface of the bottom plate, an upper electrode provided on the lower surface of the top plate, and a voltage application unit capable of applying high-frequency voltage and low-frequency voltage between the lower electrode and the upper electrode. The lower electrode includes a reservoir electrode on which the blood sample is placed, a first drive electrode provided adjacent to the reservoir electrode, and a second drive electrode provided adjacent to the first drive electrode.

[0003] In this plasma fractionation technique, the voltage application unit generates a negative electrophoretic force by applying a high-frequency voltage (2 MHz) between each drive electrode and the upper electrode. This creates an electric field in the space between the upper and lower electrodes that opposes the movement of the blood sample containing red blood cells. The voltage application unit then generates an EWOD force by applying a low-frequency voltage (1 MHz) between the reservoir electrode and the upper electrode. This maintains the shape of the blood sample. After the plasma droplets in the blood sample migrate to the second drive electrode, the voltage application unit stops applying the high-frequency voltage to the first drive electrode, positioning the plasma droplets on the second drive electrode. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Takeshi Komatsu and 3 others, "Separation of plasma from whole blood with DMF device", Abstracts of the 38th Research Meeting of the Society of Chemistry and Micro-Nano Systems, October 30, 2018, 4P04, p.97 Summary of the Invention [Problem to be solved by the invention]

[0005] To perform a blood test using blood as a measurement sample, it is common to separate blood cells by centrifugation or other methods, and then use plasma or serum for testing. This makes the testing process complicated, spurring a need for a simpler testing method. For example, a blood test for bipolar disorder patients, who are treated with lithium carbonate, is needed to measure the lithium ion concentration in their blood. It is desirable for this test to be easy to perform. Other needs include easy testing for various vitamins, such as vitamin B1, to check nutritional status and the presence or absence of various deficiencies, metal ions, such as zinc, AST and ALT, which are indicators of liver and kidney function, C-reactive protein (CRP), which is an indicator of inflammation, and HDL, LDL, and chylomicrons, which are indicators of lipid metabolism disorders.

[0006] An object of the present invention is to provide a blood testing device and a blood testing method that enable blood tests to be performed simply using whole blood, without the need for pretreatment such as centrifugation, when testing blood as a sample. [Means for solving the problem]

[0007] A blood testing apparatus according to one aspect of the present invention is a blood testing apparatus capable of separating blood cell components and plasma components from whole blood, and comprises a bottom plate, a top plate arranged above the bottom plate, a lower electrode provided on the upper surface of the bottom plate, an upper electrode provided on the lower surface of the top plate, a voltage application unit capable of applying a voltage between the lower electrode and the upper electrode, and at least one detection unit arranged on the lower electrode and capable of detecting a measurement substance in the blood, wherein the lower electrode includes a reservoir electrode in which a blood sample is placed and a plurality of drive electrodes arranged so as to be spaced apart from each other, the voltage application unit is capable of applying a low-frequency voltage between the reservoir electrode and the upper electrode and is capable of selectively applying a low-frequency voltage and a high-frequency voltage between each drive electrode and the upper electrode, and the at least one detection unit is arranged on the drive electrode.

[0008] The term "measurement substance" refers to the components contained in blood, and refers to the items traditionally used as measurement parameters in blood tests, including metal ions such as lithium and zinc mentioned above, lipids such as HDL, LDL, and chylomicrons, AST, ALT, and C-reactive protein (CRP), among others.

[0009] Furthermore, a blood testing method according to one aspect of the present invention is a blood testing method that can separate blood cell components and plasma components using the blood testing device and detects a measuring substance contained in a blood sample, and includes a separation step of separating plasma droplets from the blood sample placed on the reservoir electrode by applying a low-frequency voltage between the reservoir electrode and the upper electrode and selectively applying a high-frequency voltage and a low-frequency voltage between the plurality of driving electrodes and the upper electrode, and a detection step of detecting the measuring substance contained in the plasma by moving the plasma droplets separated in the separation step to the at least one detection unit provided on the driving electrode. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a blood testing device and a blood testing method that enable blood tests to be performed simply using whole blood, without the need for pretreatment such as centrifugation when performing tests using blood as a sample. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a diagram schematically illustrating a blood test apparatus according to one embodiment of the present invention. [Figure 2] 1A to 1C are cross-sectional views schematically illustrating a process for separating plasma from a blood sample. [Figure 3] 1A to 1C are cross-sectional views schematically illustrating a process for separating plasma from a blood sample. [Figure 4] 1A to 1C are cross-sectional views schematically illustrating a process for separating plasma from a blood sample. [Figure 5] 2 is a graph showing the analysis results obtained by the blood test apparatus shown in FIG. 1 and the measurement results obtained by a microplate reader. [Figure 6] 2 is a graph showing the analysis results obtained by the blood test apparatus shown in FIG. 1 and the measurement results obtained by a conventional method of plasma obtained by centrifuging a blood sample. DETAILED DESCRIPTION OF THE INVENTION

