JP2003302330A - Planar flow cell device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a low-cost planar flow cell device which obtains a stable flow and whose maintenance is easy. <P>SOLUTION: The planar flow cell device is composed of at least two liquid feed ports used to house at least two kinds of liquids; one waste liquid port; at least two liquid feed flow channels communicating with the liquid feed ports; a joining part used to join the liquid feed flow channels; a flow cell composed of a flow channel used to make the joining part communicate with one waste liquid port; and a liquid feed mechanism which makes the liquids flow into the flow cell by applying the same positive pressure to all the liquid feed ports or by applying a negative pressure to the waste liquid port. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a flat plate flow cell device, and more particularly to a cell or particle analysis device using a flat plate sheath flow cell.

[0002]

2. Description of the Related Art In recent years, various analyzes and reactions have been actively conducted on a microchip having a fine flow path in a flat plate made of Si, glass, resin or the like. analysis,
The amount of reagents used in the reaction is extremely small, and by disposing of a low-cost microchip as a disposable, it is possible to prevent contamination between analysis and reaction batches.

As a method for moving the liquid in the flow path on the microchip, there are roughly classified a method using an electro-immersion flow and a method for sending the liquid by pressure. In the electrosurgical flow, an electrode is inserted into each port at the end of the flow path and an appropriate electric potential is set for each to generate a potential gradient between the flow paths, which causes a flow in the liquid containing the electrolyte. A syringe pump is usually used for liquid delivery by pressure. One syringe is connected to each port, and the syringe is pushed by one or a plurality of mechanisms to perform liquid feeding.

On the other hand, there is a technique called flow cytometry as a method for analyzing cells and particles. In flow cytometry, a sample solution containing cells and particles is wrapped in a sheath flow cell in a sheath flow cell and contracted to create a thin sample solution flow in which cells and particles flow one by one. Each sample is measured by an optical or electrical method. Obtaining a stable sheath flow is very important in terms of measurement accuracy, and variations in the sample flow position and flow width within the flow cell must be suppressed.

Recently, a flow cell using a groove formed on a substrate of Si, glass or resin by a fine processing technique has been announced. As a liquid feeding method, Journal
The example described in Analytical Chemistry, Vol 71, pp4173- (1999) utilizes electrosurgical flow.
Proceedings of the Japan Society of Mechanical Engineers (Volume B) Vol. 65, No. 629, pp223- (1999
(Year), the sheath liquid is pneumatically supplied and the sample liquid is supplied into the microchannel by a syringe pump.

[0006]

The above-mentioned liquid feeding method by electro-immersion flow makes it possible to easily control the flow velocity by electrical control, but since a high electric field is applied to the liquid, the electric component Problems such as causing chemical reactions occur. In particular, when it is used as a flow cytometer for cell analysis, cell melting occurs in a high electric field. Therefore, there is a problem that the voltage applied to the liquid is limited, the liquid cannot be sent at a high flow rate, and the analysis time becomes long.

In addition, in the liquid feeding method using a syringe pump,
A syringe was required for each port, and a slight fluctuation in the feed rate of each syringe due to mechanical causes was likely to occur, and phenomena such as fluctuations in the flow rate ratio and pulsation at the confluence of the flow channels were likely to occur. In the sheath flow cell of the flow cytometer, the above phenomenon appears as a change in the flow rate ratio of the sample / sheath liquid, which destabilizes the flow of the sample liquid in the measurement section and thus affects the measurement accuracy. Further, since the syringe is in contact with the sample solution, there is a problem of contamination when handling a plurality of samples.

As described above, none of the conventional techniques is sufficient, and improvement has been desired especially for the sheath flow cell device of the flow cytometer. It is an object of the present invention to solve the above problems and provide a low-cost flat plate type flow cell device which can obtain a stable flow and can be easily maintained.

[0009]

Means for Solving the Problems As a result of studying a new liquid feeding technique for solving the above-mentioned problems, the present inventor has found that in a flat plate flow cell device, the same positive pressure is applied to all liquid supply ports. Alternatively, by applying a negative pressure to the waste liquid port, it was found that the flow rate ratio at the merging portion in the flow cell can be controlled to be constant regardless of the applied pressure fluctuation,
The present invention has been completed.

