JP5812469B2 - Cell separation chip - Google Patents

Description

  The present invention relates to a cell separation chip that separates cells to be measured from other cells.

  In recent years, immediate allergies such as food allergies have become a social problem. Immediate allergies can interfere with social life or in some cases can be life-threatening. Therefore, simplification of immediate allergy diagnosis and improvement of reliability are desired.

  Initially, in the immediate type allergy diagnosis, a load test for confirming the induction of symptoms by ingestion of causal food or the like, and a test using blood or skin of a patient were generally performed. However, these methods have disadvantages such as a heavy burden on the subject, a lot of blood required, and a high ratio of false positives and false negatives.

  Recently, an immediate allergy diagnosis has been performed by analysis at the cellular level. In immediate allergy diagnosis at the cell level, it is necessary to extract cells to be measured from a blood sample by centrifugation or filter separation. Therefore, cells that are unnecessary for measurement in a blood sample (for example, cells other than basophil cells if the measurement target is basophil cells that cause type I allergy) are magnetically modified, and magnetic beads An apparatus or the like for removal using a method is disclosed (for example, see Patent Document 1). A magnetic bead binds to an antibody specific for a cell to be measured or other cells and magnetically labels the cell.

  An apparatus for removing magnetically modified cells by spreading magnetic particles having a diameter of about 500 μm in a column and flowing blood through the column is also disclosed. Basophil cells separated by such an apparatus are stimulated with an antigen and used for measurement of histamine secreted from the cells.

JP 2006-006166 A

  In order to extract cells from blood with the above-described apparatus, a lot of blood samples, for example, a blood sample of about several ml (for example, 2 to 3 ml or more) is required for one test. Become. Moreover, since the apparatus mentioned above is used many times, there exists a possibility that the reliability of a diagnosis may fall by contamination.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a cell separation chip that enables highly reliable diagnosis while reducing the amount of sample.

In order to achieve the above object, a cell separation chip according to the first aspect of the present invention comprises:
A cell separation chip formed by attaching a member to a substrate,
As a void having a volume of microliter order provided between the substrate and the member,
A solution loading portion provided on the member with a loading port of a solution containing cells to be measured and other magnetically modified cells; and
A fine flow path extending from the solution charging portion and branching into a plurality of lines ,
A solution output unit that outputs the solution that is input to the solution input unit and flows through the fine channel;
Is provided,
Magnetic particles contained in the member for increasing the magnetic field gradient of an external magnetic field, and comprising a magnetic field gradient generating means for generating a magnetic field gradient in the fine channel that prevents the flow of the magnetically modified cells ,
In the member,
The magnetic particles are densely arranged around the fine channel .

The solution output part is
Having an air opening formed on the member;
The fine channel is formed so as to send the solution to the solution output unit side by capillary force.
It is good as well.

In the solution output part,
An inlet for introducing a substance that stimulates the cell to be measured is provided,
It is good as well.

The magnetic field gradient generating means includes
A current circuit wired in the member;
It is good as well.

The magnetic field gradient generating means includes
A small magnet embedded in the member,
It is good as well.

In the solution output part,
A metal thin film on which the cells to be measured are spotted is provided on the substrate;
It is good as well.

Incident means for causing P-polarized light to be incident on the metal thin film at an incident angle satisfying a total reflection condition from the opposite surface of the substrate on which the metal thin film is formed;
Detecting means for detecting information on the intensity of the reflected light of the P-polarized light incident on the metal thin film;
Further comprising
It is good as well.

The detection means includes
Capturing an intensity image corresponding to a two-dimensional intensity distribution of the P-polarized reflected light incident on the metal thin film;
It is good as well.

It further comprises a detecting means for detecting a change in impedance of the metal thin film that changes in accordance with a change in the attached cell to be measured.
It is good as well.

It further comprises detection means for detecting a change in polarization state between incident light and reflected light on the metal thin film,
It is good as well.

A plurality of the solution output units are provided,
It is good as well.

As the solution output part,
A first solution output unit and a second solution output unit are provided;
The magnetic field gradient generating means includes
Generating a magnetic field gradient that hinders the flow of the magnetically modified cells only in the fine flow path communicating with the first solution output section;
It is good as well.

  In the cell separation chip according to the present invention, a cell to be measured is transferred to another magnetically modified cell while a solution containing cells is fed through a fine channel which is a part of a void having a volume of microliter order. Therefore, cells to be measured can be extracted from a small amount of solution. In addition, since the cell separation chip according to the present invention can be disposable, contamination can be prevented and highly reliable diagnosis can be performed.

