JP2019117064A - Particle detector and method for detecting particles - Google Patents

Abstract

To provide a particle detector and a method for detecting particles that can continuously and separately detect particles in a wide range.SOLUTION: The particle detector includes: a particle separation flow path 110 for separating particles into a direction perpendicular to the direction of fluid flow according to the diameter of the particles; and at least two particles recovery flow paths 102a and 102b connected to the particle separation flow path 110 separately, the particles recovery flow paths 102a and 102b having particle detection parts 103a and 103b with an aperture and an electric detector.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a particle detection apparatus and a particle detection method.

  Coulter method (electrical detection zone method; hereinafter ESZ) using electrical detection as a technique capable of accurately detecting minority particle groups by measuring particles one by one, for example, see Non-Patent Document 1 )It has been known. In this method, the information (signals) obtained from each detection means correspond to each particle on a one-to-one basis, so that individual particles can be evaluated, and the percentage included in numbers is small. Although particles can be measured accurately, there is a problem that the measurable particle size range (dynamic range) is relatively narrow. For example, in ESZ, the particle diameter is calculated using an electrical signal generated when particles are allowed to pass through a small hole called an aperture, but generally the dynamic range is said to be 2 to 60% of the aperture diameter. Also, in ESZ, if particles larger than the aperture are present, the particles will clog the aperture and will not be able to be measured thereafter, so it is necessary to take measurements after removing larger particles with a filter with an aperture smaller than the aperture diameter. It becomes. However, in the filtering operation, since a considerable amount of adsorption of particles to the filter substrate occurs, some loss of the sample is a concern. In addition, there is also a problem that the possibility of obtaining a quantitative result at this point is reduced, so it is not desirable to perform the filtering operation when the particle size distribution in the sample is accurately measured.

R. W. De Blois. et al, The Review of Scientific Instruments, Volume 41, Number 7, pp 909-916 (1970)

  An object of the present invention is to provide a particle detection apparatus and a particle detection method capable of detecting a wide range of particles individually and continuously.

  In view of the above problems, the present inventors separate particles in the direction perpendicular to the flow of fluid according to their particle diameter, and then use a detector to detect particle samples with a wide distribution. I found out.

  That is, according to the present invention, a particle separation channel in which particles are separated in a direction perpendicular to the fluid flow according to the particle diameter, and two or more particle collection streams branched and connected to the particle separation channel The present invention relates to a particle detection device having a channel, wherein the particle recovery flow path includes a particle detection unit including an aperture and an electric detector.

  Further, in the present invention, the particle diameter ranges detectable by the apertures of the particle detection unit in each particle recovery channel may be different from each other, or part of the particle diameter ranges detectable by the apertures may overlap each other .

  Furthermore, in the present invention, a flow path structure in which at least one parameter of the number of the particle collection flow paths, the shape, the width, the height, and the length of the branch portion is adjusted and particles of a certain size or more are not mixed I assume. Thereby, clogging of the aperture can be avoided. In addition, by adjusting these parameters, it is possible to cause particles of a particle diameter that can be detected by the apertures of each particle collection flow channel to flow into the particle collection flow channel.

  Furthermore, in the present invention, the particle separation channel may have a channel structure using the principle of pinched flow fractionation. Specifically, in the particle separation channel, a channel structure using the principle of pinched flow fractionation can be formed. This makes it possible to separate the particles according to the particle size in the direction perpendicular to the fluid flow.

In another aspect of the present invention, there is provided a method of detecting particles contained in a fluid, comprising:
The particles are separated in the direction perpendicular to the fluid flow according to their particle size,
Divide the separated particles into two or more channels,
There is also a method characterized in that the particles are detected by an electric detector including an electrode arranged across an aperture installed in the flow path.

  In the above method, the detectable particle size range of the electric detector may be different depending on the installed flow path, and even if a part of the detectable particle size range overlaps with each other installed flow path Good. In addition, by adjusting at least one parameter of number, shape of branch, width, height, and length, and having a flow channel structure in which particles of a certain size or more do not mix, two or more particles can be obtained. The particles may be separated by dividing into channels or using the principle of pinched flow fractionation. The method of the present invention is implemented by driving the particle detection device of the present invention as an example.

  According to the present invention, samples having a wider particle size distribution can be detected accurately and continuously, and a large amount of particle samples can be measured as compared with the separation technique of batch processing.

