JP2005106778A - Filter chip, method for observing flow characteristics of cells and particles using the cells - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a filter chip used for observing the shapes of cells and/or floating particles, contained in a sample liquid passing through a fine processed flow channel provided to a substrate under suction pressure or pressure and is capable of clearing the shapes of the cells and/or floating particles small in shadow in the fine processed flow channel and passing through this flow channel and reducing the destruction of cells or the like. <P>SOLUTION: This filter chip has a difference between the upper and lower widths of the cross section, in the direction of crossing the fine flow channel of the protruded part of the fine flow channel is about 4 μm or smaller and has no acute angle shape in the surface opposite to the flow of the sample liquid. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a filter chip used for measuring flow characteristics of cells and particles, a flow characteristic observation method and a measurement apparatus using the filter chip.

  For example, erythrocytes, which are components of blood, flow through microcirculation regions such as capillaries while receiving external forces in the blood and deforming into various shapes. In particular, deformation of red blood cells is indispensable when flowing in a peripheral blood vessel having a diameter of half or less of the diameter of red blood cells (7 to 8 μm). Therefore, the ease of deformation of red blood cells (hereinafter referred to as deformability) is very important for maintaining health, and rapid and accurate measurement of deformability is desired in the diagnosis of cardiovascular diseases and the like.

  Patent Document 1 discloses a sample to be measured in measuring the flow characteristics of cells and particles by the passage speed of cells and particle suspensions passing through a microfabrication channel such as a filter or a circuit under suction pressure or pressure. Pass the sample suspension liquid or the liquid used to suspend the sample suspension liquid through the microfabrication channel before passing the suspension liquid, and then pass the sample suspension liquid continuously by passing the medium and the sample suspension liquid. Disclosed are a method for measuring flow characteristics of cells and particles, each of which continuously measures velocity, and a flow characteristic measuring apparatus based on this method.

  In the above specification, as a finely processed flow path, for example, a filter chip in which a flow path portion (V groove) is provided on silicon or a quartz substrate is disclosed. For example, a sample solution containing red blood cells is caused to flow on the filter chip due to the pressure difference, and the state of the red blood cells flowing through the V-groove is observed using a reflection microscope. According to this method, it is possible to measure erythrocyte deformability, leukocyte activity, platelet aggregation ability, liposome fluidity and microparticle fluidity by observing the passing speed of blood cells and microparticles.

  However, when the filter chip having the V-groove disclosed in the above specification is used, a shadow may be formed around the convex portion forming the V-groove during observation with a microscope, which may obstruct observation. In addition, since both ends of the convex portion have a sharp angle, there is a possibility that cells or particles in the sample liquid collide with it to be destroyed.

JP-A-11-118819

  It is an object of the present invention to provide a filter chip having a microfabricated flow path that does not cause a shadow in the microfabricated flow path when observed with a reflection microscope. Another object of the present invention is to provide a filter chip having a microfabricated flow path that is less likely to destroy cells and particles in the sample solution when the sample solution is flowed. Furthermore, the present invention relates to a method and a measuring apparatus for observing the flow characteristics of cells and particles using the filter chip.

  As a result of diligent research to solve the above problems, the present inventors have been able to prevent shadows that obstruct observation by improving the cross-sectional shape of the flow path on the filter chip, and the convexity for forming the flow path. It was found that the destruction of cells and particles in the sample liquid due to the collision with the portion can be prevented by improving the shape of the convex portion, and the present invention has been completed.

  Furthermore, the present inventors have found that knowledge that has not been obtained in the field of clinical examinations can be obtained by observing the flow characteristics of blood or the like and measuring components in the blood in parallel.

  That is, the problems of the present invention are as follows: 1) In a filter chip used for observing the shape of cells and suspended particles in a sample solution passing through a microfabrication channel under a suction pressure or pressurization with a reflective microscope, The difference between the width of the upper part and the lower part of the convex part forming the microfabricated flow path is about 4 μm or less, and the method for observing the flow characteristics of cells and / or particles using the filter chip 2) A filter chip characterized in that at least a side of the convex portion for forming the microfabricated flow channel facing the flow channel and on the flow channel entrance side is substantially an isosceles obtuse triangle or a straight line in plan view. And a method for observing the flow characteristics of cells and / or particles using the filter chip, and 3) cells using the filter chip in which the microfabricated flow path is formed in one unit and / or It was achieved and observed for flow properties of the particles, measuring device having a function that can perform in parallel measurements of components in the blood, by.

