JP2007057290A - Demarcation microchip and demarcation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a disposable inexpensive, safe and compact demarcation microchip constituted by mounting a fine particle demarcation processing means in a microchip and enabling stable demarcation processing without requiring skill, and a demarcation device. <P>SOLUTION: A pressure chamber also used as a flow channel is provided to the electromechanical transducer overlying the microchip and developed as the function of a pump or a valve to realize miniaturization. The volume of the chamber is increased and decreased by bending a wall surface in a shearing mode to change a flow. The pressure chamber is provided to a flow channel symmetrically after branching and the chamber is driven by push-pull operation so as to aim a moment when fine particles arrive at a branch point to precisely control a flow direction. Further, a piezoelectric body of a lamination structure (chevron structure) is used to realize high-speed operation as the microchip. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a fractional microchip and a fraction which identify microorganisms, cells or fine particles floating in a liquid by means such as spectroscopic analysis or image analysis, and isolate the fine particles at high speed and precisely according to the identification data. Related to the device.

  Due to advances in biotechnology, cells can be easily removed from cell tissues by digesting matrix tissues with proteolytic enzymes such as trypsin and collagenase, and further breaking the binding between cells using a chelating agent of Ca ions such as EDTA. It became possible to isolate, and experiments using live cells were widely conducted.

  Currently, the fluorescent probe is selected according to the target, and the cells to be extracted are identified by labeling the cell constituents with fluorescent label primers (DNA, RNA), fluorescent label peptides, fluorescent labeled sugar chains, fluorescent labeled antibodies, etc. In addition, detection of proteins produced in cells is performed, and the process of fractionating and selectively sorting fluorescently labeled cells is becoming increasingly important.

  Typical fractionation techniques for labeled floating cells include analysis of cell populations using a flow cytometer and extraction of cells using a cell sorter with a sorting function. (See, for example, Non-Patent Document 1).

  In particular, the latter cell sorter has established itself as a method for automatically separating a large amount of sample cells at high speed. A commercially available system continuously forms droplets with a continuous head (see, for example, Patent Document 1). The presence or absence of cells is detected by the light emission intensity while the droplet ejected from the head pulls the tail from the nozzle, and an electric charge (+ / 0 / −) is given to the droplet immediately before the tail is cut off.

  Uncharged droplets go straight and are collected as an aport solution. Among the discharged droplets, the positively and negatively charged droplets are bent in the advancing direction by a deflection electrode (a voltage of several kV is applied and opposed), + charge droplet recovery container and -charge liquid recovery The sample is dispensed into a container (for example, see Patent Document 2).

  Therefore, this apparatus is expensive due to its large-scale system configuration, and requires skill in its operation, such as adjusting the oscillation frequency, controlling the drive voltage, and stabilizing and maintaining the droplet formation state by adjusting the sheath solution flow rate. In addition, it was difficult to perform unattended operation for a long time.

  In principle, it is impossible to completely suppress mist generation. Therefore, when handling dangerous samples, it is necessary to pay sufficient attention to environmental contamination. The road was extremely contaminated and maintenance after use was complicated.

  In addition, problems have been pointed out such as damage to cells due to charging / discharging of droplets and collision with collection containers.

  On the other hand, recently, as described in Patent Document 2, a fractionation apparatus (for example, refer to Patent Document 3) applying optical manipulation technology has also been reported, but a large amount of cell sorting is required in terms of fractionation speed. There is a problem as a technique for performing the above.

On the other hand, although it is still in the research and development stage, a fractionation method in which the flow direction of the solution is controlled by an external pump using a microchip has also been reported, but there are problems in response speed and complicated valve operation and pump switching operation. Therefore, practical application is expected to be difficult (for example, see Non-Patent Document 2).
U.S. Pat. No. 4,515,274 US Pat. No. 6,281,018 JP 2004-167479 A "Flow cytometry freedom" Supervision by Hiromitsu Nakauchi Published by Shujunsha in December 2004 ISBN 4-87962-281-8 Anne Y. Fu, et al., "An Integrated Microfablicated Cell Sorter", Anal. Chem. Vol.74 (11), pp2451-2457

  It is no exaggeration to say that the problem of the cell sorter using the charging method is caused by the technique of fractionating into droplets. Needless to say, it is preferable to implement a fractionation process in a closed system in consideration of reducing the load required for stable discharge of droplets, suppressing mist generation, preventing contamination, ensuring work safety, and simplifying maintenance.