FIGS. 1 (a) and 1 (b) are diagrams showing an embodiment of the present invention, showing application of hydrodynamic filtration (HDF) to a particle separation channel 110. FIG. FIG. 1 (a) is a top view of the microchip 10, and FIG. 1 (b) is an enlarged view of the region 190 in FIG. 1 (a). FIG. 2A is a view showing an embodiment of the present invention, and shows an aspect in which two particle detection units 103 are arranged in series. FIG. 2 (b) is a diagram schematically showing how the current value being measured changes with time when one particle continuously passes through two particle detectors 103, and the upper graph shows It is measured by the upstream particle detection unit 103, and the lower graph shows that it is measured by the downstream particle detection unit 103. Here, for convenience, the positions of the vertical axes of the graphs of the two particle detection units 103 are shifted, but in actuality, the current values that become the baseline when the particles do not pass are substantially the same. FIG. 3A is a view showing an embodiment of the present invention, in which two apertures are arranged in series, and unlike FIG. 2, shows an aspect in the case where only one pair of electrodes is used. FIG. 3 (b) is a view schematically showing how the current value being measured changes with time when particles pass continuously through the two apertures. FIG. 4A is a view showing an embodiment of the present invention, and shows an aspect in the case where a plurality of particle detection units 103 are installed in parallel to one particle recovery flow channel 102. FIG. 4 (b) shows an equivalent circuit of FIG. 4 (a). FIG. 5A is a view showing an embodiment of the present invention, and shows an aspect in the case where a plurality of particle detection units 103 are installed in parallel with respect to one particle recovery channel 102, and a plurality of particles are shown. The detection part 103 shows the aspect which reduces the electrical interference between the several particle | grain detection parts 103 by applying a voltage from a downstream electrode in a fluidly. FIG. 5 (b) shows the equivalent circuit of FIG. 5 (a) and shows that a plurality of particle collection channels 102 are independent. FIG. 6 is a view showing an embodiment of the present invention, and shows an aspect in which a plurality of apertures are disposed in parallel to one particle recovery channel 102 and can be used as a lower cost apparatus. FIG. 7 shows an embodiment for manufacturing the particle detection unit 103 more inexpensively using the relay flow channel 60 and the electrode insertion port 59. FIGS. 8 (a) to 8 (c) are diagrams showing an embodiment of the present invention, showing an aspect in which HDF is applied to the particle separation channel 110. FIG. 8 (a) shows two particle recovery channels 102, FIG. 8 (b) shows three particle recovery channels 102, and FIG. 8 (c) shows particle recovery channels 102. 3 shows that the particle separation channel 110 has a branch channel 105. Fig.9 (a) is a figure which shows the flow of the fluid in the branch part 110A of FIG.8 (b), and FIG.9 (b) shows the enlarged view of the linear flow path part of Fig.9 (a). FIG. 10 (a) is a diagram showing the flow of particles in a straight flow channel under laminar flow conditions, and FIG. 10 (b) shows the trajectory of the flow of particles in the particle diffusion flow channel 110B. FIGS. 11 (a) to 11 (c) are diagrams showing an embodiment of the present invention, showing an aspect in which PFF is applied in the particle separation channel 110. FIG. 11 (a) shows two particle recovery channels 102, FIG. 11 (b) shows three particle recovery channels 102, and FIG. 11 (c) shows FIG. 11 (a). The enlarged view of the area | region 21 of (b) is shown. FIG. 12A is a view showing an embodiment of the present invention, and shows an aspect in which an asymmetric PFF is applied to the particle separation channel 110. 12 (b) and (c) show an enlarged view of the region 21 of FIG. 12 (a). FIG. 13A is a view showing an embodiment of the present invention, in which the asymmetric PFF is applied in the particle separation channel 110, and the wall surface on the narrowed channel wall surface 16b side of the expanded channel 17 is gradually enlarged Aspect of the invention. FIG.13 (b) shows the enlarged view of the area | region 21 of Fig.13 (a). FIG. 14 is a view showing an embodiment of the present invention, in which two apertures constitute the particle detection unit 103, the downstream of each aperture is connected to the outlet, and each outlet is also used as the electrode insertion port 59. The embodiment which is made to function is shown. FIG. 15A shows a schematic view of the microchip 10 used in the first embodiment. FIG.15 (b) shows the enlarged view of the area | region 21 of Fig.15 (a). FIG. 16 is a diagram showing an example of the electric signal of 0.1 μm, 0.2 μm particles detected by the particle detection unit 102 a in Example 1. FIG. 17 is a diagram showing an example of an electrical signal of 0.2 0.5 μm particles detected by the particle detection unit 102 c in the first embodiment. 18 (a) and 18 (b) are diagrams showing an example of electric signals of 0.5, 1.0, and 2.0 μm particles detected by the particle detection unit 102b in Example 1, and FIG. (A) shows 0.5 and 1.0 μm particles, and FIG. 18 (b) shows an electric signal of 2.0 μm particles. FIG. 19 shows the particle size distribution of the mixed particle sample prepared from the measurement results in Example 1. FIG. 20 shows the particle size distribution of the mixed particle sample prepared from the measurement results in Comparative Example 1. FIG. 21 shows the particle size distribution of antibody aggregate samples prepared from the measurement results in Example 2. FIG. 22 (a) is a view showing an embodiment of the present invention, in which three particle recovery channels and two in each particle recovery channel are provided after particle separation by HDF in the particle separation channel 110. The aspect provided with a particle | grain detection part is shown. FIG. 22 (b) shows an enlarged view of the area 21. FIG. 23 shows the particle size distribution of the mixed particle sample prepared from the measurement results in Example 3.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. However, the present invention can be practiced in different forms, and is not limited to only the embodiments and examples shown below.

  The details of the embodiment of the present invention will be described based on FIG. 1 which is a schematic view showing an example of the apparatus configuration of the particle detection apparatus according to the present invention. The cross section of the flow path in the microchip 10 is preferably rectangular in view of the ease of fabrication of the flow path structure, but may be a cross section such as a circle, an ellipse, or a polygon, or partially rectangular Other shapes may be used. Moreover, although it is preferable that the flow path height is uniform from the ease of preparation, the depth may differ partially.

  The sample containing the particles is introduced from the inlet 14 and sent to the downstream of the flow channel by the liquid sending portion, and the particle detection flow channel 101, the particle separation flow channel 110, the particle recovery flow channel 102a or 102b, respectively corresponding particle detection It passes through the section 103a or 103b and flows out to the outlet 104a or 104b. In the particle separation channel 110, the particles are separated in the direction perpendicular to the fluid flow according to the particle size, and the separated particles flow to the particle collection channel 102a or 102b. Electrical detection is performed in the particle detection units 103a and 103b that have arrived via the particle collection flow channel 102a or 102b. At this time, the insides of the particle detection units 103a and 103b are filled with a solution containing an electrolyte, and are connected to the electric measurement device 56 and the power supply 57 via the leads 55 respectively connected to the electrodes 54a and 54b. At the time of particle detection, a current of an arbitrary value flows by the power supply 57, and a closed circuit via the aperture 53 is formed. Further, the electric measuring device 56 is connected to the analyzing unit 61, and the detecting unit 61 calculates the detection signal obtained from the electric measuring device 56 to create a particle size distribution.

  The sample containing particles is a fluid containing particles to be measured. The particles in the present invention have a particle size in the range of 1 nm to 100 μm, preferably 10 nm to 10 μm, and include, for example, nucleic acids, proteins, vesicles, extracellular vesicles, inorganic powders, metal colloids, polymer particles, viruses, It includes cells, cell masses, protein aggregates and the like. The fluid in the present invention is a conductive fluid, preferably an aqueous solution containing an electrolyte, but a conductive oil or oil can also be used.

  The inlet 14 may have a structure that can hold a sample containing particles, and is preferably a concave structure. Further, as the material, metal, glass, or ceramics having a small amount of elution may be used, but in order to manufacture at low cost, it is preferable to be formed of a polymer material. The particle introduction channel 101 is formed between the inlet 14 and the particle separation channel 110. The particle introduction channel 101 is disposed to assist the separation of particles in the particle separation channel 110, but may be omitted to miniaturize the microchip 10.