That is, the present invention is (1) at least 2
At least two liquid supply ports for storing different types of liquids, at least two liquid supply passages communicating with the liquid supply ports, a merging portion where the liquid supply passages merge, the merging portion and one waste liquid port A flat plate-shaped member comprising a flow cell consisting of a flow path communicating with each other, and a liquid feed mechanism for flowing a liquid into the flow cell by applying the same positive pressure to all of these liquid supply ports, and a flat plate-shaped flow cell device,
(2) At least two containing at least two types of liquids
One liquid supply port, at least two liquid supply passages communicating with the liquid supply ports, a joining portion where the liquid supply passages join, and a passage that connects the joining portion and one waste liquid port. A flat plate-shaped flow cell device comprising a flat plate-shaped member including a flow cell, and a liquid feeding mechanism for flowing a liquid into the flow cell by applying a negative pressure to a waste liquid port thereof,
(3) The flat-plate flow cell device according to (1), characterized in that the liquid feeding mechanism includes a coupler that covers all the liquid supply ports in an airtight manner and a pressurizing mechanism that applies a positive pressure to the coupler. (4) The flat plate flow cell device according to (2), wherein the liquid feeding mechanism includes a coupler that hermetically covers the waste liquid port and a depressurizing mechanism that applies a negative pressure to the coupler, and (5) the liquid. The supply ports are one sample solution port for containing a sample solution containing a sample of cells or particles and at least one sheath solution port for containing a sheath solution, and the flow cell sends the sample solution so that the sheath solution wraps the sample solution. A flat plate flow cell device according to any one of (1) to (4), characterized in that it is a sheath flow cell for liquid flow, and (6) the sheath flow cell includes the sheath liquid only in the left-right direction of the sample liquid. It is equipped with a measurement mechanism that optically or electrically measures cells or particles that pass through a predetermined measurement site on the sheath flow cell, and the flow channel depth at the measurement site is 30 μm or more and 100 μm or less. The flat plate flow cell device according to (5), (7) the flat plate member is made of a transparent material, and the measurement mechanism irradiates the measurement site with a laser beam and cells or particles. And a measurement mechanism for measuring scattered light or fluorescence of the
The flat-plate flow cell device according to (5) or (6) is characterized in that it is 0 μm or less.

[0011]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT An embodiment of the present invention is shown in FIGS.
It will be described with reference to the drawings. FIG. 1 shows one embodiment of the flat plate type flow cell device of the present invention. Flat plate flow cell 1
Is made of a transparent resin substrate. Flat plate flow cell 1
Is a sample liquid port 2 for storing a sample liquid containing cells and particles
And sheath liquid ports 3 and 4 for accommodating the sheath liquid, and the ports are connected to the channels 5, 6 and 7 formed in the flat plate, respectively. The flow paths 5, 6 and 7 are joined at a junction point 8 so that the sample liquid is sandwiched by the sheath liquid from both sides and connected to the flow passage 9. The end of the flow channel 9 is connected to the waste liquid port 10. The openings of the sample liquid port 2 and the sheath liquid ports 3 and 4 are covered with a coupler 11, and airtightness is maintained between the coupler 11 and the flat plate-shaped flow cell 1. Coupler 1
1 is connected to a positive pressure source via a tube 12.
By applying a positive pressure to the coupler 11, the liquid of the sample port 2 and the sheath liquid ports 3 and 4 is swept into the flow path.

FIG. 2 shows another embodiment of the flat plate type flow cell device of the present invention. The coupler 11 is installed so as to cover the opening of the waste liquid port 10. The coupler 11 is connected to a negative pressure source via a tube 12. By applying a negative pressure to the coupler 11, the liquids of the sample liquid port 2 and the sheath liquid ports 3 and 4 are drawn into the flow path via the waste liquid port 10 and the flow paths 5, 6, 7, and 9. In both the embodiment shown in FIGS. 1 and 2, the flow of the liquid at the merging portion 8 is as shown in FIG. The liquid flow at the merging portion is made to flow at a flow velocity such that it becomes a laminar flow.
4, 15 flow in layers as shown in the figure, and the flow of the sample solution is contracted. The flow rate ratio of the sample liquid and the sheath liquid in the flow channel 9 is determined by the ratio of the pressure loss in the flow channels 5, 6, and 7, that is, the flow channel shape and the viscosity of the liquid flowing therethrough, regardless of the pressure applied to the coupler 11. Even if the pressure applied to the coupler 11 changes, the flow rate changes but the flow rate ratio does not change, and the sample liquid flow does not pulsate, and a stable flow can be obtained.

