JP7165346B2 - particle detector - Google Patents

Description

The present invention relates to a device for detecting particles.

Particles with a diameter of 0.1 to several μm, called subvisible particles, have attracted attention in the medical and pharmaceutical fields. Particles having a particle size within this range include exosomes, liposomes, virus-like particles, aggregates of antibody drugs, vaccine particles, and the like, and methods for their analysis and evaluation are eagerly awaited. There are several techniques for detecting these particles, but no method for detecting them with high accuracy is known. In the optical method, the detection limit is theoretically about 1/2 of the wavelength of the light source. , light sensor). There are currently no good light sources and detectors available at the measurement wavelengths below 400 nm, which are necessary for the detection of sub-visible particles. As a method of overcoming the limitations of optical techniques, there is a method of detecting scattered light from particles instead of directly observing particles.

As a device for detecting scattered light of particles, Aggregates Sizer (manufactured by Shimadzu Corporation), which is a device that uses a laser diffraction/scattering method (Non-Patent Document 1), and Nanosite that uses a nanotracking method (Patent Document 1). manufactured by Malvern Instruments).

Patent No. 4002577

JOURNAL OF PHARMACEUTICAL SCIENCES 104:618-626 (2015)

In a method of detecting particles while irradiating a microfluidic chip with light (for example, a laser diffraction/scattering method), the light emitted from the light source completely penetrates the chip substrate due to the roughness of the surface of the microfluidic chip. and scattered light is generated in the area where the light enters the microchannel chip. When the scattered light is incident on the detection section, there is a problem that the signal-to-noise ratio (S/N ratio) is lowered when detecting the scattered light generated from the particles flowing through the microchannel.

Accordingly, it is an object of the present invention to provide a method for detecting particles (especially sub-visible particles) using a microchannel chip by suppressing a decrease in the S/N ratio and detecting particles satisfactorily.

In order to solve the above problems, the inventors of the present invention have made intensive studies, and as a result, have invented the following particle detection device.

That is, the present invention
A first substrate and a second substrate bonded to the first substrate are provided, and a microchannel through which a fluid containing particles flows is formed between the channel structure excavated in the first substrate and the second substrate. a microfluidic chip,
a light irradiation unit that irradiates the fluid containing the particles with light from the first substrate side;
a detection unit that detects scattered light generated by the particles by irradiating the fluid with the light from the light irradiation unit;
A particle detection device comprising
The present invention relates to the apparatus, wherein scattered light generated in the first substrate by irradiating light from the light irradiation section is prevented from being detected by the detection section.

Specific examples of the above preventive measures include:
(1) disposing the light irradiator so that the scattered light generation region generated by the first substrate is out of the detection range of the detector;
(2) The first substrate is provided with a shielding plate for preventing the scattered light generated by the first substrate from being detected by the detector;
(3) applying a liquid having the same refractive index as that of the first substrate to the light incident area of the surface of the first substrate;
etc.

It is preferable to use a channel capable of classifying particles as the channel, since the particle size and number of particles can be continuously detected. Moreover, as the material of the first substrate, polydimethylsiloxane (PDMS), which is excellent in microfabrication performance, is preferable.

According to the present invention, in detecting particles using a microchannel chip, it is possible to satisfactorily detect particles (especially sub-visible particles) while suppressing a decrease in the S/N ratio.