  From this point of view, it is desirable to integrate flow paths having a fractionation function on a disposable microchip. However, as mentioned in the background art, it is difficult to put it into practical use unless measures for improving the fractionation processing speed are taken. Furthermore, providing a large number of pumps and valves outside is not preferable because it increases the size and complexity of the system, increases the cost of the apparatus, and increases the load on the operator.

  That is, an object of the present invention is to create a technique that can simplify control factors and perform stable fractionation processing without requiring skill, and enables high-speed operation by installing a pump and a valve function on a microchip. Another object of the present invention is to provide a fractionation microchip and fractionation apparatus that are safe and maintenance-free.

  In order to solve the above problems, the present invention integrates a fractionation function excluding a liquid feeding mechanism and an identification means in a microchip, and performs a flow from collection of a sample solution to discharge of a collected liquid after fractionation processing. The system can be moved by a continuous water column. Specific means will be described below.

(1) Improvement of safety and maintainability As described above, the problem is solved by performing fractionation processing in a closed system. In the present invention, the risk of contaminating the work environment and the apparatus is avoided by performing the fractionation process in the microchip. In addition, since the microchip can be disposable, there is no risk of contamination, and the piping of the apparatus main body can be exchanged, so that cleaning of tube connectors can be simplified.

(2) Simplification of control factors and equipment A sampling pump is used to collect and deliver the sample solution so that the total flow rate in the system can be kept constant regardless of temperature changes and the remaining amount of sample solution. did. Therefore, the time required from the fixed observation point of the identification area in the fractionation microchip to the branch point where the fractionation is actually performed is always constant, and can be uniquely determined by the amount of liquid fed.
Therefore, the fractionation operation at the branch point may be performed after a certain time (delay time td) after the identification data is acquired at the observation point, and the control can be simplified.
In the present invention, the function of forming a droplet or charging / deflecting is unnecessary, and an optical system other than a detection system is not required unlike a fractionation apparatus using light pressure, so that the act of adjusting the apparatus is not necessary. In addition, the size can be reduced.

(3) Fractionation speed in microchip In the present invention, the flow of liquid at the branching point is controlled to fractionate the fine particles. The chamber for realizing the pump function referred to in (4) is branched. By providing it in the vicinity of the point, the acoustic distance was shortened and a switching speed (conversion of the flow direction) that could not be realized by the switching operation of the external pump was realized.

  In addition, in the charge type cell sorter, there are many dead volumes, and when fractionating a small amount of sample, it is necessary to dilute the dispersion liquid and the operation time is sacrificed. The fractionation process can be performed in a short time.

(4) Microchip integration of pump / valve function In the present invention, the chamber that functions as a pump and a valve and also serves as a flow path is formed of an electromechanical conversion element, thereby realizing a reduction in chip size. Specifically, the chamber functions as a pressure chamber by expanding the flow channel in the groove depth direction to form a chamber and reducing the flow channel cross-sectional area of the branch point side outlet and the discharge side port. I did it.

  That is, when electric power is supplied to the electromechanical transducer so as to increase the chamber volume by bending the wall surface, the liquid flows from the branch point side and the discharge port side to compensate for the pressure drop inside the chamber. On the other hand, if power is supplied to the electromechanical conversion element so that the chamber volume is reduced by bending the wall surface, the liquid will flow out to the branch point side and the discharge port side in order to alleviate the pressure increase inside the chamber. .

  Therefore, the flow can be controlled by utilizing the above pressure change, and the fine particles can be guided to the selection flow path or the discharge flow path at the moment when they reach the branch point. These operations were realized in the microchip without installing valves or external pumps for fractionation.

