WO2017149756A1

WO2017149756A1 - Particle analysis device and particle analysis method

Info

Publication number: WO2017149756A1
Application number: PCT/JP2016/056791
Authority: WO
Inventors: 樹高倉; 悠石毛
Original assignee: 株式会社日立製作所
Priority date: 2016-03-04
Filing date: 2016-03-04
Publication date: 2017-09-08

Abstract

A particle analysis device of the present invention is equipped with a measuring unit, and a measuring container that is provided with: a substrate having a plurality of fine pores; a first flow channel, which is in contact with, on the front surface of the substrate, the fine pores, and which separates some of the fine pores from each other; and a second flow channel in contact with, on the rear surface of the substrate, the fine pores. The measuring unit measures a flowing current or electric resistance between each of a plurality of detection electrodes that are provided on the front surface of the substrate or inside of the first flowing channel, and at least one facing electrode that is provided on the rear surface of the substrate or inside of the second flowing channel.

Description

Particle analyzer and particle analysis method

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

There is Patent Document 1 as background art in this technical field. Patent Document 1 describes a resistance pulse method for measuring a change in current when a particle passes through a nanopore, as a method for measuring particles contained in a sample solution, particularly a polymer including nucleic acid molecules. Patent Document 1 describes that “the present disclosure provides an array of nanopore detectors (or sensors) for molecular detection and / or nucleic acid sequencing”.

JP-T-2015-508896

In the resistance pulse method, the particles move from the cis side filled with the sample liquid to the other transformer side through the pores with the measurement. At this time, waste liquid in which particles after measurement are accumulated is generated on the transformer side. Therefore, in order to newly measure different sample liquids using the same pores, it is desirable to replace the cis-side sample liquid with a new sample liquid and also replace and wash the transformer-side waste liquid.

In the resistance pulse method, a method of arraying a plurality of pores and a plurality of electrodes is known in order to increase the volume of a sample to be measured. In Patent Document 1, after forming a lipid bilayer directly on each of a plurality of electrodes, pores are formed in the lipid bilayer, and particles are introduced outside the lipid bilayer. Therefore, since the inside covered with the lipid bilayer membrane on each electrode becomes the transformer side, it is difficult to replace and wash the waste liquid on the transformer side. Therefore, in the configuration of Patent Document 1, the same pore cannot be washed and repeatedly measured, and a new pore is formed when the same device is used for the same measurement multiple times. There is a need. Moreover, it is necessary to form a new pore device for each measurement, and there is a problem that costs such as time and cost are required.

In view of the above problems, the present invention provides a technique that enables repeated measurement using the same pore device in a particle analyzer having an array structure.

For example, in order to solve the above problems, the configuration described in the claims is adopted. The present application includes a plurality of means for solving the above-mentioned problem. To give an example, a substrate having a plurality of pores, and a surface of the substrate that is in contact with the plurality of pores and a part of the pores. A measurement container including a first flow path formed so as to divide the gap, and a second flow path in contact with the plurality of pores on the back surface of the substrate, and the surface of the substrate or the first flow path A plurality of detection electrodes provided inside and corresponding to each of the plurality of pores; at least one counter electrode provided on the back surface of the substrate or inside the second flow path; A voltage source that applies a voltage between the plurality of detection electrodes and the counter electrode; and a measurement unit that measures a current or electric resistance flowing between each of the plurality of detection electrodes and the counter electrode. A particle analysis apparatus is provided.

Further, according to another example, a substrate having a plurality of pores and a first flow path formed so as to be in contact with the plurality of pores on the surface of the substrate and to partition between some of the pores. A first introduction step of introducing a liquid having conductivity into the first flow path of the measurement container including a second flow path in contact with the plurality of pores on the back surface of the substrate, and the measurement container A second introduction step of introducing a conductive liquid into the second flow channel, and an operation step of operating the first flow channel or particles contained in the second flow channel to pass through the plurality of pores. A plurality of detection electrodes provided on the front surface of the substrate or inside the first flow path and arranged to correspond to each of the plurality of pores; and the back surface of the substrate or the second flow path Each of the plurality of detection electrodes using at least one counter electrode provided inside Particle analysis method comprising the step of measuring the current or resistance flowing between the counter electrode.

