WO2013018497A1

WO2013018497A1 - Collector, separation method, and display method

Info

Publication number: WO2013018497A1
Application number: PCT/JP2012/067171
Authority: WO
Inventors: 伴　和夫; 藤岡　一志; 紀江松井; 隆治圓城寺; 崇志岩本
Original assignee: シャープ株式会社; フィルテクノジャパン株式会社
Priority date: 2011-07-29
Filing date: 2012-07-05
Publication date: 2013-02-07
Also published as: JP2013027366A; JP5921829B2

Abstract

A collection system (1) comprises: a collector equipped with a through-flow tube for allowing a liquid to flow therethrough and a plurality of electrodes installed at positions toward the through-flow tube; a pump (800) and tubes (400A, 400B) for carrying the liquid along a predetermined liquid flow direction in the through-flow tube; and a control device (200) for controlling the frequency of each of the electrodes of the collector. The electrodes include a first electrode and a second electrode arranged in stages from the upstream side to the downstream side in the liquid flow direction. The control device controls the frequency of the first electrode so that viable bacteria in a sample liquid are collected at the first electrode and controls the frequency of the second electrode so that dead bacteria in the sample liquid after passing through the first electrode are collected at the second electrode.

Description

Collection device, separation method, and display method

The present invention relates to a collection device, a separation method, and a display method, and more particularly, to a collection device, a separation method, and a display method for collecting bacteria in a sample solution using dielectrophoretic force and separating live and dead bacteria. .

Recently, food poisoning caused by microorganisms such as Salmonella, Staphylococcus, Clostridium botulinum, and pathogenic E. coli O-157 has become a problem. While related companies hold lectures and awareness-raising activities related to prevention and hygiene for these microorganisms, they are trying to prevent accident diffusion through expensive capital investment, and various reagents that can detect microorganisms simply and quickly. And devices have been proposed.

Concentration technology that efficiently concentrates target bacteria including proteins such as microorganisms and analyzes such concentrates is provided in the beverage and food fields such as drinking water, meat, prepared dishes, processed foods, pharmaceuticals, formulations, pharmaceuticals, Pharmaceutical and cosmetics fields such as cosmetics, clinical and medical fields such as AIDS, tuberculosis and avian influenza, bioindustry fields such as DNA, RNA, proteins and nucleic acids, and environmental measurement fields such as hot springs, water treatment and sewage treatment It is expected to play an active role in various fields such as ship ballast, bay management, and ocean measurement fields such as marine pollution.

As a method for collecting microorganisms, as disclosed in, for example, Japanese Patent Application Laid-Open No. 2007-6858 (Patent Document 1), a collection method using a dielectrophoretic force has recently attracted attention. Patent Document 1 discloses a chip for the purpose of separating microorganisms by dielectrophoresis, assuming that microorganisms with a small amount of charge can be collected at a low voltage without depending on the charges possessed by the microorganisms.

In addition, “Major life and death separation of microbes in a microfluid by dielectrophoresis” (Non-Patent Document 1) by Masato Suzuki et al. Explains the principle for separating live and dead bacteria in microorganisms.

Japanese Patent Laid-Open No. 2007-6858

By the way, in various fields as described above, there is a need to collect each of live and dead bacteria in a sample solution.

When collected using the technique of Patent Document 1, there was a problem that live bacteria and dead bacteria were not separated and could not meet the above needs.

Although Non-Patent Document 1 discloses the principle of separation, it does not disclose a method specifically implemented by an apparatus, and there is a problem that actual separation based on the principle is difficult.

The present invention has been made in view of such problems, and is a collection device and a separation method capable of collecting microorganisms in a sample solution by dielectrophoretic force and suitably separating live and dead bacteria in the microorganisms. And to provide a display method.

In order to achieve the above object, according to an aspect of the present invention, the collection device is a collection device for collecting bacteria in a sample solution, and a flow-through tube for flowing the liquid, A plurality of electrodes installed in the tube, a transport mechanism for transporting the liquid in the through-flow tube along a predetermined liquid flow direction, and a control device for controlling the frequency of each of the plurality of electrodes. The plurality of electrodes include a first electrode and a second electrode arranged in stages from the upstream side to the downstream side in the liquid flow direction. The control device controls the frequency of the first electrode so as to collect viable bacteria in the sample liquid that has flowed into the flow-through tube with the first electrode, and passes through the first electrode with the second electrode. The frequency of the second electrode is controlled so as to collect dead bacteria in the sample solution.

Preferably, the frequency at which the dielectrophoretic force generated in both the live and dead bacteria in the sample liquid in the cross-flow tube is greater than the stress exerted on the live and dead bacteria by the sample liquid transported by the transport mechanism is the first frequency. The dielectrophoretic force generated by dead bacteria in the sample liquid in the once-through tube is smaller than the stress exerted on the dead bacteria by the sample liquid transported by the transport mechanism. When the second frequency is the frequency at which the sample solution transported by the transport mechanism is greater than the stress exerted on the living bacteria, the control device sets the frequency of the first electrode as the second frequency, and the second electrode. Is the first frequency.

Preferably, the frequency at which the dielectrophoretic force generated in both the live and dead bacteria in the sample liquid in the cross-flow tube is greater than the stress exerted on the live and dead bacteria by the sample liquid transported by the transport mechanism is the first frequency. The dielectrophoretic force generated by dead bacteria in the sample liquid in the once-through tube is smaller than the stress exerted on the dead bacteria by the sample liquid transported by the transport mechanism. Assuming that the second frequency is the frequency at which the sample solution transported by the transport mechanism is greater than the stress exerted on the living bacteria, the control device sets the frequency of the first electrode to the first frequency, The frequency of the electrode is switched from the first frequency to the second frequency, and the frequency of the second electrode is set as the first frequency.

Preferably, the plurality of electrodes further include a third electrode arranged on the upstream side in the liquid flow direction with respect to the first electrode, and the control device includes a third electrode in the sample liquid flown through the flow-through tube. The frequency of the third electrode is controlled so as to collect the live bacteria and dead bacteria, and the control device controls the frequency of the third electrode, and then the live bacteria and dead bacteria collected on the third electrode The frequency of the third electrode is controlled so as to be released from the third electrode, the frequency of the first electrode is controlled so as to collect viable bacteria with the first electrode, and the second electrode is dead. The frequency of the second electrode is controlled so as to collect bacteria.

More preferably, the control device generates the frequency of the third electrode so as to collect the live bacteria and dead bacteria in the sample liquid flowing through the flow-through pipe, and generates both the live and dead bacteria in the sample liquid in the flow-through pipe. The dielectrophoretic force is set to a frequency that is greater than the stress exerted on the living bacteria and dead bacteria by the sample liquid transported by the transport mechanism.

Preferably, the plurality of electrodes are in parallel with the first electrode and the second electrode in the liquid flow direction and stepwise from the upstream side to the downstream side in the liquid flow direction, respectively. And a fourth electrode and a fifth electrode arranged in a direction orthogonal to the liquid flow direction. The control device controls the frequency of the fourth electrode so as to collect viable bacteria in the sample liquid that has flowed through the flow-through tube with the fourth electrode, and the fifth electrode collects the fourth electrode with the fourth electrode. The frequency of the fifth electrode is controlled so as to collect viable bacteria released later.