Incidentally, when a piezoelectric material is used as the electromechanical conversion element, when an electric field is applied perpendicularly to the spontaneous polarization direction Ps, a thickness-slip distortion occurs on a plane parallel to the polarization direction and perpendicular to the electric field direction. The wall can be bent using the shear deformation of the d15 mode. (Details will be described in Examples.)
In addition, since the structure of both a selection flow path and a discharge flow path is made the same, and it arrange | positions symmetrically, since both characteristics can be equalized, a pump drive sequence can be simplified.

  According to the fractionation apparatus of the present invention, since the introduction of the sample solution, the identification, and the collection of the sorted solution after the fractionation process are performed in the closed system flow path, there is a risk of the sample solution being scattered in the atmosphere. In addition, the fractionation operation can be performed without causing contamination of the apparatus or environmental pollution.

  Also, if the microparticles are sufficiently discretely diluted to flow through the microchannels, unlike conventional cell sorters, stable fractionation can be performed simply by fixing only the flow rate of the sample solution as a control factor. Therefore, it is possible to provide an easy-to-use fractionation apparatus that does not require the skill of the operator.

  Furthermore, by exchanging tubes and disposable fractional microchips used for sending and receiving liquids for each experiment, maintenance-free contamination-free fractionation is possible, and the device itself can be simplified and miniaturized. Become.

  Of course, unlike conventional cell sorters, there is no charge to the droplets, no discharge process, and no collision with the collection container.

Hereinafter, the present invention will be described in detail with reference to the drawings. The present invention is not limited to the following embodiments.
(System configuration)
FIG. 1 shows an apparatus configuration for evaluating the fractionation microchip of the present invention. A commercially available microsyringe pump 200 having a flow rate adjusting function was used for feeding the dispersion. In the normal apparatus, various types of syringes 210 can be attached and detached, and can be selected according to the sample volume and the liquid feeding speed. The dispersion liquid fed from the syringe pump is introduced into the chip from the inlet 131 of the fractionation microchip 100 via the liquid feeding capillary 220.
A detection system using fluorescence was used as the identification means. The fluorescence image of the beads excited and emitted by the mercury lamp 300 is magnified by the microscope 310 and captured by the high-speed video camera 320, and the video output signal is captured as a moving image in the central control unit 330 and image processing is performed. In addition, the image-processed video signal is output to the monitor 340 and can be played back, or a live video can be monitored.

  When the orange or yellow-green beads flowing discretely in the flow channel chip 120 are recognized in the identification area 122 serving as a fixed observation point, the central control unit 330 delays from the flow rate of the dispersion liquid. After elapse of time td (adjustable artificially), a selection or discharge (non-selection) signal is transmitted to the driver unit 350. The output of the driver unit 350 is connected to the electrode terminals 116A, 116B and 117 of the fractionation microchip via the cable 360. Parameters such as the pump drive voltage (Vdd2) and pulse width (ton) can be arbitrarily set by the driver unit 350.

In the embodiment, the selection flow path and the discharge flow path have the same structure and are symmetrically arranged, so that the selection flow path and the discharge flow path are equivalent and interchangeable. Therefore, hereinafter, the identification symbols A and B are used for distinction. If the structure and arrangement of the two are different, it can be easily handled by adjusting parameters of the drive voltage and pulse width.
The fractionated beads are fractionated into a collection container 410 through a collection tube 400 connected to the discharge port A132A and the discharge port B132B of the fractionation microchip 100.

(Evaluation methods)
Using commercially available fluorescent beads for calibration of flow cytometer (diameter of about 6 μm) as fine particles, beads that emit orange with a UV light source (Fluoresbrite Polychromatic (PC Red)) and beads that emit yellowish green (Fluoresbrite Yellow Green (YG) )) Were mixed in equal amounts to prepare a dispersion with a concentration of 0.0025 vol%, and a fractionation test was conducted.