According to the present invention, repeated measurement using the same pore device is possible in a particle analyzer having an array structure. Further features relating to the present invention will become apparent from the description of the present specification and the accompanying drawings. Further, problems, configurations and effects other than those described above will be clarified by the description of the following examples.

It is a schematic diagram of one Example of the particle | grain analyzer by this invention. It is the top view which showed an example of the shape of the 1st flow path in a particle analyzer. It is the top view which showed an example of the shape of the 1st flow path in a particle analyzer. It is a schematic diagram which shows the equivalent circuit in the particle | grain analyzer of FIG. It is a calculation result which shows the crosstalk of a detection signal. It is an example of the flow of the analysis process using a particle analyzer. It is an example of the flow of the analysis process using a particle analyzer. It is a schematic diagram which shows the measurement result containing the crosstalk of a detection signal. It is a schematic diagram which shows the analysis method of the measurement result containing the crosstalk of a detection signal. It is a schematic diagram of one Example of the particle | grain analyzer by this invention. It is the top view which showed an example of the shape of the 1st flow path in a particle analyzer. It is the top view which showed an example of the shape of the 1st flow path in a particle analyzer. It is the top view which showed an example of the shape of the 1st flow path in a particle analyzer. It is a schematic diagram of one Example of the particle | grain analyzer by this invention. It is an example of the flow of the analysis process using a particle analyzer.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. In addition, the Example described below is an example and can be modified within a range not departing from the gist of the present invention. Also, a spot color illustrated or described with one exemplary aspect may be combined with a spot color with another aspect. The components already described in the drawings of the embodiments are given the same reference numerals to clarify the correspondence.

[Example 1]
FIG. 1 is a schematic view of an embodiment of a particle analyzer according to the present invention, and is a side sectional view of a measurement container. The particle analyzer includes a measurement container 10. The measurement container 10 is formed such that a substrate 101 having a plurality of pores 100 regularly provided in the plane, and the one surface of the substrate 101 is in contact with the pores 100 and part of the pores 100 are separated. The first flow path 102 and the second flow path 103 in contact with the pores 100 on the other side of the substrate 101 are provided. Here, for convenience, the surface of the substrate 101 in contact with the first flow path 102 is referred to as “front surface”, and the surface of the substrate 101 in contact with the second flow path 103 is referred to as “back surface”.

The inlet 110 and the outlet 111 are connected to the first channel 102, and the inlet 112 and the outlet 113 are connected to the second channel 103. A conductive liquid can be injected into the first flow path 102 and the second flow path 103 using the

corresponding inlets

110 and 112, respectively. In addition, from the first flow path 102 and the second flow path 103, it is possible to discharge the liquid having conductivity using the

corresponding outlets

111 and 113, respectively.

A flow path wall 104 is provided inside the first flow path 102, and the flow path wall 104 is formed so as to separate adjacent pores 100. As an example, the flow path wall 104 may be formed of PDMS (polydimethylsiloxane).

A plurality of detection electrodes 121 are provided in the vicinity of each pore 100 inside the first flow path 102. At least one counter electrode 122 is provided inside the second flow path 103. In FIG. 1, the detection electrode 121 and the counter electrode 122 are shown to be embedded in the measurement container 10, but the present invention is not limited to this. For example, a plurality of detection electrodes 121 may be provided on the surface of the substrate 101, respectively. In addition, at least one counter electrode 122 may be provided on the back surface of the substrate 101. The detection electrode 121 and the counter electrode 122 are provided so as to be in electrical contact with the liquid introduced into the first channel 102 and the second channel 103, respectively.

The detection electrode 121 and the counter electrode 122 are connected to each other outside the first flow path 102 and the second flow path 103 by a conducting wire, thereby forming a closed circuit. This circuit includes a voltage source 130, and the voltage source 130 applies a voltage between the detection electrode 121 and the counter electrode 122. The voltage source 130 is preferably a variable voltage source. In FIG. 1, the single voltage source 130 is configured to apply the same voltage to all the detection electrodes 121. However, independent voltage sources may be provided for the detection electrodes 121. Good.

