JP4093740B2 - Fine particle sorting microchip and fine particle sorting device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The invention of this application relates to a fine particle sorting chip. More specifically, the invention of this application relates to a microchip for separating fine particles and cells, a fine particle sorting apparatus incorporating the microchip, and a method for sorting fine particles and cells.
[0002]
[Prior art and its problems]
With the development of microfabrication technology in the semiconductor industry, microchips fabricated on silicon and glass substrates have come to be widely used in analytical instruments and the like. Specifically, various microchips having electrodes, flow paths, and the like formed on a silicon or glass substrate are used in electrochemical detectors for liquid chromatography, small-sized electrochemical sensors in medical fields, and the like.
[0003]
In recent years, the production and development of micro-total-analysis systems (μ-TAS) and microreactors aimed at increasing the speed, efficiency and integration of chemical experiments, or ultra-miniaturization of analytical instruments have attracted attention. The world is actively researching.
[0004]
These μ-TASs are superior to conventional devices in that they can be measured and analyzed with a small amount of sample, can be carried, can be realized at low cost, and can be disposable. In particular, it has attracted attention as a method that is highly useful in experimental systems using expensive reagents and in screening operations for biochemical substances in small quantities and many samples.
[0005]
In addition, μ-TAS enables the elucidation of specific molecular behavior in a minute space, separation and analysis of single molecules, single particles, or single cells by applying ultrasensitive detection and ultratrace analysis. Expected to be.
[0006]
On the other hand, with the enormous amount of gene information revealed by the Human Genome Project, the importance of mass-product screening technology in the pharmaceutical field such as genome drug discovery is increasing. For example, when evaluating the effect of a specific substance on human cells, create a cell culture cell library for each organ, isolate and purify these cultured cells to control quality, or sort each cell after substance administration. Observe the state.
[0007]
Conventionally, a cell or fine particle sorter such as a cell sorter or a particle analyzer has been used for such cell sorting. However, since the conventional cell sorter is large and expensive, the situation is that it cannot be easily used in a laboratory or a medical field. In addition, since cells are sorted based on indirect information such as scattered light and fluorescence, there is a problem that the operation requires skill and low versatility. Furthermore, since the cell sorter needs to make the sorted cells into droplets, there is a problem that the size and type of cells that can be sorted are limited. On the other hand, a particle analyzer is known as a device for separating various fine particles such as powder and cells according to size and shape, but this is also based on indirect measurement information, which is expensive. Therefore, there were problems such as low versatility.
[0008]
Therefore, in recent years, an inexpensive cell sorter for separating fine particles in a laminar flow while directly observing them with a microscope has been proposed (Micro Total Analysis '98, pp. 77-80 (Kluwer Academic Publishers, 1998); Analytical Chemistry, 70 , pp.1909-1915 (1998)), however, only the elemental technology of the separation part has actually been developed, and it has not yet been put into practical use.
[0009]
The invention of this application has been made in view of the circumstances as described above, eliminates the problems of the prior art, easily and accurately separates fine particles such as cells, and inexpensive and highly versatile fine particles. It is an object to provide a sorting device.
[0010]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the invention of this application is first a flow-type fine particle sorting microchip having a fine flow path on a substrate, and at least fine particles on the substrate. A contained solution introduction channel, a sheath flow forming solution channel disposed on both sides of the introduction channel, a single channel into which the microparticle-containing solution and the sheath flow forming solution flow, and the one channel provided, the particulate measurement site for measuring the introduced fine particles, is installed downstream of those fine particle measurement site, is installed fine particles separate collection first fine particle separation flow path for the on both sides a second particle separation flow path, the first and particulate separation flow path so as to sandwich the first fine particle separation flow path is disposed between the second particle separation flow path, from the particles measurement region to particulate separation flow path braking the movement direction of the installed particles near the inflow opening of the A pair of electrodes for, have, electrodes have a sharp shape, provides a particulate fractionation microchip, characterized in that the acute angle portion is placed in the inlet near the mouth flow.
