KR100938926B1 - Cell Sorting Apparatus using Ultrasonic Wave - Google Patents

Abstract

The present invention relates to an apparatus for identifying and classifying specific target cells to be classified in a cell by using an ultrasonic beam, wherein the ultrasonic wave is applied to a cell flowing along a channel of a microfluidic flow plate and then detected from the cell. The target cells are identified according to the characteristics of the ultrasonic beam, the target cells flowing through the channel are classified by applying physical force to the target cells, and the abnormal target cells are identified and classified based on the mechanical properties such as the size, shape and structure of the cells. A novel concept of cell identification and classification is disclosed.

Cells, Sorting, Ultrasound, Channels, Microfluidics

Description

Cell sorting apparatus using cell identification by ultrasonic beam {Cell Sorting Apparatus using Ultrasonic Wave}

The present invention relates to an apparatus for identifying and classifying a specific target cell to be classified in a cell using ultrasonic waves, and more particularly, to detect from a cell by applying ultrasonic waves to a cell flowing along a channel of a microfluidic flow plate. The present invention relates to an apparatus for identifying and classifying target cells by analyzing the characteristics of the ultrasound.

Since observing cells through light microscopy, in order to quantitatively analyze and classify cells, "Fluorescence Activated Cell" is a combination of cell staining, optoelectronics, and techniques that allow cells to flow along microtubules. Sorting Apparatus) has been developed. Such a fluoroactive cell sorting device is a "fluidic system" that allows the dyed cells to flow through a predetermined tube, and an "optical system" for observing cells flowing under the control of the cell flow control system. "And an" electronic system "for processing an optical signal of an optical system into an electrical signal.

In recent years, microfluidics is an advanced technology associated with many technologies, such as engineering, physics, chemistry, microtechnology, biotechnology, etc., which deals with the fluid volume in micrometer-scale or nanometer-scale. It was developed and applied to the field of dealing with biological cells. In particular, such microfluidic technology is applied to a device for classifying biological cells contained in a fluid, and the biological cell sorting device is usefully used to accurately classify the biological cells.

Fig. 3 is a block diagram showing a conventional fluorescent active cell sorting apparatus. For example, an argon ion laser 106 that continuously emits parallel light S1 having a wavelength of 480 nm and a light power of 100 mW, and its focus. Focusing lens 107 for focusing parallel light (S1) on the cell, in order to shine the light focused at the focus of the focusing lens 107 to the cells, the cell flow chamber that locates the nozzle at the focus and discharges the cells through the nozzle ( 108, the scattering light condensing lens 110, the scattering light condensing lens 110, the scattered light condensing lens 110 to collect the scattered light (D1) to receive the focused light as the cell passes through the focus of the light detector 111 for detecting the scattered light is converted into an electrical signal ), A fluorescent condenser lens 109 that collects the fluorescence emitted by the cells passing through the focal spot after absorbing the focused light, and a light filter that partially reflects and partially transmits the fluorescence collected by the fluorescent condenser lens 109. 105, reflected from the light filter 105 A first light multiplier 104 for detecting the first reflected fluorescence and a dichroic light filter for receiving a first transmission fluorescence transmitted through the light filter 105 and selectively reflecting and partially transmitting the light according to the wavelength of the fluorescence. (103), a second photomultiplier tube (102) for detecting the second reflection fluorescence reflected from the dichroic light filter (103), and a third photomultiplier tube (2) for detecting the second passage fluorescence transmitted through the dichroic light filter (103). 101, a power supply circuit section (not shown) for supplying power to each element and controlling each element.

Here, the cell flow chamber 108 is a sample fluid tube 108-1 for receiving a sample fluid in which cells are mixed from the outside, and a sheath fluid tube 108- for receiving a sheath fluid from the outside. 2), it consists of a nozzle 108-3 which allows the cells of the cis liquor and the sample liquid to pass through the focus of the focusing lens 107.

The conventional fluorescently active cell sorting device is discharged from the nozzle 108-3, and the cell sorting unit 113 for classifying the target cells according to the optical characteristics of the cells detected by the photodetector 111, and other than the target cells. It further comprises a cell sorting vessel (not shown) containing cells (other cells).