The particle analyzer includes a measurement unit that measures current or electric resistance flowing between each of the plurality of detection electrodes 121 and the counter electrode 122. An ammeter 131 for measuring the current flowing through each detection electrode 121 is connected to the circuit as a measurement unit. Instead of the ammeter 131, an ohmmeter may be connected to the circuit. According to this configuration, the same measurement as described above can be performed by measuring the change in electrical resistance across the pores 100.

The particle analyzer includes a processing device (not shown) connected to the ammeter 131. For example, the processing device may be realized using a general-purpose computer. The computer includes at least a processor such as a CPU (Central Processing Unit), a memory, and a storage device such as a hard disk. The processing device receives the current value measured by the ammeter 131 as an input, and executes a predetermined process (for example, the contents described in FIGS. 8 and 9) on the current value. In addition, the processing device may record the current value measured by the ammeter 131 in the storage device. Further, the processing device may analyze the particles 20 based on the measured current value and output the analysis result. The processing apparatus may include a display unit such as a display, and the display unit may display the current value measured by the ammeter 131 and the analysis result.

The sample liquid is introduced into the first channel 102 or the second channel 103. Here, the sample liquid refers to a liquid in which the particles 20 are dispersed in a conductive liquid. Even if the sample liquid is introduced into either the first channel 102 or the second channel 103, the same measurement can be performed.

The particles 20 are nucleic acid molecules, protein molecules and aggregates thereof, metal nanoparticles, polymer beads, cells, and the like, and the particle size thereof is about 1 nm to 20 μm. The diameter of the pore 100 is large enough to allow the particle 20 to pass through. A pore having a diameter smaller than about 5 μm can be produced by applying a lithography technique to a semiconductor thin film. Moreover, pores having a diameter larger than about 5 μm can be produced by molding a resin material using a mold.

FIG. 2 is a top view showing an example of the shape of the first flow path in the particle analyzer shown in FIG. Two pores 100 are formed in the bottom surface of the first flow path 102, that is, in the substrate 101. The flow path wall 104 is provided so as to partially separate the two pores 100. Thus, the flow path wall 104 may be formed as a surface extending in the direction perpendicular to the flow direction when the conductive liquid is introduced into the first flow path 102. The flow path wall 104 may be formed by a structure integrated with the measurement container 10 or the substrate 101. The liquid introduced from the inlet 110 of the first flow channel 102 can go around the connecting portion 105 formed by the flow channel wall 104 and fill the entire inside of the first flow channel 102.

1 and 2, the substrate 101 is illustrated as including two pores 100, but the number of the pores 100 may be more than two. In FIG. 2, the connecting portion 105 is illustrated as having two locations, but the first flow path 102 of the measurement container 10 only needs to include at least one connecting portion.

FIG. 3 is a top view showing an example of the shape of the first flow path when the particle analyzer shown in FIG. 1 has a large number of pores. As described above, even when the plurality of pores 100 are provided, the flow path wall 104 can be provided so as to partially divide the pores 100. In the example of FIG. 3, the first flow path 102 includes a plurality of flow path walls 104. The plurality of flow path walls 104 have a cross-shaped cross section when viewed from above. The first flow path 102 is divided into 12 sections by a plurality of cross-shaped flow path walls 104. One pore 100 is disposed in each of the 12 compartments.

Next, the effect of the flow path wall 104 will be described. When the cis side or the transformer side of the plurality of pores 100 are connected via a liquid having conductivity, there is a problem that crosstalk of electrical signals occurs between the neighboring pores 100. Crosstalk here means that the electric signal intensity in the pores through which the particles have passed decreases, and at the same time, an electric signal change occurs in the neighboring pores through which the particles have not passed. According to the present embodiment, the electrical resistance between the plurality of pores 100 can be increased by the flow path wall 104, and the crosstalk can be reduced.