As described above, by connecting all the liquid ports on the upstream side of the flow or the waste liquid port on the downstream side to one pressure source by the coupler, stable operation can be achieved without using an expensive and highly accurate pump mechanism. A flow having a flow rate ratio can be realized. Since the flat plate-shaped flow cell and the coupler are separable, and the sample solution and the sheath solution to be used are in contact with the flat plate-shaped flow cell only, by disposing the flow cell as a disposable, the contamination between the samples can be eliminated. A complicated device cleaning mechanism is not required, the cost of the device can be reduced, and maintenance is easy.

Next, an example of a method for producing a flat plate type flow cell will be described with reference to FIG. Each figure represents a cross section. As shown in A, first, the flow path pattern on the photomask is transferred to the photosensitive resist 17 on the Si substrate 18 by a photolithography process. Subsequently, as shown in B, the unexposed resist is removed, the Si substrate is etched, and the groove of the flow path pattern is formed on the Si substrate. Next, as shown in C, Ni was electroformed on Si and the flow path pattern was convex.
Create the Ni plate 19. Next, as shown in D,
The flow path pattern is transferred onto the transparent resin substrate by thermocompression molding the transparent resin substrate 20 using the plate 19 as a mold. As shown in E, a portion of the resin substrate that will be the liquid port is perforated, and another resin substrate 21 is attached to the surface on which the flow path pattern is formed, thereby achieving the purpose as shown in F. A flat plate flow cell can be created.

One important application of the planar flow cell device according to the present invention is in flow cytometers. In a flow cytometer, a sheath flow cell is used to flow cells or particles as a sample one by one to an optical or electrical measurement site. The width of the sample flow at the measurement site is preferably narrowed down to the same size as the sample. One of the most commonly used applications for flow cytometers is blood cell counting. The largest leukocyte size in blood cells is about 7 to 21 μm. A flat-plate sheath flow cell, as shown in FIGS. 1 and 2, in which a sample liquid is wrapped in a sheath liquid only in a two-dimensional direction to cause a contraction is easy to create because the flow path is two-dimensional. If the channel depth is not sufficiently shallow, particles and cells are likely to pass through at the same time. In order to measure cells or particles one by one, the depth of the flow channel is 30 μm or more and 100 or more.
It is desirable that the thickness is μm or less. If it is shallower than 30 μm,
It is close to the size of white blood cells, and cells are likely to be clogged in the flow channel. If it is deeper than 100 μm, simultaneous passage of cells will occur with a non-negligible probability, resulting in a large measurement error.

FIG. 5 shows a structural example of a flow cytometer device using a flat sheath flow cell equipped with an optical measuring mechanism using laser light. The laser beam 32 oscillated from the laser 22 is collected by the lens group 23,
The measurement site on the flow path 9 of the flat plate flow cell 1 made of a transparent member is irradiated. Scattered light or fluorescence 33 is generated from the cells or particles irradiated with the laser light. The scattered light or fluorescence 33 is collimated by the lens 25 and split into two optical paths by the beam splitter 26. One is condensed by the lens 27, guided to the optical sensor 28, and used for measuring the forward scattered light. On the other hand, the optical filter 29 takes out only the light of the required wavelength, is condensed by the lens 30, is guided to the optical sensor 29, and is used for fluorescence measurement. The laser light transmitted through the flat plate flow cell 1 is cut by the light shielding plate 24 so as not to enter the optical sensor.

The flow path width of the measurement site of the sheath flow cell of the present invention which is measured by such an optical system is 60 μm or more and 5 or more.
It is preferably 00 μm or less. The reasons for limitation will be described below.
As described above, the width of the sample flow at the measurement site is narrowed down to the same size as the sample. In the case of cell measurement, the sample flow width is 10
-30 μm is suitable. Generally, laser light has an intensity distribution in the beam diameter direction. The distribution typically shows a Gaussian distribution, and when a sample flow having the above width is irradiated with such a laser beam, the light intensity hitting the sample is changed depending on the passage position of cells and particles in the cross-flow direction. Differently, it causes variations in scattered light and fluorescence measurements.

