KR100473362B1 - Apparatus and method for flow cytometry - Google Patents

Abstract

Disclosed are an apparatus and method for fine particle analysis. The disclosed assay device includes a substrate on which a focusing chamber is formed. At least one sample channel is formed in the substrate through which a sample, which is a fine particle such as a cell, is introduced into the focusing chamber. At least one focusing channel is formed in the substrate through which a sheath flow surrounding the sample flows into the focusing chamber such that the samples flow in a single line in the focusing chamber. In addition, a waste channel is formed in the substrate through which the analyzed sample is discharged from the focusing chamber. On the other hand, the waste channel may be connected to at least two classification channels for classifying the sample by the component by the pressure gradient. With such a configuration, the sample and the sheath flow can flow by the pressure gradient, so that the moving speed of the sample and the sheath flow can be increased.

Description

Apparatus and method for fine particle analysis {APPARATUS AND METHOD FOR FLOW CYTOMETRY}

The present invention relates to an apparatus and method for analyzing microparticles, and more particularly, to an apparatus and method for analyzing microparticles such as cells moving in a line along a fluid flow in a microchannel.

In the field of fine particle analysis, such as flow cytometry, it is well known to analyze cells by floating the cells in suspension with a sheath flow, which is a buffer solution. This scheme is outlined as follows.

The flow of the sheath flow is disturbed to form water droplets, and when the water droplets cut off the ends of adjacent flows, cells are contained in the droplets. Subsequently, each drop is sorted by detecting the desired cells and applying an electric field to the respective drop just before the drop drops away from the vicinity of the flow. The droplets containing the desired cells are then deflected by the electric field and collected in the collection container. During this process, it is very important to know the exact time when the droplet containing the desired cell reaches the charging region so that the specific droplet is charged while the other surrounding droplets are only slightly charged.

US patent (Registration No. 6,120,666) applying the basic flow cytometry described above is shown in FIG. Referring to FIG. 1, microchannels having a very short width enough to allow cells 120 to pass through are formed on a substrate (not shown) in a cross shape. The crosshairs of the microchannels become the focusing chamber 22. The focusing chamber 22 is imaged by an image acquisition device 160 such as a CCD camera, and the image is processed by the image control processor 150, whereby cells are analyzed.

On the other hand, a channel extending from above with respect to the focusing chamber 22 is the sample channel 100, and the cell 120 serving as the sample is introduced through the sample channel 100. A pair of channels connected to both sides of the focusing chamber 22 are the focusing channels 102 and 104, and a sheath flow flows through each of the focusing channels 102 and 104. The channel connected below the focusing chamber 22 is a waste channel 106 through which the cell 120 which has been analyzed flows.

As shown in the figure, the plurality of cells 120 introduced through the sample channel 100 are surrounded by the sheath flow flowing from both focusing channels 102 and 104 in the focusing chamber 22, thereby focusing one by one. It passes through the chamber 22. The cells 120 of this arrangement are photographed by the image acquisition apparatus 160 and then analyzed by the image control processor 150.

On the other hand, the flow of the cell 120 and the sheath flow is performed by applying an electric field to each channel (100, 102, 104, 106), by the potential difference generated between each channel (100, 102, 104, 106).

However, in the conventional flow cytometry apparatus, as described above, the cells and the sheath flow to flow using an electric field, the following problems occur.

First of all, an apparatus for applying an electric field is required for a conventional flow cytometry apparatus, which has a disadvantage that the apparatus is quite expensive due to its high cost.

In particular, since the buffer solution used as the sheath flow must be induced by the electric field, the buffer solution is necessarily limited to the conductive material. For this reason, the range which can be used as a buffer solution becomes extremely narrow.

In addition, since the sheath flow is moved with a potential difference, there is a problem that the moving speed is too slow once. Slow travel speeds can cause time-consuming ripple problems. In order to solve this problem, a strong electric field of several kV should be applied to each channel to increase the potential difference, but a strong electric field causes a new problem of separating specific substances such as proteins in cells. As a result, since the use of a strong electric field is limited, the disadvantage that the movement speed of the sheath flow is slow is still not solved.

In addition, when the fine particles have a missing portion, particularly red blood cells having a deformed shape, or when the fine particles flow in an inclined state, the laser, which is a scanning means, through the missing portion or the slanted empty space is left as it is. There was a high possibility of passing. That is, the image acquisition apparatus may not recognize the fine particles in some cases.

Accordingly, the present invention has been devised to solve all the problems of the conventional flow cytometry, and employs a pressure gradient method instead of a flow method using a potential difference, so that the flow of the sheath is not limited to the application range of the buffer solution. It is an object of the present invention to provide an inexpensive microparticle analysis apparatus and method as well as to allow a very high flow rate.

