KR102457508B1 - Device for selective sorting of specimens - Google Patents

Abstract

a substrate support stage for supporting a substrate on which a sample is stacked; a laser light source from which light for separating a target sample from among the samples is extracted; an optical shape modulator for patterning the laser light according to the shape of the separation target region in which the target sample is located; And there is provided an apparatus for selectively separating the sample from the substrate comprising a sample collection stage in which the substrate for receiving the separated target sample is located.

Description

Device for selective sorting of specimens from a substrate

The technology disclosed herein relates to a device for selectively separating a sample from a substrate.

When a high-purity material is needed from a sample containing a biomaterial, a technology for selectively separating it is required. In recent years, separation technology using a laser has been popularized, enabling efficient micro-unit separation even when the amount of material to be separated is limited or visual judgment is required to distinguish the material. However, there are many technical limitations, and in particular, it is difficult to isolate and analyze only cancer cells showing a specific morphology from a genetically heterogeneous cancer tissue slide.

The separation technique using the laser can be divided into a microstructure manipulation technique using light, a microstructure fabrication technique using light, a laser capture microdissection technique, and the like. Among them, microstructure manipulation technology using light corresponds to, for example, a technology that applies physical and chemical changes to microstructures using radiation pressure, thermal expansion, etching, and optical tweezing effects such as lasers and pulsed lasers, Microstructure fabrication technology using light corresponds to a technology that applies physical and chemical changes to a material that responds to light into microstructures, and laser capture microablation technology uses a laser to cut off a sample or substrate stacked on a substrate. It is a separation technique.

According to these separation techniques, a laser is focused on a spot to burn and cut around the material to be separated, and the separation by burning causes chemical transformation of the sample due to thermal damage to the sample. In particular, when separating a bio sample, DNA, RNA, protein, etc. are analyzed after separation, but if there is damage, accurate analysis cannot be performed.

In addition, during the laser micro-cutting process, the substrate on which the sample is fixed is moved and applied with a laser. There is a technique that separates the selected area without moving the plate on which the material is fixed by finely adjusting the path of the laser, but there is a limit to the path deformation, so when separating a large material, the position of the substrate must also be adjusted. At this time, when a laser is applied around a desired target material, an error is easy to occur if a computer-controlled motorized stage is not used, and even if a motorized stage is used, the separation speed is limited according to the speed of the motorized stage. In addition, cumulative errors occur while moving the substrate on which the sample is stacked, and unwanted parts may be separated or burned. Therefore, there is no damage to the target sample, and there is a need for a separation technology in which reliability, accuracy, and speed are secured.

US Patent No. 8722357 (Registration Date 2014/05/13) US Patent Registration No. 8715955 (Registration Date 2014/05/06) US Registered Patent No. 7968819 (Registration Date 2011/06/28)

BioTechniques, Vol. 2, No. 2 (1999) Cellular and Molecular Biology, 44 (1998)

According to one aspect of the technology disclosed herein, the method comprising: providing a substrate on which a sample is laminated; selecting one or more desired separation target regions among regions of the layer of the sample; patterning the energy source to correspond to the area to be separated; and applying the energy extracted from the patterned energy source to the separation target region to separate and recover the target sample corresponding to the separation target region from the substrate. provided

According to another aspect of the technology disclosed herein, the method comprising: providing a substrate on which a biomaterial sample is laminated; selecting one or more desired separation target regions among regions of the layer of the biomaterial sample; extracting light energy from the laser light source; shape-modulating the light extracted from the laser light source to correspond to the shape of the target sample corresponding to the separation target region; separating the target sample corresponding to the separation target area from the substrate by applying the light energy at a time so that the shape-modified light coincides with the separation target area; and recovering the separated target sample to a separate corresponding substrate.

According to another aspect of the technology disclosed herein, a substrate support stage for supporting a substrate on which a sample is laminated; a laser light source from which light for separating a target sample from among the samples is extracted; an optical shape modulator for patterning the laser light according to the shape of the separation target region in which the target sample is located; And there is provided an apparatus for selectively separating the sample from the substrate comprising a sample collection stage in which the substrate for receiving the separated target sample is located.

