JP2009274041A - Sample concentrating device and method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample concentrating device which can concentrate particles to be detected to a high concentration. <P>SOLUTION: The inner wall surface of a passage 14 is negatively charged, and positive ions 30 contained in a sample solution 28 in the passage 14 are adsorbed on the inner wall surface of the passage 14. When an electric field is generated between electrodes 18, 19 for concentration, negatively charged particles 29 in the sample solution 28 in a concentration area between the electrodes 18, 19 for concentration is forced in the reverse direction of the electric field and moved (electrophoresis), and attracted to the electrode 18 for concentration to be flocculated and to be concentrated at the electrode 18 for concentration. The positive ions 30 are moved in the electric field direction by Coulomb force to generate a flow (electroosmotic flow) of the sample solution 28. Consequently, the particles 29 are continuously supplied to the concentration area by the electroosmotic flow, and the supplied particles 29 are concentrated by the electrophoresis. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a sample concentration apparatus and a sample concentration method. Specifically, the present invention relates to a sample concentrating device for concentrating a sample containing biological materials such as bacteria, viruses, DNA, and proteins by electrophoresis and a sample concentrating method using the device.

(Overview of electrophoresis)
In the fields of biosensors and chemical sensors, sensor sensitivity is required to be improved in order to detect low-concentration substances and to compensate for a decrease in detection capability accompanying downsizing of the apparatus. For example, in a bacterial test, a method is used in which a bacterium is cultured for several days and then a target bacterium is detected using a cultivated sample. However, with this method, it takes several days to detect bacteria, and the detection sensitivity depends on the growth ability of the bacteria, so it cannot cope with a rapid test. In addition, there is a method of analyzing DNA as a method of high sensitivity and detection in a short time. However, since this method cannot distinguish between dead bacteria and live bacteria, it also detects sterilized bacteria and has a fundamental problem. is there.

  On the other hand, the concentration method using electrophoresis allows the concentration and separation of bacteria by electrophoresis, so that highly sensitive detection of bacteria can be performed by concentrating the sample, and the viable bacteria can be killed. It is expected that viable bacteria can be detected with high sensitivity. In particular, capillary electrophoresis has attracted attention in recent years because of its advantages such as high resolution, analysis in a short time, and a small amount of sample and reagent required.

  In conventional concentration systems using electrophoresis, a method called stacking that concentrates using a gel or two types of solutions has been generally used. However, in this system, the detection method for concentrated samples is limited, There is a drawback that the sample amount is limited. In addition, although there are inventions regarding concentration even in known invention examples, there are only a few examples considering the mechanism for detecting a concentrated sample, and if it is left as it is, it can be diffused until a concentrated sample is detected, or it can be detected on the sensor. I did not.

(Explanation of examples of known literature)
As a known document, for example, there is JP-A-2005-127771 (Patent Document 1). What is disclosed in this document is a microchip for gel electrophoresis of DNA samples. In this chip, an intermediate electrode is provided in the middle of the flow path, and the negatively charged DNA is collected in the vicinity of the middle where a positive voltage is applied. Then, after that, the voltage of the intermediate electrode is opened, and the positive electrode is applied by applying a plus voltage to the electrode beyond that.

  However, in the microchip of Patent Document 1, since it is necessary to use a gel, when there is a flow such as an electroosmotic flow, it cannot be applied to concentration by the same mechanism. Moreover, since a gel is used, a sample of large particles such as E. coli cannot be concentrated. Furthermore, the particles in the solution cannot be analyzed as they are, and there is a problem that the detection method is limited because the particles penetrate into the gel. In particular, compatibility with chemical sensors and SPR is poor.

  As another known document, there is JP-A-6-505593 (Patent Document 2). This document discloses the sample concentration being improved by extracting the sample buffer from the capillary with electroosmotic flow while stacking the sample components in the auxiliary buffer.

  However, in the method described in Patent Document 2, since a diluted sample solution is used, the structure becomes complicated. In addition, the overall structure is large and complicated. Furthermore, since concentration is performed by stacking, there are problems similar to general stacking concentration.

  In general, in stacking concentration, since the sample solution must be held between the electrophoresis solutions, the greatest disadvantage is that the injection amount of the sample solution is limited. That is, when the injection amount of the sample solution increases, a difference occurs in the electroosmotic flow due to the difference in electric field strength between the solution and the electrophoresis solution, and the solution is mixed and cannot be separated. This disadvantage is also the same in the method of Patent Document 2.