FIG. 4 shows an equivalent circuit when the substrate 101 has two pores in the apparatus shown in FIG. Hereinafter, the left pore 100 is referred to as a pore 1 and the right pore 100 is referred to as a pore 2 in the drawing of the apparatus of FIG.

4, the electrical resistance between the pore 1 and 2 wherein R is and its corresponding detection electrode 121, _{R I} is the electrical resistance of the liquid between the pore 1 and the pore _{2, R P1} and _{R P2} are , Corresponding to the electrical resistance in the pore 1 and the pore 2, respectively. _RP1 and _RP2 include electrical resistances from the pores 1 and 2 to the counter electrode 122, respectively, but these are usually sufficiently smaller than the electrical resistance of the pores themselves. The electrical resistance forms a bridge circuit, and the voltage V is applied by the voltage source 130. The current flowing through the detection electrode 121 corresponds to the current flowing through the electric resistance R on the equivalent circuit.

FIG. 5 shows a change in current when the electric resistance _RP1 is increased by 1% due to particles entering the pore 1 in the equivalent circuit shown in FIG.

The electrical resistance R _P1 of the pore 1 was increased by 1% from 1 MΩ (R _P1 = 1.01 MΩ). At this time, R _P2 = 1 MΩ, R = 10 kΩ, and R _I was changed to 1 kΩ, 10 kΩ, 100 kΩ, and 1 MΩ. The value of FIG. 5 shows the ratio of the current value to the original current value (before increasing the electrical resistance R _P1 by 1%). The higher the electrical resistance R _I, when the current value in a pore 1 is changed, a small change in the current value in the pore 2. For example, when the electrical resistance R _I = 1 MΩ, when the electrical resistance R _P1 of the pore 1 is increased by 1%, the current value in the pore 2 is hardly changed compared to the original current value. I understand.

The influence of cross-talk, but also current change occurs in the pores 2 which particles do not pass through, it became clear that crosstalk is reduced by increasing the electric resistance R _I between the pore 1 and 2. In particular, the electrical resistance R _I between the pores 100 is made greater than the electrical resistance between the pores 100 and its corresponding detection electrode 121, clear that can effectively reduce crosstalk became.

Therefore, the flow path wall 104 may be formed of a material and / or structure that increases the resistance between the adjacent pores 100. Preferably, when the flow path wall 104 is formed, the electrical resistance between each of the plurality of pores 100 and the corresponding detection electrode 121 is between the plurality of pores 100 and the adjacent pores 100. It is smaller than the electrical resistance of the liquid having the electrical conductivity.

According to this embodiment, by providing the flow path wall 104 in the first flow path 102, the connecting portion (liquid passage) 105 between the pores 100 is narrowed, and the electrical resistance between the pores 100 is increased. be able to. As a result, crosstalk can be reduced.

4 and 5 show an example in which the substrate 101 includes two pores 100. However, the substrate 101 includes three or more pores (for example, FIG. 3). However, the flow path wall 104 can be provided so as to obtain the same effect.

FIG. 6 is an example of a flow of an analysis process using the particle analyzer shown in FIG. As shown in FIG. 6, the particle analysis process includes a first introduction step S <b> 1 for introducing a conductive sample liquid in which particles 20 are dispersed in the first flow path 102 of the measurement container 10, and a second flow of the measurement container 10. The second introduction step S2 for introducing a conductive liquid into the path 103, and the voltage source 130 between the detection electrode 121 and the counter electrode 122 while operating the particle 20 to pass through the pore 100. And a measurement step S3 of applying a voltage and measuring a current flowing through each of the detection electrodes 121.

In the flow shown in FIG. 6, the case where the second introduction step S2 is performed after the first introduction step S1 has been described, but the first introduction step S1 may be performed after the second introduction step S2. As another example, the first introduction step S1 and the second introduction step S2 may be performed simultaneously. Moreover, although the case where the particle | grains 20 were introduce | transduced in 1st introduction | transduction process S1 was demonstrated, you may introduce | transduce the particle | grains 20 in 2nd introduction | transduction process S2 as already stated.

