KR20090073650A - Microfluidic device for sorting cells using laser ablation - Google Patents

Abstract

A microfluidic cell sorting apparatus using the laser ablation is provided to allow the target cells to be classified rapidly and precisely. A microfluidic cell sorting apparatus using the laser ablation comprises a microfluid flowing plate(20) where the sample solution containing cell and seath fluid flow; a detection laser(28) which outputs the detection laser beam; a first collection lens(29) which collects the detection laser beam to the microfluid flowing plate; a condensing lens(31) which condenses the laser beam irradiated on the cell flowing along the microfluid flowing plate; an optical detector(30) which detects the light of the collection lens to convert it into the electric signal; a first high power pulse laser(50) which outputs the pulse laser beam of high power to the microfluid flowing plate; a second collection lens(51) which collects the pulse laser beam of high power to the microfluid flowing plate; and a circuit part which receives the detection signal outputted from the optical detector to output the target cells classification control signal. The first high power pulse laser outputs the pulse laser beam of high power to the microfluid flowing plate according to the target cells classification control signal to induce the laser ablation, and applies the pressure pulse formed to the presently detected target cells(25).

Description

Microfluidic cell sorting apparatus using laser ablation {MICROFLUIDIC DEVICE FOR SORTING CELLS USING LASER ABLATION}

The present invention relates to a cell sorting technique, and more particularly, a microfluid that detects target cells flowing along a channel of a microfluidic flow plate, and classifies the detected target cells by using laser ablation. It relates to a cell sorting device.

Since observing cells through an optical microscope, "Fluorescence Activated" is a combination of cell staining, optoelectronics, and techniques that flow cells along microtubules for quantitative analysis and sorting of cells. Cell Sorting Apparatus). 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.

1 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 discharge volume and velocity of the sample and sheath liquids 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 characteristics, and fluorescence emission characteristics of the cells included in the sample solution are analyzed, and the target cells are analyzed based on the results. To sort, the argon ion laser 106 shines a continuous laser light onto 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 unit, the cell sorting unit 113 causes the mechanical operating element to perform the operation opposite to the target cell sorting operation so that the target cell is 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.

However, in the conventional fluorescence activated cell sorting device, a mechanical operating element, such as a piston, included in the cell sorting unit 113 generates a friction in a channel associated with the cell flow pipe, thereby applying a pressure pulse to a target cell flowing along the nozzle and Since the target cells are classified accordingly, the target cells cannot be classified accurately and quickly, and the mechanical life of the cell flow tube is shortened due to mechanical friction. In addition, the energy utilization efficiency is low because the cells must be continuously irradiated with high power laser light whether the cells pass through the nozzle or not. In addition, because the argon ion laser and its accompanying peripheral devices are large, heavy, low in mobility, and very expensive, the fluorescence activated cell sorter is also large, heavy, occupies a large space, and has low mobility and high cost. Therefore, there is a problem that is not widely spread.

An object of the present invention is to provide a microfluidic cell sorting apparatus for detecting target cells flowing along a channel of a microfluidic substrate, and classifying the detected target cells accurately and quickly using laser ablation.

To this end, the microfluidic cell sorting apparatus according to the present invention includes a microfluidic flow plate through which a sample liquid containing a cell and a cis liquid flow; A detection laser outputting a detection laser beam; A first focusing lens for collecting the detection laser beam on the microfluidic flow plate; A condenser lens for collecting a laser beam reflected by the cells flowing along the microfluidic flow plate; A photo detector for detecting light from the condenser lens and changing an electrical signal; A first high power pulse laser outputting a high power pulse laser beam to the microfluidic flow plate; A second focusing lens collecting the high power pulsed laser beam on the microfluidic flow plate; And a circuit unit which receives the detection signal output from the photodetector and outputs a target cell classification control signal.

Here, the first high power pulse laser outputs a high power pulse laser beam to the microfluidic flow plate in accordance with a target cell classification control signal output from the circuit unit to cause laser ablation, and thus target pressure pulses formed according to the current detected target. Added to the cells.

The microfluidic cell sorting apparatus according to the present invention can classify the target cells flowing along the channels of the microfluidic substrate by applying a pressure pulse to them, using laser ablation.

In addition, since the microfluidic cell sorting apparatus according to the present invention has relatively little failure, its lifespan is semi-permanent and accordingly, maintenance costs are low.

An embodiment of a microfluidic cell sorting apparatus using laser ablation according to the present invention will be described in detail with reference to the accompanying drawings.