1 is a cross-sectional view of one aspect of a conventional particle detection device; FIG. 1 is a plan view of one aspect of a conventional particle detection device, viewed from the first substrate side; FIG. The arrows in the figure indicate the direction of fluid flow. FIG. 2 is a plan view, viewed from the first substrate side, of one embodiment of the particle detection device of the present invention in which the light irradiation section is arranged such that the noise region is out of the detection range of the detection section; FIG. 3B is a plan view of the plan view of FIG. 3A with the light irradiation section, the first substrate, and the flow path left; In the figure, a is a line drawn perpendicular to the center line of the microchannel from the end of the light irradiation unit 400, starting at the intersection of the centerline 110 of the microchannel and the light emitted from the light irradiation unit 400. is the length of a straight line whose end point is the intersection of the center line and the center line. b in the figure is a straight line whose starting point is the end of the light irradiation unit 400 and whose end point is the intersection of a line drawn perpendicular to the center line of the microchannel from the end of the light irradiation unit 400 and the center line. length. In the figure, c is the angle formed by the center line 110 of the microchannel and the light irradiated from the light irradiation unit 400 in the plan view. 1 is a cross-sectional view of one embodiment of a particle detection device of the present invention, in which a first substrate is provided with a shielding plate; FIG. 1 is a plan view of one aspect of a particle detection device of the present invention in which a first substrate is provided with a shielding plate, viewed from the first substrate side; FIG. FIG. 2 is a cross-sectional view of one embodiment of the particle detection device of the present invention, in which the light incident area of the first substrate is coated with a liquid having the same refractive index as that of the first substrate; FIG. 2 is a plan view of one embodiment of the particle detection device of the present invention, in which the light incident area of the first substrate is coated with a liquid having the same refractive index as that of the first substrate, viewed from the first substrate side; 4 is a camera image obtained by observing scattered light and noise of particles in Example 1. FIG. 4 is a camera image obtained by observing scattered light and noise of particles in Example 2. FIG. 4 is a camera image obtained by observing the scattered light of the particles of Example 3. FIG. 4 is a camera image obtained by observing scattered light and noise of particles of Comparative Example 1. FIG. 7 is a camera image obtained by observing scattered light and noise of particles of Comparative Example 2. FIG. 10 is a graph showing the relationship between the number of observed bright spots and luminance in Example 3 (with silicone oil applied) and Comparative Example 3 (without silicone oil applied).

EMBODIMENT OF THE INVENTION Hereinafter, the form for implementing this invention is demonstrated in detail using drawing. However, the present invention can be implemented in many different forms and is not limited to the exemplary embodiments and examples shown below.

One aspect of a conventional particle detector is shown in FIG. The particle detection device shown in FIG.
A microchannel comprising a first substrate 200 and a second substrate 300 bonded to the first substrate, wherein a microchannel 100 through which a fluid containing particles flows is formed between the first substrate and the second substrate. a channel chip;
a light irradiation unit 400;
and a detection unit 600 that detects scattered light.

The light (preferably laser light) irradiated from the light irradiation unit 400 is irradiated to the fluid containing the particles 500 flowing through the microchannel 100, and travels straight without being scattered along with the scattered light 800 generated by the particles. Light 700 is separated. By detecting the scattered light 800 with the detection unit 600, the number of the particles 500 in the fluid flowing through the microchannel 100 and the positional information of the particles 500 can be detected.

A microchannel chip can be easily produced by using techniques such as molding, embossing, photolithography, soft lithography, wet etching, dry etching, nanoimprinting, laser processing, electron beam direct writing, and machining. As materials for the first substrate and the second substrate, PDMS, various polymer materials such as acrylic resin, glass, and the like can be used, and any two kinds of substrates among these materials can be used in combination. is also possible. Preferably, the material of the first substrate is PDMS. For ease of fabrication, it is preferable to set the width and depth of the microchannel to values ranging from submicrons to several hundreds of micrometers. In particular, when detecting particles having sizes on the order of nano- to micro-levels, it is preferable to set the width and depth of the micro-channel to values ranging from submicrons to several tens of micrometers. The bonding method of the first substrate and the second substrate is not particularly limited, and can be performed by a method using an adhesive, a thermocompression bonding method, an ultrasonic bonding method, or the like.

When the light irradiated from the light irradiation unit 400 in FIG. 1 is incident on the first substrate, scattered light (hereinafter also referred to as “noise”) is generated due to the roughness of the surface of the first substrate. When the scattered light generation region (hereinafter also referred to as a noise region 900) on the first substrate is in the vicinity of the scattered light generation region of the particles to be measured (see FIG. 2), the S/N ratio decreases and the particles It becomes difficult to detect the scattered light of

3A and 3B show one embodiment of the particle detection device of the present invention. The position of the light irradiation unit 400 is different from that of the particle detection device in FIG. As a result of intensive studies by the present inventors on the spatial arrangement of the light irradiation section, for example, in the plan view of the particle detection device of the present invention as seen from the first substrate side shown in FIG. It was found that noise can be excluded from the detection range by setting the angle (c in FIG. 3B) with the light emitted from the irradiation unit 400 to be more than 0 degrees and less than 180 degrees. When the angle is 0 degree or 180 degrees, the scattered light on the first substrate hits the particles flowing directly under it and is scattered. Since the scattered light from the particles generated here enters the detection range of the detection unit 600, the brightness of the entire detection range increases, and it is thought that the contrast of the scattered light of the particles in the detection range that is originally desired to be observed is lowered. . By removing noise from the detection range as described above, it is possible to suppress a decrease in the contrast of scattered light from particles in the detection range of the detection unit 600 .