Another object of the present invention is to prevent cell separation due to a strong electric field, and to significantly improve the reliability of a cell analysis result.

Still another object of the present invention is to enable accurate recognition of fine particles having a deformed shape having missing portions and to prevent the fine particles from flowing in an inclined state.

In order to achieve the above object of the present invention, the microparticle analysis apparatus according to the present invention includes a substrate on which a focusing chamber is formed. At least one sample channel is formed in the substrate through which a sample, which is a fine particle such as a cell, is introduced into the focusing chamber. At least one focusing channel is formed in the substrate through which a sheath flow surrounding the sample flows into the focusing chamber such that the samples flow in a single line in the focusing chamber. In addition, a waste channel is formed in the substrate through which the analyzed sample is discharged from the focusing chamber. On the other hand, the waste channel may be connected to at least two classification channels for classifying the sample by the component by the pressure gradient. On the other hand, the sample channel, the focusing channel and the sorting channel is connected to the container that stores the sample and the buffer solution which is the sheath flow.

The sample channel, the focusing channel and the sorting channel are provided with pressure control means for forming a pressure gradient between each channel. The pressure gradient generated by each pressure control means causes the sample and sheath flow to flow from each channel to the waste and fractionation channels.

Here, the focusing channels are preferably two symmetrically arranged, and in particular, the sheath flow flowing through each focusing channel is preferably introduced into the focusing chamber at the same pressure. This is because the pressure of the sheath flow provided on both sides must be the same, so that the sample is subjected to uniform pressure from both sides, so that the sample is disposed in the longitudinal direction along the flow direction of the sample.

 At this time, the pressure control means includes a vacuum pump connected to each vessel, a microvalve disposed between the vacuum pump and the vessel, a pressure controller for sensing the pressure in the vessel, and a pressure controller for controlling the operation of the microvalve in accordance with a signal of the pressure sensor. It includes.

On the other hand, samples moving in a row by the sheath flow in the focusing chamber by the pressure gradient by the pressure control means are captured by the image acquisition means. The image photographed by the image capturing means is transmitted to the image controller so that the image controller counts the number of samples moving in the focusing chamber and analyzes the samples. The image controller recognizes the types of samples and transmits them to the pressure controller for the classification channel, whereby the samples are classified by the respective types through the classification channel by the corresponding pressure controller.

As the image acquisition means, a light source and a photodiode may be used, or a photo multiplier tube (PMT) may be used instead of the photodiode. In addition, a general laser or a laser diode may be used as the light source.

On the other hand, the focusing channel is preferably two symmetrically arranged, in particular, the sheath flow flowing through each focusing channel is preferably introduced into the focusing chamber at the same pressure. This is because the pressure of the sheath flow provided on both sides must be the same, so that the sample is subjected to uniform pressure from both sides, so that the sample is disposed in the longitudinal direction along the flow direction of the sample. In this case, light irradiated in a direction orthogonal to the flow direction of the sample becomes orthogonal to the sample, so that light can always reach the sample.

The method of analyzing the cells while allowing the sample to flow with the sheath flow through the microchannel is as follows. First, pressure gradients are formed in the sample channel and the focusing channel and the sorting channel respectively connected to the focusing chamber. Pressure gradients formed in each channel allow the sample and sheath flow to flow through the sample and focus channels into the focusing chamber. At this time, two focusing channels are configured, and a sheath flow having the same pressure flows from each focusing channel.

In the focusing chamber, the samples are flowed one by one while surrounded by a sheath flow, which is captured and analyzed. Analyzed samples are classified into specific components by pressure gradient and collected in each sorting channel.

According to the configuration of the present invention described above, since the sample and the sheath flow flows by a pressure gradient rather than an electric field, the buffer solution used as the sheath flow does not necessarily need to be conductive, and it is very easy to increase the pressure gradient. Therefore, the flow rate of the sheath flow can be made as fast as desired. In addition, cell separation due to strong electric fields is also prevented. In particular, the sheath flow of the same pressure causes the sample to flow in a state in which the longitudinal direction coincides with the flow direction of the sample, so that a sample having any shape can be clearly recognized.

Hereinafter, an apparatus and method for analyzing fine particles according to a preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited or limited by the following examples.

2 is a configuration diagram of a fine particle analysis apparatus according to the present invention, FIG. 3 is an enlarged view showing in detail a pressure control means that is an essential part of the present invention, and FIG. 4 is a configuration diagram of an image acquisition means that is an essential part of the present invention. 5 is a diagram illustrating a flow relationship between a sample and a sheath flow.