2 is a plan view of a microfluidic cell sorting apparatus using laser ablation according to the present invention, which includes a microfluidic flow plate 20 through which a sample liquid containing a cell and a cis liquid flow, and a detection laser outputting a detection laser beam ( 28), the first focusing lens 29 for collecting the laser beam on the microfluidic flow plate 20, the condenser lens 31 for collecting the laser beam reflected on the cells flowing along the microfluidic flow plate 20, condensing lens ( 31, a photo detector 30 for detecting light of the light and changing an electric signal, a circuit unit receiving the detection signal output from the photo detector 30 and outputting a target cell classification control signal, based on the target cell classification control signal output from the circuit unit. In order to apply a pressure pulse to the target cells 25 flowing along the microfluidic flow plate 20, a high power pulsed laser 50 and a high power pulse forming a laser ablation to the microfluidic flow plate 20. Laser (5 And a second focusing lens 51 for collecting the pulsed laser beam of 0) on the microfluidic flow plate 20.

The microfluidic flow plate 20 includes a sample liquid channel 24-1 through which the sample liquid containing cells flows from the sample liquid reservoir 21, and a sheath through which the sheath liquid discharged from the cis liquid reservoirs 22 and 23 flows. Sample fluid with the fluid channels 24-2 and 24-3, the sample fluid channel 24-1 and the sheath fluid channels 24-2 and 24-3 connected, and the sheath solution wrapped around the sample fluid. Is connected to the main channel 24-4 and the main channel 24-4 through which the cis liquor flows, and receives a pulsed laser beam of the high power pulse laser 50 to be applied to a target cell flowing in the main channel 24-4. The impact output from the laser ablation forming unit 24-5 by the laser ablation forming unit 24-5 in which the laser ablation is formed, and by the laser ablation formed in the laser ablation forming unit 24-5. A target cell flow channel (24-6) through which target cells subjected to pulses (pressure pulses) flow, and other cell flow channels (24-7) through which other cells flow. The. Also included are target cell vessels 33 containing target cells, and other cell vessels 34 containing cells other than target cells (other cells).

Figure 3 is a perspective view of a microfluidic cell sorting apparatus using laser ablation according to the present invention.

As shown in the figure, the microfluidic flow plate 20 is preferably arranged to be positioned between the laser 28 and the photodetector 30. In addition, the detection laser beam from the detection laser 28 is connected to the main channel 24-4, the laser ablation forming part 24-5, the target cell flow channel 24-6, and other cell flow channels 24-. It is preferable to align the microfluidic flow plate 20, the detection laser 28, and the photodetector 30 so as to cross the point where 7) meets. A better alignment method is the point where the detection laser beam from the detection laser 28 meets the main channel 24-4 and the laser ablation formation portion 24-5 if the direction in which the sample liquid flows is set upstream. It is better to align it past the point slightly upstream. This is because the detected cells move downstream at the flow rate of the sample liquid while the circuit section, the photodetector and the detection section determine whether the cells detected from the detection light are target cells.

On the other hand, as shown by the portion A in the drawing, the high output pulse laser 50 and the focusing lens 51 have a high output pulse laser beam 52 with the laser ablation forming portion 24-of the microfluidic flow plate 20. 5) are arranged to gather. In the embodiment of the present invention, the size of the laser beam focus is about 10 microns and the depth of focus is about 20 microns.

A black metal piece 60 is further attached to the laser ablation formation 24-5 so that the high power pulsed laser beam 52 can produce laser ablation more efficiently.

Figure 4 is a schematic diagram of the circuit portion of the microfluidic cell sorting apparatus using laser ablation according to the present invention.

As shown in the figure, the microfluidic cell sorting apparatus includes a detection laser driver 41 for driving the detection laser 28, a detection unit 42 for analyzing the electrical signal output from the photodetector 30, and a high output pulse laser 50. To control the high output pulse laser driver 43 and the detection laser driver 41 driving the high output pulse laser driver 43 according to the signal output from the detector 42. The control part 40 consists of the power supply part 44 which supplies power to each element.

As described above, the operation of the microfluidic cell sorting apparatus according to the present invention made as described above is as follows.

5 and 6 is a block diagram showing the operation of classifying the target cells and other cells other than the target cells in the microfluidic cell sorting apparatus using the laser ablation according to the present invention.

With the microfluidic cell sorting device aligned as shown in Figs. It flows along flow channels 24-2 and 24-3 and sums in 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 detection laser beam passes, the detection laser beam is included in the sample liquid and shined on the cells lined up. The photodetector 30 collects the detection laser beam reflected on the cell and outputs the detected laser beam to the detection unit 42.