4 and 5 show one embodiment of the particle detection device of the present invention. It differs from the particle detector of FIG. 1 in that a shielding plate 1000 is provided on the first substrate. The shielding plate 1000 may be placed on the surface of the first substrate, or the shielding plate may be embedded in the first substrate as shown in FIGS. The shield plate can prevent noise from entering the detection range of the detection unit. The position of the shielding plate is preferably between the noise region 900 and the scattered light generation region of the particles to be measured. Preferably, the shielding plate is installed perpendicular to the direction of fluid flow in the channel as shown in FIG. The shielding plate may be embedded until reaching the surface of the first substrate in contact with the microchannel as shown in FIG. It may be embedded to the extent that it does not reach. Although the size of the shielding plate is not limited, it is preferably larger than the line width of the light used, and more preferably larger than the width of the microchannel as shown in FIG. The color of the shielding plate is preferably black in order to prevent reflection of light.

6 and 7 show one embodiment of the particle detection device of the present invention. 1 in that a liquid 1100 having the same refractive index as that of the first substrate is applied to the light incident area on the surface of the first substrate. When the first substrate is a polymer resin, a liquid having the same refractive index can be applied by using a monomer corresponding to the polymer. For example, if the first substrate is PDMS, the liquid having the same refractive index as that of the first substrate may be silicone oil, and if it is an acrylic resin, methyl methacrylate. When the first substrate is glass, a similar effect can be obtained by applying alcohol or paraffin oil having a similar refractive index. As shown in FIGS. 6 and 7, by applying a liquid having the same refractive index as that of the first substrate, it is possible to prevent the generation of scattered light due to unevenness on the surface of the first substrate. As shown in FIG. 7, the area to be coated with the liquid should be equal to or larger than the line width of the light to be used, and there is no problem even if the entire surface of the first substrate is coated.

The microchannel used in the particle detection device of the present invention is preferably a channel capable of classifying particles. As such a channel, an embodiment using a pinched flow fractionation method (see, for example, M. Yamada, M. Nakashima, and M. Seki, Anal. Chem. 76, 5465-5471 (2004)) , an embodiment using hydraulic action (see, for example, M. Yamada et al., Lab on a Chip, 5, 1233-1239 (2005)), an embodiment using deterministic separation action (for example, Keith J. Morton. et al., Proceedings of National Academic Society, Vol. 105, No21, pp. 7434-7438 (2008)), embodiments using inertial force (e.g. Mehdi Rafeie et al., Lab on a Chip, 16 , 2791-2802 (2016)) can be exemplified.

The particles to be detected in the particle detection device of the present invention are not particularly limited as long as they are particles made of substances insoluble in the sample and fluid. Examples include industrial materials such as agents, research and medical materials such as cells, DNA, proteins such as antibodies, and viruses. The size (particle size) of proteins such as antibodies is generally about several nanometers, but there is a risk of aggregation and insolubilization when exposed to mechanical or thermal stress during the production process. The size (particle size) of the insolubilized protein is about several tens of nm to several tens of μm. Insolubilized proteins of several tens of nanometers can be separated and removed by conventional column chromatography or ultracentrifugation. However, insolubilized proteins corresponding to subvisible aggregates with a particle size of about 0.1 μm to 2 μm could not be detected with high accuracy by the above-described conventional method. The particle detector of the present invention can accurately detect even sub-visible aggregates.

In the present invention, as the fluid to be introduced into the microchannel chip, a liquid insoluble to the particles may be appropriately selected according to the particles to be detected. For example, when the particles to be detected are industrial materials, the solvent used during production may be used as it is, or may be replaced with an inexpensive and harmless solvent such as water. On the other hand, when the particles to be detected are biological samples such as cells, viruses, and antibodies, it is preferable to use the solvent used during production or preparation. Specifically, when the particles to be detected are cells, the culture medium, whole blood, plasma, physiological saline, PBS (Phosphate Buffered Saline), and TBS (Tris Buffered Saline) are used to ensure the survival of the cells. etc. are preferable. Additives such as surfactants, proteins, pH adjusters, stabilizers, thickeners, preservatives, antibiotics, polymers, and monomers may be added to the fluid.

