Backflow generation mechanism in the particle detection section of the flow path structure

Abstract

To provide a mechanism that solves closing of an aperture of a channel structure.SOLUTION: For a channel structure including an aperture and a channel having an outlet on the downstream side of the aperture, a backflow generation mechanism pushes a pin structure into the outlet on the downstream side of the aperture, and brings the pin structure into contact with fluid that fills the outlet on the downstream side of the aperture to perform reverse cleaning, thereby solving closing of the aperture.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a mechanism for eliminating blockage in a particle detection section of a flow path structure.

Background Art

[0002] As a technique capable of accurately detecting a minority particle group by measuring particles one by one, the Coulter method (electrical detection zone method; hereinafter referred to as ESZ) using electrical detection is known. In this method, since the information (signal) obtained at the time of detection corresponds one-to-one to each particle, it is possible to evaluate each particle, and even particles with a small proportion can be accurately measured. In the ESZ method, the particle diameter is calculated using the electrical signal generated when particles pass through an aperture. Generally, its dynamic range is said to be 2 to 60% of the aperture diameter. In order to eliminate the narrow dynamic range, which is a drawback of the ESZ method, a method of classifying particles and detecting them by the ESZ method having different aperture diameters has been developed (for example, Patent Document: WO2018 / 147462). As a technique enabling continuous separation as in Patent Document 1, pinched flow fractionation (hereinafter referred to as PFF) using a microchannel is utilized.

[0003] In principle, the ESZ method has a problem that particle detection becomes impossible when the inside of the aperture is blocked. Blockage occurs when particles are adsorbed without passing through the aperture, or when particles having a size exceeding the aperture diameter flow into the aperture.

[0004] As a solution when the aperture is blocked, backwashing, that is, a method of creating a flow in the opposite direction to the direction in which particles flow to wash the aperture, is effective. However, the structure of the flow path (hereinafter referred to as PFF flow path) using the PFF method as in Patent Document 1 is complicated, and a method of creating a sufficient flow in the particle detection section in the flow path for backwashing has not been established.

Prior Art Documents

Patent Document

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] The problem of the present invention is to provide a mechanism for eliminating the blockage of the aperture of the flow path structure.

Means for Solving the Problems

[0007] As a result of intensive studies to solve the above problems, the present inventors have reached the present invention. That is, an aspect of the present invention is For a flow path structure including an aperture for particle detection and a flow path having an outlet on the downstream side of the aperture, Pushing a pin structure into the downstream outlet of the aperture, Contacting the fluid filled in the downstream outlet of the aperture with the pin structure and performing backwashing, A backflow generation mechanism characterized by eliminating aperture blockage.

Effects of the Invention

[0008] According to the present invention, it is possible to eliminate the blockage generated in the aperture of the flow path structure that can quantitatively evaluate minute particles in the nano- to micro-level range over a wide range.

Brief Description of the Drawings

[0009]

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Mode for Carrying Out the Invention

[0010] Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the drawings. However, the present invention can be implemented in different forms and is not limited only to the examples of the embodiments and examples shown below.

[0011] Details of embodiments of the present invention will be described based on FIGS. 1 and 3, which are schematic diagrams showing an example of a particle separation detection flow path.

[0012] As shown in FIG. 1, a fluid 100P containing particles and a particle-free fluid 100N are introduced from inlets 14a and 14b, respectively, which are fluid inlets, and are sent downstream in the flow path by a liquid feeding unit, and pass through a narrow flow path 16, a divergent flow path 17, a particle recovery flow path 102a or 102b or 102c, and corresponding particle detection units 103a or 103b or 103c, and then flow out to outlets 104a or 104b or 104c, which are fluid outlets. Particles flowing through the divergent flow path 17 flow into the particle recovery flow path 102, and electrical detection is performed at the particle detection unit 103 that has reached through the particle recovery flow path 102. At this time, the inside of the particle detection unit 103 and the outlet 104 are filled with a solution containing an electrolyte, and the electrodes 54a and 54b shown in FIG. 3 are immersed. Further, an electrical measuring instrument 56 and a power supply 57 are connected to the electrodes 54a and 54b via lead wires 55 connected thereto, respectively. When detecting particles, a current of an arbitrary value flows due to the power supply 57, and a closed circuit is formed through the aperture 53. Further, the electrical measuring instrument 56 is connected to an analysis unit 61, and the detection signal obtained from the electrical measuring instrument 56 is calculated by the analysis unit 61 to create a particle size distribution. The cross-section of the flow path in the microchip 10 is preferably rectangular for ease of manufacturing the flow path structure, but may also be circular, elliptical, polygonal, or the like, or may be partially non-rectangular. Also, the flow path height is preferably uniform for ease of manufacturing, but may be partially different in depth.