In the measurement step S3 in the flow shown in FIG. 6, electrophoresis by an electric field induced in the vicinity of the pore 100 may be used to operate the particle 20 so as to pass through the pore 100, which will be described later. Thus, pressure flow may be used. For electrophoresis, when negatively charged particles 20 are introduced into the first flow path 102, a negative voltage is applied to the detection electrode 121, and the first flow path 102 to the second flow path 103. The particles 20 may be driven. When positively charged particles 20 are introduced into the first flow path 102, a positive voltage is applied to the detection electrode 121, and the particles 20 are transferred from the first flow path 102 to the second flow path 103. What is necessary is just to drive.

FIG. 7 is another example of an analysis process flow using the particle analyzer shown in FIG. As shown in FIG. 7, in the particle analysis process, liquid is introduced into one or both of the first flow path 102 and the second flow path 103 immediately before the flow of the analysis process shown in FIG. A cleaning step S0 for cleaning is included. Thereby, it is possible to remove the substance remaining in the vicinity of the pore 100 and in the first and

second flow paths

102 and 103 by the previous measurement, and perform a more precise measurement.

The liquid used in the cleaning step S0 may be the same as the conductive liquid introduced into the first and

second flow paths

102 and 103 in the first introduction step S1 and the second introduction step S2. The cleaning liquid may have completely different properties. About 1st introduction process S1, 2nd introduction process S2, and measurement process S3, you may change an execution order similarly to the flow shown in FIG.

FIG. 8 is a diagram showing a change in current obtained in the particle analyzer shown in FIG. The vertical axis indicates the current value measured in each pore 100, and the horizontal axis indicates time. The current values of the plurality of pores 100 are displayed side by side so that the pores having the largest crosstalk are continuous. At times t ₁ and t ₂ , the particles 20 pass through the pores, and a decrease in current value is measured across a plurality of adjacent pores. The processing device may be configured to analyze an electrical signal including crosstalk between the plurality of pores 100.

For example, with respect to a series of electrical signals including such crosstalk, by searching for an extreme value in a three-dimensional graph as shown in FIG. 8, the pore through which the particle 20 has passed can be specified. . For example, the processing device receives an electrical signal from the ammeter 131 as input information and executes a process of searching for an extreme value in the three-dimensional graph. The processing apparatus may output a search result (for example, information on pores through which particles have passed).

FIG. 9 is a diagram showing the current change amount of each pore at the time when the particles pass in the current measurement data shown in FIG. The curve in the figure shows the theoretical value of crosstalk. By fitting the current change amount of each pore with a theoretical value, a signal value equivalent to that measured with a single pore is obtained from a series of data measured with multiple pores including crosstalk. Obtainable. The processing device may receive electrical signals as input information from the plurality of ammeters 131, and may perform a fitting process on the signal values of the respective pores based on the theoretical value of crosstalk. Moreover, in this analysis, you may calibrate using the data at the time of measuring a known particle | grain instead of a theoretical value.

The above particle analyzer and analysis process facilitate the exchange of the sample liquid and the injection of the cleaning liquid in the pore sensor array, and enable repeated measurement using the same pore device (the substrate 101 having the pore 100). Become. Therefore, the cost for measurement can be reduced. Further, according to the present embodiment, the crosstalk of the electric signal between the plurality of pores 100 can be reduced, and the signal including the remaining crosstalk can be analyzed.

[Example 2]
FIG. 10 is a schematic view of an embodiment of the particle analyzer according to the present invention. Constituent elements described in the above-described embodiments are denoted by the same reference numerals and description thereof is omitted. In the present embodiment, the pressure control unit 140 is connected to the apparatus described in the first embodiment.

The pressure control unit 140 can generate a pressure flow by applying a differential pressure across the pore 100 to the first channel 102 and the second channel 103. In addition, illustration of an electric circuit is abbreviate | omitted in FIG. The pressure control unit 140 is connected to the outlet 113 of the second channel 103, but is connected to the inlet 110 of the first channel 102, the outlet 111 of the first channel 102, or the inlet 112 of the second channel 103. May be. Further, the pressure control unit 140 may also be used as a pump used when liquid is introduced into the first channel 102 or the second channel 013.