  The liquid feeding unit may use a method of feeding liquid by a pressure gradient such as a syringe pump, a peristaltic pump, a pressure pump or the like, or an electroosmotic flow pump to suppress uneven velocity distribution in the flow path cross section of the microchip 10 May be used. In this case, the pipe connected from the pump is directly connected to the inlet 14 to feed the sample held in the inlet 14 by applying pressure to the sample. Alternatively, the pump may be connected to the outlet through a pipe, and the fluid in the flow path of the microchip 10 may be suctioned by applying a negative pressure to send the liquid. Furthermore, by making the liquid level of the inlet 14 higher than the liquid level of the outlet 104a or the outlet 104b, the liquid may be fed due to the liquid level difference, and in this case, the liquid feeding portion becomes unnecessary. In order to make a more quantitative measurement, it is preferable to pass the particles by a pressure gradient, and it is most preferable to use a pressure-fed pump with less pulsation.

  The flow rate of the liquid transfer unit is preferably set to an arbitrary value depending on the cross-sectional area of the flow path and the cross-sectional area of the aperture, and for example, is preferably set between 0.1 μL / hour and 1 mL / hour.

  Two or more particle collection flow paths 102 are provided in view of expanding the measurable particle size range (dynamic range). Here, the particle detection unit 103 is provided inside or downstream of the particle collection flow channel 102, and is used to detect particles flowing into the particle collection flow channel 102. At least one particle detection unit 103 needs to be provided for one particle collection flow channel 102, and two or more particle detection units 103 may be provided (FIGS. 2 to 6). Moreover, a flow path for collecting particles out of the measurable particle diameter range can also be further provided. In an aspect in which two or more particle detection units 103 are provided for one particle collection flow channel 102, the two or more particle detection units 103 may be arranged in series (FIGS. 2 and 3). For example, when two particle detection units 103 are arranged in series, it is possible to calculate the passage velocity of particles in the flow path from the interval of the signal generated from the same particle that has passed through two particle detection units 103. Since the particle passage speed is generally in proportion to the flow rate of the recovery channel 102, it is possible to estimate the flow rate of the fluid in the recovery channel. At this time, as shown in FIG. 2, it is preferable that the voltage applied to the electrodes be applied from an electrode on the upstream side of the upstream aperture 53 'and an electrode on the downstream side of the downstream aperture 53' '. When a voltage is applied from the electrode 54b 'on the downstream side of the upstream aperture 53' or the electrode 54a '' on the upstream side of the downstream aperture 53 '', a voltage drop occurs between the two electrodes and one of the apertures It is necessary to measure while considering the influence of the change of the current value on the other aperture current value. Therefore, when using the particle detection unit 103 including two or more apertures, the upstream electrode 54a 'of the upstream aperture 53' and the downstream side of the downstream aperture 53 '' as shown in FIG. The measurement may be performed using only the electrode 54 b ′ ′ of As shown in FIG. 7, an electrode insertion port 59 fluidly and electrically connected to the aperture through the relay channel 60 is provided upstream and downstream of the particle detection channel, and the electrode 54 The particles may be detected by ESZ by immersing them.

  Furthermore, in an aspect in which two or more particle detection units 103 are provided for one particle collection flow channel 102, the two or more particle detection units 103 may be arranged in parallel (FIGS. 4 to 6). According to this aspect, an excellent effect of increasing the amount of processing in proportion to the number of parallelized particle detection units 103 can be obtained. At this time, the electrodes 54 for electrical detection are disposed so as to sandwich the respective apertures, and measurement is possible if the number of particle detection units 103 is twice. On the other hand, as shown in FIG. 6, the measurement is possible even in the aspect in which one electrode is disposed on the upstream side with respect to each aperture and the number of the particle detection unit 103 is provided downstream. This embodiment is preferable from the viewpoint of cost reduction by reduction. Here, when the particle detection unit 103 is arranged in parallel and voltages are simultaneously applied to the respective apertures, if voltages are simultaneously applied to a plurality of electrode pairs disposed so as to sandwich the respective apertures, as shown in FIG. Since the equivalent circuit becomes complicated and the change in current value of one aperture affects the current value of the other aperture, it is necessary to take account of these effects. On the other hand, when it is desired to individually apply a voltage to each aperture and detect each individually, it can be implemented by making an aperture not to be measured open, without connecting to the ground or chassis, and other apertures mentioned above There will be no impact on it. At this time, a switching circuit may be used between the electrode 54 and the power supply 57 in order to bring it into an open circuit state, or it may be brought into an open circuit state by a relay system or a photo MOS system. It is preferable that the particle detection unit 103 construct a measurement system in which electromagnetic noise is not affected as much as possible at the time of measurement. In this respect, the switching circuit uses a photocurrent such as a photo MOS sensor rather than a relay method using an electromagnet. The method is preferred. However, as shown in FIG. 5, when a voltage is applied from the electrode on the downstream side of the aperture to the upstream side, the change in the current value of one aperture does not affect the current value of the other apertures. You may use.

The particle detection unit 103 includes an aperture 53 and an electric detector. The aperture 53 refers to a hole smaller than the flow passage diameter formed in the flow passage, and is defined by the particle detection flow passage 62 and the aperture forming structure 52. The cross-sectional shape of the aperture may take various shapes depending on the manufacturing process, and when it is processed by etching or laser irradiation, it takes a circular or elliptical shape, and is a polymer material such as polydimethylsiloxane (hereinafter PDMS) by photolithography and soft lithography. In the case of molding by The cross-sectional area of the aperture should be larger than the particle to be measured, but generally the particle size range that can be measured by ESZ is said to be 2 to 60% of the aperture cross-sectional area It is necessary to design according to the size of. Further, as shown in FIGS. 2, 3 and 14, the particle detection unit 103 may be configured by two apertures. Further, as shown in FIGS. 4 to 6, a plurality of particle detection units are provided to constitute a plurality of apertures, the downstream of each aperture is connected to the outlet, and each outlet also functions as the electrode insertion port 59 It is good also as composition made to do. In this case, if the volume of the apertures is approximately the same, the signals obtained from each aperture will be approximately the same. That is, it is possible to detect all the particles that have flowed into the recovery flow channel upstream of each aperture, and it can be said that this is a preferable embodiment from the viewpoint of quantitative measurement of the concentration. Since the sensitivity of particle detection by ESZ decreases in proportion to the flow path resistance between the aperture and the electrode, it is necessary to take this signal reduction into consideration when calculating the particle size from the obtained signal (equation (Refer to (1)).
The electric detector of the particle detection unit 103 mainly includes an electrode 54, an electric measuring device 56 connected to the electrode 54 via a lead 55, and a power source 57. The two electrodes 54 are disposed across the aperture 53. The electrical measuring device 56 may be any device that detects electrical characteristics, and may include a current measuring device, a voltage measuring device, a resistance measuring device, a charge amount measuring device, and a current measuring device may be used to measure ESZ. Most preferred. Further, it is preferable to detect a minute current value change by increasing the gain after current-voltage conversion using an IV amplifier in order to detect more minute particles. Also, in order to detect particles passing through the aperture without being dropped, the sampling time interval of the electric measuring device 56 is preferably sufficiently shorter than the time required for particles to pass through the aperture, and more than 10,000 times per second. It is preferable to sample, and it is more preferable to sample 20,000 times or more in 1 second.