According to another aspect of the present invention, the separation method flows through the flow path by controlling the frequency of each of the plurality of electrodes arranged stepwise from the upstream side to the downstream side in the liquid flow direction of the flow path. A method for separating live and dead bacteria in a sample solution, the step of controlling the frequency of the first electrode so as to collect the live bacteria in the sample solution with the first electrode arranged upstream in the liquid flow direction. And the frequency of the second electrode so as to collect dead bacteria in the sample liquid after passing through the first electrode with the second electrode arranged downstream of the first electrode in the liquid flow direction. Controlling.

According to still another aspect of the present invention, the display method is a method of collecting bacteria in a sample solution with a collection device, and displaying the result on the display device, wherein the collection device is a liquid flow in a flow path. The first electrode includes a plurality of electrodes arranged stepwise from the upstream side to the downstream side in the direction, and collects live bacteria in the sample solution with the first electrode arranged upstream in the liquid flow direction. The step of controlling the frequency of the first and the second electrodes arranged downstream of the first electrode in the liquid flow direction so as to collect dead bacteria in the sample liquid after passing through the first electrode. The display position in which the step of controlling the frequency of the electrode 2 and the viable bacteria in the collected sample liquid and the dead bacteria in the collected sample liquid are on the same screen is specified, and the display position is Displaying live bacteria and dead bacteria contained on the same screen of the display device.

According to this invention, microorganisms in a sample solution can be collected by dielectrophoretic force, and viable and dead bacteria in microorganisms can be suitably separated.

It is a figure which shows the specific example of a structure of the collection system concerning embodiment. It is a schematic exploded view for demonstrating the structure of the collection apparatus contained in a collection system. It is a top view of the electrode unit contained in a collection device. It is a side view of the electrode unit contained in a collection device. It is a figure which shows the specific example of the pattern (shape) of the electrode formed in an electrode unit. It is a figure which shows the specific example of the pattern (shape) of the electrode formed in an electrode unit. It is a figure which shows the specific example of the pattern (shape) of the electrode formed in an electrode unit. It is a figure which shows the specific example of the pattern (shape) of the electrode formed in an electrode unit. It is a figure which shows the specific example of the pattern (shape) of the electrode formed in an electrode unit. It is a figure which shows the specific example of the combination of an electrode pattern. It is a figure which shows the specific example of the combination of an electrode pattern. It is a figure which shows the specific example of the combination of an electrode pattern. It is a figure which shows the specific example of the combination of an electrode pattern. It is a figure which shows the specific example of the combination of an electrode pattern. It is a flowchart showing the outline | summary of the 1st operation example in a collection system. It is a figure showing the collection state in each step of the flowchart of FIG. It is a figure showing the collection state in each step of the flowchart of FIG. It is a figure showing the collection state in each step of the flowchart of FIG. It is a figure showing the collection state in each step of the flowchart of FIG. It is a figure showing the collection state in each step of the flowchart of FIG. It is a block diagram which shows the specific example of a function structure of the control apparatus contained in a collection system. It is a flowchart showing the flow of operation | movement of a control apparatus. It is a flowchart showing the outline | summary of the 2nd operation example in a collection system. It is a figure showing the collection state in each step of the flowchart of FIG. It is a figure showing the collection state in each step of the flowchart of FIG. It is a figure showing the collection state in each step of the flowchart of FIG. It is a figure showing the collection state in each step of the flowchart of FIG. It is a figure showing the collection state in each step of the flowchart of FIG. It is a figure showing the collection state in each step of the flowchart of FIG. It is the microscope picture image | photographed by the collection operation | movement using a collection system. It is the microscope picture image | photographed by the collection operation | movement using a collection system. It is the microscope picture image | photographed by the collection operation | movement using a collection system. It is the microscope picture image | photographed by the collection operation | movement using a collection system.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following description, the same parts and components are denoted by the same reference numerals. Their names and functions are also the same.

<System configuration>
FIG. 1 is a diagram illustrating a specific example of the configuration of the collection system 1 according to the present embodiment. With reference to FIG. 1, the collection system 1 includes a collection device 100 including a plurality of electrodes, and a power supply 26 therein, and controls connection between the power supply and each electrode of the collection device 100. A control device 200 for controlling application to the electrodes, a cleaning liquid chamber 300A for holding a cleaning liquid, a tube 400A for connecting the sample liquid chamber 300B for holding the sample liquid and the collection device 100, and The tube 400B for connecting the waste liquid chamber 300C for holding the waste liquid and the collection device 100, and the imaging for photographing the bacteria collected by the collection device 100 installed in the vicinity of the collection device 100 An apparatus 500 and a display apparatus 600 that is electrically connected to the imaging apparatus 500 and displays a captured image are included.

<Apparatus configuration of collection device>
The collection device 100 has an inner space as will be described later, and the liquid flows from the upstream side to the downstream side. The tube 400A is connected to the upstream side, and the tube 400B is connected to the downstream side.

The tube 400A is connected with a three-way valve 700 as an example of a mechanism for switching the liquid to flow through. The three-way valve 700 includes a valve 700A provided between the cleaning liquid chamber 300A and the tube 400A, a valve 700B provided between the sample liquid chamber 300B and the tube 400A, and between the collection device 100 and the tube 400A. There are three valves, the provided valve 700 C, and the opening and closing of each valve is controlled by the control device 200. That is, under the control of the control device 200, the cleaning liquid chamber 300A is connected to the collection device 100 via the tube 400A, or the sample liquid chamber 300B is connected to the collection device 100 via the tube 400A.

Of course, the mechanism for switching the connection between the collection device 100 and the cleaning liquid chamber 300A or the sample liquid chamber 300B is not limited to the three-way valve 700, and may be another method.

The pump 800 is connected to the tube 400B. The pump 800 is electrically connected to the control device 200, and its operation is controlled by the control device 200. That is, when the pump 800 is operated by the control of the control device 200, as shown by the arrow in FIG. 1, the collection device 100 passes through the tube 400A from the cleaning liquid chamber 300A or the sample liquid chamber 300B, and passes through the tube. A flow path reaching the waste liquid chamber 300C through 400B is formed.

FIG. 2 is a schematic exploded view for explaining the structure of the collection device 100.
With reference to FIG. 2, the collection device 100 is mainly composed of a substrate 10, an electrode part 12 including an electrode unit 11, and a water tank part 13 having a water tank wall surface 14.

The substrate 10 has a fixing mechanism (not shown) for fixing the electrode part 12 and the water tank part 13.

The electrode part 12 and the water tank part 13 are fixed on the substrate 10 by the above mechanism. Specifically, the electrode unit 12 is disposed immediately above the substrate 10, and the water tank unit 13 is disposed on the substrate 10 with the electrode unit 12 interposed therebetween.

The water tank unit 13 has a water tank wall surface 14, and the water tank unit 13 is fixed on the substrate 10 with the electrode unit 12 sandwiched therebetween, so that the substrate 10 is a bottom surface, and the substrate 10 and the water tank wall surface 14 have an inside thereof. A water tank that is a space capable of holding a liquid is formed.

