Measurement method

Abstract

To provide a measurement method using a micro fluid device for measuring floating DNA concentration in blood.SOLUTION: A measurement method measures floating DNA concentration in blood by a micro fluid device which has: a first inflow hole into which a blood specimen flows; and a second inflow hole into which a buffer liquid being a carrier of the blood specimen flows through the first inflow hole, and having a channel in which multiple micro columns are arranged, the method is configured to: add to the blood specimen, an addition agent for making clogging between the micro columns due to the DNA tangible; make the buffer liquid flow into the micro fluid device from the second inflow hole; make the blood specimen to which the addition agent is added flow into the micro fluid device from the first inflow hole; measure a blood flow area in the channel; fit the blood flow area to a correlation between a reference blood flow area which is obtained by measuring in advance using the micro fluid device, for a reference blood specimen having known floating DNA concentration and the floating DNA concentration of the reference blood specimen for estimating the floating DNA concentration of the blood specimen.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present disclosure relates to a method for measuring the concentration of floating DNA contained in a blood sample. [Background technology]

[0002] Various techniques have been disclosed for the analysis or separation of biological components using microfluidic devices. In these techniques, clogging (thrombus) between microposts is always a problem. Platelets are assumed to be the cause of such clogging, but it is difficult to completely eliminate clogging, for example by adding an anticoagulant to blood or coating the surface of the microfluidic device with an anti-adhesion coating for biomolecules, and it is highly likely that factors other than platelets are involved in the clogging.

[0003] From the above viewpoint, Patent Document 1 discloses that in a blood component separation method in which blood components are separated by a microfluidic device having an inlet located on the upstream side, an outlet located on the downstream side, and a flow path located between the inlet and the outlet, with a plurality of microposts arranged at predetermined intervals in the flow path, a DNA flocculant is added as an anti-clogging agent to a blood sample flowing in from the inlet. The technology described in Patent Document 1 makes it possible to reduce clogging in a blood component separation method using a microfluidic device without carrying out any special treatment on the microfluidic device or providing any special mechanism.

[0004] When attempting to separate relatively large-diameter cells from blood using a microfluidic device, clogging occurs between the microposts, causing an increase in the width of the blood flow downstream of the location of the clogging, which has been a problem. Regarding clogging between microposts in a microfluidic device, there is research suggesting that DNA floating in the blood becomes entangled in a manner that bridges the gaps between the microposts, and platelets accumulate around the gaps, causing clogging (Non-Patent Document 1).

[0005] Free-floating DNA in blood refers to DNA released into the blood from dead cells, and research is being conducted on it as a biomarker in the blood, similar to circulating tumor cells. In addition, the free-floating DNA concentration in the blood of cancer patients is higher than that of healthy individuals (Non-Patent Document 2), and they are more likely to develop deep vein thrombosis (Non-Patent Document 3). Therefore, it is suggested that the state of free-floating DNA in blood (i.e., concentration and length distribution) may affect diseases caused by thrombus. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2021-1850 [Non-patent literature]

[0007] [Non-Patent Document 1] T. Konishi, Y. Jingu, T. Yoshizawa, M. Irita, T. Suzuki & M. Hayase, "Mitigation of Channel Clogging in a Microfluidic Device for Capturing Circulating Tumor Cells", Int. J. Automation Technol., Vol.14(2020), pp.109-116. [Non-Patent Document 2] SA Leon, B. Shapiro, DM Sklaroff & MJ Yaros, "Free DNA in the Serum of Cancer Patients and the Effect of Therapy", Cancer Res., Vol.37(1977), pp.646-640. [Non-Patent Document 3] JF Timp, SK Braekkan, HH Versteeg & SC Cannegieter, "Epidemiology of cancer-associated venous thrombosis", Blood, Vol.122(2013), pp.1712-1723. Summary of the Invention [Problem to be solved by the invention]

[0008] An objective of the present disclosure is to provide a technique for estimating the concentration of floating DNA in blood using a microfluidic device. [Means for solving the problem]