[0013]
The invention of this application, the second, fine particle-containing solution introduction channel, a sheath flow forming solution flow path, fine measurement region, and the inner wall of the particulate separation flow path, the one that is coated with particulate anti-adhesion layer A fine particle sorting microchip is provided.
[0014]
Further, the third invention of this application, microparticles in the microparticle-containing solution is a cell, any one of the particulate fractionation microchip cells are those selected from the group consisting of microorganisms, and polymer microparticles I will provide a.
[0015]
The invention of this application, the fourth, a particulate fractionating apparatus for fractionating particles, optical at least the one of the particulate fractionation microchip above, the fine particles in the fine particle measuring site on the particle fractionation microchip A measuring means for measuring automatically or electromagnetically, a voltage applying means for applying a voltage to the electrodes on the fine particle sorting microchip, and an output means for outputting optical or electrical information of the fine particles. Provided is a fine particle sorting apparatus.
[0016]
The invention of this application also provides, in a fifth aspect , the fine particle sorting apparatus in which the measuring means is an optical microscope, and in the sixth , any one of the fine particle sorting apparatuses in which the measuring means is a fluorescent microscope. .
[0017]
Furthermore, the seventh invention of the present application, the use of any particulate separation device, to provide a microparticle fractionation method for fractionating particulates.
[0020]
DETAILED DESCRIPTION OF THE INVENTION
The invention of this application relates to a flow type fine particle sorting microchip having a fine channel on a substrate. FIG. 1 shows an outline of the microparticle sorting microchip. The microparticle separation microchip of the invention of this application is formed on a substrate (1) with at least (a) a microparticle-containing solution introduction channel (2) and (b) a sheath flow disposed at the side of the channel. A solution flow path (3), (c) a fine particle measurement portion (4) for measuring the introduced fine particles, and (d) a separate collection of fine particles placed downstream of the fine particle measurement portion (4). the fine particle separation flow path (5), for controlling the movement direction of microparticles installed near (e) inflow port from the microparticle measurement region (4) particle separation flow path (5) (51) It has a pair of electrodes (6). At this time, the substrate (1) may be any substrate as long as it can process and install a fine flow path and electrodes by various fine processing techniques, and can be selected from various materials such as silicon, glass, quartz, and plastic. For example, when fine particles are measured by optical means, a material that does not absorb in the wavelength region of the light source to be used is selected. The size of the substrate (1) is not particularly limited, and at least the size of the components (a) to (e) can be finely processed according to the size and type of fine particles such as cells to be sorted. If it is.
[0021]
In the microparticle sorting microchip of the invention of this application, the microparticle-containing solution introduction channel (2) is a channel for introducing a solution containing sample microparticles (7) such as cultured cells, blood cells, and polymer powder. Further, upstream thereof, a connector portion for connecting to a liquid feeding means such as a tank or a pump for storing the fine particle-containing solution may be provided. In such a microchip, the inner diameter of the fine particle-containing solution introduction channel (2) is not particularly limited. It is sufficient if it has a diameter capable of stably introducing the sample fine particles (7). For example, the width can be about 1 to 100 μm. The shape is not particularly limited, but a rectangular shape is preferable because it can be easily formed by a known semiconductor microfabrication technique and the like, and image distortion does not occur when performing microscopic observation described later.
[0022]
The sheath flow forming solution channel (3) forms a flow (sheath flow) that wraps both sides of the sample fine particles (7) when the sample fine particles (7) are introduced into the fine particle measurement site (4). a flow path for introducing the liquid (sheath fluid) for, may be disposed on the side of both the fine particle-containing solution introduction channel (2). The inner diameter of the sheath flow forming solution channel (3) is not particularly limited, but can be, for example, about 1 to 100 μm in width. The diameters of the fine particle-containing solution introduction channel (2) and the sheath flow forming solution channel (3) are designed so that a sheath flow is formed, or the flow rate of the fine particle-containing solution and the flow rate of the sheath solution are adjusted.