In the fractionation, the beads emitting orange light were sorted on the outlet A side, and the beads emitting yellow-green light were sorted on the outlet B side, and evaluation was made based on the success rate confirmed on the monitor screen.
Note that the values of the drive voltage (Vdd2) and the drive pulse width (ton) depend on the flow path structure and the like, but in this evaluation, good results were obtained with Vdd2 = ± 26 V and ton = 24 μs. Evaluation was conducted in a unified manner.

(Embodiment 1)
An embodiment in which a PZT monopolar plate (not a bonded structure) is used as the electromechanical coupling element will be described.

(1) Production of Actuator Chip FIG. 2 shows a production method of the actuator chip 110. For PZT, a commercially available HIP-treated material having a piezoelectric constant of about d15 = 950 pm / V was selected. A dry film 113 is laminated on the polarization + surface of a 0.8 mm thick PZT plate 111 polarized in the thickness direction to form a pattern as shown in FIG. Next, when a groove 114 having a depth of about 360 μm, a groove width of about 80 μm, and a linear portion of the groove bottom of 18 mm is formed by a dicing saw equipped with a blade having a diameter of 51.4 mm and a blade thickness of 78 μm, as shown in FIG. become. The flow path wall forming the pressure chamber had a thickness of about 60 μm.

Subsequently, the known oblique vapor deposition method is repeated twice, Ti and Au are laminated and deposited to a depth of about 180 μm from the opening surface on both sides of the groove wall, and the dry film is removed by the lift-off method. Is completed.
Six grooves are formed in the actuator chip, and the grooves 114A and 114B in FIG. 2B are flow paths through which the dispersion liquid flows. Note that a wall electrode (not shown) formed in the groove 114A and a wall electrode (not shown) formed in the groove 114B are electrically connected to the GND electrode terminal 117, and the dispersion is electrolyzed. Etc. are not generated.

In addition, two (four in total) grooves 114 adjacent to the groove 114A and the groove 114B are dummy grooves for electrode formation, and wall surface electrodes (formed on both wall surfaces of the grooves 114A and 114B serving as flow paths ( Wall electrodes 115 facing each other (not shown) are made conductive by a jumper pattern 118 and connected to electrode terminals A116A and B116B, respectively.
Therefore, the connection is made such that the wall forming the groove 114A and the wall forming the groove 114B can be bent independently.

  In addition, you may coat | cover protective films, such as parylene, as needed from the state of FIG.2 (c). In that case, the film on the surface of the electrode terminal must be removed in a separate process.

(2) Production of Base Substrate FIG. 3 shows a base substrate 130 of a fractional microchip. Since there are many cases where excitation is performed by ultraviolet rays, in the embodiment, a mirror-polished quartz plate having a thickness of about 0.52 mm is selected as a material. However, the present invention is not limited to this. After forming an opening at a predetermined position with a dry film, the electrode terminal notch 134 and five holes were processed by sandblasting. Further, the inlet 131 is finished into a round hole with a diameter of about 100 μm by secondary processing, and the after-branch inlets 133A and 133B and the outlets 132A and 132B into which the dispersion after branching flows are each finished into a round hole with a diameter of about 70 μm. .

(3) Manufacture of channel chip The channel chip 120 is shown in FIG. In this example, a mold is made using SU-8 (for structure formation) resist, which is a well-known technique in the production of microchannels, and the mold is taken with polydimethylsiloxane (PDMS) to obtain a thickness of 0.5 mm, high A channel chip having a T-shaped channel 121 having a thickness of 25 μm and a width of 50 μm was produced. Of course, any material may be used as long as it has characteristics (for example, translucency, smoothness, etc.) allowed by the identification means.

(4) Assembly of fractionation chip FIG. 5 shows an assembly diagram of the fractionation microchip of the present invention. The epoxy adhesive is thinly applied to one side of the base substrate, and the portions where the groove bottoms of the grooves 114A and 114B of the actuator chip intersect the surface coincide with the after-branch introduction ports 133A and 133B and the discharge ports 132A and 132B of the base substrate. Adhere and cure.
Next, the non-adhesive surface of the base substrate and the flow channel forming surface of the PDMS flow channel chip are activated with oxygen plasma for a short time, and then the three ends of the T-shaped flow channel are formed on the base substrate. Crimping is performed accurately so as to coincide with the rear inlets 133A and 133B and the inlet 131, and permanent bonding is performed. The fractionated microchip 100 was completed through the above steps.