The detector 42 compares an electric signal value corresponding to the optical signal detected by the photodetector 30 with an electric signal value of a target cell set in advance, and outputs the compared value to the controller 40. If the control unit 40 determines that the comparison value received from the detection unit 42 is an electric signal value corresponding to the target cell, the control unit 40 outputs the target cell classification control signal to the high output pulse laser driver 43 to classify the currently detected cells. do. The high power pulse laser driver 43 transmits the target cell classification driving signal to the high power pulse laser 50 in accordance with the target cell classification control signal received from the control unit 40.

As shown in FIG. 5, the high output pulse laser 50 that receives the drive signal from the high output pulse laser driver 43 has a high output pulse laser beam at the laser ablation forming part 24-5 of the microfluidic flow plate 20. Shine the light. Then, the sheath liquid staying in the laser ablation forming portion 24-5 absorbs the energy of the high power laser beam and suddenly expands its volume. The volume of the sheath liquid thus suddenly expanded forms a pressure pulse (shock pulse). The pressure pulse is applied to the currently detected target cells 25 so that the target cells 25 move to the target cell flow channels 24-6. Thus, the target cells 25 that flow along the target cell flow channel 24-6 are stored in the target cell container 33. Here, the black metal piece 60 allows the high power pulsed laser beam to absorb energy more efficiently into the sheath solution in the first laser ablation formation 24-5.

On the other hand, when the optical signal detected by the photodetector 30 is not the corresponding optical signal of the target cell, the controller 40 does not output any control signal to the high power laser driver 50. Then, as shown in FIG. 6, the cis-liquid containing the currently detected cells is not affected by the pressure pulses from the laser ablation forming unit 24-5, so that other cell flows formed in the direction of the main channel in a straight line are formed. Flows straight into channels 24-7. That is, the other cells contained in the cis liquor maintain the flow direction of the main channel and move to the other cell flow channels 24-7 and then stored in the other cell vessel 34.

Therefore, the microfluidic cell sorting apparatus according to the present invention accurately and quickly because it sorts the target cells and other cells included in the sample solution by applying a pressure pulse to the cells flowing along the channel of the microfluidic substrate using laser ablation. Can be classified.

In the embodiment of the present invention, the manufacturing method of the microfluidic flow plate is not described in detail, but the microfluidic flow plate is formed of a bottom substrate and a cover substrate, and after the square or circular channels are formed on the bottom substrate, It is manufactured by attaching a cover plate. In addition, the microfluidic flow plate is made of a thin silicon material, a glass material, or a polymer material. In addition, the main channel 24-4 and the first laser ablation formation part 24-5 may be blocked with a thin film.

The laser according to the present invention was implemented as a semiconductor laser.

On the other hand, although the embodiment of the microfluidic cell sorting apparatus using the laser ablation according to the present invention has been implemented with a structure having only one laser ablation forming unit, as another embodiment, the laser ablation currently implemented around the main flow channel The second laser ablation forming portion may be further formed at a symmetrical position with respect to the forming portion. In this case, a second high power pulse laser and a second high power pulse laser driver are further provided. In the operation method, the first high power pulse laser is operated when the currently detected cell is the target cell, and the second high power pulse laser is operated when the currently detected cell is the other cell.

In this case, a second high output pulse laser for outputting a high output pulsed laser beam to the second laser ablation forming unit, and a second high output pulse laser beam for collecting the high output pulsed laser beam of the second high output pulsed laser to the second laser ablation forming unit. And a third focusing lens, wherein the first high power pulsed laser causes laser ablation to the first laser ablation forming portion to apply pulse pressure to the target cells, and the second high power pulsed laser pulses to other target cells. Cells are sorted by causing laser ablation to the second laser ablation forming portion to apply pressure. Preferably, the second laser ablation forming portion further includes a black metal piece.

In another embodiment, laser ablation formation 24-5, target cell flow channels 24-6, and other cell flow channels 24-7 form stages in the microfluidic flow plate. It can also be implemented to classify the target cells corresponding to the number of stages.

The embodiment according to the present invention has been described with reference to the drawings. However, the present invention is not limited to the above-described embodiments, and various modifications can be made by those skilled in the art without departing from the gist of the present invention appended to the claims.

1 is a block diagram of a conventional fluorescent active cell sorting apparatus.

Figure 2 is a plan view of a microfluidic cell sorting apparatus using laser ablation according to the present invention.

Figure 3 is a perspective view of a microfluidic cell sorting apparatus using laser ablation according to the present invention.

Figure 4 is a schematic diagram of the circuit portion of the microfluidic cell sorting apparatus using laser ablation according to the present invention.

5 is a block diagram showing an operation of classifying a target cell by the microfluidic cell sorting apparatus using laser ablation according to the present invention.