  The analysis unit 61 can include an arithmetic device for calculating the measurement result, and a recording medium for recording the measurement result or the calculation result derived therefrom. Alternatively, the computing device and the recording medium may be integrated with the electrical measurement device 56 or may be an external device connectable to the electrical measurement device 56. The data recorded on the recording medium includes the sampled current value, the change in the current value generated when the particle passes, and the particle diameter, the number of particles, the particle concentration, the detection time or the measurement calculated from the change in the current value. It includes the elapsed time from the start time.

When using hydrodynamic filtration (HDF) in the separation method used in the particle separation channel 110, the end of the upstream part of the particle separation channel 110 is connected to the particle introduction channel 101, and the fluid flows out It may be connected to the particle collection flow path 102 via the branched part 110A at the end of the downstream part (FIG. 8). At this time, the branch portion 110A needs to be fluidly connected to at least two or more particle collection channels 102 in order to separate particles, and the downstream hydrodynamic resistance including the particle It is necessary to set the cross-sectional area and volume of each flow path and aperture in consideration. For example, when three recovery channels 102 are provided as shown in FIG. 8B and FIG. 9, assuming that the flow rates flowing to the respective recovery channels are Qa, Qb and Qc, the ratio of the respective flow rates is the width w of the channels. It is calculated from the height h and the length L, and specifically, the flow rate in the straight flow path is calculated according to the equation (2) from the Hagen-Poiseuille equation.

Further, as shown in the enlarged view 9 (b), the velocity distribution in the flow path of the microchip under laminar flow conditions is parabolic, and is generally represented by u (r) in the equation (3) (at this time, w 0 Is the radius of the tube, r is the distance from the center of the tube, μ is the viscosity, L is the length of the tube, and ΔP is the pressure drop.

  The ratio of the areas Sa, Sb, and Sc in the parabola divided by the arbitrary distances w1 and w2 from the channel wall is equal to the ratio of the flow rates Qa, Qb, and Qc flowing to the respective recovery channels. At this time, among particles present in the flow channel, particles whose center or center of gravity of the particles is present closer to the flow channel wall than w1 flow into the collection flow channel 102a, and are closer to the flow channel wall than w2. The particles present in flow into the recovery flow channel 102b, and the particles present between w1 and w2 flow into the recovery flow channel 102c. Therefore, the radius of the largest particle flowing to the recovery flow channels 102a and 102b is w1 and w2, respectively. Therefore, when the cross-sectional shape of the aperture is circular, the radius of the aperture needs to be w1 or w2 or more. When the cross-sectional shape is substantially circular or elliptical, the minimum radius passing through the center or center of gravity of the substantially circular or elliptical shape needs to be w1 or w2 or more. In addition, when the cross-sectional shape of the aperture is rectangular, it is necessary for each of the two pairs of opposing sides to be at least twice as long as w1 or w2. In addition, when the sectional shape of the aperture is a polygon, the radius of the inscribed circle needs to be w1 or w2 or more.

  Here, a mode may be adopted in which a plurality of branch portions 110A (not shown in FIG. 8) are provided at locations other than the end of the particle separation channel 110, and one or more branch channels 105 are provided (FIG. 8 (c)). . The branch flow channel 105 may be provided only on one flow channel wall side, or may be provided on both sides of the flow channel wall. Further, one or more branch channels 105 may be joined simultaneously in the downstream recovery channel 102, or may be gradually joined.

  Furthermore, the particle diffusion channel 110B may be formed on the fluid upstream side of the branch portion 110A (not shown in FIG. 10) of the particle separation channel 110 (FIG. 10 (b)). The fluid upstream side of the particle diffusion channel 110B is connected to the particle introduction channel 101, and the fluid downstream side is connected to the branch 110A. Further, in the case where the particle introduction channel 101 is not used, the fluid upstream side of the particle diffusion channel 110B is directly connected to the inlet.

  The particle diffusion channel 110B preferably has a structure in which the width or height, or both, of the channel is expanded from the fluid upstream side to the downstream side. This is different from the preferred embodiment in that there are a plurality of particle recovery channels in the channel width direction of the particle diffusion channel 110B or in the height direction. In the case of using a chip flow path forming technology, a structure in which the width of the flow path is expanded is preferable in terms of the easiness of the production thereof.

  In the particle diffusion channel 110B, the Brownian motion of the particle per unit time, that is, the fact that the diffusion distance is inversely proportional to the square root of the particle diameter, the particle flows as it flows through the particle diffusion channel 110B. Since the particles diffuse in the expanding direction of the flow path according to the diameter, in the vicinity of the expanded wall surface of the flow path, the probability of existence of particles having a small particle size increases, and a concentration effect is obtained. Here, the angle θa at which the flow path wall surface a and the particle diffusion flow path wall surface a are connected and the angle θb at which the flow path wall surface b and the particle diffusion flow path wall surface b are connected in FIG. Should be less than °. Further, under conditions of high flow rate such that the flow velocity in the flow channel surrounded by the flow channel wall surface a and the flow channel wall surface b is 1 m / sec or more, the occurrence of vortex flow in the enlarged portion may be concerned. It is preferable to feed the liquid under relatively low flow conditions such that it is less than the above, or to make the angles θa and θb larger than 90 °. On the other hand, when the particles flow into the particle diffusion channel 110B, the particles carry an inertial force in the direction in which the particles flow according to their weight (the direction in which the branch 110A is present in the hydrodynamic downstream direction). receive. In other words, when the particle density is the same, the larger the particle diameter, the more the particle with a smaller particle diameter will be present in the flow path wall, since the inertial force will be closer to the center of the channel. Contribute to the improvement of Further, the angles θa and θb may be asymmetrical, and one wall surface may be connected in a substantially straight line, and only the other wall surface opposed may be enlarged and connected at an angle of less than 180 °.