[0009] An embodiment of the present disclosure is a method for measuring the concentration of DNA suspended in blood using a microfluidic device having an inlet located on the upstream side, an outlet located on the downstream side, and a flow path located between the inlet and the outlet, and a plurality of microposts arranged at a predetermined interval in the flow path. In the measurement method, each row of the microposts in the width direction is provided at a position shifted by the same distance in the same direction in the width direction from the previous row. The inlet includes a first inlet through which a blood sample flows in, and a second inlet through which a fluid medium that serves as a carrier for the blood sample flows in across the first inlet. An additive for making the clogging between the microposts by DNA visible is added to the blood sample, and while the fluid medium is flowing into the microfluidic device from the second inlet, the blood sample to which the additive has been added is flowing into the microfluidic device from the first inlet, and the blood flow area in the flow path is measured. The floating DNA concentration of the blood sample is estimated by applying the blood flow area to the correlation between a reference blood flow area previously measured using the microfluidic device for a reference blood sample with a known floating DNA concentration and the floating DNA concentration of the reference blood sample.

[0010] The additive is preferably an anticoagulant that inhibits the function of platelets. A specific example of the anticoagulant is tirofiban. In addition to tirofiban, it is also preferable to add N-acetyl-L-cysteine, an antioxidant, to the blood sample as an additive.

[0011] Furthermore, the spacing between adjacent microposts in the width direction is preferably 13 to 40 μm, more preferably 15 to 35 μm, and even more preferably 20 to 25 μm. Effect of the Invention

[0012] According to the present disclosure, a technique is provided for estimating the concentration of suspended DNA in blood using a microfluidic device. [Brief description of the drawings]

[0013] [Figure 1] FIG. 2 is a plan view that illustrates a microfluidic device used in the measurement method of the embodiment. [Diagram 2] FIG. 2 is an enlarged plan view showing a micropost. [Diagram 3] Schematic diagram of a blood sample without additives flowing through a microfluidic device. [Figure 4] Schematic diagram of a blood sample containing additives flowing through a microfluidic device. [Diagram 5] FIG. 1 is a scatter plot showing the relationship between the blood flow area observed when a blood sample containing no additives was passed through a microfluidic device and the concentration of suspended DNA in the blood sample. [Figure 6] FIG. 1 is a scatter plot showing the relationship between the blood flow area observed when a blood sample containing an additive was passed through a microfluidic device and the concentration of suspended DNA in the blood sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. The drawings mentioned below are merely schematic drawings, and the shape and size ratios between the various parts do not necessarily reflect the actual product. Furthermore, symbols commonly used in the various drawings indicate common members or parts even if not mentioned in the description of the drawings below. Furthermore, the numerical values ​​mentioned below are merely examples, and the scope of the present disclosure is not limited thereto. Furthermore, in this disclosure, the numerical ranges expressed by the numerical values ​​before and after "~" include the numerical values ​​before and after them.

[0015] FIG. 1 is a schematic diagram showing a microfluidic device 10 used in the blood component separation method of the present embodiment in a plan view. In this microfluidic device 10, an inlet 11 is located on the upstream side, an outlet 12 is located on the downstream side, and a flow path 13 is located between the inlet 11 and the outlet 12. The inlet 11 includes a first inlet 11A through which a blood sample 30 (see FIG. 3) flows, and a second inlet 11B through which a flow medium for flowing the blood sample 30 downstream flows. The flow medium is a liquid that serves as a carrier for the blood sample 30, such as an appropriate buffer solution. The second inlet 11B branches off on the upstream side of the first inlet 11A and joins on the downstream side across the first inlet 11A. The flow path 13 is a portion through which the blood sample 30 flows downstream together with the flow medium, and a plurality of microposts 20 (see FIG. 2), which will be described later, are arranged at predetermined intervals. The outlet 12 includes a first outlet 12A located on the left side in the flow direction, and a second outlet 12B located on the right side in the flow direction.