[0013] The flow path structure may include a plurality of apertures, and in that case, it is preferable to have one or more outlets on the downstream side of the apertures.

[0014] As the material of the flow path structure, various polymer materials such as polydimethylsiloxane (PDMS) and acrylic, glass, silicone, ceramics, various metals such as stainless steel, semiconductors, etc. can be used, and it is also possible to use a combination of any two types of substrates among these materials. However, in order to manufacture and provide the flow path itself at low cost, it is preferable to use a polymer material at least partially.

[0015] If the flow path is made of a stretchable material such as PDMS, the above-described pin structure is preferably made of a hard material, for example, various polymer materials such as acrylic, and various metals such as glass, silicone, ceramics, and stainless steel. Conversely, if the flow path is made of the hard material, a stretchable material such as PDMS or butadiene-styrene rubber can be used to press in the liquid.

[0016] The fluid in the present invention is an aqueous solution, preferably an aqueous solution containing a surfactant, but may contain salts. Further, a water-soluble organic solvent or a water-insoluble organic solvent can be used, and they may contain a surfactant or salts.

[0017] When the pin structure is press-fitted using a jig for pressing in the pin structure as shown in FIG. 9, it is preferable because it is simple and has good reproducibility. In order to prevent a gap from being formed when the pin structure is press-fitted into the outlet 104, the size of the pin structure preferably has the same cross-sectional area as the outlet 104, but the cross-sectional area of the pin structure may be larger as long as it can be press-fitted into the outlet 104. When using a jig, a plurality of pin structures may be provided. If the flow path is made of a stretchable material such as PDMS, the above-described pin structure is preferably made of a hard material, for example, various polymer materials such as acrylic, and various metals such as glass, silicone, ceramics, and stainless steel. Conversely, if the flow path is made of the hard material, a stretchable material such as PDMS or butadiene-styrene rubber can be used to press in the fluid.

[0018] When the pin structure is press-fitted into the outlet 104, it is preferable that the press-fitting condition is such that the fluid 100N filled in the outlet after the press-fitting contacts the pin structure. For adjusting the press-fitting condition, the amount of the fluid 100N filled in the outlet 104 may be adjusted, or the press-fitting distance of the pin structure may be adjusted.

[0019] The fluid 100N filled in the outlet is an aqueous solution in the present invention, preferably an aqueous solution containing a surfactant, but may contain salts. Further, a water-soluble organic solvent or a water-insoluble organic solvent can also be used, and they may contain a surfactant or salts.

Example

[0020] (Example 1) Using general photolithography and soft lithography techniques, the microchip 10 shown in FIG. 1 was fabricated. The specific procedure is the same as the method described in the examples of Patent Document 1. At this time, for each flow path of the microchip 10, the height of the flow path 13 is 4.5 μm for all except the particle detection parts 103a and 103c, and at the ends of the flow path 13, inlets 14a, 14b, outlets 104a, 104a', 104b, 104b', 104c, 104c', 23 (each hole diameter 1.9 mm, hole height 5 mm) penetrating the upper surface of the substrate 11 are provided. Further, the flow path 13 includes a branch flow path 18a (width 20 μm, length 1.5 mm), a branch flow path 18b (width 40 μm, length 500 μm), a narrow flow path 16 (width 6 μm, length 20 μm), a diverging flow path 17 (24b angle 135 degrees, maximum diverging flow path width 600 μm, length 0.5 mm), a drain flow path 22 (width 500 μm, length 1.7 mm), a particle recovery flow path 102a (width 75 μm, length 4 mm), a particle recovery flow path 102c (width 140 μm, length 7.5 mm), and a particle recovery flow path 102b (width 512 μm, length 3.75 mm). Also, the two apertures of the particle detection part 102a are both 1 μm in width, 0.4 μm in height, and 10 μm in length, the two apertures of the particle detection part 102c are both 2 μm in width, 0.8 μm in height, and 10 μm in length, and the two apertures of the particle detection part 102b are both 3.5 μm in width, 4.5 μm in height, and 20 μm in length.