  When HDF is used in the separation method used in the particle separation channel 110, the particle size ranges that can be detected by the particle detection units 103a and 103b in FIG. 8 may be completely the same, but samples having a wide particle size range are measured. It is preferable to set different particle size ranges so that it is possible to detect particles that could not be separated if part of them overlaps with each other, and more robust results can be obtained. preferable. As shown in FIG. 8A, when there are two particle collection flow paths 102, the particle diameter range that can be detected by the particle detection units 103a and 103b makes the particle detection unit 103a a large particle diameter range, and the particle detection unit 103b may be set to a small particle size range, or vice versa. However, based on the HDF theory, it is necessary to set the aperture not to be blocked by particles.

  In the concentration conversion from the particle count number, the flow rate flowing to each particle collection channel 102 is calculated using the set flow rate of the liquid sending unit and equation (1), so the particle count number per measurement time By dividing by this, the particle concentration is calculated.

  Subsequently, an example of the configuration in the case of using a flow path utilizing the principle of pinched flow fractionation (PFF) in the particle separation flow path 110 is shown in FIG. When using a flow path utilizing the principle of PFF, the particle separation flow path 110 includes a branch flow path 18 a, a branch flow path 18 b, a narrowed flow path 16, and an enlarged flow path 17. The inlets 14a and 14b holding fluid 100P containing particles and fluid 100N not containing particles are fluidly connected to the branch flow channel 18a and the branch flow channel 18b, respectively. The branch flow channel 18 a and the branch flow channel 18 b merge downstream thereof and are fluidly connected to the upstream side of the narrowed flow channel 16. Thereafter, particle separation is performed in the enlarged flow path 17 connected to the downstream of the narrowing flow path 16 based on the principle of PFF. The channel width of the expanded channel 17 is constant at a predetermined position, and the particles are collected by the collection channels 102 a and 102 b fluidly connected downstream of the expanded channel 17 and connected downstream of the collection channel. The particles are detected by the detection units 103a and 103b. Here, although any number of recovery channels may be installed, it is preferably 5 or less, and most preferably 3 or less, in view of downsizing and cost reduction of the channels.

  Moreover, you may form the expansion flow path 17 based on the principle of asymmetrical PFF. In this case, as shown in FIGS. 12 and 13, the drain channel 22 may be provided in the expanded channel 17, and this embodiment is more preferable in terms of improving the particle separation performance. The drain channel 22 is connected to the outlet 23 so that fluid can be drained or collected. It is preferable to design that 50% or more of the fluid flows into the drain channel, and it is more preferable to design so that 70% or more of the fluid may flow.

  Furthermore, in the structure in which the narrowed channel 16 and the expanded channel 17 are connected, as shown in FIG. 12C, the channel width may be expanded stepwise at the expansion start point, as shown in FIG. Alternatively, as shown in FIG. 13 (b), the channel width may be gradually expanded. When the channel width is gradually expanded, the wall surface 17b of the expanded channel may be expanded to form a straight line to form the slope 40 or may be expanded to form a curve. When the channel width of the expanded channel 17 is expanded stepwise or linearly, it is between the wall surfaces 16a and 16b of the narrowed channel and the wall surfaces 17a and 17b of the expanded channel 17 following the wall surface. The enlarged flow path can be defined by the angles 24a and 24b of. For example, angles 24a and 24b in the case of stepwise expansion are 90 degrees. When the wall surface of the expanded flow path 17 linearly expands gradually, the angles 24 a and 24 b are represented by 90 ° to 180 °. The wall angles 24a, 24b of the enlarged area may independently be any angle between 90 ° and 180 °, for example 120 °, 135 °, 150 °, 180 °. However, even if the angle 24b of the enlarged flow path wall 17b with respect to the narrowing flow path wall 16b is 90 ° or less, it is a partial structure and the structure is such that it is substantially gradually expanded If this is the case, there is no problem because most of the fluid gradually flows in the direction of the outlet 23.

  At least two of the plurality of particle collection flow channels 102 connected downstream of the expanded flow channel 17 need to be provided, and it is preferable to optionally increase the number according to the particle diameter range to be measured. Each particle collection flow channel 102 has at least one particle detection unit 103, and a plurality of particle collection channels may be provided.

  The expanded channel 17 may have a function to further promote the separation ability of particles by giving the function as the particle diffusion channel 110B described in the paragraph of HDF, but in this case, the expanded channel The length needs to be long enough for the particles to diffuse, and at least 1 μm or more. The flow path length of the expanded flow path 17 is determined to an arbitrary value by the expanded flow path width and the angles 24 a and 24 b.

  In addition, a plurality of particle recovery channels 102 may be provided directly downstream of the narrowing channel 16 without using the expanded channel.

  When the particle separation channel 110 uses a channel that utilizes the principle of PFF, the diameter of the particles that can be present increases from the side of the narrowing channel wall 16a toward the side 16b, for example, as shown in FIG. In the case of a), it is preferable to set the cross-sectional area of the aperture so that the particle detection unit 103a detects small diameter particles and the particle detection unit 103b detects large diameter particles. In the case of FIG. 11C, the particle detection unit 103a detects small diameter particles, the particle detection unit 103c detects medium diameter particles, and the particle detection unit 103b detects large diameter particles. It is preferable to set That is, it is preferable to set the cross-sectional area of the aperture so as to be able to detect smaller particles as the particle detection unit 103 is closer to the narrowed flow passage wall surface 16a. For example, in the case of detecting particles of 0.1 to 2.0 μm, in the case of FIG. 11 (c), FIG. 12 and FIG. 13, the particle detecting unit 103a detects particles of 0.1 to 0.3 μm in diameter. In order to set the aperture cross-sectional area of 0.4 μm ^ 2 (a rectangular one with a width of 1 μm and a height of 0.4 μm or a circular one with a radius of 0.36 μm), 2) An aperture cross section of 2 μm ^ 2 (a rectangular one with a width of 2 μm and a height of 1 μm or a circular one with a radius of 0.8 μm) is installed to detect particles of 2 to 0.8 μm, In order to detect particles having a diameter of 0.4 to 2.0 μm, the particle detection unit 103 b has an aperture cross section of 14 μm 2 (a rectangular one having a width of 3.5 μm and a height of 4 μm, or a circular shape having a radius of 2.1 μm) ) May be installed.