In order to suppress this variation, a method of increasing the laser beam diameter to make the curve of the Gaussian distribution smooth is generally used. However, if the beam diameter is too large, unnecessary scattered light from the flow cell other than the sample increases, and the measurement signal decreases. Particularly, when a part of the laser light hits the flow path wall of the flat plate type flow cell, the scattered light remarkably increases. FIG. 6 shows the results of actually measuring the scattered light intensity (background) in the absence of the sample when the laser position was moved in the transverse direction of the flow channel. In the figure, a and b indicate the position of the flow path wall, and at point c, the increase of scattered light due to the influence of the flow path wall has finally disappeared. a
The distance W from to point c is almost equal to the beam diameter. From this result, it was found that the flow path width at the measurement site is preferably at least twice the laser beam diameter.

On the other hand, as the flow channel width increases, the amount of sheath liquid flowing therein also increases. In the present invention, since the sheath liquid is stored in the sheath liquid port of the flat plate type flow cell, the increase in the amount of the sheath liquid is not preferable and therefore the remarkably large flow channel width is also not preferable. As described above, it is necessary to determine the flow channel width in consideration of measurement variations, unnecessary scattered light from the flow cell, and the amount of sheath liquid. Table 1 shows the results of evaluating the CV values of the measurement signals of particles of the same size with several combinations of the channel width, the beam diameter, and the sample flow width. At the same time, the amount of sheath liquid (ratio to the amount of sample liquid) required by the combination of the channel width and the sample flow width is also described.

[0020]

[Table 1]

As a result of the above examination, the preferred flow path width of the measurement site of the flat sheath flow cell of the present invention is 60 μm.
It has been found that it is not less than m and not more than 500 μm. Channel width 6
If it is smaller than 0 μm, the usable laser beam diameter is 3
It becomes smaller than 0 μm, and as a result, the CV value of the measurement signal exceeds 6%, which hinders the identification of the sample. Flow width 500
C of measurement signal when μm and laser beam diameter is 250 μm
Even when the sample flow width is 30 μm, the V value is 1% or less, which is sufficiently low, and the expansion of the beam diameter beyond that causes a serious problem such as a decrease in SN. Also, the amount of sheath liquid becomes large, and a large sheath liquid port is required. Therefore, the channel width is 500μ
There is no effect to make it more than m.

More preferably, the flow path width of the measurement site is 10
It is preferably 0 μm or more and 400 μm or less. Channel width 100 μm,
When the laser beam diameter is 50 μm, the CV value of the measurement signal is less than 3%, which enables more accurate analysis. Channel width 4
At 00 μm, the required amount of sheath fluid is 40 for the sample.
It is less than double, and can be stored in a small sheath fluid port. More preferably, the channel width is 150 μm or more and 300 μm or less. With a channel width of 150 μm and a laser beam diameter of 75 μm, the CV value is 1%, which enables more accurate measurement. When the channel width is 300 μm or less, the required amount of sheath liquid is 30 times or less that of the sample, and the sheath liquid port can be stored in a smaller sheath liquid port, so that the flat plate flow cell can be downsized.

[0023]

EFFECTS OF THE INVENTION At least two liquid supply ports for storing at least two types of liquids, one waste liquid port, at least two liquid supply passages communicating with the liquid supply ports, and the liquid supply passages are joined. In a flat plate-shaped flow cell including a merging section that forms a flow path and a flow cell that connects the merging section and one waste liquid port, the same positive pressure is applied to all the liquid supply ports, or
By applying a negative pressure to the waste liquid port, the flow rate ratio of each liquid at the merging portion in the flow passage is determined by the shape of the flow passage from each liquid supply port to the merging portion and the viscosity of the liquid, and is applied. It does not change even if the pressure fluctuates. Therefore, a stable flow can be obtained with a constant flow rate of each liquid at the merging portion.
Especially in the sheath flow cell of the flow cytometer, it greatly contributes to the improvement of the measurement accuracy.