[0021] To the outlet 104 of the microchip 10 with the aperture blocked (see Fig. 4), 10 μL of a 1×PBS solution (phosphate buffered saline) containing 0.05% (v / v) Tween 20 as fluid 100N (hereinafter referred to as PBS-T) was introduced, and the distance from the bottom surface of the outlet 104 to the gas-liquid interface was set to 3.5 mm. A pin structure was press-fitted into the outlet 104 with the jig shown in Fig. 9. At this time, the distance from the tip of the pin structure after press-fitting to the bottom surface of the outlet 104 was set to 3.0 mm, and it was press-fitted in a state where the fluid 100N filled in the outlet 104 was in contact with the pin structure. When the aperture 53 was observed under a microscope after press-fitting, it was confirmed that the blockage of the aperture 53 was eliminated (see Fig. 5).

[0022] (Comparative Example 1) Similar to Example 1, for the microchip 10 with the aperture blocked, 7 μL of PBS-T as fluid 100N was introduced into the outlet 104, and the distance from the bottom surface of the outlet 104 to the gas-liquid interface was set to 2.5 mm. A pin structure was press-fitted into the outlet 104 with the jig shown in Fig. 9. At this time, the distance from the tip of the pin structure after press-fitting to the bottom surface of the outlet 104 was set to 3.0 mm, and it was press-fitted in a state where there was a 0.5 mm gap and the gas-liquid interface of the fluid 100N filled in the outlet 104 was not in contact with the pin structure. When the aperture 53 was observed under a microscope after press-fitting, it was confirmed that the blockage of the aperture 53 was not eliminated (see Fig. 6).

[0023] (Comparative Example 2) Similar to Example 1, for the microchip 10 with the aperture blocked, 5 μL of PBS-T as fluid 100N was introduced into the outlet 104, and the distance from the bottom surface of the outlet 104 to the gas-liquid interface was set to 1.8 mm. A pin structure was press-fitted into the outlet 104 with the jig shown in Fig. 9. At this time, the distance from the tip of the pin structure after press-fitting to the bottom surface of the outlet 104 was set to 3.0 mm, and it was press-fitted in a state where there was a 1.2 mm gap and the gas-liquid interface of the fluid 100N filled in the outlet 104 was not in contact with the pin structure. When the aperture 53 was observed under a microscope after press-fitting, it was confirmed that the blockage of the aperture 53 was not eliminated (see Fig. 7).

Explanation of Reference Signs

[0024] Particles that block the 8 apertures 10 Microchip 11 Substrate 12 Substrate 13 Flow path 14a, 14b Inlet side ports 16 Narrow flow path 16a Sample liquid side narrow flow path wall surface 16b Sheath liquid side narrow flow path wall surface 17 Diverging flow path 17a Sample liquid side diverging flow path wall surface 17b Sheath liquid side diverging flow path wall surface 18a, 18b Inlet side branches 22 Drain flow path 23 Drain outlet 50 Particles 51 Direction in which the particles flow 52 Aperture formation structure 53 Aperture 54a, 54b Electrodes 55 Conductive wire 56 Electrical measuring instrument 57 Power supply 60 Relay flow path 61 Analysis unit 62 Particle detection flow path Fluid containing 100P particles Fluid not containing 100N particles 102a~c Particle recovery flow paths 103a~c Particle detection parts 104a~c Outlets

Claims

For a flow path structure including a narrow flow path for pinch flow fractionation (PFF), an aperture for particle detection, and a flow path having an outlet on the downstream side of the aperture, pushing a pin structure into the outlet on the downstream side of the aperture, bringing the fluid filled in the outlet on the downstream side of the aperture into contact with the pin structure for backwashing, a backflow generation mechanism characterized by eliminating aperture blockage.

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