  As another known document, there is JP-T-2002-517751 (Patent Document 3). What is disclosed in this document is a method in which a microchip or a capillary is used to form an electrode at the inlet, and a component substance is concentrated near the inlet by a balance between the flow of the solution and electrophoresis by the electrode. is there. This solution flow includes pressure flow, electroosmotic flow and the like.

  However, in the method of Patent Document 3, since a very high voltage of 14 kV is applied to the entrance electrode, there is a problem in terms of safety in use, and the application is also limited. In addition, since the electrode is provided outside the flow path, the region in which the component substance is electrophoresed is widened, so that the concentration efficiency is poor, and it cannot be used at a low voltage. In addition, since the concentration is performed outside the flow path, the concentration position of the component substance is not accurately determined, and the detection may be inaccurate. Furthermore, when a sensor unit is provided in the flow path, some means is required to move the component substances to the sensor unit.

  Another known document is JP-A-2005-87868 (Patent Document 4). What is disclosed here is that two types of solutions having different electrical conductivities are fed from both ends of a T-shaped channel by electroosmotic flow, and target particles are brought to either side and separated at the solution interface. It is a method.

  However, in this method, it is necessary to use two types of solutions, which is not convenient. Above all, this method is a particle separation method, not a concentration method.

  Another known document is JP-A-2002-233792 (Patent Document 5). In what is disclosed here, a plurality of electrodes are arranged along the flow path direction on the inner surface of the flow path, an electric field is applied to the electrodes arranged in the cross section direction of the flow path, and the particles are attracted to the electrodes and captured. ing.

  However, the one disclosed in Patent Document 5 is also a particle separation mechanism and does not concentrate the particles. Moreover, it does not describe the point of detecting the supplemented particles or the point of moving the supplemented particles.

  Another known document is JP-A-2001-504937 (Patent Document 6). What is disclosed here is that an electric field is applied from the outside of the flow channel, charged particles are brought to the flow channel wall, and the pH is adjusted to be the isoelectric point of the target particle. Only particles are allowed to pass through without approaching the particle wall, thereby separating the particles.

  However, the method disclosed in Patent Document 6 is also a method for separating particles, and is not a method for concentrating particles. Moreover, in this method, it is necessary to adjust pH for every substance, and it takes time and effort.

JP 2005-127771 A JP-T 6-505593 Special table 2002-517751 gazette Japanese Patent Laying-Open No. 2005-87868 JP 2002-233792 A JP 2001-504937 A

  The present invention has been made in view of the technical problems as described above, and the main object of the present invention is to detect with a simple structure by utilizing electrophoresis and electroosmotic flow. It is an object of the present invention to provide a sample concentrator capable of concentrating component substances to be concentrated at a high concentration.

  The sample concentrator of the present invention has a flow path connecting a sample solution supply section and a recovery section, and includes at least two electrodes for concentrating charged component substances of the sample solution to be detected. The inner wall surface of the flow path is charged with the same polarity as the component substance.

  The at least two electrodes for concentrating the charged component substance of the sample solution are electrodes for concentrating (aggregating) the charged component substance by sucking the charged component substance by generating an electric field between the electrodes. It is. In order to increase the concentration of the component substance, it is desirable that one electrode is used to suck and concentrate the component substance.

  In the sample concentrator of the present invention, since the inner wall surface of the flow path is charged with the same polarity (plus or minus) as the component substance, ions having the opposite polarity to the component substance in the sample solution Adsorbed on the wall. Therefore, when an electric field is generated between the electrodes for concentration, the charged component substances are attracted in the same direction as the electric field or in the opposite direction, and are collected and concentrated on a certain electrode for concentration. Further, the ions adsorbed on the inner wall surface of the flow path are moved by receiving a force in the direction opposite to the component substance by the electric field, thereby causing a flow in the sample solution. Therefore, the sample solution in which the component substance is sucked by the electrode and is reduced in quantity is discharged from the concentration area, and a new sample solution containing the component material is sent to the concentration area. Therefore, the component substance is continuously supplied to the concentration area by the sample solution and further concentrated, so that the component substance can be concentrated to a high concentration.

  An embodiment of the sample concentrator according to the present invention is characterized in that the cross section of the flow path has a flat shape whose height is smaller than its width. In such an embodiment, since the height of the flow path is low, the sample solution in the flow path can be made to flow uniformly by the movement of ions. On the other hand, since the width of the flow path is wide, the flow rate of the sample solution flowing through the flow path can be increased.