A connection port 15A for connecting the tube 400A and a connection port 15B for connecting the tube 400B are arranged on the side of the collection device 100 in contact with the water tank. FIG. 1 shows an example in which the tube 400A and the tube 400B are connected to both ends of the rectangular parallelepiped collection device 100 as an example. In FIG. 2, as another example, the connection port 15A and the tube 15A are connected to one end. An example in which the connection port 15B is arranged and the tube 400A and the tube 400B are connected to one end is shown.

The tube 400A connected to the cleaning liquid chamber 300A or the sample liquid chamber 300B is connected to the connection port 15A, and the pump 800 connected to the tube 400B is operated by connecting the tube 400B to the connection port 15B. Then, a liquid flow in the direction from the connection port 15A toward the connection port 15B is generated.

An electrode unit 11 in which a plurality of electrodes are arranged is formed on the surface of the electrode portion 12 on the water tank side. Since the liquid is held in the water tank, the electrode disposed in the electrode unit 11 is in contact with the liquid, and an electric field is generated in the liquid in the water tank.

3A and 3B are diagrams for illustrating a specific example of the electrode arrangement of the electrode unit 11. FIG. FIG. 3A is a view of the electrode unit 11 as viewed from the water tank portion 13 toward the substrate 10 and is a top view. FIG. 3B is a view seen in the direction of arrow IIIB in FIG. 3A, and this view is a side view. Moreover, the arrow F in the figure represents the liquid flow direction.

As shown in FIG. 3A, the electrode unit 11 includes a first electrode 11A, a second electrode 11B, a third electrode 11C, a fourth electrode 11D, and a fifth electrode 11E as a plurality of electrodes in the liquid flow direction. Arranged in stages. In the present invention, it may be arranged in at least two stages. In the following description, an example in which the liquid flow direction is arranged in three stages will be described as an example.

In the following description, the most upstream electrode position in the liquid flow direction is the first stage, the electrode position adjacent to the first stage downstream is the second stage, and the electrode position adjacent to the second stage downstream is This is called the third stage. The first stage has one electrode having a width close to the entire liquid flow width, and the second stage and the third stage each have two electrodes arranged substantially perpendicular to the liquid flow and having a width approximately half of the liquid flow width. Are arranged. A more detailed arrangement example is illustrated in FIG.

That is, referring to FIG. 3A and FIG. 3B, the first electrode 11A, which is one electrode on the first stage, and the second electrode 11B and the third electrode 11C, which are two electrodes on the second stage, Two electrodes, a fourth electrode 11D and a fifth electrode 11E, are arranged on the stage.

4 to 8 are diagrams showing specific examples of electrode patterns (shapes) formed on the electrode unit 11 respectively.

For example, the electrode pattern may be a vertical comb shape as shown in FIG. 4, a parallel comb shape as shown in FIG. 5, or “U” as shown in FIG. 6. ", A cross-shaped diagonal shape as shown in FIG. 7, or a comb-shaped diagonal shape as shown in FIG.

In the vertical comb shape shown in FIG. 4 and the parallel comb shape shown in FIG. 5, n vertical electrodes are arranged in parallel at equal intervals, and n electrodes of the same shape are alternately combined from the facing side. As a result, a comb-shaped electrode pattern having a gap number of 2n−1 is formed. For example, one electrode may have an electrode width of 50 μm or 100 μm, and an electrode interval (gap) may be 10 μm.

As will be described later, in the collection device 100, the bacteria are collected by being held on the electrodes by the dielectrophoretic force. In the case of a vertical comb-shaped electrode pattern, since the electrodes are arranged perpendicular to the liquid flow direction, the collected bacteria are firmly held by the electrodes due to the dielectrophoretic force. Therefore, the vertical comb-shaped electrode shown in FIG. 4 is preferably used for any of the first electrode 11A to the fifth electrode 11E.

As will be described later, in the collection operation of the collection device 100, the bacteria held by the electrodes are moved (released) to other electrodes along the liquid flow direction. In the case of a parallel comb-shaped electrode pattern, since the electrodes are arranged parallel to the liquid flow direction, the collected bacteria move smoothly to another electrode (release) compared to the vertical comb-shaped electrode pattern. ). Therefore, the parallel comb electrodes shown in FIG. 5 are preferably used for any of the first electrode 11A to the fifth electrode 11E.

In the “U” shape shown in FIG. 6, n “U” shape electrodes are arranged in parallel at equal intervals and are alternately combined from the opposite side. As a result, an “U” -shaped electrode pattern having a gap number of 2n−1 is formed. For example, one electrode may have an electrode width of 100 μm and an electrode interval (gap) of 10 μm.

In the case of the “U” -shaped electrode pattern shown in FIG. 6, there are a portion where the electrodes are arranged in parallel to the liquid flow direction and a portion where the electrodes are arranged perpendicular to the liquid flow direction. On the other hand, it is possible to move and aggregate bacteria from a place arranged in parallel to a place arranged vertically. As will be described later, in the collection operation of the collection device 100, the bacteria are collected at the observation position on the downstream side while moving the bacteria in the liquid flow direction. Therefore, the “U” -shaped electrode shown in FIG. 6 is suitably used for the first electrode 11A disposed upstream from the branch from the second electrode 11B to the fifth electrode 11E.

In the crossed diagonal electrode pattern shown in FIG. 7, one diagonal electrode and n−1 “V” -shaped electrodes are arranged in parallel at equal intervals, and n identical electrodes alternate from the opposite side. Are combined. As a result, an electrode pattern having a gap number of 2n−1 is formed. For example, one electrode may have an electrode width of 50 μm or 100 μm, and an electrode interval (gap) may be 10 μm.

In the case of the crossed diagonal electrode pattern shown in FIG. 7, bacteria can be concentrated to the central portion where the diagonal electrode is located by the same phenomenon as the “U” -shaped electrode pattern. Therefore, the crossed diagonal electrode shown in FIG. 7 is also preferably used for the first electrode 11A arranged upstream from the branch from the second electrode 11B to the fifth electrode 11E.

In the comb-shaped diagonal electrode pattern shown in FIG. 8, n “L” -shaped electrodes are arranged in parallel at equal intervals. As a result, an electrode pattern having an n-1 gap number is formed. For example, one electrode may have an electrode width of 50 μm or 100 μm, and an electrode interval (gap) may be 10 μm.

The bacteria held between the electrodes move to the lower part of the electrode according to the direction of the electrodes, and the bacteria held on the upstream side of the electrode move to the lower part of the electrode and then move and accumulate in parallel with the liquid flow direction. Therefore, compared with the vertical comb-shaped electrode pattern shown in FIG. 4, the bacteria collected in a narrower range can be aggregated, and is preferably used to collect the bacteria in the imaging range described later. .

The shapes of the electrodes 11A to 11E of the electrode unit 11 can be variously configured by combining the shapes shown in FIGS. In the present invention, the shapes of the first electrode 11A to the fifth electrode 11E are not limited to specific shapes, and the illustrated shapes can be variously combined. Then, an imaging region in the imaging device 500 is set on the electrode, and the imaging device 500 is installed so that the region is the imaging region.

9 to 13 are diagrams showing specific examples of combinations of shapes of the first electrode 11A to the fifth electrode 11E. Of course, the combination of the shapes of the first electrode 11A to the fifth electrode 11E in the present invention is not limited to only those shown in FIGS. Note that an arrow F in FIGS. 9 to 13 represents the liquid flow direction.

