JP7404639B2 - Method of filling fluid into channel structure - Google Patents

Description

The present invention relates to a method for filling fluid into a channel structure.

The Coulter method (electronic detection zone method; hereinafter referred to as ESZ) using electrical detection is known as a technique capable of accurately detecting minority particle groups. In the ESZ method, the particle diameter is calculated using an electrical signal generated when a particle passes through an aperture, and its dynamic range is generally said to be 2 to 60% of the aperture diameter. In order to eliminate the narrow dynamic range that is a drawback of the ESZ method, a method has been developed in which particles are classified and detected using the ESZ method having different aperture diameters (for example, Patent Document 1).

As a technology that enables continuous separation as in Patent Document 1, pinched flow fractionation (hereinafter referred to as PFF) using a microchannel is used. Since the PFF method uses hydrodynamic characteristics, it is not possible to perform the desired classification unless bubbles occur in the flow channel or the flow channel is completely filled with liquid. However, the flow channel structure using the PFF method Because of the complexity of the solution, it has been difficult to completely fill the inside of the channel with the solution by introducing the solution from the inlet.

WO2018/147462 publication

An object of the present invention is to provide a method for efficiently filling a fluid into a channel structure having a constricted portion without leaving any air bubbles.

In order to solve the above-mentioned problems, the present inventors have made extensive studies and have arrived at the present invention.

That is, one aspect of the present invention is
The present invention is characterized in that a channel structure having an inlet connected to a channel having a constricted portion is filled with fluid from a channel end hole other than the inlet.

Further, another aspect of the present invention is
For a channel structure including an inlet connected to a channel having a constricted region, an outlet connected only to the channel having the constricted region, and a filling outlet connected to a channel other than the channel having the constricted region , characterized in that the fluid is filled from the filling outlet.

According to the present invention, it becomes possible to efficiently fill a fluid without leaving any air bubbles into a channel structure that allows quantitative evaluation of nano- to micro-sized particles over a wide range of areas.

1 is a drawing of a flow path used in Example 1 and Comparative Examples 1 and 2. (a) is an overall view of the flow path, (b) is an enlarged view of region 21, and (c) is an enlarged view of region 103. (a) is a diagram showing the filling method in Comparative Example 1, (b) is an enlarged photograph of 105 after filling, and (c) is an enlarged photograph of 106 after filling. (a) is a diagram showing the filling method in Comparative Example 2, and (b) is an enlarged photograph of 107 after filling. This is an example of a pin for press fitting. This is an example of a jig equipped with a pin for easily pressurizing liquid.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described in detail using the drawings. However, the present invention can be implemented in different forms and is not limited to the embodiments and examples shown below.

A flow path having a narrowed portion is a flow path whose width is relatively narrower than that of the flow path connected to a certain flow path, or a flow path where the width of the flow path is partially narrowed in the middle of the flow path. Refers to the flow path. Examples of the flow path structure having an inlet connected to a flow path having a constricted area include a flow path structure that systemizes PFF (hereinafter sometimes abbreviated as PFF flow path), hydrodynamic filtration method, Examples include channel structures that systemize the field flow fractionation method (membrane portion is regarded as a constricted channel) and the ESZ method (aperture region). For example, in a PFF channel, a constricted channel is formed by merging a channel for a sample liquid (sample channel) and a channel for a sheath fluid (sheath channel), and an enlarged channel is connected to the constricted channel. , it is necessary in principle to have at least an outlet channel connected to the expanded channel, and the narrowed channel has a narrower channel width than the sample channel and the sheath channel. The channel corresponds to a channel having a constricted portion in the present invention. That is, by filling the PFF channel with fluid from the end (exit) of the outlet channel, it becomes possible to fill the fluid efficiently without leaving any bubbles.

In the case of a microchip 10 equipped with a particle separation detection flow path such as a combination of a PFF flow path and an ESZ method as shown in FIG. The structure is such that the inlet branches 18a, 18b, the constricted channel 16, the enlarged channel 17, the drain channel 22, the particle recovery channels 102a, 102b, 102c, and the particle detection channels 103a, 103b, 103c connect between the holes. (See Figures 1(a) and (b)). Apertures 53 are present in the particle detection channels 103a, 103b, and 103c (see FIG. 1(c)). Since the width of the particle detection channel is partially narrowed by the aperture, the particle detection channel corresponds to a channel having a narrowed portion in the present invention. That is, the outlets 104a, 104b, and 104c are outlets that are connected only to the flow path that has the constricted region, and the outlet 23 is connected to the drain flow path 22 (which is fluidly connected to the constricted flow path 16 and the enlarged flow path 17). If the fluid is filled from the outlet 23, it becomes possible to efficiently fill the fluid without leaving any air bubbles.

Methods for filling fluid include liquid transfer using a pressure gradient such as a syringe pump, peristaltic pump, or pressure pump, liquid transfer using an electroosmotic flow pump, liquid transfer using a liquid level difference, and liquid injection using a pin as shown in Figure 4. It is also possible to press fit using a structure. Further, as a method of introducing the fluid using a pin, it is preferable to use a jig to press in the pin as shown in FIG. 5 because it is simple and has good reproducibility.