[0012] An embodiment of the present invention will be described with reference to the drawings. In the drawings referred to below, identical or corresponding components are designated by the same numbers. Although lithium ions in blood are used as an example of a substance to be measured, by changing the reagent that is adapted to the detection unit to a reagent that corresponds to the substance to be measured, it is possible to measure a substance in blood according to the purpose.

[0013] For example, to measure lithium ions in plasma, the measurement reagent "ESPA Li II (Nipro Corporation)" containing tetraphenylporphyrin can be used. Other reagents available include "ESPA Zn II (Nipro Corporation)" for measuring zinc, "ESPA TP (Nipro Corporation)" for measuring total protein, "ESPA ALB(G) (Nipro Corporation)" for measuring albumin, "ESPA UA-FS (Nipro Corporation)" for measuring uric acid, "ESPA UN Liquid (Nipro Corporation)" for measuring urea nitrogen, "ESPA CRE Liquid II (Nipro Corporation)" for measuring creatinine, "ESPA GLU(HK) Liquid (Nipro Corporation)" for measuring glucose, "ESPA IP Liquid II (Nipro Corporation)" for measuring inorganic phosphorus, "ESPA Ca (Nipro Corporation)" for measuring calcium, and "ESPA UA-FS (Nipro Corporation)" for measuring uric acid. For measuring total cholesterol, "ESPA Cu" (Nipro Corporation) can be used; for measuring triglycerides, "ESPA TCHO-FS" (Nipro Corporation) can be used; for measuring triglycerides, "ESPA TG-FS" (Nipro Corporation) can be used; for measuring phospholipids, "ESPA PL Liquid" (Nipro Corporation) can be used; for measuring LDH cholesterol, "ESPA LDH Liquid" (Nipro Corporation) can be used; for measuring AST, "ESPA GPT Liquid II" (Nipro Corporation) can be used; for measuring ALT, "ESPA GGT Liquid" (Nipro Corporation) can be used; and for measuring creatinine kinase, "ESPA CK Liquid II" (Nipro Corporation) can be used. For measuring indoxyl sulfate in plasma, "Indoxyl Sulfate Measurement Reagent 'Nipro'" (Nipro Corporation) can be used.

[0014] FIG. 1 is a diagram that schematically shows a blood test apparatus according to one embodiment of the present invention. FIG. 2 is a cross-sectional view that schematically shows a process for separating plasma from a blood sample. As shown in FIGS. 1 and 2, blood test apparatus 1 includes bottom plate 100, top plate 200, lower electrode 300, upper electrode 400 (see FIG. 2), voltage application unit 500 (see FIG. 2), and at least one detection unit 600. In this embodiment, blood test apparatus 1 includes a plurality of detection units 600. Note that upper electrode 400 and voltage application unit 500 are not shown in FIG. 1.

[0015] The bottom plate 100 is made of, for example, a glass substrate. The top plate 200 is disposed above the bottom plate 100. The top plate 200 is made of, for example, a glass substrate. The gap between the top plate 200 and the bottom plate 100 is set to, for example, 50 μm.

[0016] The lower electrode 300 is provided on the upper surface of the bottom plate. The lower electrode 300 is made of, for example, a transparent electrode film (ITO). The lower electrode 300 has a reservoir electrode 310 and a plurality of drive electrodes 320.

[0017] A blood sample S is placed on the reservoir electrode 310. The reservoir electrode 310 is formed in a rectangular shape. In Fig. 1, the blood sample S is patterned with dots, and in Figs. 2 to 4, red blood cells R in the blood sample S are shaded.

[0018] The plurality of drive electrodes 320 are arranged side by side at intervals. Each drive electrode 320 is formed in a rectangular shape. The outer shape of each drive electrode 320 is smaller than the outer shape of the reservoir electrode 310. The size of each drive electrode 320 is, for example, 1 mm 2 The plurality of driving electrodes 320 includes a plurality of connecting electrodes 322 and a plurality of branch electrodes 324.