[0023]
In the fine particle sorting microchip of the invention of this application, the fine particle measurement portion (4) is a portion for optically or electromagnetically measuring the fine particles. Specifically, it is a part for irradiating the sample fine particle (7) with light in a specific wavelength region from the microscopic optical measurement system and observing the substantial image and the fluorescent image of the sample fine particle (7). Or it is good also as a site | part for observing the moving speed and migration speed of sample microparticles | fine-particles (7) with a CCD camera etc. FIG. Further, two or more sets of microelectrodes may be incorporated in the fine particle measurement site (4), an alternating voltage as low as about 100 mV may be applied between the electrodes, and the impedance flowing between the electrodes may be measured. In such a particle measurement site (4), each solution flows from the particle-containing solution introduction channel (2) and the sheath flow forming solution channel (3), and sample particles are introduced. Therefore, the fine particle measurement site (4) is desirably a single flow channel where the fine particle-containing solution introduction flow channel (2) and the sheath flow forming solution flow channel (3) are joined. It is preferable that the diameter is larger, for example, a width of about 5 to 150 μm. The height of each channel is not particularly limited. However, the distance (thickness) from the bottom surface or the top surface of the chip to the flow path is as thin as about 50 to 500 μm due to the relationship with the focal length of the lens when observing fine particles or intracellular microstructure with a microscope. There is a need to.
[0024]
Furthermore, particulate fractionation microchip of the invention of this application state, and are not having the fine particle separation flow path for separate collection installation particulate downstream of the particulate measurement region (4) (5), Figure 1 As shown in FIG. 2, the first fine particle sorting channel (5b) and the second fine particle sorting channels (5a, 5c) installed on both sides thereof are provided at the center. The fine particle sorting channel (5) may include one or more channels for waste liquid.
[0025]
The fine particle-containing solution introduction flow path (2), the sheath flow forming solution flow path (3), the fine particle measurement site (4), and the fine particle sorting flow path (5) as described above can be used for the substrate (1) by photolithography or the like. Although it may be used as processed by semiconductor microfabrication technology, some sample particles are likely to adhere to the inner walls of these channels, and there is a large difference in the movement speed of the particles between the walls and the center of the channels. Since some of them occur, it is preferable to form a fine particle adhesion preventing layer. Specifically, a fine particle adhesion preventing layer can be formed by coating a polymer such as gelatin or polyethylene glycol.
[0026]
Particulate fractionation microchip of the invention of this application also a pair of fine particles measuring sites (4) particle separation flow path (5) inflow port to the (51) for controlling the movement direction of the installed particles near The electrode (6) is provided. Such electrode (6) gives rise to an electric field in the vicinity of the inflow port (51) by applying a voltage, controls the flow of particulates into the inflow port by the interaction (51) between the electric field and the fine particles Is to do. Electrode (6) is not particularly limited size, shape may or concentrate the electric field near the inflow opening (51), so that the gradient of the inhomogeneous electric field can or maximized, as shown in FIG. 1 It shall have an acute angle shape. The electrode (6) material is appropriately selected from various common electrode materials such as gold, silver, platinum, and Al. The number of electrodes (6) needs at least one pair (two) to generate an electric field, but can be increased according to the number of fine particle sorting channels (5). By applying a voltage to the electrodes (6), as can be individually controlled, it can be respectively controlled variable-not inflow into the inflow port (51).
[0027]
The fine particle sorting microchip as described above may be manufactured by any method, but can be easily manufactured by various known microfabrication techniques including semiconductor lithography and etching.
[0028]
The invention of this application further provides a fine particle sorting apparatus for sorting fine particles. Such a particulate separation device is at least
(I) the fine particle sorting microchip,
(Ii) a measurement means for optically or electromagnetically measuring fine particles at a fine particle measurement site on the fine particle sorting microchip;
(Iii) voltage application means for applying a voltage to the electrode on the microparticle sorting microchip;
(Iv) It has an output means for outputting optical or electrical information of fine particles.