The light irradiation unit that can be used in the method of the present invention may be appropriately selected according to the properties of the particles to be detected. The light irradiation part is not particularly limited as long as it can irradiate light with high directivity, and mercury lamps, tungsten lamps, fluorescent lamps, light emitting diodes, sodium lamps, xenon lamps, etc. can be used, but laser light sources (for example, green laser light sources) can be used. is preferred. In addition, when the particles to be detected are dyed with a fluorescent dye, a luminescent dye, or the like, it is preferable to use a light source corresponding to the excitation wavelength of the fluorescence. Moreover, it is preferable to use a light source of around 500 nm from the viewpoint of the output of the light source and the sensitivity of the detection section.

The detection unit used in the particle detection device of the present invention is not particularly limited, but examples include cameras, optical sensors, and microscopes. The detection unit may be arranged on the first substrate side (that is, the space located in the direction from the second substrate to the first substrate and in which the light irradiation unit is arranged). It may be arranged on the substrate side (that is, a space located in the direction from the first substrate to the second substrate and in which the light irradiation section is not arranged). Preferably, the detector is arranged on the second substrate side as shown in FIGS.

The present invention will be described in more detail below using examples and comparative examples, but the present invention is not limited to these examples.

As particles to be detected in the fluid, polystyrene standard particles 3100A (manufactured by ThermoFisher) as 0.1 μm particles and polystyrene standard particles 3500A (manufactured by ThermoFisher) as 0.5 μm particles were used. A 1×PBS solution (phosphate buffer) containing 0.05% (v/v) Tween 20 was used as the fluid containing particles to be detected. A 1×PBS solution containing 0.05% (v/v) Tween 20 was used after removal of foreign matter using a syringe filter with a pore size of 0.1 μm (manufactured by Merck Millipore) prior to the experiment.

In the following examples and comparative examples, an inverted microscope IX71 (Olympus) was used, and a 520 nm green LED light source (PGL-F-520-20 mW, manufactured by Changchun New Industries Optoelectronics Tech. Co., Ltd.) was used as the light irradiation unit. was used to irradiate the observation area, and the light scattered by the particles was observed using a digital CMOS camera ORCA-FLASH (Hamamatsu Photonics Co., Ltd.) as a detector.

Production Examples Production of Microchannel Chip Microchips used in the following examples and comparative examples were produced using general photolithography and soft lithography techniques. Specific procedures are shown below.

After dropping a photoresist SU-8 3005 (Microchem) onto a 4-inch bare silicon wafer (Filtec Co., Ltd.), a photoresist thin film was formed using a spin coater (MIKASA). A channel pattern is formed on a photoresist film using a mask aligner (Ushio Inc.) and a chrome mask with an arbitrary pattern, and the channel pattern is developed using SU-8 Developer (Microchem). A template of the desired channel was made.

Subsequently, a mixture of an uncured siloxane monomer and a polymerization initiator (weight ratio: 10:1) prepared using SYLGARD SILICONE ELASTOMER KIT (Dow Corning Toray) was poured into the prepared mold and heated at 80°C for 2 hours. By heating, a polydimethylsiloxane (PDMS) having the shape of the channel transferred was produced. The hardened PDMS was carefully peeled off from the mold, shaped into an arbitrary size with a cutter, and then a puncher was used to form inlet ports and outlets of the channels. After surface treatment of the exfoliated PDMS and slide glass (Matsunami Glass Co., Ltd.) with an oxygen plasma generator (Meiwa Forsyth Co., Ltd.), the PDMS and the slide glass were bonded together to prepare a microchip.

Example 1
In FIG. 3B, a was 14 cm, b was 5 mm, and the light irradiation part was installed so that the height from the surface of the first substrate (PDMS substrate) to the end of the light irradiation part was 10 cm. While irradiating with light, 0.2 μL of fluid containing particles (0.5 μm particles) was introduced into the microchannel. Next, a phosphate buffer solution was fed at a flow rate of 2 μL/h, and the microscope image of FIG. 8 was obtained. The noise area was far from the detection range, and the scattered light of particles could be clearly detected.

Example 2
A black shielding plate was embedded until it reached the surface of the first substrate in contact with the channel so as to be perpendicular to the direction in which the fluid flows in the channel of the microchannel chip manufactured above (Fig. 4 ). In FIG. 3B, a was 14 m, b was 5 mm, and the light irradiation part was installed so that the height from the surface of the first substrate to the end of the light irradiation part was 10 cm. A fluid containing particles (0.5 μm particles) was sent to the microchannel while irradiating light in the same manner as in Example 1, and the microscope image of FIG. 9 was obtained. The shielding plate prevented noise from entering the detection range, and the scattered light of the particles could be clearly detected.