[0019] The multiple connecting electrodes 322 are arranged so as to be continuously aligned from a position adjacent to the reservoir electrode 310. In this embodiment, the multiple connecting electrodes 322 are arranged so as to be aligned in a straight line. However, the arrangement of the multiple connecting electrodes 322 is not limited to a straight line, and as long as they are aligned continuously, they may be branched left and right or cross-shaped, or two or more branch electrodes may intersect to form a straight line again.

[0020] Each branch electrode 324 branches off from the connecting electrode 322. The branch electrodes 324 are arranged at positions spaced apart from one another in the arrangement direction of the connecting electrodes 322. Five branch electrodes 324 are shown in FIG.

[0021] 2 to 4, a coating layer 302 made of parylene C is provided on the surface of the lower electrode 300. A resist layer 304 made of SU-8 is provided on this coating layer 302, and a coating layer 306 made of polytetrafluoroethylene is provided on the resist layer 304.

[0022] The upper electrode 400 is provided on the lower surface of the top plate 200. The upper electrode 400 is made of, for example, a transparent electrode film (ITO). On the surface of the upper electrode 400, a coating layer 402 made of polytetrafluoroethylene is provided.

[0023] The voltage application unit 500 is capable of applying a voltage between the lower electrode 300 and the upper electrode 400. The voltage application unit 500 is capable of applying a low-frequency voltage (for example, 1 MHz) between the reservoir electrode 310 and the upper electrode 400, and is also capable of selectively applying a low-frequency voltage or a high-frequency voltage (for example, 1.5 MHz to 2 MHz) between each drive electrode 320 and the upper electrode 400.

[0024] Here, the frequency range of the high-frequency voltage will be explained. Because high frequencies can cause hemolysis, plasma separation was investigated and tested at frequencies between 100 kHz and 2 MHz. At frequencies between 100 kHz and 900 kHz, red blood cells and plasma simultaneously moved across the driven electrode 320, making separation of plasma from the blood sample S difficult. On the other hand, at frequencies above 1 MHz, red blood cells moved away from the driven electrode 320 due to the non-uniform strong electric field generated on the driven electrode 320, while plasma was guided toward the driven electrode 320. In the test, plasma was successfully separated from the blood sample S by applying a high-frequency voltage to two or three driven electrodes 320 (separation efficiency 99%), confirming that higher frequencies affect the induced migration of red blood cells. From this test, 1.5 MHz was determined to be the optimal frequency for the high-frequency voltage.

[0025] The detection unit 600 is capable of detecting a analyte contained in the blood sample S. The detection unit 600 is disposed on the branch electrode 324. The detection unit 600 is fabricated, for example, by impregnating a filter paper with a measurement reagent for the analyte and then drying the measurement reagent. The diameter of the detection unit 600 is set to, for example, 2 mm. The detection unit 600 is also capable of detecting metal ions (lithium ions and zinc ions) as the analyte. For example, when detecting lithium ions contained in the blood sample S as the analyte, F28 tetraphenylporphyrin is preferably used as the measurement reagent.

[0026] Next, a blood testing method using the above-described blood test apparatus 1 will be described. This blood testing method includes a separation step and a detection step. To prevent evaporation of blood sample S, this testing method is preferably performed in an environment of, for example, 2 cSt silicone oil.

[0027] In the separation process, a low-frequency voltage is applied between the reservoir electrode 310 and the upper electrode 400, and a high-frequency voltage and a low-frequency voltage are selectively applied between the plurality of drive electrodes 320 and the upper electrode 400. Specifically, as shown in FIG. 2, a low-frequency voltage V is first applied between the reservoir electrode 310 on which the blood sample S is placed and the upper electrode 400.LF is applied.

[0028] In this state, a high frequency voltage V is applied to the first connecting electrode 322a adjacent to the reservoir electrode 310 and the second connecting electrode 322b adjacent to the first connecting electrode 322a. HF As a result, an electric field E that opposes the movement of the blood sample S is generated in the space between the connecting electrodes 322a, 322b and the upper electrode 400.

[0029] Then, a low-frequency voltage V is applied between the reservoir electrode 310 and the upper electrode 400. LF is applied, the high frequency voltage V HF The application of the high frequency voltage V is stopped, and the high frequency voltage V is applied only between the second connecting electrode 322b and the upper electrode 400. HF is applied, which separates a plasma droplet P from the blood sample S placed on the reservoir electrode 310.