[0016] The flow channel 13 is formed in a flat plate shape with the flow direction as the longitudinal direction, and a micropost array in which a plurality of microposts 20 are arranged is formed from the bottom surface of the flow channel 13, as shown in an enlarged view in Fig. 2. The microposts 20 have a triangular shape with their apexes facing upstream in a plan view.

[0017] The size and intervals of the microposts 20 can be set appropriately depending on the size of the microfluidic device 10 and the particle size of the measurement target. In this embodiment, the length α of one side of the microposts 20 is 10 to 20 μm, preferably 13 to 17 μm.

[0018] In the flow channel 13, as shown in Fig. 2, the longitudinal rows of the microposts 20 are arranged so as to incline leftward (upward in the figure) toward the flow direction (rightward in the figure). In other words, each widthwise row of microposts 20 is provided at a position shifted the same distance in the same width direction from the previous row. Specifically, each micropost 20 is shifted leftward in the flow direction by a width β from the immediately preceding micropost 20. In this embodiment, this width β is 2.00 to 3.50 µm, and preferably 2.50 to 3.00 µm.

[0019] In the flow channel 13, the distance γ from the apex of each micropost 20 to the apex of the adjacent micropost 20 is 30 to 50 μm, preferably 35 to 45 μm. Furthermore, the distance δ in the width direction between adjacent microposts 20 is 13 to 40 μm, preferably 15 to 35 μm, and more preferably 20 to 25 μm.

[0020] The microfluidic device 10 of FIG. 1 is used as follows. That is, when a blood sample 30 (see FIG. 3) is flowed from the first inlet 11A while a fluid medium is flowed from the second inlet 11B, a laminar flow is formed downstream with the blood sample 30 at the center and the fluid medium on both sides of the blood sample 30. Here, if particles with a relatively large diameter (e.g., tumor cells) are present in the blood sample 30, such particles are swept downstream in the direction in which the microposts 20 are inclined. As a result, such particles reach the first outlet 12A at the tip of the inclination of the microposts 20. On the other hand, other particles with a relatively small diameter reach the second outlet 12B at the opposite side of the inclination of the microposts 20. Here, the microfluidic device 10 is designed so that the shape, size, and interval of the microposts 20 are appropriate to separate particles of a desired size.

[0021] FIG. 3 is a schematic diagram showing a state where clogging occurs between the microposts 20. If no clogging occurs between the microposts 20, the blood sample 30 should flow downstream in the flow channel 13 while maintaining a constant width. However, if clogging occurs, the width of the blood flow region 40 downstream from the position where the clogging occurs will expand as shown in FIG. 3. This clogging is caused by platelets, but also occurs when floating DNA in the blood gets entangled between the microposts 20. Therefore, in the measurement method of the present disclosure, the floating DNA concentration in the blood is measured using the above-mentioned microfluidic device 10.

[0022] Specifically, an additive is first added to the blood sample 30 to make visible the clogging between the microposts caused by DNA. The "additive" refers to a reagent or substance added to the blood sample 30 in order to eliminate factors other than DNA that may cause clogging between the microposts and to evaluate the clogging between the microposts purely caused by DNA. An example of such an additive is an anticoagulant for inhibiting clogging caused by platelets. By inhibiting the action of platelets with the anticoagulant as an additive, it can be evaluated that the clogging occurring between the microposts 20 is caused by DNA floating in the blood.

[0023] As for the anticoagulant, any reagent or substance generally used as an anticoagulant can be used as an additive in the measurement method of the present disclosure. An example of an anticoagulant that can be used is tirofiban. In addition to tirofiban, it is preferable to further add N-acetyl-L-cysteine ​​as an antioxidant as an additive. The concentrations of the reagents added to the blood sample 30 are, for example, 5 to 100 mM for tirofiban and 1 to 100 μM for N-acetyl-L-cysteine.