FIG. 9 shows an example in which all of the first electrode 11A to the fifth electrode 11E are formed in a comb-shaped electrode pattern. In this configuration, when viable bacteria are collected on the fourth electrode 11D and dead bacteria are collected on the fifth electrode 11E by controlling the frequency with respect to these electrodes, as shown in FIG. 9, orthogonal to the liquid flow direction. By setting a region P1 straddling the fourth electrode 11D and the fifth electrode 11E of the third stage to be an imaging region in the imaging device 500, the living bacteria collected by the fourth electrode 11D and the fifth electrode 11E are captured. The collected dead bacteria are photographed at the same time.

Depending on other frequency control, live bacteria may be collected on the second electrode 11B, and dead bacteria may be collected on the fourth electrode 11D. In this case, an area P2 that straddles the second electrode 11B of the second stage and the fourth electrode 11D of the third stage arranged in the liquid flow direction is set as an imaging area in the imaging apparatus 500, whereby the second electrode 11B The live bacteria collected and the dead bacteria collected on the fourth electrode 11D are photographed simultaneously.

FIG. 10 shows an example in which the first electrode 11A is formed in an “U” -shaped electrode pattern, and the second electrode 11B to the fifth electrode 11E are formed in a comb-shaped electrode pattern. In this configuration, when viable bacteria are collected on the fourth electrode 11D and dead bacteria are collected on the fifth electrode 11E by controlling the frequency with respect to these electrodes, the fourth electrode of the third stage orthogonal to the liquid flow direction. By setting a region P3 straddling 11D and the fifth electrode 11E as a photographing region in the imaging device 500, live bacteria collected on the fourth electrode 11D and dead germs collected on the fifth electrode 11E are photographed simultaneously. Is done.

Depending on other frequency control, live bacteria may be collected on the second electrode 11B, and dead bacteria may be collected on the fourth electrode 11D. In this case, an area P4 that straddles the second electrode 11B of the second stage and the fourth electrode 11D of the third stage arranged in the liquid flow direction is set as an imaging area in the imaging apparatus 500, whereby the second electrode 11B The live bacteria collected and the dead bacteria collected on the fourth electrode 11D are photographed simultaneously.

FIG. 11 shows an example in which the first electrode 11A to the third electrode 11C are formed in a comb-shaped electrode pattern, and the fourth electrode 11D and the fifth electrode 11E are formed in a comb-shaped oblique electrode pattern. In this configuration, when viable bacteria are collected on the fourth electrode 11D and dead bacteria are collected on the fifth electrode 11E by controlling the frequency with respect to these electrodes, as shown in FIG. 11, orthogonal to the liquid flow direction. By setting the region P5 at the tip of the comb-shaped diagonal where the bacteria are gathered, as the imaging region in the imaging device 500, the region straddling the fourth electrode 11D and the fifth electrode 11E of the third stage Live bacteria collected on the four electrodes 11D and dead bacteria collected on the fifth electrode 11E are photographed simultaneously.

In FIG. 12, the first electrode 11A is formed in an “U” -shaped electrode pattern, the second electrode 11B and the third electrode 11C are formed in a comb-shaped electrode pattern, and the fourth electrode 11D and the fifth electrode 11E are formed in a comb-shaped oblique electrode pattern. Represents an example. In this configuration, when viable bacteria are collected on the fourth electrode 11D and dead bacteria are collected on the fifth electrode 11E by controlling the frequency with respect to these electrodes, as shown in FIG. 12, orthogonal to the liquid flow direction. By setting an area P6 at the tip of the comb-shaped diagonal where the bacteria are gathered, as an imaging area in the imaging device 500, the area spans the fourth electrode 11D and the fifth electrode 11E of the third stage. Live bacteria collected on the four electrodes 11D and dead bacteria collected on the fifth electrode 11E are photographed simultaneously.

In FIG. 13, the first electrode 11 A is formed with a cross-shaped diagonal electrode pattern, the second electrode 11 B and the third electrode 11 C are comb-shaped electrode patterns, and the fourth electrode 11 D and fifth electrode 11 E are formed with a comb-shaped diagonal electrode pattern. An example is shown. In this configuration, when viable bacteria are collected on the fourth electrode 11D and dead bacteria are collected on the fifth electrode 11E by controlling the frequency with respect to these electrodes, as shown in FIG. 13, orthogonal to the liquid flow direction. By setting the region P7 at the tip of the comb-shaped oblique tip where the bacteria are concentrated, as the imaging region in the imaging device 500, the region straddling the fourth electrode 11D and the fifth electrode 11E of the third stage Live bacteria collected on the four electrodes 11D and dead bacteria collected on the fifth electrode 11E are photographed simultaneously.

<Device configuration of control device>
Referring to FIG. 1 again, control device 200 has, as a device configuration, a CPU (Central Processing Unit) 20 for controlling the entire device, a memory 21 for storing a program executed by CPU 20, a power source, and the like. 26, a switch 22 for switching the connection between the power supply 26 and the first electrode 11A to the fifth electrode 11E, a communication I / F (interface) 24 for communicating with the three-way valve 700, and a pump 800. The communication I / F 23 and the communication I / F 25 for communicating with the imaging apparatus 500 are included.

The CPU 20 reads out and executes a program stored in the memory 21 in accordance with an instruction input from an instruction input unit (not shown) such as a switch, and causes the collection device 100 to execute a collection operation described later. At that time, the frequency of each of the first electrode 11A to the fifth electrode 11E is controlled.

An example of electrode frequency control performed by the CPU 20 is PWM control (pulse width modulation). If PWM control is performed, the switch 22 corresponds to a transistor switch or the like, and the CPU 20 controls the frequency of each of the first electrode 11A to the fifth electrode 11E by controlling the switching timing of the switch 22, that is, the pulse width. .

Of course, the frequency control of each of the first electrode 11A to the fifth electrode 11E by the CPU 20 is not limited to PWM control, and may be another control method. In that case, the control device 200 includes a device configuration corresponding to the control method.

Further, the CPU 20 outputs a control signal to the three-way valve 700 and the pump 800. Further, a control signal is output to the imaging apparatus 500 to control photographing with the imaging apparatus 500.

In addition, the control apparatus 200 may be comprised with a general computer as an example. Therefore, other device configurations not shown in FIG. 1 may be included.

<Apparatus configuration of imaging apparatus>
Further, referring to FIG. 1, the imaging apparatus 500 has, as its device configuration, a CPU 50 for controlling the entire apparatus, a memory 51 for storing a program executed by the CPU 50, a CCD (Charge Coupled Device), and the like. Imaging unit 52, communication I / F 53 for communicating with control device 200, and communication I / F 54 for communicating with display device 600.

The CPU 50 receives a control signal from the control device 200 via the communication I / F 53, and reads and executes a program stored in the memory 51 according to the control signal. In accordance with the execution of the program, the CPU 50 controls the photographing unit 52 to perform a photographing operation.

As described above, the imaging device 500 is set so as to include any position of the electrodes as an imaging region from above the first electrode 11A to the fifth electrode 11E included in the collection device 100. And imaging | photography of this imaging | photography area | region is performed by imaging | photography at the timing according to the control signal from the control apparatus 200. FIG.