[0030] In the detection step after the separation step, the plasma droplets P separated in the separation step are moved to the detection unit 600 provided on the driving electrode 320. Specifically, the plasma droplets P are guided from the connecting electrode 322 to the branch electrode 324, and reach the detection unit 600 arranged on the branch electrode 324. As a result, the measurement substance contained in the plasma droplets P is detected by the detection unit 600.

[0031] As described above, in the blood test apparatus 1 of this embodiment, the detection unit 600 is provided on the driving electrode 320, so that the measured substance contained in the blood sample S can be easily detected by moving the plasma droplet P separated from the blood sample S on the reservoir electrode 310 to the detection unit 600 on the driving electrode 320.

[0032] Regarding the above blood testing method, the following two experiments were conducted. (Experimental Example 1) The blood sample S used was 5 μL of a mixture of human whole blood and lithium standard solutions of various concentrations (0.0, 0.4, 0.8, 1.2, 1.6, 2.0 mM) at a volume ratio of 18:1 (0.0, 0.4, 0.8, 1.2, 1.6, 2.0 mM lithium ion / 0.5% Pluronic® F-127).

[0033] The detection unit (colorimetric detection paper device) 600 was prepared as follows: 2 The filter paper (Advantech No. 1) was moistened with 350 μL of a lithium ion measurement reagent (0.168 mg / mL F28 tetraphenylporphyrin, Nipro Corporation), dried, and then punched into a circle with a diameter of 2 mm.

[0034] In the separation process for separating plasma droplets from the blood sample S, a 1.5 MHz high-frequency voltage V is applied to the upper electrode 400 and each driving electrode 320 with 5 μL of the blood sample S placed on the reservoir electrode 310. HF was approved.

[0035] In the detection step, the plasma droplets (0.2 μL) separated in the separation step were transported to the detection unit 600. The detection of the lithium ion concentration by the detection unit 600 in this detection step was performed by image analysis of the magenta intensity of the entire detection unit 600.

[0036] As shown in FIG. 5, the coefficient of determination was 0.99 or greater for both the image analysis results (Example 1) and the measurement results of blood sample S using a microplate reader (λ = 546 nm, 37°C for 10 minutes). The coefficient of determination (LOD) for the image analysis results was 0.26 mM, and the coefficient of determination (LOD) for the measurement results using the microplate reader was 0.051 mM. These results demonstrate that blood test apparatus 1 can rapidly and accurately detect blood lithium ions in the range of 0.4 to 1.2 mM required in clinical settings using a small amount of blood. Furthermore, in the blood test method using blood test apparatus 1, the introduction of plasma droplets into all detection units (paper devices) 600 was completed within 20 minutes.

[0037] (Experimental Example 2) The results of the image analysis (Example 2) were compared with the results (Comparative Example) of measuring plasma obtained by centrifuging the blood sample S used in Experimental Example 1 using a conventional method (a method using "ESPA LiII (Nipro Corporation)"). As a result, as shown in Figure 6, it was confirmed that the two results were in good agreement.

[0038] Furthermore, since the correlation coefficient between the two was less than 1 and greater than 0.7, there was a correlation between the image analysis results and the concentration of lithium ions added as a standard solution, confirming that this blood test method is appropriate as a measurement method.

[0039] The conventional method is as follows: Human whole blood was centrifuged at 3000 rpm for 5 minutes, and the supernatant (plasma) was used as the sample. Plasma samples containing lithium ions were obtained by adding various concentrations of lithium ions to human whole blood and then centrifuging the mixture. Measurements were performed according to the ESPA LiII kit protocol: plasma samples (3 μL) and color reagent (F28TPP, 180 μL) were mixed in a 96-well plate, incubated at 37°C for 10 minutes in a microplate reader (SpectraMax i3x, Molecular Devices, Sunnyvale, CA, USA), and then the absorbance (λ = 546 nm) was measured.

[0040] It should be noted that the embodiments disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the description of the above embodiments, and further includes all modifications within the meaning and scope of the claims.

[0041] For example, a plurality of reservoir electrodes 310 may be provided on the bottom plate 100, and a drive electrode 320 may be provided adjacent to each reservoir electrode 310, with the detection unit 600 provided on each drive electrode 320.