1 is a process flow diagram illustrating an embodiment of a method for selectively separating a sample from a substrate.
2 is a process schematic diagram showing a method for separating a biomaterial according to an embodiment of the technology disclosed herein.
3 is a process schematic diagram showing a method for separating a biomaterial according to another embodiment of the technology disclosed herein.
4 is a schematic diagram for explaining the role of the sacrificial layer in more detail in the embodiment of FIG. 3 .
5 is a comparison between the conventional laser micro-cutting method (A) and the laser separation method (B) according to an embodiment of the present invention.
6 is an actual image showing the result of removing the sacrificial layer according to the spot size when a rectangular laser beam spot size-controlled by an optical modulator is irradiated on a substrate.
7 shows micrographs before and after separation by laser irradiation of a blood smear (A) and a tissue section (B).
8 is a process schematic diagram showing a method for separating a biomaterial according to another embodiment of the technology disclosed herein.
9 is a diagram illustrating an apparatus for selectively separating a sample from a substrate according to an embodiment of the technology disclosed herein.

Hereinafter, embodiments of the technology disclosed herein will be described in detail with reference to the drawings. The embodiments introduced below are provided so that the disclosed ideas can be sufficiently conveyed to those skilled in the art. Accordingly, the technology disclosed in this specification is not limited to the embodiments described below and may be embodied in other forms. And in the drawings, the width, length, thickness, etc. of the components may be exaggerated for convenience. In the description of the drawings as a whole, descriptions were made from an observer's point of view, and when it is said that one component is on another component, this includes not only the case directly above the other component, but also the case where there is another component in the middle.

1 is a process flow diagram illustrating an embodiment of a method for selectively separating a sample from a substrate.

Referring to FIG. 1 , a substrate on which a sample is laminated is provided in step S110. The sample is not particularly limited thereto, but in relation to the problems described in the background art, the sample may be a bilological material. The biomaterial may be derived from nature or synthesized. Specifically, the biomaterial may be at least one selected from the group consisting of tissues, blood, cells, DNA, RNA, proteins, exosomes, metabolites, and biopsy specimens. Typically, the biomaterial may be a cell or tissue having a size of several um to several hundreds of um.

The biomaterial is provided on a substrate using a technique such as stamping, rolling, smear, capillary, microfluidics, pipetting dispensing, etc. can be

The substrate is not particularly limited as long as it provides a surface for supporting the biomaterial, but is selected from the group consisting of slide glass, microbeads, nanoparticles, nanostructures, capillaries, microfluidic supports, pore structures, sponge structures, and dendrimers. It may be one or more of these, or a substrate whose surface is partially or entirely functionalized with one or more of a chemical functional group, DNA, RNA, and protein. The substrate may be made of glass, silicon, or a polymer material. For example, the substrate may be generally slide glass. Alternatively, the substrate may be a microarray substrate on which a biological sample such as DNA or protein is integrated.

As a specific example, the substrate may be a laser-transmissive substrate, and the laser light applied to the substrate may include pulsed laser light.

The substrate on which the sample is stacked may include a sample on which the sample is stacked on the substrate or, if necessary, even a sample on which a part of the sample is in the substrate.

In step S120, one or more desired separation target regions are selected among regions of the sample layer. The sample may be fixed on a substrate through a covalent bond or adsorption, or may be stacked without being fixed.

The separation target region may be selected in various ways. For example, a desired region may be selected from among the stacked sample regions with information such as image observation, fluorescence signal, or coordinate information.

In step S130, the energy source is patterned to correspond to the region to be separated.

The energy source may include, without limitation, ultrasonic waves, ultraviolet rays, visible rays, infrared rays and electron beams. The energy source may preferably be a laser light source in the ultraviolet region. For patterning the energy source, various modulators such as Light Shape Modulator (LSM), Spatial Light Modulator (SLM), and Acousto-optic Modulator (AOM) may be used. can For example, when using a photoforming modulator, the cross-sectional area of laser light can be enlarged using a lens, and then the pattern can be adjusted using a slit or a micromirror array.

In the energy extracted from the patterned energy source, the cross-sectional area of the original energy path is shape-modulated to correspond to the shape of the target sample corresponding to the separation target region. As a result, a desired target sample can be selected and separated using the shape-transformed energy.