[0029]
In the fine particle sorting apparatus of the invention of this application, examples of the measuring means for optically or electromagnetically measuring fine particles at the fine particle measuring portion on the fine particle sorting microchip include the above-described microscopic optical measurement system and CCD camera. For example, when a microscopic optical measurement system is used, the sample fine particles (7) can be irradiated with light in a specific wavelength region, and a solid image of the sample fine particles (7) can be observed at the same wavelength as the irradiated light. Further, the fluorescent image of the sample fine particle (7) may be observed in a longer wavelength region, and it may have a function for comparing the substantial image and the fluorescent image or an image processing function. On the other hand, if a CCD camera is used as the measuring means, the moving speed and migration speed of the sample fine particles (7) can be observed. Thereby, the influence on the migration speed of the sample fine particles (7) due to voltage application can be measured, and the separation timing can be determined. Of course, the measuring means may be other than these, for example, means for irradiating a semiconductor laser and measuring passing light with a photodiode.
[0030]
In the fine particle sorting apparatus of the invention of this application, the voltage applying means for applying a voltage to the electrode on the fine particle sorting microchip may be any device that can apply a DC / AC voltage. By applying a voltage in the vicinity of the inflow port (51), when the electric field is generated, the charge by the dielectric polarization to the microparticle surface appears, particles are driven. Since the magnitude of the generated electric field varies depending on the electrode shape, a shape that maximizes the gradient of the non-uniform electric field may be selected as described above. In addition, the same phenomenon can be seen whether the voltage is direct current or alternating current. However, in the case of direct current voltage, superposition of dielectrophoresis and electrophoresis is observed, whereas in alternating voltage, the electrophoretic force is averaged due to the reversal of the electric field polarity. Therefore, only dielectrophoresis independent of the charge of the fine particles is observed. Therefore, it is preferable to apply an alternating voltage. Furthermore, such a voltage may be provided with a mechanism capable of controlling the on / off timing based on information on the fine particles selected by the measuring means, and the voltage application may be controlled.
[0031]
As the output means for outputting optical or electrical information of the fine particles, as described above, software or a monitor for imaging and analyzing a solid image or a fluorescent image by microscopic observation, or sample fine particles by applying voltage Examples include a monitor for observing the state of electrophoresis in (7). Furthermore, a frequency response measuring device (impedance analyzer), an ion sensitive field effect transistor (ISFET), a voltmeter, an ammeter, etc. are illustrated.
[0032]
The fine particle sorting apparatus as described above may have at least the above (i) to (iv), but in addition, a storage tank for storing the fine particle-containing solution and a sheath solution for storing the sheath solution. You may have a storage tank and also the liquid feeding means for introduce | transducing these solutions into a microparticle separation microchip. Moreover, you may have a collection tank, a waste liquid tank, etc. for collect | recovering the classified fine particles. Furthermore, such a fine particle sorting apparatus may have a plurality of the fine particle sorting microchips described above and can repeat the sorting operation in multiple stages under the same conditions or multiple conditions.
[0033]
Further, in the fine particle sorting apparatus of the invention of this application, the miniaturization is achieved by refining each component part such as a measuring means, a voltage applying means, an output means, and each storage tank by a known fine processing technique. This makes it possible to easily perform fine particle sorting in the field such as medical diagnosis, which has been difficult with conventional cell sorters and particle analyzers. For example, if an analysis system such as an immunoassay device (Japanese Patent Application No. 2000-131833) reported by the inventors of this application is constructed on and connected to the microparticle sorting microchip of the invention of this application, it is highly accurate. The usefulness as a cell analysis / sorting system is also increased.
[0034]
The invention of this application also provides a fine particle sorting method for sorting fine particles using the fine particle sorting apparatus as described above. Specifically, for example, a solution containing the sample fine particles (7) is introduced from the introduction flow path (2), a sheath flow is formed, and the fine particles are introduced into the measurement site (4). There is a fine particle separation method in which an alternating voltage is applied to the electrode (6) after the electromagnetic measurement, and the fine particles are guided to the fine particle separation flow path (5) by the repulsive dielectrophoretic force generated.