First, referring to FIG. 2, cross-shaped fine channels 210, 220, 230, and 240 are formed on a substrate (not shown). The microchannels 210, 220, 230, and 240 are very small, such as 100 μm × 50 μm, which can be easily manufactured through a plastic micro-processing method in which a mold made of silicon is poured, a polymer solution is poured into the mold, and then cured in an oven.

The cross-shaped fine channels 210, 220, 230, and 240 each have unique functions. First, the cross-shaped cross section becomes the focusing chamber 200 which is an image photographing area. The channel 210 continued from above with respect to the focusing chamber 200 becomes a sample channel. The sample S, which is a fine particle such as a cell, is introduced into the focusing chamber 200 through the sample channel 210.

A pair of channels 220 and 230 connected to both sides of the focusing chamber 200 are focusing channels, and the sheath flow H flows through the focusing channels 220 and 230. As described above, the sheath flow H surrounds the sample S so that the samples S are moved in line in the focusing chamber 200 one by one. In particular, according to the present invention, as will be described later, since the sample (S) and the sheath flow (H) flows by a pressure gradient rather than a potential difference, the buffer solution used as the sheath flow (H) is not limited to a conductive material. .

Meanwhile, the channel 240 connected below the focusing chamber 200 becomes the waste channel 240 through which the analyzed sample S flows. The waste channel 240 is most preferably positioned in line with the sample channel 210 for smooth flow of the sample S and the sheath flow H. The waste channel 240 is divided into several, in this embodiment, two classification channels 250 and 260. The classification of the sample S by the dividing channels 250 and 260 is also made by the pressure gradient which is a feature of the invention, which will be described in detail later.

The containers 310, 320, 330, 350, and 360 are connected to each of the channels 210, 220, 230, 250, and 260 except for the waste channel 240. That is, the container 310 connected to the sample channel 210 is a sample container, and the containers 320 and 330 connected to both focusing channels 220 and 230 are buffer solution containers in which a buffer solution for the sheath flow is stored. In addition, the containers 350 and 360 connected to the respective sorting channels 250 and 260 are sorting containers in which the sample S is collected by type.

As described above, the sample (S) and the sheath flow (H) is flowed by a pressure gradient, pressure control means for this is provided for each channel (210,220,230,250,260). The configuration of the pressure control means for each of the channels 210, 220, 230, 250, and 260 is the same, and the configuration of the pressure control means is shown in detail in FIG. 3.

The pressure control means shown in FIG. 3 is provided in the right focusing channel 230, and as shown, a vacuum pump 432 is connected to the buffer solution container 330. A microvalve 433 for fine control of vacuum pressure is installed between the vacuum pump 432 and the buffer solution container 330. A pressure sensor 431 for measuring the pressure in the buffer solution container 330 is attached to the top of the buffer solution container 330.

A pressure controller 430 that controls the operation of the microvalve 433 by the signal of the pressure sensor 431 is installed on the upper portion of the buffer solution container 330. As shown in FIG. 2, the pressure controllers 410, 420, 430, 450, and 460 are installed in the respective channels 210, 220, 230, 250, and 260 to independently control the flow rates of the sample S and the sheath flow H flowing through the channels 210, 220, 230, 250, and 260.

In particular, pressure controller 430 is used to provide equal pressure to both focusing channels 220, 230. When the same pressure is provided to both focusing channels 220 and 230, the sheath flow H flowing from both sides flows into the focusing chamber 200 at the same pressure. Therefore, since the sample S flowing along the center receives the same pressure from both sides from the sheath flow H on both sides, the longitudinal direction of the sample S coincides with the flow direction of the sample S. FIG. That is, the sample S which flowed obliquely is also naturally arranged along the flow direction by the sheath flow H of both sides.

Meanwhile, the position at which the sample S is analyzed is inside the focusing chamber 200. The image acquiring means 500 photographs the samples S flowing in the focusing chamber 200 one by one in a state surrounded by the sheath flow H.

As illustrated in FIG. 4, the image acquiring means 500 includes a light source 510, a front angle light scattering sensor 520, and a side angle light scattering configured to detect light scattered from the sample S by being emitted from the light source 510. Sensor 530, and a photodiode (not shown) for detecting the signal detected by each sensor (520,530). It is preferable to use a laser diode as the light source 510 instead of an expensive general laser.