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

If the flow path is made of a stretchable material such as PDMS, the pin structure described above is preferably made of a hard material, for example, if it is made of various polymeric materials such as acrylic, glass, silicone, ceramics, various metals such as stainless steel are preferred. On the other hand, if the channel is made of the above-mentioned hard material, the liquid can be press-injected by using a stretchable material such as PDMS or butadiene-styrene rubber.

The fluid in the present invention is an aqueous solution, preferably an aqueous solution containing a surfactant, but may also contain a salt. Moreover, oil or oil can also be used.

Example 1
A microchip 10 shown in FIG. 1 was manufactured using general photolithography and soft lithography techniques. The specific procedure is the same as the method described in Examples of Patent Document 1. At this time, for each channel of the microchip 10, the height of the channel 13 is set to 4.5 μm for all channels except for the particle detection section 103a and the particle detection section 103c. Inlets 14a, 14b, outlets 104a, 104a', 104b, 104b', 104c, 104c', and 23 (each hole diameter 2 mm) were provided. 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 narrowed flow path 16 (width 6 μm, length 20 μm), and an expanded flow path. 17 (24b angle 135 degrees, channel width at maximum expansion 600 μm, length 0.5 mm), drain channel 22 (width 500 μm, length 1.7 mm), particle collection channel 102a (width 75 μm, length 4 mm), A particle recovery channel 102c (width 140 μm, length 7.5 mm) and a particle recovery channel 102b (width 512 μm, length 3.75 mm) were used. The two apertures of the particle detection unit 102a are both 1 μm wide, 0.4 μm high, and 10 μm long, and the two apertures of the particle detection unit 102c are both 2 μm wide, 0.8 μm high, and long. The particle detection unit 102b has two apertures each having a width of 3.5 μm, a height of 4.5 μm, and a length of 20 μm.
7 μL of Milli-Q water was introduced into the outlet 23 as a liquid of 100N. The introduced liquid was press-fitted using the jig shown in FIG. 5, and it was confirmed that the liquid was uniformly press-fitted inside.

Comparative example 1
Into the microchip used in Example 1, 7 μL each of Milli-Q water was introduced as a 100N liquid into the inlets 14a and 14b. The degree of pressure injection of the introduced liquid was observed under a microscope, and it was confirmed that the liquid was not uniformly pressed into the interior (see FIGS. 2(b) and 2(c)).

Comparative example 2
Into the microchip used in Example 1, 7 μL of Milli-Q water was introduced into the outlet 104a as a 100N liquid. Introduced liquid When the introduced liquid was press-fitted using the jig shown in Figure 5 and the condition of the press-in was observed under a microscope, it was confirmed that the constricted area (aperture) was clogged with dust and could not be used as a flow path (see Figure 3(b)). .

8 Particles that blocked the aperture 10 Microchip 11 Substrate 12 Substrate 13 Channels 14a, 14b Inlet side ports 16 Constricted channel 16a Wall surface of the narrowed channel on the sample liquid side 16b Wall surface of the narrowed channel on the sheath liquid side 17 Enlarged channel 17a Sample liquid side Expansion channel wall surface 17b Sheath liquid side narrow expansion channel wall surface 18a, 18b Inlet side branch 19 Enlargement start point 21 Channel 22 Drain channel 23 Outlet 24b Angle 40 Slope portion 50 Particle 53 Aperture 54, 54a, 54b Electrode 62 Particle detection flow Channel 100N Fluid 102a-c Particle recovery channel 103a-c Particle detection section 104a-c Outlet 105 Particle recovery channel 106 Particle recovery channel 107 Aperture A

Claims (2)

A liquid filling method for filling a flow path structure with a particle separation detection flow path in which a particle detection portion is combined with a PFF flow path without leaving any air bubbles , the method comprising:
The flow path structure is
an inlet side branch flow path to which a plurality of inlet side flow paths are connected ;
a narrowed channel to which the inlet side branch channel is connected;
an enlarged flow path connected to the narrowed flow path;
a plurality of recovery channels connected to the enlarged channel and having an aperture portion for particle detection;
a drain flow path connected to the enlarged flow path;
Equipped with
The inlet side flow path is a flow path having a hole that is an opening used as an inlet for flowing liquid into the flow path structure,
The narrowed channel is a channel that functions as a part of the PFF channel,
The recovery flow path is a flow path that has a hole that is an opening used as an outlet for taking out the liquid from the flow path structure, and has a narrowed portion with respect to the expanded flow path,
The drain flow path has a hole that is an opening used as a part for taking in and out liquid from the flow path structure, and has a hole that is an opening used as a part for taking liquid in and out from the flow path structure, and has a hole in the narrowed flow path and the recovery flow path connected to the expanded flow path. a flow path wider than the narrowed portion included ;
Filling the flow path structure with liquid from an opening of the drain flow path through the drain flow path, which is the widest flow path among the flow paths having an opening connected to the flow path structure. A liquid filling method characterized by: 2. The liquid filling method according to claim 1, wherein the filling is performed by press-fitting the liquid with a pin through a hole that is an opening of the drain flow path .