By applying pressure by the coupler structure, except for the flat plate-shaped flow cell, there is no contact with the liquid, and by making the flow cell disposable, it is possible to prevent contamination between batches, and it becomes unnecessary to clean the apparatus.
In a flat-plate sheath flow cell in which the sheath liquid wraps the sample flow two-dimensionally, by setting the channel depth of the measurement site to 30 μm or more and 100 μm or less, clogging of the sample in the channel is prevented and measurement accuracy is improved. Can be made. In a cell / analyzer using a flat-plate sheath flow cell equipped with an optical measurement mechanism using laser light, by setting the flow path width of the measurement site to 150 μm or more and 500 μm or less, unnecessary scattered light from the flow cell is reduced, The sheath liquid can be saved.

[Brief description of drawings]

FIG. 1 is a perspective view showing a flat sheath flow cell and a liquid feeding mechanism showing an example of the present invention.

FIG. 2 is a perspective view showing a flat sheath flow cell and a liquid feeding mechanism showing another example of the present invention.

FIG. 3 is a top view showing a flow state of a merging portion of the sheath flow cell according to the example of the present invention.

FIG. 4 is a process drawing schematically showing a process of producing a flat plate-shaped flow cell of the present invention by using a sectional view of the cell.

FIG. 5 is a schematic view showing an arrangement of optical systems according to an example of the present invention.

FIG. 6 is a diagram showing a change in scattered light intensity (a state without a sample) when the position of the laser beam is changed in the direction crossing the flow path.

[Explanation of symbols]

1 Flat plate flow cell 2 Sample solution port 3 Sheath liquid port 4 Sheath liquid port 5 Flow path connected to the sample solution port 6,7 Flow path connecting to sheath fluid port 8 confluence section 9 Flow path connecting to waste liquid port 10 Waste liquid port 11 couplers 12 tubes 13 Sample flow 14, 15 sheath liquid flow 16 Photomask 17 Resist 18 Si substrate 19 Ni 20 Resin substrate 1 21 resin substrate 2 22 laser 23 lens groups 24 Light shield 25 Collimating lens 26 Beam splitter 27 Condensing lens 28 Optical sensor 29 Optical filter 30 condenser lens 31 Optical sensor

Claims (7)

[Claims] 1. At least two liquid supply ports for accommodating at least two types of liquids, at least two liquid supply passages communicating with the liquid supply ports, and a confluence portion where the liquid supply passages merge, From a flat plate-shaped member including a flow cell including a flow path that connects the confluence portion and one waste liquid port, and a liquid feeding mechanism that causes the liquid to flow into the flow cell by applying the same positive pressure to all the liquid supply ports. Flat plate flow cell device. 2. At least two liquid supply ports for accommodating at least two types of liquids, at least two liquid supply channels communicating with the liquid supply ports, and a confluence section where the liquid supply channels merge. A flat plate-shaped flow cell including a flat plate-shaped member including a flow cell including a flow path that connects the confluence portion and one waste liquid port, and a liquid feeding mechanism that applies a negative pressure to the waste liquid port to flow the liquid into the flow cell apparatus. 3. The flat-plate flow cell according to claim 1, wherein the liquid feeding mechanism includes a coupler that hermetically covers all liquid supply ports and a pressurizing mechanism that applies a positive pressure to the coupler. apparatus. 4. The flat plate type flow cell device according to claim 2, wherein the liquid feeding mechanism comprises a coupler that hermetically covers the waste liquid port and a depressurizing mechanism that applies a negative pressure to the coupler. 5. The liquid supply port is one sample liquid port for storing a sample liquid containing a sample of cells or particles, and at least one sheath liquid port for storing a sheath liquid, and the flow cell is a sheath liquid sample. The flat plate flow cell device according to any one of claims 1 to 4, which is a sheath flow cell that sends a liquid so as to wrap it. 6. A measurement mechanism for optically or electrically measuring cells or particles passing through a predetermined measurement site on a sheath flow cell by sending a sheath liquid in a sheath flow cell so as to wrap the sample solution only in the left-right direction. The plate-shaped flow cell device according to claim 5, wherein the flow path depth at the measurement site is 30 μm or more and 100 μm or less. 7. A flat plate-shaped member is made of a transparent material, a measuring mechanism comprises a laser light source for irradiating a measurement site with laser light, and a measuring mechanism for measuring scattered light or fluorescence from cells or particles. 7. The flat plate type flow cell device according to claim 5, wherein the flow path width is 60 μm or more and 500 μm or less.