The image data obtained by photographing is transmitted to the display device 600 via the communication I / F 54. Display device 600 displays a screen based on screen data received by a CPU (not shown). Further, when the display device 600 is a computer on which an analysis program is installed, the CPU (not shown) may execute the analysis program to analyze the image data.

<Principle of separation method>
The collection device 100 collects biological particles in a sample solution by attaching them to an electrode using the principle of dielectrophoresis.

Here, a separation method using the principle of dielectrophoresis will be described.
Dielectrophoresis is a phenomenon in which induced dipole moment is generated by utilizing a difference in electrical properties such as dielectric constant between a biological particle and a medium (such as water), and the particle is generated by the dielectrophoretic force generated by the balance. Refers to a phenomenon in which is attached to or separated from the electrode.

It is known that the dielectrophoretic force generated in each particle of normal organisms (viable bacteria) without damaged cell membranes and particles of dead organisms (dead bacteria) shows different tendencies depending on the frequency of the generated electric field. It has been.

Specifically, up to H1 [kHz], the dielectrophoretic force generated for both live and dead bacteria increases as the frequency increases, and the dielectrophoretic force generated for live bacteria always outweighs the dielectrophoretic force generated for dead bacteria. .

After H1 [kHz], the dielectrophoretic force generated for both living and dead bacteria decreases with increasing frequency while maintaining the magnitude relationship of the generated dielectrophoretic force, but the rate of decrease is relative to the increase in frequency. It becomes moderate. On the other hand, the rate of decrease in dielectrophoretic force generated in dead bacteria is greater than the rate of decrease in dielectrophoretic force generated in live bacteria. In the vicinity of H2 [MHz], the dielectrophoretic force generated in dead bacteria decreases until it becomes approximately 1/3 times the dielectrophoretic force generated in live bacteria. In the vicinity of H3 [MHz], the dielectrophoretic force generated in killed bacteria becomes zero.

The collection device 100 separates live and dead bacteria using a change according to the frequency of the dielectrophoretic force. H1, H2, and H3 depend on the width and shape of the electrodes and the distance between the electrodes. Then, when experiment was conducted using Escherichia coli with the collection apparatus 100 which the inventor made as a prototype for confirmation, the above H1, H2, and H3 are about 100 [kHz], about 1 [MHz], and about 4 [MHz], respectively. Met.

That is, by generating an electric field having a frequency of about 100 [kHz] with respect to the sample liquid while the sample liquid is held in the water tank, both live and dead bacteria can be attached to the electrode and collected. it can. This frequency will be referred to as “first frequency” in the following description. The first frequency is a frequency at which the dielectrophoretic force generated for both live and dead bacteria in the sample liquid is greater than the stress exerted on the live and dead bacteria when the sample liquid is conveyed by the operation of the pump 800. More specifically, it indicates a frequency of about 100 [kHz] as described above.

On the other hand, by generating an electric field with a frequency of 4 [MHz], which is a frequency at which the dielectrophoretic force generated in killing bacteria at a frequency higher than the first frequency is 0, only viable bacteria adhere to the electrode. Only live bacteria will be collected. This frequency will be referred to as “second frequency” in the following description. The second frequency is such that the dielectrophoretic force generated in the dead bacteria in the sample liquid is smaller than the stress exerted on the dead bacteria when the sample liquid is conveyed by the operation of the pump 800, and the dielectric generated in the live bacteria in the sample liquid. It can be said that the electrophoretic force is a frequency that causes the sample liquid to be greater than the stress exerted on the living bacteria when the sample liquid is conveyed by the operation of the pump 800. Specifically, as described above, a frequency of about 4 [MHz] is used. Point to.

In addition, an electric field of the second frequency is generated in the sample liquid to collect only viable bacteria first, and an electric field of the first frequency is generated for the remaining sample liquid from which the viable bacteria have been removed. Since only dead bacteria adhere, only dead bacteria are collected.

By performing frequency control on this electrode in order from the upstream electrode of the liquid flow, it is possible to finally collect and separate live and dead bacteria on the downstream electrode where the imaging region is set. .

In addition, an electric field of the first frequency is generated in the sample solution to collect viable bacteria and dead bacteria, and then the frequency is changed from the first frequency to the second frequency, thereby collecting the collected live bacteria and dead bacteria. Our live bacteria are kept collected and dead bacteria are released.

By controlling the frequency of this electrode at the upstream electrode of the liquid flow, it becomes possible to collect only dead bacteria at the downstream electrode. Bacteria and dead bacteria can be separated and collected.

<Overview of operation>
FIG. 14 is a flowchart showing an outline of a first operation example in the collection system 1. 15 to 19 are diagrams showing the collection state at each step of the flowchart of FIG. In FIG. 15 to FIG. 19, the bacterium represented by the solid line represents the bacterium collected by the electrode, and the bacterium represented by the dotted line represents the bacterium not collected by the electrode. Moreover, the electrode represented by the thick line represents that a voltage is applied.

Referring to FIG. 14, first, the sample liquid is allowed to flow through the collection device 100 (# 11). FIG. 15 shows the state of collection at that time, and shows a state in which a sample liquid containing live and dead bacteria has been transported into the water tank.

In # 11, the flow of the sample solution is started, and the live and dead bacteria are collected in the first stage (# 12). Here, a voltage is applied to the first electrode 11A arranged on the first stage at the first frequency described above.

FIG. 16 shows the collection state at that time. That is, referring to FIG. 16, when a voltage is applied to the first electrode 11 A of the first stage at the first frequency described above, the electric field in the sample liquid passing thereover is within a range affected. Viable and dead bacteria adhere to the first electrode 11A and are collected.

After # 12, the cleaning liquid is allowed to flow while maintaining the voltage of each electrode (# 13). FIG. 17 shows the collection state at that time. That is, referring to FIG. 17, only the living and dead bacteria collected on the first electrode 11 A remain on the electrode by washing the inside of the water tank with the washing liquid while maintaining the voltage of each electrode, that is, on the electrode, Other bacteria are eliminated.

Thereafter, live and dead bacteria are released from the first stage while continuing the flow of the cleaning solution (# 14). Here, the voltage of the first electrode 11A of the first stage is turned off.

FIG. 18 shows the state of collection at that time, and live and dead bacteria collected on the first electrode 11A in # 11 dissociate from the first electrode 11A. The living and dead bacteria dissociated from the first electrode 11A are directed to the second stage on the downstream side along the flow of the cleaning liquid.

At # 14, live and dead bacteria are released from the first stage, and live bacteria are collected at the second stage downstream (# 15), and dead bacteria are collected at the third stage downstream (# 16). Here, the voltage is applied to the second electrode 11B or the third electrode 11C of the second stage at the second frequency described above, and the fourth electrode 11D or the fifth electrode 11E of the third stage on the downstream side is applied to the second electrode 11B or the third electrode 11C. A voltage is applied at the first frequency.