[0042] [Aspect] It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0043] A blood test apparatus according to one aspect of this disclosure is a blood test apparatus that detects metal ions contained in blood, and includes a bottom plate, a top plate arranged above the bottom plate, a lower electrode provided on the upper surface of the bottom plate, an upper electrode provided on the lower surface of the top plate, a voltage application unit capable of applying a voltage between the lower electrode and the upper electrode, and at least one detection unit arranged on the lower electrode and capable of detecting a measurement substance in the blood, wherein the lower electrode includes a reservoir electrode in which a blood sample is placed and a plurality of drive electrodes arranged in a row at intervals from each other, the voltage application unit is capable of applying a low-frequency voltage between the reservoir electrode and the upper electrode, and is capable of selectively applying a low-frequency voltage and a high-frequency voltage between each drive electrode and the upper electrode, and the at least one detection unit is arranged on the drive electrode.

[0044] In this blood test device, a detection unit is provided on the driving electrode, so that the measured substance contained in the blood sample can be easily detected by moving fractionated plasma from the blood sample on the reservoir electrode to the detection unit on the driving electrode.

[0045] Furthermore, it is preferable that the plurality of driving electrodes include a plurality of connecting electrodes arranged in a continuous line from a position adjacent to the reservoir electrode, and a plurality of branch electrodes each branching from the connecting electrodes, and that the at least one detection unit includes a plurality of detection units arranged on each branch electrode.

[0046] In this aspect, by using the same type of detection unit as the multiple detection units, it is possible to increase the number of inspections and improve the inspection accuracy, or by using multiple detection units that detect different objects, it is possible to inspect multiple inspection objects.

[0047] Furthermore, it is preferable that the at least one detection unit is capable of detecting metal ions as the measurement substance.

[0048] Furthermore, a blood testing method according to one aspect of the present invention is a blood testing method that can separate blood cell components and plasma components using the blood testing device and detects metal ions contained in a blood sample, and includes a separation step of separating plasma droplets from the blood sample placed on the reservoir electrode by applying a low-frequency voltage between the reservoir electrode and the upper electrode and selectively applying a high-frequency voltage and a low-frequency voltage between the plurality of driving electrodes and the upper electrode, and a detection step of detecting the measured substance contained in the plasma droplets by moving the plasma droplets separated in the separation step to at least one detection unit provided on the driving electrode. [Explanation of symbols]

[0049] 1 Blood testing device, 100 bottom plate, 200 top plate, 300 lower electrode, 310 reservoir electrode, 320 driving electrode, 322 connecting electrode, 324 branch electrode, 400 upper electrode, 500 voltage application unit, 600 detection unit, S blood sample, P plasma.

Claims

1. A blood test device capable of separating blood cell components and plasma components from whole blood, A bottom plate and a top plate disposed above the bottom plate; a lower electrode provided on an upper surface of the bottom plate; an upper electrode provided on a lower surface of the top plate; a voltage application unit capable of applying a voltage between the lower electrode and the upper electrode; At least one detection unit is provided on the lower electrode and is capable of detecting a measurement substance in blood; The lower electrode is a reservoir electrode into which the blood sample is placed; a plurality of drive electrodes arranged in a spaced relationship to one another; the voltage application unit is capable of applying a low-frequency voltage between the reservoir electrode and the upper electrode, and is also capable of selectively applying a low-frequency voltage and a high-frequency voltage between each drive electrode and the upper electrode; The blood test apparatus, wherein the at least one detection unit is disposed on the drive electrode.

2. The plurality of drive electrodes are A plurality of connecting electrodes arranged in a continuous line from a position adjacent to the reservoir electrode; a plurality of branch electrodes each branching from the connecting electrode; The blood test apparatus according to claim 1 , wherein the at least one detecting unit includes a plurality of detecting units arranged on each branch electrode.

3. The blood test apparatus according to claim 1 , wherein the at least one detection unit is capable of detecting metal ions as the measurement substance.

4. A blood testing method that can separate blood cell components and plasma components by using the blood testing device according to any one of claims 1 to 3 and detect a measurement substance contained in a blood sample, comprising: a separation step of separating plasma droplets from the blood sample placed on the reservoir electrode by applying a low-frequency voltage between the reservoir electrode and the upper electrode and selectively applying a high-frequency voltage and a low-frequency voltage between the plurality of drive electrodes and the upper electrode; a detection step of detecting the measurement substance contained in the plasma droplets by moving the plasma droplets separated in the separation step to the at least one detection unit provided on the driving electrode.

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