The conventional fluorescently active cell sorting apparatus configured as described above sorts the cells through the following process.

First, as shown in the drawing, in a state in which all the components are aligned, the sample liquid containing the stained cells (target cells) is subjected to a dyeing process with a fluorescent dye in advance, and the sample fluid tube 108-1. Accordingly, the cis liquor, which enters the cell flow chamber 108 and simultaneously flows around the sample liquid, also enters the cell flow chamber 108 along the sheath fluid tube 108-2. Then, the surface liquid and the sheath liquid are discharged to the outside through the nozzle 108-3. The sample liquid flows along the central axis of the nozzle, and the sheath liquid flows around the sample liquid. In other words, the cells surrounded by the sample liquid flow along the nozzles without departing from the central axis of the nozzles. The amount and rate of discharge of the sample and sheath fluids are controlled by the internal pressures of the sample and sheath fluid tubes.

As such, when the sample solution and the sheath solution flow through the nozzle, the size, shape, structure, composition, light scattering properties, and fluorescence emission characteristics of the cells included in the sample solution are analyzed, and the target cells are classified based on the results. To do this, the argon ion laser 106 shines a continuous laser light on the nozzle 108-3 so that cells passing through the nozzle can receive the laser light. Then, the cell material (nucleus) of the cells subjected to the focused argon ion laser light generates scattered light (D1) and emits fluorescence (F1) due to the fluorescent dye stained on the cell material (nucleus). The scattered light D1 enters the photodetector 111 through the scattered light condensing lens 110. At the same time, the fluorescence F1 is collected by the fluorescence condensing lens 109 to become focused fluorescence in the form of a bundle of light.

The focused fluorescence enters the light multipliers 104, 102, and 101 through reflection and transmission in the light filter 105 and the dichroic light filter 103, respectively. Then, the controller (not shown) processes the signals output from the photomultiplier pipe (104, 102, 101), and transmits the target cell classification control signal to the cell sorter 113. The cell sorting unit 113 operates a mechanical operating device (not shown) such as a piston according to the target cell sorting control signal, and is currently discharged from the nozzle 108-3. Sort the cells.

The cell sorting unit 113 is composed of a target cell flow tube and other cell flow tubes connected to the mechanical operating element, the nozzle 108-3. Upon receiving the target cell sorting control signal from the control unit, the cell sorting unit 113 operates a mechanical operating element to operate the target cell to flow from the nozzle 108-3 to the target cell flow tube. On the other hand, if the target cell sorting control signal is not received from the control section, the cell sorting section 113 causes the mechanical operating element to perform the operation opposite to the target cell sorting operation so that the target cells are released from the nozzle 108-3. Acts to flow into other cell flow tubes. Therefore, the cell classification unit 113 classifies target cells and other cells.

In the conventional fluorescently activated cell sorting apparatus, the cell is classified according to the size, shape, or structure of the cell because the cell is identified by the optical signal and the detected optical characteristics.

However, abnormal cells that are sick or have problems with normal cells are not classified by the optical properties such as the size, shape, and structure of the cells, but rather by the characteristics of the cell composition, that is, by the mechanical properties such as the strength, firmness or elasticity of the cells. In many cases, there is a problem in that cell sorting by other factors other than such optical properties cannot be properly performed. Therefore, the cell detection method based on the conventional optical characteristics has a limit in accurately identifying and classifying target cells.

In particular, according to a recent study, cancer cells and normal cells are very similar in shape, and thus have similar optical characteristics, while cancer cells are much softer than normal cells, so they focus on dynamic differences such as elasticity of such membranes. This will suggest ways to easily identify and classify abnormal cells such as cancer cells.

The present invention has been made to solve and supplement the problems of the prior art as described above, it is possible to accurately identify and quickly classify the target cells to be classified accordingly by extracting the mechanical properties of the cells using ultrasonic waves The aim is to provide a new concept of cell sorting.

In addition, an object of the present invention is to propose a method for identifying cells with abnormalities, such as cancer cells, which could not be easily identified by optical properties, using ultrasound.

In addition, the present invention has a similar optical characteristics to normal cells, such as cancer cells, it is difficult to identify the cells using a conventional optical system, focusing on the mechanical differences such as elasticity of cells to effectively identify and classify abnormal cells The purpose is to present a plan.