By applying the energy extracted from the patterned energy source to the separation target region in step S140, the target sample corresponding to the separation target region is separated from the substrate and recovered.

In one embodiment, the patterned application of the energy may be performed without relative movement between the position of the substrate and the position to which the energy is applied. That is, for example, for large-area patterning, the energy irradiation range can be expanded by using a lens or the like without using a motorized stage in particular.

In the application of the energy, the wavelength, the magnitude, the number of applications and the application time of the energy may be controlled so that the target sample is not damaged. For example, the target sample can be separated by applying the energy only once.

In one embodiment, the application of the energy may be performed in such a way that the entire uniform energy is transmitted along the outline of the separation target region, so that the separation of the target sample is instantaneously performed in one stroke. The outline of the separation target region may be a closed path surrounding the separation target region. In this case, for example, a target sample can be quickly separated by one energy application (single shot). In particular, when the area to be separated is large, this method is preferable because it can be accurately and quickly separated.

In another embodiment, the application of the energy may be performed on the entire area occupied by the target sample. In this case, since damage may occur when energy is directly applied to the target sample, particularly the biomaterial, it is preferable to interpose the sacrificial layer between the substrate and the sample layer. The material of the sacrificial layer is not particularly limited as long as it is a material that can absorb energy and can be easily separated from the substrate by application of the energy.

As described above, when energy is applied to a sample in a manner according to various embodiments disclosed in the present specification, it is separated from the shape of the target sample at once, so that the probability of damage is reduced. This is different from the cutting method like laser micro-cutting.

Meanwhile, in a preferred embodiment, an additional guide beam guiding the location of the region to be separated and the location of the patterned point to which the energy is applied coincide may be irradiated to the sample layer. Since the energy used for separation of the sample is high, separation with high reliability and precision is possible by examining the irradiation position and shape using a guide beam in advance.

2 is a process schematic diagram showing a method for separating a biomaterial according to an embodiment of the technology disclosed herein. Referring to (A) of FIG. 2 , it can be seen that the laser light extracted from the light source is changed in shape while passing through the optical shape controller, and the shape-modified laser magnified through the lens is applied to the biomaterial. More specifically, referring to FIG. 2 (B), a cross section in the vertical direction to the path of the laser light before patterning (shown in a circle) and a cross section in the vertical direction to the path of the laser light after patterning (closed) shown in the form of a path). In this way, when light is modulated and applied to the substrate according to the shape of the outline (ie, closed path) of the desired region to be separated among regions of the layer of the sample stacked on the substrate, it is accurately irradiated with a single shot. A biomaterial sample having a desired shape may be isolated.

According to the method described above, by applying a patterned laser by selecting only a shape having a closed path defined by the outline of the separation target region, not the entire separation target region, it provides the advantage of selectively separating a target biomaterial sample without damage. can do. Furthermore, when energy is applied, uniform energy is transmitted along the outline of the region to be separated, so that the region to be separated, that is, the target sample is detached at once.

3 is a process schematic diagram showing a method for separating a biomaterial according to another embodiment of the technology disclosed herein. Referring to FIG. 3 , in separating one or more biomaterials of interest, a modified laser light is applied, but the laser is applied within a range corresponding to the inside of the area rather than the outline of the area of the biomaterial of interest. In this case, since thermal damage may occur when a laser is directly applied to the biomaterial as described above, it is preferable to interpose the sacrificial layer between the substrate and the sample layer.

4 is a schematic diagram for explaining the role of the sacrificial layer in more detail in the embodiment of FIG. 3 .

Preferably, the sacrificial layer is made of a controlled material so that sufficient energy can be transferred to the separation target region to be separated from the substrate without causing damage to the separation target region when energy is applied. The sacrificial layer increases separation efficiency by absorbing energy and at the same time reduces the total amount of energy applied to the target sample, thereby minimizing damage to the target sample.

The sacrificial layer may be made of a light-transmitting metal oxide or a light-transmitting plastic material. The sacrificial layer may be glass having increased energy absorption by reducing transmittance or increasing absorbance, or silicon having increased energy absorption by decreasing transmittance or increasing absorbance. In addition, the sacrificial layer may be coated on a solid surface such as glass or silicon, and may exist inside a solid such as glass or silicon, but is not limited thereto. The material constituting the sacrificial layer is preferably a material having no optical distortion so as to easily check whether the laser light is accurately applied to the location of the target sample.