  When the filter chip with the microfabricated flow channel of the present invention is used, the projections forming the flow channel do not cause shadows in the flow channel that obstruct the observation. The shape of the cells and / or particles when passing through the flow path can be clearly observed.

  In addition, since at least the side where the cells and / or particles flow into the convex portion forming the flow path is formed to be flatter than the obtuse isosceles triangle in plan view, the cells and / or particles are on this side. The risk of being destroyed by collision is greatly reduced.

  By using the measuring apparatus of the present invention that can perform the flow characteristics of cells and / or particles by the fitter chip of the present invention and the quantification of specific components in blood in parallel, the correlation between these data indicates that It is possible to obtain new findings in clinical tests that could not be obtained.

  The filter chip according to the present invention has a basic planar shape as shown in FIG. That is, a group of convex portions having a substantially constant shape and arranged at almost equal intervals are formed on the substrate by fine processing. By suction or pressurization, the sample liquid containing cells and / or particles passes through the gap between the convex portions. For fine processing, for example, a known method in the field of manufacturing semiconductor chips can be used. As the substrate, any material that can be finely processed, such as glass, quartz, and silicon, can be used.

  The size and interval of the convex portions can be variously selected according to the properties of the cells and / or particles to be observed. For example, in FIG. 1, the widths of the flow paths of three filter chips are 2 μm, 6 μm, and 10 μm from the left, respectively.

  The filter chip of the present invention is characterized by the shape of the convex part, and in the cross-sectional shape of the convex part in the direction perpendicular to the fine flow path, the difference between the upper width and the lower width is about 4 μm or less. In other words, the wall surface parallel to the fine channel rises from the substrate at an angle close to perpendicular.

  FIG. 2 schematically shows a cross-sectional shape in a direction perpendicular to the fine flow path of the convex portion forming the fine flow path in the filter chip conventionally used in this field and the filter chip according to the present invention. In the filter chip according to the present invention, the difference in width between the upper and lower bases of the convex portion is small.

  FIG. 3 shows a photomicrograph when whole blood is flowed using a filter chip conventionally used in this field and the filter chip according to the present invention. The width of the channel measured at the top of the convex part is the same. As is clear from the above, in the filter chip according to the present invention, the shadow of the convex portion falling in the flow channel is small, so that cells and / or particles in the flow channel can be easily and clearly observed.

  Moreover, FIG. 3 represents the difference in the planar shape of the convex part in two types of filter chips. As is apparent from FIG. 3, the convex portion of the conventional filter chip has a sharp corner on the side facing the flow of the sample solution, and there is a risk that cells and / or particles in the sample solution will collide with this and be damaged. is there. On the other hand, in the filter chip according to the present invention, since the side facing the sample liquid flow is substantially linear, this fear is greatly reduced.

  However, the side opposite to the flow of the sample solution does not need to be linear, and may protrude outside the convex portion in a plan view up to a substantially obtuse isosceles triangular shape with the side as an oblique side. The vertices of the obtuse isosceles triangle are preferably rounded.

  The sides of the convex portions along the flow of the sample liquid do not need to be linear, and may be formed so that the planar shape of the convex portions is approximately a rhombus, a drum shape, or the like depending on the purpose.

  Further, in order to be able to use the filter chip without distinguishing the inlet and outlet of the sample solution, it is preferable that the surface of the convex portion opposite to the surface facing the flow of the sample solution has the same shape. .

  In any shape, the convex portion is preferably rounded so as not to have a corner in plan view.

  In the filter chip according to the present invention, the interval between the convex portions, that is, the width of the fine channel and the height of the convex portion, that is, the depth of the fine channel can be appropriately selected according to the object to be measured. The convex portions are formed so that the width of the fine channel is in the range of about 1 to 20 μm and the depth is about 1 to 20 μm. Furthermore, they do not have to be constant on a single filter chip and may vary. Thereby, a lot of information about the flow characteristics of cells and / or particles can be obtained using a single filter chip.