(5) Operation Principle FIG. 6 shows the push-pull operation principle of the fractionation microchip. 6B is a schematic diagram viewed from the direction of the arrow by cutting along the alternate long and short dash line in the sketch of FIG. 6A. Further, since the selected flow path and the discharge flow path are equivalent, they are identified by A and B, and the pattern of the drive voltage applied to GND common to the terminals A and B in FIG. Further, the movement of the wall at that time is shown in the center, and the flow of the liquid in the flow path chip is indicated by the arrow on the left.

  Here, a description will be given of a case where beads that emit orange fluorescence flow to the A side when they flow. FIG. 6B shows a state in which the beads do not flow to the branch point (<td), and no voltage is applied to both the terminals A and B, and the wall is not deformed (normal state). It has become. In this state, the flow path on the A side and the flow path on the B side share a flow rate that is half of the liquid feeding amount.

  Next, FIG. 6C shows a state where the beads have arrived at the branch point (≈td). A negative voltage is applied to the terminal A so that the A-side chamber volume on the suction side increases, and at the same time the extrusion side. A positive voltage is applied to the terminal B so as to reduce the B-side chamber volume. When the drive voltage is properly selected, the wall can continue to be displaced (usually several tens of microseconds depending on the chamber shape), but it is possible to create a state where all the dispersion flows into the A side. Guided to the road. That is, a state in which the B-side flow path is instantaneously closed without a valve is created.

FIG. 6 (d) shows a return stage after the beads are taken into the A-side flow path after passing through the branch point. In order to return the A-side chamber to the normal state, it is set to the GND level (0V) and at the same time B The side chamber is also set to the GND level (0 V) in order to return to the normal state. In this case, the A-side flow path is closed, and all of the dispersion liquid flows into the B-side flow path.
FIG. 6E shows a state where the steady state is restored. By repeating such an operation, the traveling direction of the beads that have reached the branch point can be controlled.
The above operation cycle is sufficiently faster than the flow rate of the beads, but the concentration should be adjusted appropriately so that the subsequent beads do not reach the branch point during bead loading. There must be.

  Table 1 shows the selection evaluation results (Example 1) in the case of the push-pull operation shown in this example.

（実施の形態２）
実施の形態１の場合で、端子Ｂに電圧を印加せずにプッシュ動作（押し出し動作）を省略し、プル動作（吸い込み動作）のみを行なった場合の選別評価結果（実施例２）を同じく表１に示す。

(Embodiment 2)
In the case of the first embodiment, the selection evaluation result (Example 2) when the push operation (push-out operation) is omitted without applying the voltage to the terminal B and only the pull operation (suction operation) is performed is also shown in Table 1. It is shown in 1.

(Embodiment 3)
In the case of Embodiment 1, the pulling operation (suctioning operation) is omitted without applying a voltage to the terminal A, and the selection evaluation result (Example 3) when only the pushing operation (extruding operation) is performed is also shown in Table 1. It is shown in 1.

(Embodiment 4)
FIG. 7A to FIG. 7C show an embodiment in which the polarization direction of the actuator chip is antiparallel and stacked in series (chevron structure). However, the chip structure is the same as that of the first embodiment. As the material PZT, a laminated PZT 112 having a thickness of 180 μm on the front side PZT 112A and a thickness of 620 μm on the back side PZT 112B was used. The difference from Example 1 (1) is that electrodes are formed on the entire wall surface, and can be implemented by depositing Ti and Au by a sputtering method with good throwing power. The subsequent manufacturing procedure is the same.