FIG. 19 shows the collection state at that time. As an example, a voltage is applied to the second electrode 11B arranged side by side along the liquid flow at the above-described second frequency, and the above-described voltage is applied to the fourth electrode 11D. An example in which a voltage is applied at the first frequency is shown. That is, at the above-mentioned second frequency, no dielectrophoretic force is generated in dead bacteria, or only a dielectrophoretic force smaller than the flow force of the cleaning liquid is generated, so that only viable bacteria are collected on the second electrode 11B. Become. Although the dielectrophoretic force is generated in both the live and dead bacteria at the first frequency described above, the fourth electrode 11D is obtained by collecting the live bacteria and removing them from the liquid on the upstream side. Only dead bacteria will be collected.

In addition, since the width of the second electrode 11B is approximately half of the width of the first electrode 11A, the viable bacteria of approximately half of the viable and dead bacteria released from the first electrode 11A of the first stage are the first. The dead bacteria of approximately half of the live and dead bacteria that are collected by the second stage 11B and released from the first electrode 11A are collected by the fourth electrode 11D of the third stage. Become.

<Functional configuration>
FIG. 20 is a block diagram illustrating a specific example of a functional configuration of the control device 200 for causing the collection system 1 to perform the above-described operation. Each function shown in FIG. 20 is a function mainly formed on the CPU 20 when the CPU 20 of the control device 200 reads out and executes a program stored in the memory 21. However, at least a part may be realized by a hardware configuration such as an electric circuit.

Referring to FIG. 20, the control device 200 includes an input unit 201 for receiving an instruction input for starting the collection operation from an instruction input unit (not shown) such as a switch (not shown), and the above-described # 11 according to the disclosure of the collection operation. A control signal is output to the three-way valve 700 via the communication I / F 23 according to the determination by the determination unit 202 for determining each stage of # 16 to # 16, and the opening and closing of the three-way valve 700 is controlled. A valve control unit 203 for controlling the operation of the pump 800 by outputting a control signal to the pump 800 via the communication I / F 24 according to the determination of the determination unit 202, Frequency control for controlling the frequency of each of the first electrode 11A to the fifth electrode 11E by controlling the switching timing of the switch 22, that is, the pulse width, according to the determination by the determination unit 202. Including the 205, the imaging control unit 206 for controlling the imaging in the imaging device 500 by outputting a control signal to the imaging apparatus 500 via the communication I / F25 in accordance with the determination at decision 202.

<Operation flow>
FIG. 21 is a flowchart showing an operation flow of the control device 200 for causing the collection system 1 to perform the collection operation represented in the first operation example. The operation shown in the flowchart of FIG. 21 is realized by causing the CPU 20 of the control device 200 to read out and execute a program stored in the memory 21 to exhibit each function shown in FIG.

Referring to FIG. 21, in step S101, the CPU 20 controls the three-way valve 700 for passing the sample liquid # 11. That is, the valve 700A on the cleaning liquid chamber 300A side is closed, and the sample liquid chamber 300B and the

valves

700B and 700C on the collection device 100 side are opened. Thereby, the sample liquid chamber 300B is connected to the collection device 100 via the tube 400A.

Next, in step S103, the CPU 20 operates the pump 800. Thereby, the sample liquid held in the sample liquid chamber 300B flows into the collection device 100 through the tube 400A, flows through the water tank, and is conveyed to the waste liquid chamber 300C through the tube 400B.

Next, in step S105, the CPU 20 performs frequency control on the electrodes for collecting live and dead bacteria in the first stage. That is, a voltage is applied to the first electrode 11A arranged in the first stage at the first frequency described above, and the voltage application to the electrodes arranged in the second stage and the third stage is turned off. As a result, as shown in FIG. 16, live and dead bacteria within the range affected by the electric field generated by the first electrode 11A in the flowing sample are attached to the first electrode 11A and collected.

Next, in step S107, the three-way valve 700 for cleaning liquid flow of # 13 is controlled. That is, the valve 700A on the cleaning liquid chamber 300A side and the valve 700C on the collection device 100 side are opened, and the valve 700B on the sample liquid chamber 300B side is opened. Accordingly, the cleaning liquid chamber 300A is connected to the collection device 100 via the tube 400A.

In this state, the liquid in the tube is conveyed by the pump operated in step S103, so that the cleaning liquid held in the cleaning liquid chamber 300A flows into the collection device 100 through the tube 400A and flows through the water tank. And it is conveyed to the waste liquid chamber 300C through the tube 400B.

At this time, because the frequency state controlled in the above step S105 is maintained, as shown in FIG. 17, the viable and dead bacteria collected on the first electrode 11A remain on the first electrode 11A, Surrounding bacteria are eliminated.

Next, in step S109, the CPU 20 performs frequency control on the electrodes for collecting live bacteria in the second stage and collecting dead bacteria in the third stage. That is, in the case of the above-described example, after the application of the voltage at the first frequency to the first electrode 11A is turned off, the electrode is applied at the second frequency to the second electrode 11B arranged in the second stage, and the first An electrode is applied at a first frequency to the fourth electrode 11D arranged in three stages.

As a result, as shown in FIG. 18, the live and dead bacteria collected from the first electrode 11A are released, and are carried downstream by riding on the liquid flow of the cleaning liquid. Then, as shown in FIG. 19, live bacteria are collected by the second electrode 11B, and dead bacteria are collected by the fourth electrode 11D on the downstream side thereof.

Next, in step S111, the CPU 20 outputs a control signal to the image capturing apparatus 500 to perform photographing within a preset photographing range. In this case, as the imaging range, it is only necessary to set a region P2 that straddles the second electrode 11B and the fourth electrode 11D as shown in FIG. 9, and the imaging apparatus 500 captures the range in accordance with the control signal. Thus, live bacteria collected on the second electrode 11B and dead bacteria collected on the fourth electrode 11D are photographed simultaneously.

<Effect of Embodiment>
In the collection device 100 according to the present embodiment, the viable bacteria are collected by applying a voltage of the above-mentioned second frequency, which is about 4 [MHz], to the electrode on the upstream side of the liquid flow, and the viable bacteria are excluded. The dead bacteria are collected by applying a voltage of the above-mentioned first frequency of about 100 [kHz] to an electrode on the downstream side of the transported sample liquid. That is, normally, only by applying a voltage of the first frequency, live bacteria and dead bacteria are collected without being separated, but in the collection device 100, the live bacteria and dead bacteria described above are collected. By collecting the viable bacteria first on the upstream side using the difference in the dielectrophoretic force with respect to the frequency, it becomes possible to collect dead bacteria on the downstream side. It becomes possible to separate and collect dead bacteria.

Furthermore, the electrodes are arranged in three stages with respect to the liquid flow direction described above, and once the live and dead bacteria are collected by the first stage electrodes and then released, the second and third stage electrodes are used. The bacteria to be collected are collected at a position from the electrode in the liquid flow of the cleaning liquid, that is, near the bottom of the water tank along the electrode in the above example. Therefore, the released bacteria are transported within a range that can be influenced by the electric field at the electrodes of the second stage and the third stage, and the probability of being collected by these electrodes is remarkably increased. In addition, since an amount of bacteria corresponding to the collection ability at the downstream electrode is previously collected at the upstream electrode, it is possible to efficiently collect viable and dead bacteria at the downstream electrode. it can.

In the above example, the electrodes are arranged in three stages, and the live and dead bacteria are once collected by the first stage electrode on the most upstream side, but this collection is not necessarily essential, and at least upstream It is only necessary to collect live bacteria in the second stage and to collect dead bacteria in the third stage on the downstream side. Alternatively, as will be described later, only dead bacteria can be collected on the downstream side by collecting only dead bacteria after collecting the live and dead bacteria on the upstream side.