FIG. 1A is a perspective view of a cell separation measurement chip according to Embodiment 1 of the present invention. FIG. 1B is a side view of the cell separation measurement chip. It is a figure which shows typically the magnetic field produced in a fine flow path. It is a schematic diagram which shows the detailed structure of the optical measurement part of FIG. 1 (B). It is a graph which shows an example of the incident angle dependence of the intensity | strength of reflected light. FIGS. 5A to 5C are examples of temporal changes in the image of the reflection intensity image when the living cells are not stimulated. FIGS. 6A to 6C are examples of temporal changes in the image of the reflection intensity image when a living cell is stimulated. It is a flowchart of the cell measurement operation | movement using the cell separation measuring device chip | tip of FIG. 1 (A). It is a perspective view of the cell separation measuring chip concerning Embodiment 2 of the present invention.

  Embodiments of the present invention will be described in detail with reference to the drawings.

(Embodiment 1)
First, a first embodiment of the present invention will be described.

  FIG. 1A shows a perspective view of a cell separation measurement chip 100 as a cell separation chip according to this embodiment, and FIG. 1B shows a side view of the cell separation measurement chip 100. Yes.

  As shown in FIGS. 1A and 1B, the cell separation measurement chip 100 is formed by bonding a substrate 1 and a member 2 together.

  The substrate 1 is a glass substrate. As this glass substrate, for example, S-LAL-10 glass is employed. The refractive index of this glass is 1.72.

  The member 2 is made of, for example, PDMS (polydimethylsiloxane). A gap is provided between the substrate 1 and the member 2. This void as a whole has a volume of microliter order.

  This void can be divided into a solution charging unit 3, measurement units 4 </ b> A and 4 </ b> B as solution output units, and a fine flow path 5.

  The solution inlet 3 is provided with an inlet of a solution containing cells to be measured and other magnetically modified cells on the member 2. The solution charging part 3 penetrates the lower surface from the upper surface of the member 2. The lowermost part of the solution charging part 3 has a chamber structure in which a liquid pool is formed.

  The measurement units 4A and 4B communicate with the solution input unit 3 through the fine flow path 5. In the measurement units 4A and 4B, a metal thin film 6 on which cells to be measured are spotted is provided on the substrate 1. The metal thin film 6 will be described later.

  In the measurement units 4 </ b> A and 4 </ b> B, an air release portion communicating with the fine flow path 5 is formed on the member 2. This atmosphere opening part is an inlet for introducing a stimulating substance for the cell to be measured. The stimulating substance put in from the atmosphere opening part stimulates the cell to be measured attached to the metal thin film 6 as it is.

  The fine channel 5 communicates the solution charging unit 3 with the measuring units 4A and 4B. The fine channel 5 is a functional channel that separates cells to be measured and magnetically modified cells. In this cell separation measurement chip 100, a plurality of fine flow paths 5 are provided. Each fine channel 5 is formed so as to send the solution to the measurement units 4A and 4B by capillary force. That is, when a solution is introduced into the solution introduction unit 3, the solution is sent to the measurement units 4 </ b> A and 4 </ b> B through the microchannel 5 by the capillary force of the microchannel 5. In addition, the length of each fine channel 5 is the same. In this way, it is possible to prevent the solution from reaching one measurement unit early and the solution from being delivered to the other measurement unit.

  The member 2 includes a plurality of magnetic particles 7 such as iron sand and ferrite. The diameter of the magnetic particle 7 is, for example, about 500 μm. The magnetic particles 7 are arranged in the vicinity of the fine flow path 5.

  Here, consider a case where the cell separation measurement chip 100 is placed in a magnetic field in a direction perpendicular to the flow path direction of the fine flow path 5, as shown in FIG. In this state, when a magnetically modified cell modified with a magnetic bead having a diameter of 2 to 3 μm flows in the microchannel 5, the magnetically modified cell is affected by the magnetic field and is prevented from flowing. It adheres to the inner wall of 5.

The force F received by the magnetically modified cell from the magnetic field is as follows.

The magnetic particle 7 included in the member 2 enlarges the magnetic field gradient portion represented by the following expression in the above expression (1).

Thereby, the force which a magnetic modification cell receives from a magnetic field becomes large.