To this end, the cell sorting apparatus according to the present invention includes a microfluidic flow plate having a channel through which a microfluid including a cell is formed, an ultrasonic output unit for applying an ultrasonic beam to the cell flowing through the channel, and an ultrasonic beam applied to the cell. An ultrasonic detection unit for detecting the scattered ultrasonic beams, a control unit for converting the detection signal detected by the ultrasonic detection unit into an electrical signal, and then identifying whether it is a target cell by comparing with a preset value, and outputting a cell classification control signal accordingly; And a cell classification unit for classifying target cells flowing in a channel according to a classification control signal output from the controller.

In addition, in the present invention, the microfluidic flow plate includes a sample liquid channel through which the sample liquid including cells discharged from the sample liquid reservoir, a cis liquid channel through which the cis liquid discharged from the cis liquid reservoir flows, and the sample liquid channel and the sheath. The main flow channels are combined with the fluid channels and the cis liquor is wrapped around the sample fluid, and other cell flow channels connected to the main flow channels are connected to the main flow channels. And a target cell flow channel branched from the main flow channel so that the target cell flows to the target cell flowing through the flow channel from the cell sorting unit by the force applied from the cell sorting unit.

In addition, the cell sorting unit in the present invention is characterized in that it is composed of any one of mechanical, electrical, optical or thermodynamic elements capable of applying a pressure pulse to the target cell.

According to the present invention as described above, there is an effect that can identify and classify the target cells flowing along the channel of the microfluidic substrate using ultrasonic waves.

In addition, the cell sorting apparatus according to the present invention has an effect that it is possible to provide a new concept of cell identification apparatus that can identify and classify abnormal cells based on mechanical properties such as size, shape and structure of cells.

Hereinafter, a cell sorting apparatus using cell identification by an ultrasonic beam according to the present invention will be described in detail with reference to the accompanying drawings.

1 and 2 is a block diagram of a cell sorting apparatus using cell identification by the ultrasonic beam according to an embodiment of the present invention.

The cell sorting apparatus shown in FIGS. 1 and 2 includes a microfluidic flow plate 20 having a channel through which a microfluid including a cell flows, and an ultrasonic output unit 10 that applies an ultrasonic beam to cells flowing through the channel of the microfluidic flow plate. ), An ultrasonic detector 30 which detects the transmitted and scattered ultrasonic beam after the ultrasonic beam is applied to the cell, and converts the detection signal detected by the ultrasonic detector into an electrical signal and compares the target cell with a preset value. A control unit 40 for identifying a and outputting a cell classification control signal according to the identified data, and a cell classification unit for applying a physical force to the channel so that the target cells flowing in the channel are classified according to the cell classification control signal output from the controller ( 50).

As shown in FIG. 2, the microfluidic flow plate 20 includes a sample liquid channel 24-1 through which a sample liquid including cells discharged from the sample liquid reservoir 21 flows, and a cis liquid reservoir 22, 23. Cis liquor channels 24-2 and 24-3 through which the cis liquor discharged from the same flows, and the sample liquid channel 24-1 and the cis liquor channels 24-2 and 24-3 are connected to each other. Is applied to the main flow channel 24-4 through which the sample liquid and the cis liquid flow, and the target cells flowing through the main flow channel 24-4 in the state where the cis liquor is wrapped. And a target cell flow channel (24-6) through which target cells branch and flow by the losing force, and other cell flow channels (24-7) through which other cells flow.

The ultrasonic output unit 10 is for applying an ultrasonic beam to a channel through which a fluid including a cell flows. The ultrasonic output unit 10 focuses an ultrasonic wave generated by the ultrasonic generator 12 and an ultrasonic wave generated from the ultrasonic generator into a channel through which a cell flows. And a first focusing lens 14 for giving.

The ultrasonic detector 30 is for detecting the characteristics of the ultrasonic beam transmitted from the cell by the ultrasonic beam applied to the cell flowing in the channel, and focuses the ultrasonic beam transmitted through the cell to the second focusing lens 32. And a scattering beam detection unit 38 for focusing and detecting the ultrasonic beam scattered from the cell by the third focusing lens 36.