The pressure control unit 140 may be a pump that directly sends liquid, or a pump that generates a differential pressure by operating a syringe filled with gas. Further, the pressure control unit 140 may be a mechanism that generates a differential pressure by giving a height difference between the liquid level of the first flow path 102 and the liquid level of the second flow path 103. The differential pressure between the first channel 102 and the second channel 103 is typically in the range of 200 hPa or less, preferably in the range of 100 hPa or less.

The pressure control unit 140 is operated so that the flow path into which the particles 20 are introduced has a higher pressure than the other flow path. That is, when the particles 20 are introduced into the first flow path 102, the pressure control unit 140 operates so that the pressure in the first flow path 102 is higher than the pressure in the second flow path 103. In order to perform such an operation, for example, in the example shown in FIG. 10, the inlet 110 of the first flow path 102 or the outlet 111 of the first flow path 102 is opened to the atmospheric pressure, and negative pressure is applied to the pressure control unit 140. Apply pressure.

In Example 1, the driving force for the particles 20 to pass through the pores 100 is an electric field. However, when the particle 20 does not have a sufficient surface charge, or when the diameter of the pore 100 is about 1 μm or more and the electric field strength in the vicinity of the pore 100 is small, the electrophoretic force becomes small, and the efficiency In particular, the particles 20 cannot be driven. According to the present embodiment, even in these cases, it is possible to efficiently drive the particle 20 by the pressure flow generated by the pressure control unit 140 and perform resistance pulse measurement.

[Example 3]
FIG. 11 is a top view showing an example of the shape of the first channel 102 in one embodiment of the particle analyzer according to the present invention.

In this embodiment, the plurality of flow path walls 104 are arranged along the flow direction when the conductive liquid is introduced. With this configuration, the first flow path 102 includes a plurality of divided flow paths 106. At least one pore 100 is disposed in each divided flow path 106. In the example of FIG. 11, three pores 100 are arranged in each divided flow path 106. The ends of the divided flow paths 106 are connected by a connecting portion 105.

In addition, it is preferable that the division | segmentation flow path 106 is extended along the flow direction when the liquid which has electroconductivity is introduce | transduced into the 1st flow path 102 from a viewpoint that the flow of a liquid is not prevented. For this configuration, the plurality of flow path walls 104 are formed to be parallel to the flow direction when the conductive liquid is introduced into the first flow path 102. Further, when a plurality of pores 100 are arranged in each divided flow channel 106, the pores 100 are arranged in a line along the flow direction when a conductive liquid is introduced into each divided flow channel 106. Preferably they are arranged.

According to the present embodiment, by arranging the pores 100 in one row along the divided flow channel 106, the number of pores closest to the fine pores 100 arranged in the divided flow channel 106 is two. The number of pores closest to the pore 100 arranged at the end of the divided flow channel 106 is three at the maximum. Thereby, compared with the shape of the 1st flow path 102 illustrated in FIG. 3 of Example 1, for example, even when it has many pores, the number of the pores nearest to each pore 100 can be reduced. it can. Therefore, crosstalk can be further reduced.

[Example 4]
12 and 13 are top views showing an example of the shape of the first channel 102 in an embodiment of the particle analyzer according to the present invention.

In the present embodiment, the first flow path 102 is a flow path having no branch, and a plurality of pores are arranged in a line along the flow direction when the conductive liquid is introduced into the first flow path 102. Has been placed. In the example of FIG. 12, the first flow path 102 has a structure that is folded back in a zigzag shape. In the example of FIG. 13, the first flow path 102 has a spiral structure.

According to the present embodiment, by arranging the pores in a line along the first flow path 102, the number of pores closest to each pore 100 becomes two at the maximum. This corresponds to a shape in which the connection part 105 at the end of the divided flow path 106 is omitted from the first flow path 102 illustrated in FIG. Such a shape of the first flow path 102 can reduce the number of pores that are closest to each of the pores 100 even when the plurality of pores 100 are provided. Therefore, it is possible to reduce crosstalk more effectively.