  In order to form the member 2 (made of PDMS) containing the magnetic particles 7, two liquids are mixed to form a PDMS stock solution, and then the liquid PDMS stock solution is poured into a mold at 80 degrees. Usually, it is baked and hardened. At this time, the member 2 is produced by arranging the magnetic particles 7 in the mold and pouring and forming a PDMS stock solution.

  When the magnetic particles 7 are contained in PDMS, bubbles are easily generated inside. Therefore, it is desirable to perform vacuum deaeration after pouring the PDMS stock solution into the mold.

Further, PDMS containing magnetic particles 7 has a property of warping when baked and hardened. To avoid this,
(1) The density of the magnetic particles 7 arranged on the bottom surface of the mold is adjusted. (2) A weight is placed on the top surface to be baked and hardened.

  In the case of (1), before pouring the PDMS stock solution into the mold so that the magnetic particles 7 are arranged in the vicinity of the fine flow path 5 of the member 2, in a place corresponding to the vicinity of the fine flow path 5 in the mold. The magnetic particles 7 may be densely arranged. In addition, an electric circuit is wired in a location corresponding to the vicinity of the fine flow path 5 of the mold, a current is passed through the electric circuit to generate a magnetic field around it, and the magnetic particles 7 are attracted by the magnetic field, The PDMS stock solution may be poured into a mold.

  In the completed member 2, the magnetic particles 7 are disposed in the vicinity of the fine flow path 5, and the flowing sample is easily affected by a magnetic field gradient.

  The size of the magnetic particle 7 is currently 450 μm, but it is expected that the function is sufficiently exhibited even with different sizes. The size of the magnetic particle 7 can be appropriately adjusted according to the cross-sectional area and length of the fine channel 5.

  As shown in FIG. 1B, the cell separation measurement chip 100 further includes an optical measurement unit 10. The optical measurement unit 10 includes a light source 11, a polarizing plate 12, a prism 13, an objective lens 14, and an imaging unit 15.

  The optical measurement unit 10 is a sensor that utilizes a surface plasmon resonance (SPR) phenomenon. The SPR phenomenon is a phenomenon in which when light is incident on the metal thin film 6 under a total reflection condition, the light causes a resonance phenomenon with a plasmon on the metal surface at a certain angle (resonance angle), and the reflected light is attenuated. The resonance angle is very sensitive to the refractive index of the substance on the metal thin film 6, and the change in the refractive index on the metal thin film 6 can be known by detecting the change in the resonance angle.

  FIG. 3 shows a detailed configuration of the optical measurement unit 10.

  The light source 11 is a semiconductor laser, for example. This semiconductor laser oscillates and outputs laser light having a wavelength of, for example, 630 nm. The laser light output from the light source 11 is converted into a parallel light beam by a collimator lens (not shown) or the like and enters the polarizing plate 12. As the light source 11, a red or white LED (Light Emitting Diode) or the like may be used.

  The polarizing plate 12 converts the incident laser light into a linearly polarized parallel light beam and emits it. This linearly polarized light becomes P polarized light with respect to an interface F between the substrate 1 and the metal thin film 6 described later. The light source 11 and the polarizing plate 12 correspond to the incident means.

  The prism 13 receives the parallel light flux that has been changed to P-polarized light by the polarizing plate 2. As the prism 13, for example, S-LAL-10 glass is employed in the same manner as the substrate 1. That is, the substrate 1 and the prism 13 have the same refractive index. Since the substrate 1 and the prism 13 are in contact with each other, the laser light (P-polarized light) incident on the prism 13 enters the substrate 1 and travels straight.

  The metal thin film 6 is, for example, a gold film. In addition, thin films such as Ag, Cu, Zn, Al, and K can also be used as the metal thin film 6. The thickness of the metal thin film 6 is, for example, 50 nm, and the sensing size is, for example, about 1 mm square. The metal thin film 6 is formed on the substrate 1 by vapor deposition, for example. The laser light incident on the substrate 1 is incident on the interface F between the substrate 1 and the metal thin film 6 at an incident angle θ = 56 ° that satisfies the total reflection condition and causes the surface plasmon resonance phenomenon, for example. If the metal thin film 6 is not installed, the laser light is totally reflected at the interface F.

  The laser light reflected by the interface F is emitted from the substrate 1 and the prism 13 and enters the objective lens 14. The objective lens 14 refracts and emits laser light. For the objective lens 14, a lens having a longer rear focal length than a front focal length is used. Therefore, the objective lens 14 enlarges the object image at a predetermined magnification and forms an image on the image plane. The laser light emitted from the objective lens 14 reaches the imaging surface of the imaging unit 15.