The fractionation chip configured in this example can set the driving voltage lower than that in the first embodiment, and is effective when a larger chamber volume change is required. Table 1 shows the results of sorting evaluation according to this embodiment (Example 4).
Summarizing the above examples, it has been found that although the pulling operation (suction operation) or the push operation (push-out operation) can be performed separately, the push-pull operation is preferable in order to increase the fractionation speed. It has become clear that a piezoelectric material having a bonded structure (chevron structure) may be employed to further increase the fractionation speed.

  By the way, although evaluation using fluorescent beads was performed in the above-described embodiment, the same applies to a dispersion liquid (mixed phase liquid) in which suspended cells, bacteria, microorganisms, micelles, colloids, inorganic fine particles, etc. are suspended. Needless to say, it can be handled. In this embodiment, fluorescence fractionation is taken as a representative example. However, for example, fractionation based on the shape and size of microparticles based on an image, or fractionation based on differences in the amount of movement of microorganisms is possible. Yes, it does not limit the fractionation parameters.

It is a figure explaining the block diagram of the fractionation apparatus of this invention. It is a figure which shows the preparation methods of the actuator chip | tip of this invention. It is a figure which shows the base substrate of this invention. It is a figure which shows the flow-path chip | tip of this invention. It is a set figure of the fractionation microchip of the present invention. It is a figure which shows the principle of operation of the fractionation microchip of this invention. It is a figure which shows the preparation methods of an actuator chip at the time of using the bonding material of this invention.

Explanation of symbols

100 Fraction microchip 110 Actuator chip 111 PZT plate 112 Laminated PZT plate 112A Front side PZT plate 112B Back side PZT plate 113 Dry film 114 Groove 114A Groove A
114B Groove B
115 Wall electrode 116 Electrode terminal 116 A Electrode terminal A
116B Electrode terminal B
117 GND electrode 118 Jumper pattern 120 Channel chip 121 T-shaped channel 122 Identification area 123 Branch point 124 Branched channel 124A Branched channel A
124B Flow path B after branching
130 Base board 131 Inlet port 132 Outlet port 132A Outlet port A
132B outlet B
133 After-branch inlet 133A After-branch inlet A
133B Post-branch inlet B
134 Notch for electrode terminal 200 Micro syringe pump 210 Micro syringe 220 Liquid feeding capillary 300 Mercury lamp 310 Microscope 320 High-speed video camera 330 Central control unit 340 Monitor 350 Driver unit 360 Cable 400 Collection tube 410 Collection container

Claims (7)

An identification area for optically identifying the fine particles from the outside, which is a flow path in which the dispersion liquid in which the fine particles to be fractionated are suspended in the liquid is diverted from the inflow side to the outflow side and flows. A flow path that includes the branch point located downstream of the identification area, a collection flow path that communicates with the branch point and takes in the fine particles selected based on the identification data, and a discharge flow path that releases the non-selected fine particles. With
A flow path component of at least one of the sampling flow path and the discharge flow path is deformed, and the flow direction of the dispersion liquid at the branch point is controlled by changing the flow path volume. A fractionation microchip characterized by sorting.   2. The fractionated microchip according to claim 1, wherein a part of the flow path constituting member is constituted by an electromechanical conversion element, and the flow path volume is changed by energization.   The fractional microchip according to claim 2, wherein the electromechanical transducer is a piezoelectric body and is driven in a shear mode.   2. The fractionated microchip according to claim 1, wherein the piezoelectric body has a chevron structure in which polarization directions are antiparallel and are stacked in series, and is driven in a shear mode.   5. The fine particles are fractionated by a push-pull operation by deforming the flow passage walls of the sampling flow passage and the discharge flow passage so as to cause opposite volume changes. A fractional microchip according to claim 1.   The fractional microchip according to any one of claims 1 to 5, wherein a flow path structure downstream of the branch point has an equivalent and symmetric structure.   The fractionation microchip according to any one of claims 1 to 6, a pump for feeding the dispersion to the fractionation microchip, and an identification unit for identifying the fine particles in the delivered dispersion. And a central control unit that controls the flow path volume and the flow direction of the dispersion based on the identification data obtained by the identification means.

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