Again, in FIG. 2, an image captured by the image acquisition unit 500 having such a configuration is transmitted to the image controller 600 so that the image controller 600 counters the sample S flowing in the focusing chamber 200. At the same time, the type of the sample S is determined. When the image controller 600 determines the type of the sample S, for example, red blood cells and white blood cells in the blood, the determination signal is immediately transmitted to the pressure controllers 450 and 460 for classification. Each of the pressure controllers 450 and 460 adjusts the vacuum pressures of the sorting vessels 350 and 360 according to the discrimination signal so that the red blood cells and the white blood cells are classified by the sorting channels 250 and 260.

Hereinafter, a method of analyzing a sample by the flow cytometry apparatus according to the present embodiment configured as described above will be described in detail. First, it is assumed that blood is used as a sample in this analysis method, and blood is classified into only two types of red blood cells and white blood cells.

First, a dye is added to the blood, and a dye that stains only red blood cells is added. This blood is placed in the sample vessel 310 and the buffer solution is placed in each buffer solution vessel 320, 330. Then, a vacuum pressure is applied to the sample vessel 310 and the buffer solution vessels 320 and 330 using the pressure controllers 410, 420 and 430 for the sample and the sheath flow. In particular, the same buffer pressure is applied to the buffer solution containers 320 and 330. In addition, the vacuum pressure is applied to each of the classification vessels 350 and 360 by using the pressure controllers 450 and 460 for each classification. At this time, the vacuum pressure of each of the sorting vessels 350 and 360 is higher than that of the sample vessel 310 and the buffer solution containers 320 and 330 so that the pressure of the sample container 310 and the buffer solution containers 320 and 330 is higher than the sorting container 350 and 360. Set higher than pressure. This pressure gradient is achieved by each pressure controller 410, 420, 430, 450, 460 controlling the opening angle of the microvalve.

When the pressure gradient as described above is formed, blood and the sheath flow flows to the focusing chamber 200. Since the sample channel 210 is disposed at the center and the pair of focusing channels 220 and 230 are disposed at both sides, the blood S flows in the focusing chamber 200 as shown in FIG. 5. It is surrounded by and flows one by one. In particular, since the blood S receives the same pressure from both sides, the longitudinal direction of the blood S is aligned along the flow direction of the blood S. That is, since the laser irradiated from the light source 510 is orthogonal to the flow direction of the blood S, the laser is not irradiated to the blood S according to the shape of the blood S or the flow state of the blood S. Will disappear.

The flow of the blood S and the sheath flow H are photographed by the image acquisition means 500. More specifically, the laser irradiated from the light source 510 is scattered while colliding with the blood (S), the scattered laser is detected by each light scattering sensor (520,530), this detection signal is detected by the photodiode . The image signal detected by the photodiode is transmitted to the image controller 600 to analyze the image received by the image controller 600. That is, the number of blood flowing through the focusing chamber 200, that is, the number of red blood cells and white blood cells, is counted, and an analysis of whether a disease is performed is also performed. In particular, the image controller 600 distinguishes the dyed red blood cells from the unstained white blood cells, and transmits the distinguishing signal to the pressure controllers 450 and 460 for classification.

Completed blood is discharged to the waste channel 240. At this time, for example, if the left sorting container 350 is a red blood cell container and the right sorting container 360 is a white blood cell container, and the type of blood flowing through the waste channel 240 is red blood cells, it is transmitted from the image controller 600. By the signal, each of the flow dividing pressure controllers 450 and 460 is set so that the left flow dividing vessel 350 becomes lower than the right flow dividing vessel 360. Thus, the red blood cells that are currently flowing are collected in the left sorting vessel 350 through the left sorting channel 250. If the current flowing blood is white blood cells, the flow will be reversed. Therefore, the pressure of each sorting vessel 350, 360 is continuously changed in accordance with the sorting signal from the image controller 600.

Meanwhile, in the present exemplary embodiment, a pair of classification channels 250 and 260 are connected to the waste channel 240, but the classification channel is not necessarily required for the microparticle analysis apparatus according to the present invention. That is, the analysis of the fine particles is virtually completed by photographing the fine particles in a state in which the fine particles are flowed one by one and analyzing the fine particles, and the classification function is additional, so that the classification channel is deleted and the waste channel 240 described above. The pressure control means may be connected directly.

In addition, the microparticle analysis device according to the present invention can easily perform the diagnosis of sexually transmitted diseases, and in particular can be used for the purpose of separating maternal nucleated red blood cells for fetal diagnosis.

As described above, according to the present invention, by allowing the sample and the sheath flow to flow by the pressure gradient instead of the potential difference as in the prior art, the cost of the analyzer can be significantly reduced first.