  Moreover, when using the flow path using the principle of PFF in the particle separation flow path 110, it is preferable to set the flow rate set by the liquid transfer unit between 0.1 μL / hour and 1 mL / hour, and include particles. Preferably, the flow rate of the fluid 100N is twice or more than that of the fluid 100P containing particles. How many times the flow rate of the fluid 100N containing no particles is greater than that of the fluid 100P containing particles depends on the channel width of the narrowed channel 16 and the diameter of the particles to be separated, for example, separation If the diameter of the desired particle is 1⁄4 of the channel width of the narrowing channel 16, the flow rate of the particle-free fluid 100N is preferably at least three times higher than that of the particle-containing fluid 100P. When the diameter of the particles is 1/10 of the channel width of the narrowed channel 16, it is preferable that the flow rate of the fluid 100N containing no particles is 9 or more times the flow rate of the fluid 100P containing particles. That is, when the channel width of the narrowing channel 16 is N times the diameter of the particle to be separated based on the principle of PFF, the flow rate of the particle-free fluid 100N is higher than that of the particle-containing fluid 100P. It is preferable that N-1 times or more. With the flow rate ratio, particles to be separated will slide on the wall surface 16a of the narrowing channel, so particle separation based on the PFF principle becomes possible.

  Furthermore, when using the PFF principle in the particle separation channel 110, it is not necessary to cause all the particles contained in the fluid 100P containing particles to slide on the narrowed channel wall surface 16a, and relatively large particles are used. Only particles having a diameter may be made to slide on the narrowed channel wall surface 16a. For example, in the case of FIG. 11 (b), FIG. 12, and FIG. 13, if particles smaller than the maximum particle diameter detectable by the particle detection unit 103a that detects particles of small particle diameter are made to Even if particles smaller than the maximum particle diameter are not aligned with the narrowing channel wall surface 16a, the distribution can be created by the particle detection unit 103a based on the principle of ESZ. That is, even if there are particles that could not be separated by PFF, the particle size distribution can be created in the particle detection unit, so that the excellent effect that ESZ complements the separation ability of PFF can be obtained.

  In addition, even under flow conditions where only particles with a particle diameter greater than or equal to the maximum particle diameter detectable by the particle detection unit 103a that detects particles with small particle diameters can flow to the narrowing channel wall surface 16a, the above-mentioned HDF theory Based on this, by setting the flow channel resistance so that particles of a particle diameter that the aperture of the particle detection unit 103a blocks does not flow in, measurement of a sample having a wide particle diameter distribution, which is an object of the present invention, becomes possible. That is, separation of medium and large particles by PFF, separation of small particles by HDF, and precise particle size distribution by ESZ enable measurement of samples with a wide range of particle size distributions. The excellent effect of At this time, the expanded flow path 17 in PFF also plays the role of the above-mentioned particle diffusion flow path 110B, so that the excellent effect of promoting the separation of the small particle diameter particles having a large diffusion distance per unit time can be obtained. . Further, in this embodiment, the small particle diameter flows into the particle detection unit 103c, and in some cases also flows into the particle detection unit 102b, the small particle diameter flowing into the particle detection unit 103a includes the particle sample. It becomes part of the fluid 100P. If the length of the expanded channel 17 which plays the role of the particle diffusion channel 110B is a distance sufficient for the small particle diameter to diffuse, the inflow amount is the particle collection channel 102a and the downstream channel Because it is proportional to the flow rate calculated from the structure, it can be quantified from the calculated value. Here, “the distance sufficient for small-sized particles to diffuse” in the length of the expanded channel 17 means that the sum of the flow rate of the fluid 100N containing no particles and the flow rate of the fluid 100P containing particles is 0.1 μL. If it is the range of / hour to 1 mL / hour, 1 micrometer or more is preferable, More preferably, 100 micrometers or more are preferable.

Production Example: Production of Particle Detection Device The microchip 10 provided with the embodiment of the particle detection device according to the present invention was produced using general photolithography and soft lithography techniques. The concrete procedure is shown as follows.

  After dropping a photoresist SU-8 3005 (Microchem) onto a 4-inch bare silicon wafer (Philtech Inc.), a photoresist thin film was formed using a spin coater (MIKASA). At this time, according to the target film thickness, the diluent Cyclopentanone (Tokyo Ohka Kogyo Co., Ltd.) was added to SU-8 3005. Subsequently, a flow path pattern is formed on a photoresist film using a mask aligner (USHIO Inc.) and a chromium mask on which an arbitrary pattern is formed, and the flow path pattern is developed using SU-8 Developer (Microchem). In this way, a mold for the flow path desired to be used was produced.

  Subsequently, uncured LSR 7070 FC (Momentive Performance, Inc.) was poured into the produced mold and heated at 80 ° C. for 2 hours to produce polydimethylsiloxane (PDMS) to which the shape of the flow path was transferred. The cured PDMS was carefully peeled from the mold and formed into an arbitrary size with a cutter, and a puncher was used to form the inlet and outlet of the flow path. The surface of the peeled PDMS and slide glass (Matsunami Glass Co., Ltd.) was surface-treated with an oxygen plasma generator (Meiwa Pfosis Co., Ltd.), and then the PDMS and slide glass were attached to each other to produce a microchip 10.

Electrical Detection Example: Electrical Detection of Particles by ESZ The prepared microchip 10 was placed on a substrate, and electrodes were connected to a plurality of particle detection units 103 in the microchip 10. The electrodes are composed of a pair of platinum wires, and one of the electrodes is connected to a programmable current amplifier CA 5350 (NFC circuit company) via a conductor, connected to a PC via an AD converter, and transmitted digital signals It analyzed by LabView. The other of the electrodes connected to the particle detection unit 103 was connected to a 9 V dry cell via a lead.

  Each inlet was connected to a pressure pump P-PumpBasic (Dolomite) via a Teflon tube and fed at a constant flow rate.