Figure 6 is a block diagram showing the operation of classifying other cells other than the target cell in the microfluidic cell sorting apparatus using the laser ablation according to the present invention.

Description of the main symbols in the drawings

33, 34, 136-1, 136-2: Cell sorting vessel

20: microfluidic flow plate

21: sample reservoir

22, 23: sheath solution reservoir

24-1: sample channel

24-2, 24-3: sheath solution channel

24-4: main flow channel

24-5: Laser Ablation Formation

24-6: target cell flow channel

24-7: other cell flow channels

25, 124: target cell

28: detection laser

29, 51, 107: focusing lens

30, 111: photodetector

31: condenser lens

50: high power pulse laser

101, 102, 104: photomultiplier

103: dichroic beam-splitter

105: beam-splitter

106: Argon ion laser system

108: flow chamber

108-1: sample fluid pipe

108-2: sheath fluid pipe

108-3: nozzle

123: other cells

109: fluorescent light collecting lens

110: scattered light collecting lens

113: cell sorting unit

Claims (8)

A microfluidic fluid flow plate 20 through which a sample solution including a cell and a cis solution flow; A detection laser 28 for outputting a detection laser beam; A first focusing lens 29 for collecting the detection laser beam on the microfluidic flow plate 20; A condenser lens 31 collecting the laser beams reflected on the cells flowing along the microfluidic flow plate 20; A photo detector (30) for detecting the light of the condenser lens (31) to change an electrical signal; A first high power pulse laser (50) for outputting a high power pulse laser beam to the microfluidic flow plate (20); A second focusing lens 51 collecting the high power pulsed laser beam on the microfluidic flow plate 20; And A circuit unit which receives a detection signal output from the photodetector 30 and outputs a target cell classification control signal, Here, the first high power pulsed laser 50 outputs a high power pulsed laser beam to the microfluidic flow plate 20 according to a target cell classification control signal output from the circuit unit to cause laser ablation. Microfluidic cell sorting apparatus using laser ablation, characterized in that for applying the pressure pulse formed according to the target cell 25 currently detected. The method of claim 1, wherein the microfluidic flow plate A sample liquid channel 24-1 through which a sample liquid containing cells flows from the sample liquid reservoir 21; Sheath liquor channels 24-2 and 24-3 through which sheath liquor exits the sheath liquor reservoirs 22 and 23; The main flow channel (24-), which is connected to the sample liquid channel (24-1) and the sheath liquid channels (24-2, 24-3) and flows the sample liquid and the cis liquid, with the sheath liquid wrapped around the sample liquid. 4); A first laser ablation formation unit 24-5 connected to the main channel 24-4 and to be applied with a target cell flowing through the main channel 24-4, through which the pressure pulse is emitted; A target cell flow channel 24-6 through which target cells receiving the pressure pulse flow; And Microfluidic cell sorting apparatus using laser ablation, characterized in that it comprises other cell flow channels (24-7) through which other cells other than the target cells flow. The microfluidic cell sorting apparatus using laser ablation according to claim 2, further comprising a target cell container containing the target cells and another cell container containing other cells. 3. The microfluidic cell sorting device using laser ablation according to claim 2, wherein the first laser ablation forming part further comprises a black metal piece. The circuit circuit according to any one of claims 1 to 4, wherein the circuit unit A detection laser driver 41 for driving the detection laser; A detector 42 for analyzing the electrical signal output from the photodetector 30; A first high power pulse laser driver 43 for driving the first high power pulse laser 50; The controller 40 controls the detection laser driver 41 and controls the first high power pulse laser driver 43 according to the signal output from the detector 42 to operate the first high power pulse laser 50. ; And Microfluidic cell sorting apparatus using laser ablation, characterized in that it comprises a power supply unit 44 for supplying power to each element. The microfluidic flow plate of claim 2, wherein the microfluidic flow plate further includes a second laser ablation formation portion at a position symmetrical with the first laser ablation formation portion 24-5 about the main channel 24-4. Microfluidic cell sorting apparatus using a laser ablation, characterized in that provided. The method of claim 6, A second high power pulse laser outputting a high power pulse laser beam to the second laser ablation forming unit; And The second laser ablation forming unit further includes a third focusing lens for collecting the high power pulsed laser beam of the second high power pulsed laser, The first high power pulsed laser causes laser ablation to the first laser ablation forming portion to apply pulse pressure to the target cell, and the second high power pulsed laser to generate a second laser ablation to apply pulse pressure to other target cells. Microfluidic cell sorting apparatus using laser ablation, characterized in that it causes laser ablation. The microfluidic cell sorting apparatus using laser ablation according to claim 6 or 7, wherein the second laser ablation forming unit further comprises a black metal piece.

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