  The imaging unit 15 is, for example, a CCD (Charge Coupled Device) image sensor or a CMOS (Complementary Metal Oxide Semiconductor) image sensor. The imaging unit 15 receives the laser light reflected by the interface F. The imaging surface of the imaging unit 15 and the interface F are in a conjugate relationship. Therefore, an intensity image corresponding to the two-dimensional intensity distribution of the reflected light of the parallel light beam incident on the interface F of the prism 13, that is, a reflection intensity image is formed on the imaging surface of the imaging unit 15.

  This reflected intensity image is magnified by, for example, 2 to 40 times by the objective lens 14. The imaging unit 15 captures the reflection intensity image. The imaging unit 15 outputs an image signal corresponding to the reflected intensity image.

  The image signal output from the imaging unit 15 is input to a computer (not shown). The computer displays an image signal or analyzes a cell based on the image signal.

  The optical measurement unit 10 visualizes the relative intensity distribution of the refractive index on the metal thin film 6 by using the difference in the resonance angle for each substance having a different refractive index. When the living cells C1 and C2 are cultured on the metal thin film 6, the refractive index is different from the portion where the living cells C1 and C2 are not attached.

  The graph of FIG. 4 shows an example of the incident angle dependence of the intensity of the reflected light received by the imaging unit 15. In this graph, the horizontal axis indicates the incident angle θ of the parallel light flux to the interface F, and the vertical axis indicates the intensity of the reflected light at the incident angle θ. FIG. 4 shows three characteristic curves (a) to (c).

  The characteristic curve (a) is a curve showing the incident angle dependence of the intensity of the reflected light in a state where nothing is placed on the metal thin film 6 (a state where there are no living cells C1 and C2). According to this, the intensity of reflected light is most attenuated at an incident angle of 56 ° due to the surface plasmon resonance phenomenon. This incident angle of 56 ° is called a resonance angle. In the present embodiment, the incident angle of the laser beam to the interface F is set to 56 ° so that the intensity of the reflected light when the living cells C1 and C2 are not in contact is the darkest.

  The characteristic curve (b) is a curve showing the incident angle dependence of the reflected light intensity when the living cells C1 and C2 are set on the metal thin film 6 and the living cells C1 and C2 are not yet stimulated. Since the living cells C1 and C2 are dielectrics, the dielectric constant (refractive index) changes around the metal thin film 6 with which the living cells C1 and C2 abut, and the incident angle dependence of the reflected light intensity is a characteristic curve ( The characteristic curve (b) is shifted from a), and the resonance angle is also shifted from 56 ° to θ1.

  The characteristic curve (c) shows that the living cells C1 and C2 are set on the metal thin film 6, and the living cells C1 and C2 are stimulated by the antigen contained in the liquid that has reached the measurement units 4A and 4B, and respond to the stimulation. It is a curve which shows the incident angle dependence of the reflected light intensity in the state which carried out. When the living cells C1 and C2 are stimulated and respond to the stimulation, the dielectric constant (refractive index) of the living cells C1 and C2 further changes, and the incident angle dependence of the intensity of the reflected light is characteristic from the characteristic curve (b). The curve (c) is shifted and the resonance angle is also shifted from θ1 to θ2.

  In this embodiment, the incident angle of the parallel light flux on the interface F is fixed at 56 °. Therefore, focusing on the incident angle of 56 °, the intensity of the reflected light is I1 and is the darkest when the living cells C1 and C2 are not in contact with the metal thin film 6. Further, when the living cells C1 and C2 adhere to the metal thin film 6, the intensity of the reflected light becomes I2, which is higher than I1 by ΔI1. Furthermore, when the living cells C1 and C2 attached to the metal thin film 6 are stimulated by an antigen or the like and respond to the stimulation, the intensity of the reflected light increases from I2 by ΔI2 to I3, and becomes stronger.

  As described above, in the image of the reflection intensity image captured by the imaging unit 15, the place where the living cells C1 and C2 do not exist becomes dark, the place where the cells exist becomes bright, and the living cells C1 and C2 become bright. The activated area becomes brighter.