For example, when the target sample is a biomaterial such as a cell, the energy may be an infrared laser to prevent damage from occurring. At this time, it is preferable that the sacrificial layer is evaporated by the infrared wavelength laser while transmitting visible light so as not to interfere with image observation of the biomaterial. The sacrificial layer may preferably be made of a metal oxide. The metal oxide is, for example, indium tin oxide (ITO), indium zinc oxide (IZO), zinc oxide (ZnO), indium zinc tin oxide (IZTO), cadmium tin oxide (CTO) or indium gallium zinc oxide (IGZO). However, the present invention is not limited thereto.

It is preferable to apply the energy directly to the sacrificial layer to prevent damage to the target sample. For example, in the application of the energy, the target sample may be separated from the substrate due to the total evaporation of the sacrificial layer portion as energy having sufficient energy density to evaporate the sacrificial layer portion where the target sample is located is applied. It is preferable that only a portion of the upper portion of the sacrificial layer to which the energy source is applied is evaporated without evaporating the entire located sacrificial layer, and the target sample is deposited on the remaining sacrificial layer and separated from the substrate.

The wavelength band of the applied energy may be an infrared, visible, or ultraviolet region, and a laser pulse may be used. As useful energy, the pulsed laser may have a wavelength of 10 to 10,000 nm, preferably 20 to 5,000 nm, more preferably 100 to 2,000 nm. Since most commercial pulse lasers operate in the above range, it is easy to implement a system and may be suitable for a substrate using a sacrificial layer.

The pulse laser may have a pulse duration of 1as to 1ms, preferably 1fs s to 100nm. When energy is applied by the pulse laser at the pulse irradiation time, the target sample on the substrate may be less damaged.

For example, when the pulse laser is applied to a desired point on the substrate, pulse laser ablation or radiation pressure discharge occurs to evaporate all or an upper layer of the sacrificial layer, and the target sample loaded on the sacrificial layer is combined with or alone with the sacrificial layer. separated from the entire board. As a result, the separated target sample falls in a direction opposite to the direction to which the pulse laser is applied.

Thereafter, analysis may be performed with the separated sample. For example, for gene analysis of specific cancer cells in a tissue section, the entire genome of an isolated sample may be amplified and a sequencing library may be prepared and sequenced using a next generation sequencer. In this way, by selectively isolating a desired sample in the above manner, it is possible to perform genetic analysis of specific cancer cells in a tissue section.

5 is a comparison between the conventional laser micro-cutting method (A) and the laser separation method (B) according to an embodiment of the present invention. Referring to FIG. 5A , a process of separating target cells from a cell population on a substrate such as slide glass or a polymer substrate by a conventional laser microcutting method is shown. In this case, a damaged area inevitably occurs in the process of the laser beam spot burning the target cell to a width equal to the spot diameter. That is, after the target cell is separated by cutting it, the probability that the separated part is a target cell having a damaged area increases. In addition, due to the thickness of the laser spot, a part of unwanted normal cells that are not the target cells also come off, and consequently, it is difficult to separate the pure target cells.

Referring to FIG. 5B , target cells are separated by an indirect laser catapulting method in which a shape-modified laser is irradiated with a sacrificial layer interposed between the substrate and the cells. When a laser is irradiated into the target cell region with the sacrificial layer interposed therebetween, the target cell may come off with evaporation of the sacrificial layer without damage to the target cell region due to the presence of the sacrificial layer. In this case, since the target cells are separated from the cell population at once as the target cells are cut along the cell wall region of the target cells by one application of the laser, other normal cell areas do not mix or include damaged areas.

6 is an actual image showing the result of removing the sacrificial layer according to the spot size when a rectangular laser beam spot size-controlled by an optical modulator is irradiated on a substrate. In FIG. 6 , the left side is the shape of a laser beam spot size-adjusted with an optical modulator, and the right side is a result of irradiating a laser beam after actually laminating a sacrificial layer on the substrate. The lower right is a cross-sectional view taken along the A-B cutting line.