The ultrasonic detection signals detected by the transmission beam detector 34 and the scattering beam detector 38 are sent to the controller 40, and the controller 40 converts values corresponding to the detection signals detected by the detector into electrical signals. do.

The value converted into the electrical signal by the controller 40 is compared with the electrical signal value of the target cell which is preset and stored, and identifies whether the corresponding cell to which the ultrasound beam is applied is the target cell according to the compared value. In this way, a classification control signal for classifying the target cells according to the value identified by the controller is output and transmitted to the cell classification unit 50.

The cell sorting unit 50 drives the physical force to classify the target cell into the target cell flow channel according to the sorting control signal received from the controller 40, and is selectively driven only when the cell sorting unit is identified as the target cell.

In the present invention, the cell sorting unit 50 for driving the physical force is driven to apply mechanical, electrical, optical, thermodynamic, or other physical force to the target cell, for example, by using a mechanical element such as a piston, or Electromagnetic force or a suitable one among various things such as a piezoelectric element (PZT) may be selected and used. In the preferred embodiment of the present invention, a piezoelectric element (PZT) 52 is used, which will be described below with reference to the piezoelectric element.

Next, the operation of the cell sorting apparatus according to the present invention will be described.

As shown in Figure 2, the sample solution and the cis solution containing the cells are respectively the sample fluid flow channel (24-1) and sheath fluid flow channels (24-2, 24-3) of the microfluidic flow plate 20 It flows along and sums in the main flow channel 24-4. The sample liquid is surrounded by the sheath liquid and flows along the main flow channel 24-4 so that it does not cause friction with the walls of the main flow channel 24-4.

When the sample liquid flowing at the predetermined speed along the main flow channel 24-4 reaches the point where the ultrasonic beam passes, the ultrasonic beam is included in the sample liquid and applied to the cells lined up.

In this case, the part to which the ultrasonic beam is applied may be applied to the branch point 25 where the main flow channel 24-4 is divided into the target cell flow channel 24-6 and the other cell flow channel 24-7, but the controller Since the cells continue to flow while analyzing whether the corresponding cells are the target cells by analyzing the signals detected in the system, it is preferable to install the part where the ultrasonic beam is applied slightly upstream from the branch point in consideration of this. The more precise location of the ultrasonic beam may be determined by considering the time taken by the controller to analyze the detection signal and identifying the target cell and the speed at which the cell flows through the main flow channel.

The transmission beam detection unit 34 collects and detects the ultrasonic beams transmitted through the cells, and the scattering beam detection unit 38 collects and detects the ultrasonic beams scattered and scattered on the cells.

The detection signal detected by the detectors 34 and 38 is sent to the controller 40, and the controller 40 converts the signal detected by the detector into an electrical signal and then compares the converted value with a preset value. Identify whether the cell in response to the signal is the target cell.

When the value obtained by the signal detected by the detector corresponds to the target cell is identified, the controller transmits a control signal to the piezoelectric element 52 for driving a pressure pulse to the target cell in order to classify the target cell. A pressure pulse is applied to the target cell by driving the piezoelectric element 52, whereby the cell is moved to the target cell flow channel 24-6 by the pressure pulse, whereby the target cell is classified, and the target cell is a target cell vessel. It is contained in (27).

On the other hand, if it is identified that the value obtained by the signal detected by the detection unit is not the value corresponding to the target cell, the control unit does not output a control signal for driving the piezoelectric element, and thus no pressure pulse is applied to the cell. The cells flow into the other cell flow channels 24-7 along the main channel flow direction and are contained in the other cell vessel 29.

On the other hand, as the recent research on the mobility and metastasis of cancer cells, the interest in the dynamic reorganization of the cytoskeleton is increasing, according to the physical properties of tissue cells, in particular the elasticity (elasticity) of Changes are emerging as markers for determining cancer.

According to recent studies, metastatic cancer cells are known to reduce stiffness, and thus, a technique for distinguishing metastatic cancer cells from normal cells by measuring the softness of the cells is being studied.

In particular, when diagnosing metastatic cancer, since cancer cells and normal cells look very similar under a microscope, there is a problem that conventional diagnostic methods have a high risk of not being detected even when the cancer cells are actually present in body fluids.