  In addition, the angle of incidence θ = 56 ° has the greatest contrast between the location where the live cells C1 and C2 exist and the location where the live cells C1 and C2 do not exist. This is apparent from the fact that in FIG. 4, the resonance angle is 56 ° when the living cells C1 and C2 are not present, and the reflected light is most attenuated. That is, if the incident angle is the same as the resonance angle θ = 56 °, Δl1 can be maximized, and the contrast of the image is increased.

  5 (A) to 5 (C) show changes in the image of the reflection intensity image after 0 minutes, 10 minutes, and 20 minutes in a state where the living cells C1 and C2 are not stimulated with the antigen. ing. 6 (A) to 6 (C) show changes in reflection intensity images after 0 minutes, 10 minutes, and 20 minutes after stimulation of living cells C1 and C2 with antigen. ing. As is clear from a comparison between FIGS. 5A to 5C and FIGS. 6A to 6C, when the living cells C1 and C2 react to an external stimulus, the living cells C1 and C2 It can be seen that the luminance of the portion corresponding to is changing.

  By using this optical measuring unit 10, it is possible to observe the stimulation response (refractive index change) for each cell.

  Two optical measuring units 10 may be provided corresponding to the measuring units 4A and 4B, respectively.

  Next, the operation of the cell separation measurement chip 100 according to this embodiment will be described. Here, the operation in the case of using basophil cells as cells to be measured will be described. Basophil cells are contained in human leukocytes at around 0.5%. Its diameter is about 10 μm.

  FIG. 7 shows a flowchart of a cell measurement operation using the cell separation measurement chip 100.

  First, pretreatment is performed on a blood sample (step S1). More specifically, after separating and removing red blood cells from the blood sample, white blood cells other than basophil cells in the blood sample are modified with magnetic beads (step S1). Here, magnetic beads are used that bind to surface antigens present in other cells in the blood rather than basophil cells.

  Subsequently, the cell separation measurement chip 100 is installed at a predetermined position (step S2). As a result, as shown in FIG. 2, a downward magnetic field is generated in the fine flow path 5, and the optical measurement unit 10 is set to a position as shown in FIG. Due to the magnetic particles 7, the magnetic field gradient in the fine flow path 5 becomes larger.

  In this state, the blood sample magnetically modified in step S1 is input from the input port of the solution input unit 3 using a pipette or the like (step S3). The blood sample input into the solution input unit 3 enters the fine flow channel 5 through the liquid reservoir of the solution input unit 3, and advances through the fine flow channel 5 by the capillary force generated in the fine flow channel 5.

  While traveling through the microchannel 5, the blood sample receives the magnetic field shown in FIG. As a result, the magnetically modified cells other than basophil cells receive an upward force and stay under the magnetic particles 7 on the upper surface of the fine channel 5, and only the basophil cells are present in the measurement units 4A and 4B. To reach. The basophil cells that have reached the measurement parts 4A, 4B are released from the capillary force and settle down, and adhere to the metal thin film 6. As a result, most of cells (preferably 80% or more) fixed to the metal thin film 6 are basophil cells.

  Subsequently, a stimulating substance, that is, an antigen is introduced from the atmosphere opening parts of the measurement parts 4A and 4B (step S4). Thereby, the input antigen is combined with the surface antibody of basophil cells attached to the metal thin film 6 to cause an antigen-antibody reaction.

  Subsequently, measurement is performed using the optical measurement unit 10 (step S5). The measurement may be started before the antigen is introduced in step S4. Laser light irradiation is started from the light source 11, and the imaging unit 15 captures a two-dimensional image of living cells attached to the metal thin film 6 based on the reflected light at the interface F. This two-dimensional image is displayed or analyzed by a computer (not shown), and the characteristics of basophil cells can be analyzed based on the analysis result.

  As described above in detail, when the cell separation measurement chip 100 according to the present embodiment is used, the basophil cell to be measured is transferred to another magnetic field while the blood sample is fed through the fine channel 5. Since the cells can be separated and extracted from the modified cells, the cells to be measured can be extracted from a small amount of blood sample. The present inventor has confirmed that sufficient diagnosis is possible using basophil cells contained in 5 μl of blood.

  Further, if this cell separation measurement chip 100 is used, cell separation → fixation → measurement can be performed in the same chip, so that contamination can be prevented and highly reliable measurement can be performed. it can. In addition, since the cell separation measuring chip 100 itself can be made disposable, contamination can be prevented and a highly reliable diagnosis can be performed.