7 shows micrographs before and after separation by laser irradiation of a blood smear (A) and a tissue section (B). In FIG. 7 , each A and B left photo is before separation of the target sample, and the right picture is after separation of the target sample. 7A shows a process in which one target cell in a dotted line region is selectively separated, and FIG. 7B shows a process in which a plurality of desired regions from among tissue sites are separated.

8 is a process schematic diagram showing a method for separating a biomaterial according to another embodiment of the technology disclosed herein. Referring to FIG. 8 , as described above with reference to FIG. 4 , a method in which a laser light is deformed and applied so that it enters within a region of a biomaterial to be separated, except for a region of a biomaterial of non-interest, or a method of applying a shape of a desired region as shown in FIG. 3 . The desired biomaterial is separated by cutting it according to the outline shape. Here, in the present embodiment, a method of using a separate guide beam to check the position and shape of the laser irradiation in advance is shown.

That is, a separate guide beam is placed in the separation target region to match the position of the point where energy is applied, and the guide beam is guided to pass through the optical shape modulator like the laser beam. The guide beam may be irradiated before or during the application of the patterned energy, and it is preferable to have low energy and a low irradiation time so as not to cause damage to the area to be separated.

Referring to FIG. 8 , a half mirror is used to position the laser perpendicular to an optical path through which the laser passes, and a reference light to indicate a portion to be irradiated with the laser is reflected, thereby proceeding in the same path as the laser. At this time, the shape and irradiation position of the laser beam modified through the optical shape modulator can be known in advance through the guide beam. When the projected position of the irradiated guide beam on the substrate and the position of the patterned energy source on the substrate are different from each other by a predetermined distance, the spaced positions may be adjusted from each other. As a result, accurate sample separation is possible.

The recovery of the target sample separated from the substrate may be performed as a process in which the target sample is transferred to a separate corresponding substrate or reservoir as a recovery container. The transfer to the corresponding substrate or storage container is a necessary process to separate the separated target sample and use it for other reactions. The corresponding substrate may have various shapes, and for example, may include a plurality of wells for accommodating a detached target sample. The storage container may include a container manufactured for the purpose of causing or observing a physical or chemical reaction. In addition, the storage container may include a container manufactured for storage of the target sample.

9 is a diagram illustrating an apparatus for selectively separating a sample from a substrate according to an embodiment of the technology disclosed herein. 9, the apparatus 200 for selectively separating a sample from a substrate includes a substrate support stage 210 for supporting a substrate on which a sample is stacked, a sample collection stage 220 for collecting the separated target sample, A laser light source unit 230 from which light is extracted for separating the sample from the substrate, a light shape modulator 240 for patterning the laser light according to the shape of a separation target region in which the target sample is located, and a pattern and a guide light source unit 250 for irradiating the guide beam to the same position on the substrate to which the laser light reaches.

It is preferable that the substrate support stage 210 moves the substrate on which the sample is stacked to the position of the sample to be separated by moving it along the X, Y, and Z axes.

The sample collection stage 220 moves a substrate (eg, a well plate or other storage container, etc.) capable of collecting the separated sample in the X, Y, and Z axes to move it to a position where the laser-separated sample can be collected. It is preferable to do

The laser light source unit 230 irradiates a laser of a band (ultraviolet ray, visible ray, infrared ray, etc.) capable of separating the sample. In this case, it is preferable to control the amount of energy that does not affect the sample while separating it. The laser may be a continuous laser or a pulsed laser. In the case of a pulsed laser, it is preferable to select a pulse length that can apply sufficient energy to separate the sample.

It is preferable to control the shape of the optical shape modulator 240 by matching the passing laser light and the guide beam to the position and size of the target sample. The shape can be adjusted by blocking the direction of the laser light and the guide beam by using a slit, or the shape can be adjusted by reflecting only necessary light using a digital micromirror device (DMD). Lenses can also be used to increase or decrease the shape of the laser light and guide beam. When irradiating the laser light and the guide beam at the same time, it is preferable that the guide beam and the laser light travel in the same direction so that the light of the same shape is overlapped and output.

The guide light source unit 250 emits a guide beam that travels in the same optical path as the laser emitted from the laser light source unit 230 so that the position and size at which the laser is irradiated to the sample can be viewed in real time. For example, by using a half mirror, the laser and the guide beam can be controlled to travel in the same optical path. In this case, it is preferable that the amount of energy applied to the light source is controlled to such an extent that it does not affect the target sample.