[0035]
In the fine particle sorting method of the invention of this application as described above, any fine particles may be sorted. Any material that can be separated by shape, size, property, etc., particularly those that can be separated by dielectrophoresis, may be used. Specifically, powders such as polymers, cells such as blood cells and cultured cells, or cells or microorganisms Is exemplified.
[0036]
Hereinafter, embodiments will be described with reference to the accompanying drawings, and embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail.
[0037]
【Example】
<Example 1> Preparation of fine particle sorting microchip A 20 mm square, 0.5 mm thick synthetic quartz wafer (11) was washed with HPM (HCl: H ₂ O ₂ : DW = 1: 1: 4, 60 ° C.) for 10 minutes. , DW rinse, DHF (concentration 10%) cleaning 10 minutes, DW rinse, N ₂ blow, and a Cr mask was deposited by 1.5 μm using a magnetron sputtering apparatus.
[0038]
Next, photolithography (photoresist OMR-85: Tokyo Ohka Kogyo Co., Ltd., contact exposure machine: MJB3: Carl Sousse), Cr wet etching (Ce (NH ₄ ) ₂ (NO ₃ ) ₆ : HClO ₄ : DW = 65.8g: 17.2 The capillary pattern was transferred onto the mask by ml: 400 ml, room temperature), and the photoresist was ashed by O ₂ plasma (ICP = 500 W, pressure 40 mTorr, flow rate 100 sccm, treatment time 2 minutes). Thereafter, APM (NH ₄ OH: H ₂ O ₂ : H ₂ O = 1: 1: 4, 60 ° C.) washes for 10 minutes, DHF (concentration 5%) was washed for 5 minutes, DW rinse, and N ₂ blow.
[0039]
Next, capillary grooves are formed in the quartz wafer by dry etching (ICP = 500W, Substrate bias = 15W, pressure 10mTorr, flow rate 200sccm), and Cr mask is formed by SPM (H ₂ SO ₄ : H ₂ O ₂ = 3: 1 , Boiled) washed, HPM (HCl: H ₂ O ₂ : DW = 1: 1: 4, 60 ° C.) washed for 10 minutes, DW rinse, DHF (concentration 5%) washed for 5 minutes, DW rinse, N ₂ Processed by blow.
[0040]
In order to provide electrodes on both sides of the capillary, a Cr mask was deposited with a thickness of 0.1 μm by magnetron sputtering, the mask was aligned by photolithography, and the electrode pattern was transferred to the resist.
[0041]
After wet etching of Cr, dry etching of quartz glass with SF ₆ was performed to produce a trench for embedding an electrode. After removing the Cr mask, the resist in the electrode embedding portion was removed by mask alignment by photolithography.
[0042]
Finally, Cr and Pt are deposited in this order by magnetron sputtering and lift-off is performed. Then, another quartz glass with a sample inlet and electrode terminal is bonded to 1% hydrofluoric acid, taken out, and taken in the atmosphere. A pressure bonding of 1.3 MPa was applied for pressure bonding.
<Example 2> Operation evaluation of microparticle sorting microchip According to the procedure of Example 1, as shown in FIG. 1, a cell introduction channel (2) and a sheath flow forming solution channel (3) are formed on a substrate (1). A microparticle sorting microchip having a cell collection channel (5b) sandwiched between a pair of electrodes (6) located downstream thereof and disposal channels (5a, 5c) on both sides thereof was produced.
[0043]
In this microchip, by forming a sheath flow, the cells are efficiently sent to the inflow port (51) , and the repulsive dielectrophoretic force generated by applying an alternating electric field to the electrodes is used to dispose of the cells.
[0044]
Using a sheep erythrocyte suspension diluted with phosphate-diluted saline (PBS) at pH 7.4 and purified to 1 × 10 ⁸ / ml as a sample, the inner wall of each flow path of the microparticle sorting microchip is buffered with gelatin veronal. The operation of this microchip was confirmed by coating with gelatin with a solution (GVB).