Sample preparation example: When using standard particles When using standard particles as particles in fluid 100P containing particles to be separated :
Polystyrene standard particles 3100A (manufactured by ThermoFisher) as 0.1 μm particles Polystyrene standard particles 3200A (manufactured by ThermoFisher) as 0.2 μm particles
Polystyrene standard particles 3500A (manufactured by ThermoFisher) as 0.5 μm particles
Polystyrene standard particles 4009A (manufactured by ThermoFisher) as 1.0 μm particles
Polystyrene standard particles 4202A (manufactured by ThermoFisher) as 2.0 μm particles
Was used. As a fluid 100 P containing particles to be separated, a 1 × PBS solution (phosphate buffer solution) containing 0.05% (v / v) Tween 20 was used. The 1x PBS solution containing 0.05% (v / v) Tween 20 was used after removing foreign substances using a syringe filter (manufactured by Merck Millipore) with a pore size of 0.1 μm before the experiment.

Sample preparation Example: When antibody aggregates were used A mouse monoclonal antibody prepared specifically was prepared to 0.66 mg / mL with a PBS solution containing 0.05% (v / v) Tween 20 and dried The antibody aggregates were artificially produced by heating at 90 ° C. for 15 minutes.

Example 1: After particle separation by AsyPFF, three particle recovery flow paths and particle detection units installed in each particle recovery flow path by two particle detection units, according to particle detection devices 0.1, 0.2, Detection of 0.5, 1.0, and 2.0 μm Standard Particle Mixed Samples The microchip 10 shown in FIG. 15 is manufactured as a particle detection channel based on the above-described production example, and is based on the above-described sample preparation example. Prepared. 0.1, 0.2, 0.5, 1.0, 2.0 μm standard particles are each brought to a concentration of 5 μg / mL with 1 × PBS solution containing 0.05% (v / v) Tween 20. Prepared. Therefore, the concentration of the entire polystyrene particles contained in the sample was adjusted to 25 μg / mL.
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 unit 103a and the particle detection unit 103c, and the end of the flow passage 13 penetrates the upper surface of the substrate 11. The inlets 14a and 14b, and the outlets 104a, 104a ', 104b, 104b', 104c, 104c 'and 23 (the diameter of each hole is 2 mm) are provided. Further, the flow path 13 is a branch flow path 18a (width 20 μm, length 1.5 mm), branch flow path 18 b (width 40 μm, length 500 μm), constriction flow path 16 (width 6 μm, length 20 μm), enlarged flow path 17 (enlargement angle 135 degrees, flow path width 600 μm at maximum expansion, length 0.5 mm), drain flow path 22 (width 500 μm, length 1.7 mm), particle collection flow path 102 a (width 75 μm, length 4 mm), The particle collection flow channel 102 c (width 140 μm, length 7.5 mm) and the particle collection flow channel 102 b (width 512 μm, length 3.75 mm) were used. The two apertures of the particle detection unit 102a both have a width of 1 μm, a height of 0.4 μm, and a length of 10 μm, and the two apertures of the particle detection unit 102c each have a width of 2 μm, a height of 0.8 μm, and a length The two apertures of the particle detection unit 102b each have a width of 3.5 μm, a height of 4.5 μm, and a length of 20 μm. The shape of each particle detection unit is the same as in FIG. 14, and the k value calculated in equation (1) is 2 from the resistance ratio of the aperture and the flow path from the aperture to the outlet where the electrode is inserted. .6.

  Using the microchip 10 described above, send the particle suspension prepared to the inlet 14a at a flow rate of 2.5 μL / hour, and add 1 × PBS containing 0.05% (v / v) Tween 20 to the inlet 14b. The solution was fed at a flow rate of 10 μL / hour. Subsequently, the particles flowing into each of the three particle collection channels 102 were detected for one minute based on the above-described electric detection example. FIG. 16 shows a part of the measurement of the current value change of the particle detection unit 103a of the particle collection flow channel 102a at this time, and the measurement of the current value change of the particle detection unit 103c of the particle collection flow channel 102c. A part is shown in FIG. 17, and a part of a state of measurement of a current value change of the particle detection unit 103b of the particle collection flow channel 102b is shown in FIG. 16, 0.1 and 0.2 μm standard particles in the particle detection unit 103a, 0.2 and 0.5 μm standard particles in the particle detection unit 103c in FIG. 17, and 1.0 in the particle detection unit 103b and FIG. A change in the current value of the 2.0 μm standard particle was confirmed.

In addition, it was as shown in FIG. 19 when it was put together into one histogram as the measurement result, and it was confirmed that the peaks were separated even in the mixed particles. The concentration of 0.1 μm standard particles obtained from the measurement results is 5.41 μg / mL, the concentration of 0.2 μm standard particles is 3.52 μg / mL, and the concentration of 0.5 μm standard particles is , 6.29 μg / mL, the concentration of 1.0 μm standard particles was 5.02 μg / mL, and the concentration of 2.0 μm standard particles was 5.92 μg / mL. The average particle size of 0.1 μm standard particles obtained from the measurement results is 0.107 μm, and the average particle size of 0.2 μm standard particles is 0.192 μm, and the average particle size of 0.5 μm standard particles is The diameter was 0.52 μm, the average particle diameter of the 1.0 μm standard particles was 0.98 μm, and the average particle diameter of the 2.0 μm standard particles was 2.05 μm.
From the above, even with a sample having a wide distribution of 0.1 to 2 μm using the present invention, a plurality of apertures having different cross-sectional areas, that is, a particle detection unit 103 having a plurality of detectable particle diameter ranges, Particle size measurement is possible.

Comparative Example 1 Detection of 0.1, 0.2, 0.5, 1.0, and 2.0 μm Standard Particle Mixed Sample Using Particle Detection Device by Laser Diffraction Scattering (LD) As in Example 1 A mixed particle sample was prepared and measured by Aggregates Sizer (Shimadzu Corporation).
As shown in FIG. 20 showing the measurement results, the peaks of 0.2 and 2 μm disappeared and the peaks of 0.1 μm increased, so that it was confirmed that accurate measurement with the mixed particle sample was difficult.

Example 2: Detection of antibody aggregate sample by a particle detection device that performs particle detection by three particle recovery flow paths and two particle detection units installed in each particle recovery flow path after particle separation by AsyPFF Example The antibody aggregate produced based on the sample preparation example was similarly measured using the same microchip 10 as 1.
As shown in FIG. 21 showing the measurement results, it was confirmed that measurement was possible even in antibody aggregates.