As described above, according to the method for selectively separating a sample from the substrate disclosed herein, the biomaterial can be separated by irradiating light only once, so that the separation rate can be remarkably increased. In addition, there is no need for detailed movement of the stage on which the material is fixed, eliminating the need for expensive additional devices such as motorized stages or optical path transforming devices. In addition, since there is no error due to the movement of the motorized stage, the separation accuracy is increased. In addition, there is little damage to the biomaterial by using a sacrificial layer or by separating it at once along the outline of the area to be separated. Moreover, the part to be irradiated with the laser can be checked in advance using an additional guide beam, so the reliability and precision of the separation method are very high.

Since the technology disclosed herein has much higher throughput and separation efficiency than the already commercialized cell separation technology using a laser, it is possible to accelerate related technologies suitable for cancer diagnosis and biomarker discovery.

Although the embodiments of the technology disclosed in the present specification have been described in detail above, those of ordinary skill in the art can apply the technology disclosed in the present specification several times without departing from the spirit and scope of the technology disclosed herein. It will be understood that it can be implemented by transforming it into a branch.

Claims (15)

a substrate support stage for supporting a substrate on which a sample is stacked;
a laser light source from which light for separating a target sample from among the samples is extracted;
an optical shape modulator for patterning the laser light according to the shape of the separation target region in which the target sample is located; and
In an apparatus for selectively separating a sample from a substrate comprising a sample collection stage on which a substrate for receiving the separated target sample is located,
The laser light is an infrared pulse laser light source,
A sacrificial layer is further interposed between the layer of the sample and the substrate,
The sacrificial layer is an apparatus for selectively separating a sample from a substrate made of a translucent metal oxide that is evaporated by the infrared pulse laser light source.
The method of claim 1,
The optical shape modulator selectively separates the sample from the substrate to pattern the laser light according to the shape of the outline of the separation target region.
3. The method of claim 2,
An apparatus for selectively separating a sample from a substrate in which an outline of the separation target region is a closed path surrounding the separation target region.
The method of claim 1,
An apparatus for selectively separating a sample from a substrate, which is performed without relative movement of a position of the substrate and a position to which the patterned laser light is applied.
The method of claim 1,
The patterned laser light is an apparatus for selectively separating a sample from a substrate in which the size of the laser light, the number of times of application, and the application time are controlled so that the target sample is not damaged.
The method of claim 1,
An apparatus for selectively separating a sample from a substrate from which the target sample is detached at once by allowing the patterned laser light to transmit uniform energy as a whole along the outline of the area to be separated.
The method of claim 1,
An apparatus for selectively separating a sample from a substrate further comprising a guide light source for irradiating the guide beam to the same position on the substrate to which the patterned laser light arrives.
8. The method of claim 7,
The apparatus for selectively separating the sample from the substrate, wherein the guide beam is applied before or during the application of the patterned laser light.
The method of claim 1,
The substrate is an apparatus for selectively separating the sample from the substrate which is made of a light-transmitting material.
The method of claim 1,
The sample is a device for selectively separating a sample from a substrate that is a biomaterial.
The method of claim 1,
The laser light source unit and the optical shape modulator are installed on a surface opposite to the surface of the substrate on which the sample is laminated.
The method of claim 1,
Further comprising a sacrificial layer between the sample and the substrate,
wherein the sacrificial layer is made of a controlled material so that sufficient energy to be separated from the substrate can be transmitted to the separation target region without causing damage to the separation target region when the patterned laser light is applied. A device for selectively separating a sample.
13. The method of claim 12,
The sacrificial layer is an apparatus for selectively separating a sample from a substrate made of a light-transmitting metal oxide or a light-transmitting plastic material.
13. The method of claim 12,
A sample from the substrate recovering the target sample corresponding to the separation target area by separating the target sample corresponding to the separation target area from the substrate together with the sacrificial layer by focusing the energy extracted from the patterned laser light on a portion of the sacrificial layer adjacent to the separation target area A device that selectively separates
The method of claim 1,
The sample collection stage is a device for selectively separating a sample from a substrate having a separate corresponding substrate to which the target sample is transferred.

Images