[0045]
When no voltage is applied to the electrodes, the cells flow into the collection channel (5b). However, when an alternating voltage of Vp-p = 20V and 1200kHz is applied in synchronization with the moment when the cells reach the vicinity of the inlet (51) , the cells repel and are disposed on both sides of the collection channel (5b). It was selectively sent to the flow path (5a, 5c).
[0046]
In addition, it was confirmed by fluid simulation that the liquid flowing in the cell introduction flow path was suppressed by the sheath flow on both sides, and most of the liquid flowed to the collection flow path port. Further, it has been confirmed that the operation of the waste flow path (5a, 5c) is stabilized by making it wider than the region where the gradient of the electric field generated around the electrode (6) is large.
[0047]
Thus, by applying the microparticle sorting microchip of the present invention to the cell sorting, the cells subjected to the dielectrophoretic force are discarded, and the unaffected cells are taken into the collection channel (5b), and are collected in the cells. It can be collected without damage or irritation.
[0048]
From the above, it was shown that a microparticle sorting microchip using repulsive dielectrophoretic force can be used for sorting biological cells. In addition, by using this chip as a core technology, a cell collection system having an advanced cell determination processing system that was impossible with a conventional cell sorter is realized. It is highly useful in academic and medical research.
[0049]
【The invention's effect】
As described above in detail, according to the present invention, there are provided a microparticle sorting microchip capable of easily and accurately sorting microparticles, and an inexpensive and versatile microparticle sorting apparatus having the microchip. The microparticle sorting apparatus of the present invention can be miniaturized, and by using this, various microparticles such as cells, fungus bodies, microorganisms, and polymer microparticles can be easily and accurately separated.
[Brief description of the drawings]
FIG. 1 is a schematic view illustrating a microparticle sorting microchip according to the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Substrate 2 Particle-containing solution introduction channel, cell introduction channel 3 Sheath flow forming solution channel 4 Particle measurement site 5 Particle separation channel 5a Particle sorting channel, disposal channel 5b Particle sorting channel, collection channel 5c Fine particle separation channel, waste channel 51 Inlet 6 Electrode 7 Sample fine particle 8 Waste liquid tank

Claims (7)

A flow-type fine particle sorting microchip having a fine flow path on a substrate, and at least a fine particle-containing solution introduction flow path on the substrate,
A sheath flow forming solution channel disposed on both sides of the introduction channel;
One flow path into which the fine particle-containing solution and the sheath flow forming solution flow;
A particulate measurement site for measuring the introduced particulates provided in the one flow path ;
Located downstream of this fine particle measurement site, and the second particle separation flow path installed fine particles separate collection first fine particle separation flow path for the on both sides thereof,
Is installed between the first particle separation flow path and the second particle separation flow path so as to sandwich the first fine particle separation flow path, disposed from the microparticles measuring zone in the vicinity of the inflow port to the fine separation flow path A pair of electrodes for controlling the moving direction of the fine particles;
The has, electrodes have a sharp shape, particle fractionation microchip, characterized in that the acute angle portion is placed in the inlet near the mouth flow. Fine particle-containing solution introduction channel, a sheath flow forming solution flow path, fine measurement region, and the inner wall of the particulate separation flow path, the microparticles fractionation microchip of claim 1 that has been coated with fine particles antiadhesive layer. The microparticle sorting microchip according to claim 1 or 2 , wherein the microparticles in the microparticle-containing solution are selected from the group consisting of cells, fungus bodies, microorganisms, and polymer microparticles . A microparticle sorting apparatus for sorting microparticles, wherein at least the microparticle sorting microchip according to any one of claims 1 to 3,
A measurement means for optically or electromagnetically measuring fine particles at a fine particle measurement site on the fine particle sorting microchip;
Voltage applying means for applying a voltage to the electrode on the microparticle sorting microchip;
A fine particle sorting apparatus comprising output means for outputting optical or electrical information of fine particles. 5. The fine particle sorting apparatus according to claim 4, wherein the measuring means is an optical microscope. The fine particle sorting apparatus according to claim 4, wherein the measuring means is a fluorescence microscope. A fine particle sorting method for sorting fine particles using the fine particle sorting device according to claim 4.

Images