Example 3: After particle separation by HDF, three particle recovery flow paths and particle detection units installed in each particle recovery flow path by two particle detection units: 0.1, 0.5, 0.5 Detection of 2.0 μm Standard Particle Mixed Sample The microchip 10 shown in FIG. 22 was prepared as a particle detection channel based on the above-described production example and was prepared based on the above-described sample preparation example. 0.1, 0.5, and 2.0 μm standard particles were prepared with a 0.05% (v / v) Tween 20-containing 1 × PBS solution to a concentration of 5 μg / mL, respectively. Therefore, the concentration of the whole polystyrene particles contained in the sample was adjusted to 15 μg / mL.

  At this time, for each flow path of the microchip 10, the height of the flow path 13 is 4.0 μm except for the particle detection portion 103a and the particle detection portion 103c, and the end portion of the flow path 13 penetrates the upper surface of the substrate 11. The inlet 14a, the outlet 104a, 104a ', 104b, 104b', 104c, 104c 'and 23 (the diameter of each hole is 2 mm) are provided. Further, the flow channel 13 includes a particle introduction flow channel 101 (width 20 μm, length 1.5 mm), a narrow flow channel 16 (width 6 μm, length 20 μm), an enlarged flow channel 17 (enlargement angle 135 °, flow channel width at maximum expansion) 800 μm, length 0.5 mm), particle collection flow channel 102 a (width 75 μm, length 4 mm), particle collection flow channel 102 c (width 140 μm, length 7.5 mm), particle collection flow channel 102 b (width 512 μm, length) It was 3.75 mm). The two apertures of the particle detection unit 102a both have a width of 1 μm, a height of 0.4 μm, and a length of 5 μm, and the two apertures of the particle detection unit 102c each have a width of 3 μm, a height of 1.2 μm, and a length The two apertures of the particle detection unit 102b each have a width of 3.5 μm, a height of 4.0 μm, and a length of 20 μm. The shape of each particle detection unit is the same as in FIG. 14, and the k value calculated in equation (1) is 3 from the resistance ratio of the aperture and the flow path from the aperture to the outlet where the electrode is inserted. .0.

  Using the microchip 10 described above, send the particle suspension prepared to the inlet 14a at a flow rate of 2.5 μL / hour, and add 1 × PBS containing 0.05% (v / v) Tween 20 to the inlet 14b. The solution was fed at a flow rate of 4 μL / hour. Subsequently, the particles flowing into each of the three particle collection channels 102 were detected for one minute based on the above-described electric detection example. At this time, 0.1 μm standard particles are confirmed as in the measurement of the current value change of the particle detection unit 103a of the particle collection flow channel 102a, and the current value of the particle detection unit 103c of the particle collection flow channel 102c The state of measurement of the change was confirmed to be 0.5 μm standard particles as in FIG. 17, and the state of measurement of the current value change of the particle detection unit 103b of the particle collection channel 102b was 2.0 μm standard particles as in FIG. confirmed.

The measurement results are summarized in one histogram, as shown in FIG. 23, and it was confirmed that the peaks were separated even in the mixed particles. The concentration of 0.1 μm standard particles obtained from the measurement results is 4.41 μg / mL, the concentration of 0.5 μm standard particles is 3.66 μg / mL, and the concentration of 2.0 μm standard particles is , 6.08 μg / mL. The average particle size of 0.1 μm standard particles obtained from the measurement results is 0.112 μm, and the average particle size of 0.5 μm standard particles is 0.470 μm, and the average particle size of 2.0 μm standard particles is The diameter was 2.09 μm.
From the above, even with a sample having a wide distribution of 0.1 to 2 μm using the present invention, a plurality of apertures having different cross-sectional areas, that is, a particle detection unit 103 having a plurality of detectable particle diameter ranges, Particle size measurement is possible.

DESCRIPTION OF SYMBOLS 10 microchip 11 substrate 11a substrate 11b lower surface 13 flow path 14, 14a, 14b inlet 16 narrowing flow path 16a sample liquid side narrowing flow path wall surface 16b sheath liquid side narrowing flow path wall 17 expanded flow path 17a sample liquid side expanded flow path wall surface 17b Sheath fluid side narrow expansion channel wall surface 18a, 18b Inlet side branch channel 19 Expansion start point 21 area 22 drain channel 23 outlet 24a, 24b Angle 25a, 25b Angle 40 Slope portion 50 Particle 51 Particle flow direction 52 Aperture formation structure 53 aperture 54, 54a, 54b electrode 55 lead 56 electric measuring instrument 57 power source 58 conductive solution 59 electrode insertion port 60 relay flow path 61 analysis unit 62 particle detection flow path 100 fluid 100 P fluid 100 N fluid 101 particle introduction flow path 102 ac Particle recovery flow 103a~c particle detector 104a~c outlet 110 particles separation channel 110A bifurcation 110B particle diffusion channel

Claims (10)

  Particle detection including a particle separation channel in which particles are separated in a direction perpendicular to a fluid flow according to the particle diameter, and two or more particle collection channels branched and connected to the particle separation channel The particle detection device according to claim 1, wherein the particle collection flow path includes a particle detection unit including an aperture and an electric detector.   The particle detection device according to claim 1, wherein the particle diameter ranges detectable by the apertures of the particle detection units in the respective particle collection flow paths are different from each other.   The particle detection device according to claim 1 or 2, wherein a part of the particle size ranges detectable by the aperture of the particle detection part in each particle collection channel overlap each other.   At least one parameter of the number, shape, width, height, and length of the branch portion of the particle collection channel is adjusted, and it is a channel structure in which particles of a certain size or more are not mixed. The particle detection device according to any one of claims 1 to 3, wherein   The particle detection device according to any one of claims 1 to 4, wherein the particle separation flow channel or a flow channel using a principle of pinched flow fractionation is formed in the particle separation flow channel. A method of detecting particles contained in a fluid, comprising:
The particles are separated in the direction perpendicular to the fluid flow according to their particle size,
Divide the separated particles into two or more channels,
Detecting the particles with an electrical detector comprising an electrode arranged across an aperture located in the flow path.   The method according to claim 6, characterized in that the detectable particle size range of the electrical detector is different depending on the installed flow path.   Method according to claim 6 or 7, characterized in that parts of the detectable particle size range of the electrical detector overlap each other by means of the installed flow channels.   At least one parameter of the number of flow channels, the shape, width, height, and length of the bifurcations is adjusted, and particles are made to have a flow channel structure in which particles of a certain size or more do not mix. The method according to any one of claims 6 to 8, wherein the flow path is divided into the above channels.   10. A method according to any one of claims 6 to 9, characterized in that the separating step separates particles using the principle of pinched flow fractionation.