<Other examples>
In addition, the collection method demonstrated above is an example, Comprising: The other collection method using the above-mentioned principle is also mentioned.

FIG. 22 is a flowchart showing an outline of a second operation example in the collection system 1 as another example. FIGS. 23 to 28 are diagrams showing the collection state in each step of the flowchart of FIG. In FIGS. 23 to 28, the bacteria represented by the solid line represent the bacteria collected on the electrode, and the bacteria represented by the dotted line represent the bacteria not collected on the electrode. Moreover, the electrode represented by the thick line represents that a voltage is applied.

Referring to FIG. 22, first, the sample solution is caused to flow through the collection device 100 (# 21). Here, the same control as in step S101 described above is performed.

In the second operation example, the flow of the sample solution is started at # 21 and viable bacteria are collected on the second stage (# 22). Here, a voltage is applied to one of the second electrode 11B and the third electrode 11C arranged on the second stage at the above-described second frequency.

23 and 24 show the collection state at that time. That is, referring to FIG. 23, here, the voltage is applied to the third electrode 11C of the second stage at the second frequency described above, and the voltage of the second electrode 11B of the second stage is turned off. In this state, the operation of # 21 is performed, so that the sample liquid containing life and death bacteria is conveyed into the water tank.

Referring to FIG. 24, since the width of the third electrode 11C in the direction perpendicular to the liquid flow direction is approximately half of the entire width, the third electrode 11C is approximately half the total width of the transported sample liquid. Viable bacteria among viable and dead bacteria in the transported sample liquid adhere to the third electrode 11C and are collected.

After # 22, while maintaining the frequency state at # 22, live bacteria are collected at the first stage (# 23), and dead bacteria are collected at the other electrode of the second stage downstream thereof. (# 24). Here, a voltage is applied to the first electrode 11A disposed on the first stage at the above-described second frequency, and a voltage is applied to the other electrode disposed on the second stage at the above-described first frequency.

FIG. 25 shows the collection state at that time. That is, referring to FIG. 25, since the width of the first electrode 11A in the direction orthogonal to the liquid flow direction is approximately the entire width, viable bacteria among the live and dead bacteria in the transported sample liquid are the first electrode 11A. It is attached to and collected.

In a state where viable bacteria in the sample liquid are collected and removed on the upstream side of the liquid flow, the sample liquid is conveyed to the second stage on the downstream side. At this time, although a voltage is applied to the third electrode 11C at the second frequency, viable bacteria in the sample liquid already transported are collected in the first stage. No more viable bacteria are collected. On the other hand, since a voltage is applied to the second electrode 11B at the first frequency, dead bacteria, which are bacteria contained in the sample solution, adhere to the second electrode 11B and are collected.

After # 24, the cleaning liquid is allowed to flow while maintaining the voltage of each electrode (# 25). FIG. 26 shows the collection state at that time. That is, with reference to FIG. 26, the inside of the water tank, that is, the top of the electrode is washed with the cleaning liquid while maintaining the voltage of each electrode, so that the live bacteria collected on the first electrode 11A and the second electrode 11B are collected. Only the collected dead bacteria and the live bacteria collected on the third electrode 11C remain on the electrode, and other bacteria are excluded.

Thereafter, live and dead bacteria are released from the second stage while continuing the flow of the cleaning solution (# 26). Here, the voltages of the second electrode 11B and the third electrode 11C, which are both electrodes of the second stage, are turned off.

At the same time, live and dead bacteria are collected in the third stage downstream (# 27). Here, a voltage is applied at the first frequency to both the fourth electrode 11D and the fifth electrode 11E which are both electrodes of the third stage.

FIG. 27 shows the state of collection at that time, and dead bacteria collected on the second electrode 11B and live bacteria collected on the third electrode 11C in # 26 are dissociated from the electrodes. The viable and dead bacteria dissociated from these electrodes travel to the third stage on the downstream side along the flow of the cleaning liquid. At this time, dead bacteria are conveyed in the liquid flow direction on the second electrode 11B side and viable bacteria are on the third electrode 11C side. Therefore, when a voltage is applied to both electrodes of the third stage at the first frequency, bacteria in the cleaning liquid conveyed on the respective electrodes are collected. That is, dead bacteria dissociated from the second electrode 11B are collected in the fourth electrode 11D arranged adjacent to the downstream along the liquid flow of the second electrode 11B, and along the liquid flow of the third electrode 11C. Viable bacteria dissociated from the third electrode 11C are collected at the fifth electrode 11E arranged adjacent to the downstream.

In the case of the example shown in FIGS. 23 to 28, as shown in FIGS. 11 to 13, the fourth electrode 11D and the fifth electrode 11E are formed in a comb-shaped diagonal electrode pattern. Therefore, in this example, after the operation of # 27, by maintaining the flow of the cleaning liquid for a predetermined time, the bacteria collected along the “L” -shaped electrode move, and gradually the maximum in the liquid flow direction. It will be collected downstream, that is, at the tip. FIG. 28 shows the state of collection at that time, and as shown in FIG. 27, after the live and dead bacteria are collected on each electrode of the third stage, the live and dead bacteria are collected at the tip of each electrode.

In this state, the imaging device 500 captures an area at the tip of the comb-shaped diagonal as an imaging area, so that live bacteria collected on the fourth electrode 11D and dead bacteria collected on the fifth electrode 11E are simultaneously photographed. .

Even when such an operation is performed, the present collection system 1 can separate and collect viable and dead bacteria.

As another example, after applying a voltage at the first frequency of the first electrode 11A of the first stage in # 23 and collecting both live and dead bacteria in the sample solution, the first electrode 11A is collected in # 24. The frequency may be switched from the first frequency to the second frequency, and the voltage of the electrode on the other side of the second stage on the downstream side may be applied at the first frequency. As a result, the live bacteria among the live and dead bacteria collected by the first electrode 11A are held as they are, and only the dead bacteria are released, and the released dead bacteria are transferred to the other electrode of the second stage on the downstream side. It will be collected.

In other words, the specific collection operation in the collection system 1 is not limited to the operation shown in the first operation example described above, and the dielectrophoretic force with respect to the frequencies of viable bacteria and dead bacteria. If the action is to collect the live bacteria first on the upstream side using the difference and collect the dead bacteria by performing the collection operation on the downstream side, the live bacteria are separated and collected. Any operation is possible.

<Introduction of experimental results>
The inventors of the present application use the collection system 1 to separate and collect live bacteria and dead bacteria, and photograph the collected state with the imaging device 500.

Hereafter, the micrographs actually taken are introduced. In these micrographs, the same position of the electrode unit 12 of the collection device 100 is the imaging range. Also, the image was taken after the same frequency control collection operation.

FIG. 29 is a photomicrograph of an electrode in the imaging range before the start of collection. In FIG. 29, the thick line extending in the vertical direction in the center of the imaging range represents between different electrodes, and different electrodes are imaged on the right and left sides of FIG. Further, the gap in each electrode is photographed as a thin line extending in the vertical direction on each of the left and right electrodes. At this stage, no bacteria are attached to any gap, and no bacteria are collected at any electrode.