[Example 5]
FIG. 14 is a top view showing an example of the shape of the first channel 102 and a method for introducing a conductive liquid in an embodiment of the particle analyzer according to the present invention.

The shape of the first flow path 102 of the present embodiment is the same as that of the example shown in the fourth embodiment, but a droplet generation unit 150 is connected to the front stage of the inlet 110 of the first flow path 102. The droplet generation unit 150 generates a droplet 151 in a state where the particles 20 are included in a conductive liquid. The droplet generation unit 150 introduces the generated droplet 151 into the first channel 102 from the inlet 110. Between two adjacent droplets 151 are filled with oil droplets or bubbles 152. Therefore, the droplet generation unit 150 may be configured to introduce the droplets 151 and the oil droplets or bubbles 152 into the first flow path 102 alternately.

The oil droplet or bubble 152 may be a liquid or gas that is not mixed with the droplet 151 and does not have conductivity. In addition, when the droplet 151 is introduced at a large flow rate using a pressure flow, and the volume of the droplet 151 changes greatly before and after the measurement, bubbles are used so as to allow the volume change between the droplets 151. Is preferred.

According to the present embodiment, a hydrophobic liquid that does not have gas or conductivity between the plurality of droplets 151 along the flow direction when the conductive liquid is introduced into the first flow path 102. Divided by. When the space between the plurality of droplets 151 is filled with a hydrophobic liquid or gas having no electrical conductivity, the electrical resistance between the pores 100 becomes infinite. As a result, the crosstalk between the pores 100 can be completely removed while the liquid can be replaced and washed using the first flow path 102.

FIG. 15 is an example of a flow of an analysis process using the particle analyzer shown in FIG. As shown in FIG. 15, the particle analysis process is a process in which a part of the particle analysis process shown in FIG. 6 is replaced. The particle analysis process includes a droplet generation step S4 in which the droplet generation unit 150 generates a liquid droplet 151 having conductivity including the particles 20, and a conductive liquid including the particles 20 in the first flow path 102. A droplet introducing step S5 for introducing the liquid droplet 151, a second introducing step S2 for introducing a conductive liquid into the second flow path 103 of the measurement container 10, and the particles 20 passing through the pores 100. A measurement step S3 in which a voltage is applied between the detection electrode 121 and the counter electrode 122 using the voltage source 130 and the current flowing through each of the detection electrodes 121 is measured. In the measurement step S <b> 3, the droplet 151 is positioned so as to be in contact with the pore 100 and the detection electrode 121 in the first channel 102.

In the flow shown in FIG. 15, the case where the second introduction step S2 is performed after the droplet introduction step S5 has been described, but the droplet introduction step S5 may be performed after the second introduction step S2. As another example, the droplet introduction step S5 and the second introduction step S2 may be performed simultaneously. Moreover, although the case where the particle | grains 20 were introduce | transduced in droplet production | generation process S4 was demonstrated, you may introduce | transduce the particle | grains 20 in 2nd introduction | transduction process S2 as already stated.

The present invention is not limited to the above-described embodiments, and includes various modifications. The above embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described. Also, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment. Moreover, the structure of another Example can also be added to the structure of a certain Example. Further, with respect to a part of the configuration of each embodiment, another configuration can be added, deleted, or replaced.

The functions of the above processing apparatus may be realized by software by interpreting and executing a program that realizes each function by the processor. Information such as programs, tables, and files for realizing each function can be stored in various types of non-transitory computer-readable media. As the non-transitory computer-readable medium, for example, a flexible disk, a CD-ROM, a DVD-ROM, a hard disk, an optical disk, a magneto-optical disk, a CD-R, a magnetic tape, a nonvolatile memory card, a ROM, and the like are used. Moreover, you may implement | achieve the function of said processing apparatus, etc. by hardware, for example by designing part or all with an integrated circuit.