  The lateral width of the convex portion, that is, the pitch at which the fine flow path is formed is not particularly limited as long as fine processing is possible.

  With the device according to the present invention, that is, the device capable of performing the measurement of the fluid flow characteristics using the filter chip according to the present invention and the measurement of the components in the blood in parallel, knowledge that has not been obtained in the clinical laboratory field has been obtained. This will be described in detail in the Examples.

  The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples.

  Using a device MC-FAN (manufactured by Hitachi Haramachi Electronics Co., Ltd.) that observes and measures blood flow, the deformation of cells such as red blood cells when passing through a fine channel is observed, and whole blood, plasma, and serum The flow of liquid such as was measured. At this time, a filter chip (hereinafter referred to as a custom chip) in which the difference in the vertical width of the convex portions forming the fine flow path according to the present invention is about 4 μm or less was used. The custom chip was created with the following specifications.

  FIG. 3 shows the results of measuring the flow rate of whole blood using the chip 6 shown in Table 1 and the conventional filter chip and observing the deformation of red blood cells as an image. As is apparent from this figure, it can be seen that when the chip of the present invention is used, the shadow formed in the flow path by the convex portion is reduced, and the shape of red blood cells passing through the flow path can be clearly observed.

  The difference in the shape of the convex portion in plan view between the custom chip and the conventional filter chip is as shown in FIG.

  Using the chip 2 to the chip 10 shown in Table 1 and the MC-FAN used in Example 1, using heparin lithium as an anticoagulant, whole blood collected from a healthy male, and obtained by centrifugation Plasma and physiological saline (saline) flow were measured. The measurement results are shown in FIG.

  From FIG. 4, the larger the flow path width, the larger the common blood, plasma, and saline consumption overspeed. When the ratio of the plasma passage speed and the whole blood passage speed is taken, it is surprising that the flow path width is 4 μm. In the above case, the ratio is constant at about 2, and it can be seen that the ratio increases rapidly when the flow path width is 3 μm or less.

  Since the depth of the flow path of these chips is 4.5 μm, it is necessary to deform when an erythrocyte having a size of 8 μm normally passes through the flow path having such a small cross section. According to this method, the width of the fine channel through which red blood cells can pass can be clearly defined, and based on this, the ease of deformation of red blood cells in the sample solution can be determined more than when the passage speed of red blood cells is measured alone. Can also be compared clearly.

  Using the chip 6 shown in Table 1, using the MC-FAN used in Example 1, heparin lithium as an anticoagulant and obtained by centrifuging whole blood collected from 12 healthy males Plasma and saline flow were measured. For whole blood, the hematocrit value (Hct value) was determined with a hematocrit tube. An example of the measurement result is shown in FIG.

  From FIG. 5, the passage speed of whole blood is highly dependent on the Hct value. The higher the Hct value, the slower the passage speed, and even in plasma with a Hct value of 0, there is a difference in specimens in the passage speed. It was found that the difference was maintained, and that the specimen difference disappeared when the rate ratio between plasma and whole blood was taken, and the passage speed of whole blood was determined by the plasma speed and Hct value of Hct value 0. From this, it can be seen that by using the method of the present invention, it is possible to clearly measure what factor determines the flow of whole blood.

  For the plasma used in Example 3, the component amount was further measured using a clinical automatic analyzer 7170 (Hitachi, Ltd.), and the value was compared with the plasma passage speed. The results are shown in FIG. In the horizontal axis of FIG. 6, TCHO indicates total cholesterol, LDL indicates low density cholesterol, HDL indicates high density cholesterol, TP indicates total protein, ALB indicates albumin, and TG indicates neutral fat. The value of low density cholesterol was determined by the Friedwald equation from the total cholesterol concentration, high density cholesterol concentration, and neutral fat concentration.

  From FIG. 6, the plasma passage speed has a negative correlation with total cholesterol concentration, low density cholesterol concentration, and total protein concentration, and no correlation with high density cholesterol concentration, albumin concentration, and neutral fat concentration. I understand that.

  When the results of FIG. 5 and FIG. 6 are considered together, it is clear that the speed at which whole blood passes through the fine channel may be determined by the Hct value, total cholesterol concentration, low density cholesterol concentration, and total protein concentration. I was able to. As is apparent from the results of FIG. 6, it can be seen that by using the method of the present invention, the factors that determine the speed at which whole blood, plasma, serum, etc. pass through the fine channel can be clarified.