  In addition, according to the present embodiment, the measurement unit 4A, 4B is formed with the atmosphere opening portion communicating with the fine flow path 5 on the member 2. The fine channel 5 is formed so as to send a blood sample to the measurement units 4A and 4B by capillary force. This eliminates the need for an external pump for separating and spotting cells and spotting them, thereby enabling a short-time measurement and reducing the cost required for allergy diagnosis and the like.

  Further, according to the present embodiment, since the measurement is performed using the two-dimensional SPR measurement device, the diagnosis is highly reliable and quick.

  Such a cell separation measurement chip 100 is small in size, and can perform from cell separation to measurement by simply dropping a blood sample. Therefore, the cell separation measurement chip 100 is simple and easy to use, so that it can be practically used in a clinical field.

  Moreover, according to this Embodiment, the magnetic particle 7 contained in the member 2 and which enlarges the magnetic field gradient of an external magnetic field is used as a magnetic field gradient generation means. For example, iron sand or ferrite can be used as the magnetic particles 7, so that the manufacturing cost of the cell separation measurement chip 100 can be reduced.

  Further, according to the present embodiment, a plurality of fine channels 5 having the same length are provided. In addition, a plurality of measurement units 4A and 4B communicating with one solution charging unit 3 through each fine channel 5 are provided. In this way, different stimuli can be simultaneously applied to the same measurement target cell, so that the time required for diagnosis can be shortened.

  The number of measurement units may be three or more. The larger the number of measurement units, the better. In this way, by using the microfabrication technique, it becomes easy to make a multichannel such as providing a plurality of measurement units.

(Embodiment 2)
Next, a second embodiment of the present invention will be described.

  FIG. 8 shows the configuration of the cell separation measurement chip 100 according to the second embodiment. As shown in FIG. 8, in this cell separation measurement chip 100, the magnetic particles 7 are arranged in the vicinity of the fine channel 5 communicating with the measurement unit 4B, but the fine channel 5 communicating with the measurement unit 4A. Is different from the cell separation measurement chip 100 according to the first embodiment in that the magnetic particles 7 are not disposed in the vicinity of the cell.

  As a result, only basophil cells are sent to the measurement unit 4B and adhere to the metal thin film 6, but all cells in the blood sample are sent to the measurement unit 4A. It adheres to the metal thin film 6. In this way, it is possible to observe the difference in antigen reaction in basophils with and without cells other than basophils by introducing the same stimulating substance into the measurement units 4A and 4B. It becomes.

  In each of the above embodiments, the magnetic field gradient generating means is the magnetic particle 7 contained in the member 2 and increasing the magnetic field gradient of the external magnetic field, but the present invention is not limited to this. For example, the magnetic field gradient generating means may be a current circuit wired in the member 2. The current circuit may be conducted to generate a magnetic field as shown in FIG. In this case, an external magnetic field as shown in FIG. 2 is not necessary. Moreover, you may make it embed a small magnet in the member 2 as a magnetic field gradient generation means. Also in this case, an external magnetic field as shown in FIG. 2 is not necessary.

  In the above embodiments, the member 2 is formed of PDMS. However, the present invention is not limited to this. For example, the member 2 may be formed of a resin such as an acrylic resin or glass. In short, any substance that can contain the magnetic particles 7 and the like may be used.

  Moreover, in each said embodiment, although the optical measurement part 10 imaged the two-dimensional image of the cell, this invention is not limited to this. For example, it is sufficient to detect the intensity of reflected light to the interface F.

  In each of the above embodiments, the characteristics of the measurement target cell are detected using the SPR (surface plasmon resonance) phenomenon. However, the present invention is not limited to this. For example, a change in impedance of the metal thin film 6 that changes with changes in cells may be detected, or the refractive index of the metal thin film 6 that changes with changes in cells changes the incident light to the metal thin film 6. Alternatively, measurement may be performed using ellipsometry that detects a change in the polarization state between the reflected light and the reflected light.

  In addition, the cells to be measured are not limited to basophil cells. For example, it may be other cells in the blood such as monocytes, lymphocytes, neutrophils, eosinophils, or may be cells other than blood.

  Also, a plurality of solution charging portions 3 can be provided. As described above, since a plurality of solution input parts and measurement parts can be provided on one chip by the microfabrication technique, multichanneling is facilitated, and a plurality of samples can be simultaneously examined. Usually, there are about 10 types of main allergies in the human body, and if 10 measuring units are provided, 10 types of allergies can be examined at once, and the diagnosis time is significantly shortened.