Various methods may be proposed as a method for measuring the elasticity of cancer cells, but the present inventors measure the extent to which ultrasonic waves are transmitted or scattered after applying ultrasonic waves to the cells, thereby determining normal cells and cancer cells according to the measured data. Suggest ways to identify and classify.

When normal cells become cancer cells, the shape of the cells and the internal structure of the cells are changed, and the hard surface becomes smooth. Although the cancer cells are very similar in shape to the normal cells, the elasticity is large, and thus, the cancer cells are detected using ultrasound to measure elasticity. It can be distinguished and classified.

According to an embodiment of the present invention, by applying an ultrasonic beam to the channel through which the cells extracted from the human body flows and detects the ultrasonic beam transmitted through the cells and scattered from the cells to measure the elasticity of the cells.

In the cell sorting apparatus according to the present invention, the criteria of cancer cells and normal cells according to the extent to which ultrasound is transmitted and scattered are preset and stored, and the extent of ultrasound is transmitted and scattered differently according to the degree of elasticity of the cells to be identified. As a result, cancer cells and normal cells are distinguished accordingly.

The value measured by the ultrasonic detection is compared with a preset value to identify whether it is a normal cell or a cancer cell. When the cell to be identified is identified as a cancer cell, a piezoelectric element installed at a branching point branched from the main flow channel is used. By applying pressure pulses to cancer cells, cancer cells can be sorted by moving from the main flow channel to the cancer cell flow channel where only the cancer cells branch.

According to the present invention, it is possible to easily identify whether cancer cells, which are not easily distinguished from normal cells in the past, and whether metastatic cancer cells with high mobility are present in the liquor, as well as sorting cancer cells from normal cells.

In addition, if the present invention is applied, it may be a technology that can be applied to the treatment of cancer by identifying and classifying abnormal cells such as cancer cells rather than normal cells easily.

As described above, the present invention relates to an apparatus for identifying and classifying target cells according to the mechanical properties such as the strength and elasticity of the cells using ultrasonic waves, and is not limited to the above-described embodiments. Various substitutions, changes and variations are possible without departing from the spirit and scope of the invention.

1 is a block diagram of a cell sorting apparatus according to the present invention.

2 is a block diagram of a cell sorting apparatus according to the present invention.

Figure 3 is a block diagram of a conventional fluorescent active cell sorting device.

Description of the main symbols in the drawings

10: ultrasonic output unit

20: microfluidic flow plate

30: ultrasonic detection unit

40: control unit

50: cell sorting unit

Claims (3)

A microfluidic flow plate 20 including a channel through which microfluids including cells are formed; An ultrasonic output unit (10) for applying an ultrasonic beam to the cells flowing in the channel; An ultrasonic detector 20 for detecting the transmitted and scattered ultrasonic beam of the ultrasonic beam applied to the cell; A control unit 40 for converting the detection signal detected by the ultrasonic detection unit into an electrical signal and comparing the preset value with a preset value to identify whether the target cell is a target cell, and output a cell classification control signal accordingly; And And a cell sorting unit (50) for classifying the target cells flowing in the channel according to the sorting control signal outputted from the control unit. According to claim 1, wherein the microfluidic flow plate 20 A sample liquid channel (24-1) through which the sample liquid, including cells discharged from the sample liquid reservoir, flows; Sheath solution channels 24-2 and 24-3 through which the sheath solution discharged from the sheath solution reservoir flows; A main flow channel 24-4 through which the sample liquid and the cis liquid flow in a state in which the sample liquid channel and the cis liquid channel are combined to surround the sample liquid; Other cell flow channels 24-7 connected in series with the main flow channel, A target cell flow channel (24-6) which is connected to the main flow channel and branched from the main flow channel so that the target cell flows with other cells by a force applied from the cell sorting unit to the target cell flowing in the main flow channel. Cell sorting apparatus using cell identification by the ultrasonic beam, comprising a. The method of claim 1 or 2, wherein the cell sorting unit 50 is any one of mechanical, electrical, optical or thermodynamic elements capable of applying pressure pulses to a target cell. Cell sorting device.

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