[0024] Then, while flowing the fluid medium into the microfluidic device 10 from the second inlet 11B, the blood sample 30 to which the additive has been added is caused to flow into the microfluidic device 10 from the first inlet 11A. Then, the area (blood flow area) of the blood flow region 40 (see FIG. 3) generated in the flow channel 13 is measured. There are various possible methods for measuring the blood flow area. For example, an image of the flow channel 13 is taken, and the part of the taken image that corresponds to the blood flow region 40 and the other parts are binarized, and the area of ​​the part that corresponds to the blood flow region 40 can be measured. This area may be expressed, for example, as the number of pixels on the image.

[0025] Here, if the blood flow region 40 generated in the blood sample 30 to which no additive is added is shown in FIG. 3, the blood flow area of ​​the blood flow region 40 generated in the blood sample 30 to which an additive is added is reduced as shown in FIG. 4. This is because the addition of the additive eliminates clogging caused by components inhibited by the additive. That is, the blood flow region 40 shown in FIG. 3 is generated as a result of clogging between the microposts 20 caused by both platelets and floating DNA. On the other hand, when the additive is an anticoagulant, clogging caused by platelets is eliminated, and as a result of clogging between the microposts 20 caused by floating DNA, a blood flow region 40 smaller than the blood flow region 40 in FIG. 3 is generated as shown in FIG. 4.

[0026] The floating DNA concentration of the blood sample 30 can be estimated by fitting the measured blood flow area into a predetermined correlation. This correlation can be obtained as follows. First, a reference blood sample having a known floating DNA concentration is caused to flow into the microfluidic device 10 in the same manner as above, and the area of ​​the blood flow region 40 generated in the flow channel 13 is measured. This area is referred to as the reference blood flow area. Here, the floating DNA concentration in the reference blood sample can be measured by an existing DNA concentration measurement method.

[0027] For multiple reference blood samples, the reference blood flow area is measured as described above, and the floating DNA concentration and the reference blood flow area are plotted on a graph to obtain a regression line as a correlation. By fitting the measured blood flow area to this regression line, the corresponding floating DNA concentration can be estimated as the floating DNA concentration of the blood sample 30.

[0028] It is also possible to use a reagent or substance that inhibits clogging caused by DNA (hereinafter referred to as a "DNA inhibitor") as an additive to be added to the blood sample 30. In other words, the blood flow area measured with an additive can be subtracted from the blood flow area measured without an additive for the same blood sample to estimate the floating DNA concentration paradoxically. However, this method requires measuring the blood flow area twice for the same blood sample 30. Therefore, from the viewpoint that only one measurement is required for each blood sample 30, it is desirable to add an anticoagulant as an additive to the blood sample 30, as described above. EXAMPLES

[0029] (1) Microfluidic Devices The microfluidic device used in the examples was the microfluidic device 10 shown in Fig. 1, with a flow channel 13 having a width of 1 mm and a length in the flow channel direction of 10 mm. For the microposts in the flow channel, the sizes of the α, β, γ, and δ portions shown in Fig. 2 were as shown in Table 1 below.

[0030] [Table 1]

[0031] (2) Blood samples Blood samples were collected from 17 healthy individuals. For each blood sample, 30 mM of the anticoagulant tirofiban (CAS number: 144494-65-5, Sigma-Aldrich) and 3 μM of the oxidizing agent N-acetyl-L-cysteine ​​(CAS number: 616-91-1, Sigma-Aldrich) were added as additives in the examples, and neither was added in the comparative examples. Phosphate-buffered saline (PBS) to which fetal calf serum (FCS) was added to a concentration of 0.5% was used as the fluid medium.

[0032] (3) Blood flow area measurement The blood specimen and the fluid medium of the above-mentioned Examples or Comparative Examples were flowed into the microfluidic device shown in the above-mentioned embodiment at flow rates of 15 μL / min and 800 μL / min, respectively, using a syringe pump (homemade).

[0033] While the blood sample and the flow medium were flowing in, still images of the flow path were taken through a microscope every 4 minutes. After the still images were binarized, the number of pixels in the area corresponding to the blood flow was counted and used as the blood flow area.