10 measurement container 20 particle 100 pore 101 substrate 102 first flow path 103 second flow path 104 flow path wall 105 connecting part 106 divided flow path 110 first flow path inlet 111 first flow path outlet 112 second flow path inlet 113 Second channel outlet 121 Detection electrode 122 Counter electrode 130 Voltage source 131 Ammeter 140 Pressure control unit 150 Droplet generation unit 151 Droplet 152 Oil droplet or bubble

Claims

A substrate having a plurality of pores; a first flow path formed so as to contact the plurality of pores on the surface of the substrate and partition between some of the pores; A measurement vessel having a second flow path in contact with the pores of
A plurality of detection electrodes provided on the surface of the substrate or in the first flow path and arranged to correspond to each of the plurality of pores;
At least one counter electrode provided on the back surface of the substrate or in the second flow path;
A voltage source for applying a voltage between the plurality of detection electrodes and the counter electrode;
A particle analyzer comprising: a measuring unit that measures a current or an electric resistance flowing between each of the plurality of detection electrodes and the counter electrode.
The particle analyzer according to claim 1, wherein
The electrical resistance between each of the plurality of pores and the corresponding detection electrode is smaller than the electrical resistance of the liquid having conductivity between each of the plurality of pores and the adjacent pore. Particle analyzer characterized by.
The particle analyzer according to claim 1, wherein
The particle analyzer further comprising a pressure control unit for controlling a pressure difference between the first channel and the second channel.
The particle analyzer according to claim 1, wherein
The particle analyzer according to claim 1, wherein the first flow path includes a plurality of divided flow paths, and at least one pore is disposed in each of the divided flow paths.
The particle analyzer according to claim 4,
The particle analyzer according to claim 1, wherein the plurality of divided flow paths extend along a flow direction when a liquid having conductivity is introduced into the first flow path.
The particle analyzer according to claim 1, wherein
The first flow path is a flow path having no branch, and the plurality of pores are arranged in a line along a flow direction when a conductive liquid is introduced into the first flow path. A particle analyzer characterized by that.
The particle analyzer according to claim 1, wherein
A liquid droplet generator that generates a plurality of liquid droplets having electrical conductivity;
The particle analyzer is characterized in that the plurality of droplets are divided by a hydrophobic liquid having no gas or conductivity.
The particle analyzer according to claim 1, wherein
A particle analyzer further comprising a processing device for analyzing an electrical signal including crosstalk between the plurality of pores.
The particle analyzer according to claim 8, wherein
The processing apparatus performs a fitting process on the signal value of the pore based on a theoretical value or known data of crosstalk of electrical signals between the plurality of pores. .
A substrate having a plurality of pores; a first flow path formed so as to contact the plurality of pores on the surface of the substrate and partition between some of the pores; A first introduction step of introducing a liquid having conductivity into the first flow path of the measurement container having a second flow path in contact with the pores of
A second introduction step of introducing a conductive liquid into the second flow path of the measurement container;
An operation step of operating the particles contained in the first channel or the second channel so as to pass through the plurality of pores;
A plurality of detection electrodes provided on the front surface of the substrate or in the first flow path and arranged to correspond to the plurality of pores; and the back surface of the substrate or the interior of the second flow path. A particle analysis method including a measurement step of measuring a current or an electric resistance flowing between each of the plurality of detection electrodes and the counter electrode using at least one counter electrode provided on the electrode.
The particle analysis method according to claim 10,
The electrical resistance between each of the plurality of pores and the corresponding detection electrode is smaller than the electrical resistance of the liquid having conductivity between each of the plurality of pores and the adjacent pore. A particle analysis method characterized by the above.
The particle analysis method according to claim 11,
A particle analysis method, further comprising a cleaning step of introducing a liquid into one or both of the first flow path and the second flow path to perform cleaning.
The particle analysis method according to claim 10,
The particle analyzing method, wherein the operation step includes controlling a pressure difference between the first flow path and the second flow path.
The particle analysis method according to claim 10,
The first introduction step includes
Generating a plurality of droplets of conductive liquid;
Introducing a gas or a hydrophobic liquid having no electrical conductivity between the plurality of droplets.
The particle analysis method according to claim 10,
The particle analysis method further comprising the step of analyzing an electrical signal including crosstalk between the plurality of pores.