  Further, based on the correlation between the plasma component amount and the plasma flow rate shown in FIG. 6 and the Hct dependence of the whole blood flow rate shown in FIG. 5, the Hct value, total cholesterol concentration, and total protein concentration in whole blood are shown. To estimate the flow rate of whole blood. Compare the estimated whole blood flow rate with the actual measured whole blood flow rate. If the actual measured whole blood flow rate is significantly slow, It is possible to obtain clinical findings that could not be considered by the information of clinical tests so far, such as platelet activity and erythrocyte activity increased and blood flow deteriorated.

  This correlation need not be limited to just comparing the flow rate of whole blood and the amount of components in plasma, but can be applied to a wide range of clinical tests, such as combining the platelet count and the amount of components in plasma.

  As described above, the apparatus according to the present invention, that is, the apparatus having the function of performing the measurement of the fluid flow characteristics and the blood components in parallel using the filter chip according to the present invention in one unit is used for clinical examination. New knowledge can be obtained in the field.

  By using the filter chip according to the present invention, the components contained in these samples can be determined by measuring the speed at which a liquid sample such as whole blood or plasma passes through the filter chip. Moreover, the ease of deformation of erythrocytes can be compared, and these results can be applied to clinical examinations.

The top view which shows the convex part shape of the filter chip | tip (custom chip | tip) which becomes this invention. The schematic diagram which shows the difference in the convex part shape of a custom chip | tip and the conventional filter chip | tip. The top view which shows the convex part shape of a custom chip | tip and the conventional filter chip | tip. The left side is a conventional filter chip, and the right side is a custom chip. Flow rate of whole blood, plasma and physiological saline through channels measured using custom tips with different channel widths. Flow rate of whole blood, plasma, and physiological saline passage measured using a custom chip (chip 6 in Table 1) having a channel width of 6.0 ± 0.5. Whether or not there is a correlation between the flow rate of plasma flow path and the amount of components in plasma measured using the chip 6 of Table 1.

Claims (8)

In a filter chip used for observing the shape of cells and / or suspended particles contained in a sample liquid passing through a microfabricated flow path provided on a substrate under suction pressure or pressurization, the fine flow path is a substrate A filter chip formed by a plurality of convex portions provided on the top, wherein a difference in width between an upper portion and a lower portion of a cross section of the convex portion in a direction orthogonal to the fine flow path is about 4 μm or less. The length, depth, and pitch of the microfabricated flow path are kept constant in one filter chip, and the width is changed stepwise within a range of 1 μm to 20 μm. The filter chip as described. The length, width and pitch of the microfabricated flow path are kept constant in one filter chip, and the depth is changed stepwise within a range of 1 μm to 20 μm. The filter chip as described. In a filter chip used for observing the shape of cells and / or suspended particles contained in a sample solution passing through a microfabricated flow path provided on a substrate under suction pressure or pressurization, the fine flow path is The side in plan view of the convex portion formed by a plurality of convex portions provided on the substrate and facing the flow direction of the sample liquid is substantially an obtuse isosceles triangle or a straight line perpendicular to the flow direction. A featured filter chip. In the convex part that is a polygon in which the apex angle of the substantially obtuse isosceles triangle or the obtuse angle isosceles triangle is not formed, at least one of the corners is rounded in a plan view. The filter chip according to claim 4. Using the filter chip according to any one of claims 1 to 5, under the suction pressure or pressurization, the passing speed of whole blood passing through the microfabricated flow path of the filter chip and the passing speed of plasma and / or serum are determined. A method for measuring blood flow characteristics, characterized by measuring continuously and / or intermittently and determining a ratio or inverse ratio of the passage speed of whole blood and the passage speed of plasma and / or serum. Using the filter chip according to any one of claims 1 to 5, the passing speed of whole blood and / or plasma and / or serum passing through the microfabricated flow path of the filter chip is continuously applied under suction pressure or pressurization. And / or intermittently measuring the blood flow characteristics. A measurement apparatus capable of performing measurement of fluid flow characteristics using the filter chip according to claim 1 and measurement of components in blood in parallel.

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