[0034] (4) DNA concentration measurement The floating DNA concentration of each blood sample was measured using a Quantus™ Fluorometer (Promega) after purifying DNA using the Maxwell® RSC Instrument and the Maxwell® RSC Large Volume ccfDNA Plasma Kit.

[0035] As a result, the relationship between the floating DNA concentration in blood and the blood flow area for the blood samples of the Comparative Example and the Example is plotted in graphs in Figures 5 and 6, respectively, along with the regression line. In both Figures 5 and 6, the horizontal axis represents the floating DNA concentration in blood (unit: pg / μL) and the vertical axis represents the blood flow area (unit: 10 5 In Figures 5 and 6, plots with the same values ​​on the horizontal axis are from the same blood sample.

[0036] Comparing Figure 5 and Figure 6, for all blood samples, the blood flow area is lower in Figure 6 than in Figure 5. This means that for all blood samples, the additive action eliminates clogging caused by platelets, resulting in clogging caused only by suspended DNA becoming apparent.

[0037] Here, the regression line for the blood sample of the comparative example shown in FIG. 5 is as shown in the following formula (1), and the correlation coefficient (r) is 0.316.

[0038] y=84469x+454087 (1)

[0039] The regression line for the blood samples of the example shown in FIG. 6 is as shown in the following formula (2), and the correlation coefficient (r) is 0.638.

[0040] y=98664x+289266 ···(2)

[0041] Solving the above equation (2) for x gives the following equation (2)'.

[0042] x = (1.0135 × 10 -5 )y-2.9318···(2)′

[0043] From the above, it was also found that the use of the additive increased the correlation between the concentration of free DNA in the blood and the blood flow area.

[0044] From the above, for a blood sample with an unknown floating DNA concentration, tirofiban and N-acetyl-L-cysteine ​​are added as additives, and the blood is allowed to flow into the microfluidic device of this embodiment. The measured blood flow area (y) is then applied to the above formula (2)' to estimate the floating DNA concentration (x) in the blood of the blood sample. [Industrial Applicability]

[0045] The present disclosure can be used in a measurement method for measuring the concentration of floating DNA contained in a blood sample. [Explanation of symbols]

[0046] 10 Microfluidic Devices 11 Inlet 11A First inlet 11B Second inlet 12 Outlet 12A First outlet 12B Second outlet 13 Flow Path 20 Microposts 30 Blood Samples 40 Blood flow area

Claims

1. A method for measuring a concentration of DNA suspended in blood using a microfluidic device having an inlet located on an upstream side, an outlet located on a downstream side, and a flow path located between the inlet and the outlet, the flow path having a plurality of microposts arranged therein, comprising: Each row of the microposts in the width direction is arranged at a position shifted by the same distance in the same direction in the width direction from the previous row, the inlet comprises a first inlet through which a blood sample flows, and a second inlet through which a fluid medium serving as a carrier for the blood sample flows across the first inlet, adding an additive to the blood sample to make visible the clogging between the microposts caused by DNA; While flowing the fluid medium into the microfluidic device through the second inlet, flowing the blood sample to which the additive has been added into the microfluidic device through the first inlet; measuring a blood flow area in the flow channel; A measurement method in which the floating DNA concentration of a reference blood sample is estimated by applying the blood flow area to a correlation between a reference blood flow area previously measured using the microfluidic device for a reference blood sample having a known floating DNA concentration and the floating DNA concentration of the reference blood sample.

2. The measurement method according to claim 1 , wherein the additive is an anticoagulant.

3. The method of claim 2 , wherein the anticoagulant is tirofiban.

4. The measurement method according to claim 3 , further comprising N-acetyl-L-cysteine ​​as the additive.

5. 5. The measurement method according to claim 1, wherein the widthwise spacing between adjacent microposts is 13 to 40 μm.

6. The measurement method according to claim 5, wherein the interval is 15 to 35 μm.

7. The measurement method according to claim 6, wherein the interval is 20 to 25 μm.

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