In addition, since the buffer solution used in the sheath flow does not necessarily need to be conductive, the use range of the buffer solution is also almost unlimited.

In particular, the phenomenon in which fine particles such as cells are separated by a strong electric field as in the related art is fundamentally prevented by the present invention, so that the reliability of the analysis result is greatly improved.

In addition, since the samples are arranged in a direction orthogonal to the direction of irradiation of the laser under the same pressure from the sheath flows on both sides, the case of not recognizing the sample is surely prevented.

In addition to the effects described above, the greatest advantage of the present invention is that it is very easy to set the difference of the pressure gradient, so that the flow rate control of the sample and the sheath flow is facilitated, and in particular, the moving speed of the sample and the sheath flow can be made as fast as desired by the user. Is there. As a result, there is a very large effect that the analysis time is significantly shorter than before.

In the above, the fine particle analysis apparatus and method according to a preferred embodiment of the present invention have been described and illustrated, but the present invention is not limited to the above-described embodiments without departing from the gist of the present invention as claimed in the following claims. It will be understood by those skilled in the art that the present invention may be modified in various ways.

1 is a block diagram showing a conventional flow cytometry device.

2 is a block diagram of a fine particle analysis device according to the present invention.

Figure 3 is an enlarged view showing in detail the pressure control means that is an essential part of the present invention.

4 is a block diagram of an image acquisition means that is an essential part of the present invention.

5 is a view showing a flow relationship between a sample and a sheath flow;

-Explanation of symbols for the main parts of the drawings-

200: focusing chamber 210: sample channel

220,230 focusing channel 240 waste channel

250,260 Classification Channel 310,320,330,350,360 Container

410,420,430,450,460: Pressure controller 431: Pressure sensor

432: vacuum pump 433: micro valve

500: image acquisition means 510: light source

520,530: light scattering sensor 600: image controller

Claims (16)

delete delete delete delete delete delete An apparatus for analyzing a sample that is a fine particle flowing along the microchannel formed on the substrate, A focusing chamber formed on the substrate; A sample channel coupled to the focusing chamber; At least one focusing channel connected to the focusing chamber and having a sheath flow flowing around the sample introduced into the focusing chamber through the sample channel to flow the samples one by one; A waste channel coupled to the focusing chamber; At least two classification channels branched to the waste channel, the samples being classified by type; Pressure control means for forming a pressure gradient between each channel to cause said sample and sheath flow to flow through each focusing channel through a focusing chamber; Image acquisition means for photographing samples flowing in a line in the focusing chamber; And An image controller for analyzing the sample image photographed by the image acquisition means, and transmitting a determination signal regarding the type of the analyzed sample to the pressure control means for the classification channel; The pressure control means for the sorting channel changes the pressure gradient between the sorting channels from time to time according to the discrimination signal regarding the sample type of the image controller so that the analyzed sample flows to different sorting channels according to the kind. Fine particle analysis device characterized in that the classification by type. The method of claim 7, wherein The focusing channel is a fine particle analysis device, characterized in that two arranged symmetrically with respect to the focusing chamber. The method of claim 8, And the same pressure is provided to the two focusing channels by the pressure control means so that both sheath flows introduced into the focusing chamber through the two focusing channels have the same pressure. The method of claim 7, wherein The pressure control means A vacuum pump connected to each channel; A microvalve installed between the vacuum pump and the channel; A pressure sensor attached to each channel; And And a pressure controller for controlling the microvalve according to the signal of the pressure sensor. The method of claim 7, wherein And the sample channel and the waste channel are arranged in a straight line. The method of claim 7, wherein The image acquisition means A laser diode for irradiating a laser to the sample; And And a fine particle analysis device comprising a photodiode for detecting the laser scattered from the sample. A method of analyzing a sample, which is a fine particle flowing along a microchannel formed on a substrate, Forming a pressure gradient between a sample channel and a focusing channel and a waste channel respectively connected to a focusing chamber formed in the substrate; Using a pressure gradient formed in each channel to flow a sample and a sheath flow through a focusing chamber to a waste channel through the sample channel and the focusing channel; Analyzing the samples flowing in a row in the focusing chamber surrounded by sheath flows one by one; And And after the analyzing step, classifying the sample flowing along the waste channel by type through at least two classification channels branched to the waste channel. The method of claim 13, The pressure gradient forming step And symmetrically arranging the two focusing channels about the focusing chamber and providing the same pressure to each of the focusing channels. delete The method of claim 15, The classification step Recognizing the type of sample in the analysis step; And changing the pressure gradient between the classification channels from time to time according to the recognized type of the sample, thereby flowing a sample to each classification channel for each type.