  Moreover, in each said embodiment, although the microchannel 5 sent the solution to measurement part 4A, 4B with capillary force, this invention is not limited to this. An external pump may be connected to the solution charging unit 3 and the external pump may be operated to send the solution to the measurement units 4A and 4B.

  The “cell” used in the present specification is defined in the same way as the broadest meaning used in the field, and may be any kind of cell of an animal. Moreover, it may be a naturally occurring cell or an artificially modified cell (for example, a fused cell or a genetically modified cell). “Live cell” refers to a living cell. Most preferably, it represents a living cell derived from a human (Homo sapience).

  “Stimulation” means binding of a ligand to a cell surface receptor, environmental change such as temperature or pH, mechanical stimulation, electrical stimulation, etc., and cell activity (for example, intracellular signal transduction system) All stimuli that act on the activation of

  In short, the present invention is useful not only for immediate allergy diagnosis but also for cell analysis widely.

  “Magnetic particles” refer to substances such as iron and sand iron that are magnetized by being placed in a magnetic field and have the properties of a magnet.

  Various embodiments and modifications can be made to the present invention without departing from the broad spirit and scope of the present invention. The above-described embodiments are for explaining the present invention and do not limit the scope of the present invention. In other words, the scope of the present invention is shown not by the embodiments but by the claims. Various modifications within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.

  The present invention is suitable for immediate allergy diagnosis.

DESCRIPTION OF SYMBOLS 1 Board | substrate 2 Member 3 Solution injection | throwing-in part 4A, 4B Measurement part 5 Fine flow path 6 Metal thin film 7 Magnetic particle 10 Optical measurement part 11 Light source 12 Polarizing plate 13 Prism 14 Objective lens 15 Imaging part 100 Cell separation measurement chip C1, C2 Living cell

Claims (12)

A cell separation chip formed by attaching a member to a substrate,
As a void having a volume of microliter order provided between the substrate and the member,
A solution loading portion provided on the member with a loading port of a solution containing cells to be measured and other magnetically modified cells; and
A fine flow path extending from the solution charging portion and branching into a plurality of lines ,
A solution output unit that outputs the solution that is input to the solution input unit and flows through the fine channel;
Is provided,
Magnetic particles contained in the member for increasing the magnetic field gradient of an external magnetic field, and comprising a magnetic field gradient generating means for generating a magnetic field gradient in the fine channel that prevents the flow of the magnetically modified cells ,
In the member,
The magnetic particles are densely arranged around the fine channel,
Cells separation chip. The solution output part is
Having an air opening formed on the member;
The fine channel is formed so as to send the solution to the solution output unit side by capillary force.
The cell separation chip according to claim 1. In the solution output part,
An inlet for introducing a substance that stimulates the cell to be measured is provided,
The cell separation chip according to claim 1 or 2, wherein The magnetic field gradient generating means includes
A current circuit wired in the member;
The cell separation chip according to any one of claims 1 to 3, wherein: The magnetic field gradient generating means includes
A small magnet embedded in the member,
The cell separation chip according to any one of claims 1 to 3, wherein: In the solution output part,
A metal thin film on which the cells to be measured are spotted is provided on the substrate;
The cell separation chip according to any one of claims 1 to 5 , wherein: Incident means for causing P-polarized light to be incident on the metal thin film at an incident angle satisfying a total reflection condition from the opposite surface of the substrate on which the metal thin film is formed;
Detecting means for detecting information on the intensity of the reflected light of the P-polarized light incident on the metal thin film;
Further comprising
The cell separation chip according to claim 6 . The detection means includes
Capturing an intensity image corresponding to a two-dimensional intensity distribution of the P-polarized reflected light incident on the metal thin film;
The cell separation chip according to claim 7 . It further comprises a detecting means for detecting a change in impedance of the metal thin film that changes in accordance with a change in the attached cell to be measured.
The cell separation chip according to claim 6 . It further comprises detection means for detecting a change in polarization state between incident light and reflected light on the metal thin film,
The cell separation chip according to claim 6 . A plurality of the solution output units are provided,
Cell separation chip according to any one of claims 1 to 1 0, characterized in that. As the solution output part,
A first solution output unit and a second solution output unit are provided;
The magnetic field gradient generating means includes
Generating a magnetic field gradient that hinders the flow of the magnetically modified cells only in the fine flow path communicating with the first solution output section;
Cell separation chip according to claim 1 1, wherein the.