Example 3
Silicone oil (manufactured by Shin-Etsu Chemical Co., Ltd.) was applied to the light incident area on the surface of the first substrate of the microchannel chip manufactured above. In FIG. 3B, a is 14 cm, b is 5 mm, and the light irradiation part was installed so that the height from the surface of the first substrate to the end of the light irradiation part was 10 cm. A fluid containing particles (0.1 μm particles) was fed while irradiating light in the same manner as in Example 1, and a microscope image of FIG. 10 was obtained. The noise generated in the first substrate was prevented by the silicone oil, and the scattered light of the particles could be clearly detected.

Comparative example 1
The microchannel chip used in Example 1 was used. In FIG. 3B, a is 14 cm, b is 0 mm, and the light irradiation section was installed so that the height from the surface of the first substrate to the end of the light irradiation section was 10 cm. Under the same conditions as in Example 1, a fluid containing particles was fed into the microchannel while being irradiated with light, and a microscope image of FIG. 11 was obtained. The scattered light of the particles was hidden by the noise and could not be clearly identified.

Comparative example 2
A microchannel chip having the same shape as that used in Example 2 was used except that the shielding plate was not provided. Under the same conditions as in Example 2, a fluid containing particles was fed into the microchannel while being irradiated with light, and a microscope image of FIG. 12 was obtained. The scattered light of the particles was hidden by the noise and could not be clearly identified.

Comparative example 3
The microchannel chip used in Example 3 was used. Under the same conditions as in Example 3 except that silicone oil was not applied to the light incident area of the first substrate surface of the microchannel chip, the fluid containing the particles was sent to the microchannel while irradiating with light. FIG. 13 shows the relationship between the number of bright spots detected by the detector and the luminance in Example 3 and Comparative Example 3. FIG. The brightness values were obtained by importing the detected image into software (ImageJ; distributed by the National Institutes of Health, USA), first obtaining a coordinate list of maximum values by Find Maxima, and then obtaining the brightness value of each coordinate by Measure. Compared to Example 3 (solid line), Comparative Example 3 (dashed line) has more noise (bright spots on the high-brightness side), so the number of observations of scattered light from particles (bright spots on the low-brightness side) is relatively reduced. I found out.

100: Microchannel 110: Center line of microchannel 200: First substrate 300: Second substrate 400: Light irradiation unit 500: Particles 600: Detection unit 700: Light 800: Scattered light generated by particles 900: Noise region 1000: shield plate 1100: liquid having the same refractive index as the first substrate

Claims (4)

A microchannel comprising a first substrate and a second substrate bonded to the first substrate, wherein a microchannel through which a fluid containing particles flows is formed between the first substrate and the second substrate. a chip;
a light irradiation unit that irradiates the fluid containing the particles with light from the first substrate side (where the irradiation unit is not arranged at a position where the light is irradiated perpendicularly to the first substrate) ;
a detection unit that detects scattered light generated by the particles by irradiating the fluid with the light from the light irradiation unit;
A particle detection device comprising
(i) to (iii) below:
(i) The light irradiator is arranged such that the angle between the center line of the microchannel and the light emitted from the light irradiator is greater than 0 degrees and less than 180 degrees;
(ii) A shielding plate is provided perpendicular to the direction in which the fluid flows in the channel of the microchannel chip and between the noise region and the scattered light generation region of the particles to be measured on the first substrate. and
(iii) a liquid having the same refractive index as that of the first substrate is applied to a light incidence area from the light irradiation unit on the surface of the first substrate;
characterized by comprising one or more means selected from
The apparatus according to the above, wherein scattered light generated by the first substrate is prevented from being detected by the detection unit by irradiating the light from the light irradiation unit. The device according to claim 1 , wherein the microchannel is a channel capable of classifying particles. 3. A device according to claim 1 or 2 , wherein the material of said first substrate is polydimethylsiloxane. When the material of the first substrate is polydimethylsiloxane, the liquid is silicone oil; When the material of the first substrate is acrylic resin, the liquid is methyl methacrylate; or 3. The device according to claim 1 or 2, wherein the liquid is alcohol or paraffin oil when the material of is glass.

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