FIG. 30 is a photomicrograph of the electrode in the imaging range after the collection operation was performed with the bacteria in the sample solution being 100% dead. Since bacteria are attached to the gap of the right electrode in FIG. 30, it can be seen that the right electrode is an electrode collecting dead bacteria.

FIG. 31 is a photomicrograph of the electrode in the imaging range after the collection operation was performed with the bacteria in the sample solution as 100% viable bacteria. Since bacteria are attached to the gap of the left electrode in FIG. 31, it can be seen that the left electrode is an electrode in which viable bacteria are collected.

FIG. 32 is a photomicrograph of the electrodes in the imaging range after the collection operation was performed with 50% dead bacteria and 50% viable bacteria in the sample solution. From the micrograph in FIG. 32, the same number of bacteria are attached to the gap between the left and right electrodes. From the imaging results of FIGS. 30 and 31, dead bacteria are collected on the right electrode in FIG. 32 and live bacteria are collected on the left electrode, and are collected at a ratio close to the ratio of the bacteria in the sample solution. I understand that. That is, from this collection experiment, it was found that the collection system 1 can generally maintain the ratio of viable and dead bacteria in the sample solution and separate and collect them.

The embodiment disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

1 collection system, 10 substrate, 11 electrode unit, 11A first electrode, 11B second electrode, 11C third electrode, 11D fourth electrode, 11E fifth electrode, 12 electrode part, 13 water tank part, 14 water tank wall surface, 15A , 15B connection port, 20 CPU, 21, 51 memory, 22 switch, 23, 24, 25, 53, 54 communication I / F, 26 power supply, 50 CPU, 52 photographing unit, 100 collection device, 200 control device, 201 Input unit, 202 determination unit, 203 valve control unit, 204 pump control unit, 205 frequency control unit, 206 imaging control unit, 300A cleaning liquid chamber, 300B sample liquid chamber, 300C waste liquid chamber, 400A, 400B tube, 500 imaging device, 600 Display device, 700 three-way valve, 700A, 700 , 700C valve, 800 pump, P1, P2, P3, P5, P6, P7 area.

Claims

A collection device for collecting bacteria in a sample solution,
A once-through tube for allowing liquid to flow through;
A plurality of electrodes installed in the cross-flow tube;
A transport mechanism for transporting the liquid along a predetermined liquid flow direction in the flow-through pipe;
A control device for controlling the frequency of each of the plurality of electrodes,
The plurality of electrodes include a first electrode and a second electrode arranged in stages from the upstream side to the downstream side in the liquid flow direction,
The control device controls the frequency of the first electrode so as to collect viable bacteria in the sample liquid that has flowed into the flow-through tube with the first electrode, and the first electrode with the second electrode. The collection apparatus which controls the frequency of a said 2nd electrode so that the dead microbe in the said sample liquid after passing the electrode of this may be collected.
A frequency at which the dielectrophoretic force generated in both the live and dead bacteria in the sample liquid in the flow-through tube is greater than the stress exerted on the live and dead bacteria by the sample liquid transported by the transport mechanism. The first frequency,
The dielectrophoretic force generated in killed bacteria in the sample liquid in the flow-through tube is smaller than the stress exerted on the dead bacteria by the sample liquid transported by the transport mechanism, and the dielectric generated in live bacteria in the sample liquid When the second frequency is a frequency that causes the migration force to be greater than the stress exerted on the viable bacteria by the sample liquid transported by the transport mechanism,
The collection device according to claim 1, wherein the control device sets the frequency of the first electrode as the second frequency, and sets the frequency of the second electrode as the first frequency.
A frequency at which the dielectrophoretic force generated in both the live and dead bacteria in the sample liquid in the flow-through tube is greater than the stress exerted on the live and dead bacteria by the sample liquid transported by the transport mechanism. The first frequency,
The dielectrophoretic force generated in killed bacteria in the sample liquid in the flow-through tube is smaller than the stress exerted on the dead bacteria by the sample liquid transported by the transport mechanism, and the dielectric generated in live bacteria in the sample liquid When the second frequency is a frequency that causes the migration force to be greater than the stress exerted on the viable bacteria by the sample liquid transported by the transport mechanism,
The control device switches the frequency of the first electrode from the first frequency to the second frequency after setting the frequency of the first electrode to the first frequency, and the second frequency The collection device according to claim 1, wherein the frequency of the electrode is the first frequency.
The plurality of electrodes further includes a third electrode arranged upstream of the first electrode in the liquid flow direction,
The control device controls the frequency of the third electrode so as to collect viable and dead bacteria in the sample liquid that has flowed into the flow-through tube with the third electrode,
After the control device controls the frequency of the third electrode, the control device sets the frequency of the third electrode so as to release live bacteria and dead bacteria collected by the third electrode from the third electrode. Control the frequency of the first electrode so as to collect viable bacteria with the first electrode, and the frequency of the second electrode so as to collect dead bacteria with the second electrode The collection device according to claim 1, wherein the collection device is controlled.
The control device sets the frequency of the third electrode so as to collect viable bacteria and dead bacteria in the sample liquid that have flowed through the flow-through pipe, and viable bacteria and dead bacteria in the sample liquid in the flow-through pipe. The collection device according to claim 4, wherein a dielectrophoretic force generated together is set to a frequency that is greater than a stress exerted on the live bacteria and the dead bacteria by the sample liquid transported by the transport mechanism.
The plurality of electrodes are parallel to the liquid flow direction with the first electrode and the second electrode, stepwise from the upstream side to the downstream side in the liquid flow direction, respectively. A fourth electrode and a fifth electrode arranged in a direction orthogonal to the second electrode and the liquid flow direction;
The control device controls the frequency of the fourth electrode so as to collect viable bacteria in the sample liquid that has flowed into the flow-through tube with the fourth electrode, and the fourth electrode with the fourth electrode. The collection device according to claim 1, wherein the frequency of the fifth electrode is controlled so as to collect the viable bacteria released after being collected by the electrode.
A method for separating live and dead bacteria in a sample liquid flowing in the flow path by controlling the frequency of each of a plurality of electrodes arranged stepwise from the upstream side to the downstream side in the liquid flow direction of the flow path. ,
Controlling the frequency of the first electrode so as to collect viable bacteria in the sample solution with the first electrode arranged upstream in the liquid flow direction;
The second electrode so as to collect dead bacteria in the sample liquid after passing through the first electrode by a second electrode arranged downstream of the first electrode in the liquid flow direction. Controlling the frequency of the separation method.
A method of collecting bacteria in a sample solution with a collection device and displaying the result on a display device,
The collection device includes a plurality of electrodes arranged stepwise from the upstream side in the liquid flow direction of the flow path to the downstream side,
Controlling the frequency of the first electrode so as to collect viable bacteria in the sample solution with the first electrode arranged upstream in the liquid flow direction;
The second electrode so as to collect dead bacteria in the sample liquid after passing through the first electrode by a second electrode arranged downstream of the first electrode in the liquid flow direction. Controlling the frequency of
Specify a display position where live bacteria in the collected sample liquid and dead bacteria in the collected sample liquid are on the same screen, and live bacteria and dead bacteria included in the display position On the same screen of the display device.