Another embodiment of the sample concentrator according to the present invention is such that the charged charge of the component substance to be detected is e, the viscosity of the sample solution is η, the average radius of the component substance to be detected is r, and the dielectric constant of the sample solution Ε, zeta potential ζ, and when the concentration electrode is arranged at a distance x with respect to the flow rate of the sample solution driven by the electric field when it is assumed that an electric field is applied to the entire flow path, When the ratio of the flow rate of the sample solution driven by the electric field generated between the electrodes for concentration is f (x),
e / (6πηr)> f (x) · (εζ / η)
It is characterized by the relationship of If this relational expression is established, when an electric field is generated between the electrodes for concentration, the rate at which the component substance is obtained by the electric field becomes larger than the flow rate of the sample solution. Therefore, the component substance can be concentrated on the electrode for concentration without flowing into the sample solution.

  Still another embodiment of the sample concentrating device according to the present invention includes at least two electrodes for feeding the component substance in the same direction together with the sample solution in the flow channel in the flow channel or in the vicinity of the flow channel. In preparation. According to such an embodiment, an electric field is generated between the electrodes for concentration to concentrate the component substances, and then an electric field is generated between the electrodes for liquid feeding to cause the sample solution to flow, thereby concentrating the concentrated components. The substance can be transported with the sample solution flow. For example, by moving the concentrated component substance from the concentration position to the sensor installation position, the component substance can be detected easily and with high sensitivity.

  Yet another embodiment of the sample concentrating device according to the present invention is characterized in that a distance between the liquid feeding electrodes is longer than a distance between the concentrating electrodes. When the same voltage is applied between the electrodes for liquid feeding and between the electrodes for concentration, an electric field is generated between the electrodes for liquid feeding to move the component substances in the same direction as the sample solution, and between the electrodes for concentration. This is because the distance between the electrodes for liquid feeding needs to be longer than the distance between the electrodes for concentration in order to generate the electric field and move the component substances in the direction opposite to the sample solution.

  Yet another embodiment of the sample concentrator according to the present invention is characterized in that the liquid feeding electrode is provided in the supply section and the recovery section. It is because the distance between the electrodes for liquid feeding can be enlarged by providing the electrodes for liquid feeding in a supply part and a collection | recovery part.

  Yet another embodiment of the sample concentrator according to the present invention is characterized in that a part of the electrode for concentration also serves as the electrode for liquid feeding. According to this embodiment, the number of electrodes can be reduced.

Still another embodiment of the sample concentrator according to the present invention is that the dielectric constant of the sample solution is ε, the zeta potential is ζ, the viscosity of the sample solution is η, the charged charge of the component substance to be detected is e, Where r is the average radius of the constituent materials
εζ / η> e / (6πηr)
It is characterized by the relationship of If this relational expression is established, when an electric field is generated between the electrodes for liquid delivery, the rate at which the component substance is obtained by the electric field becomes smaller than the flow rate of the sample solution. Therefore, the component substance is transported in the same direction as the sample solution.

  The sample concentration method according to the present invention includes a flow path connecting a supply section and a recovery section of a sample solution, and includes at least two electrodes for concentrating the constituent substances of the target sample solution in the flow path, At least two electrodes for delivering the component substance in the same direction together with the sample solution in the channel are provided in the channel or in the vicinity of the channel, opposite to the charged component substance of the sample solution to be detected A sample concentrating method using a sample concentrating device in which the inner wall surface of the flow path is charged with the polarity of, wherein an electric field is generated between the concentrating electrodes, and the component substance is applied to any one of the electrodes A step of concentrating and generating a flow of the sample solution in a direction opposite to the suction direction of the component substances; and an electric field is generated between the liquid-feeding electrodes to generate the sample solution in the flow path. Together create a flow, it is characterized by comprising a step of moving the component material in the same direction as the flow of the sample solution.

  According to the sample concentration method of the present invention, the component substance can be concentrated to a high concentration by the first step, and the component substance concentrated by the second step is moved to a desired position along the flow path. Can do.

  A first detection device according to the present invention includes the sample concentration device according to the present invention and a sensor disposed in a region between the concentration electrodes of the sample concentration device. According to such a detection apparatus, the component substance concentrated by the sample concentration apparatus can be immediately detected at the position, and the detection efficiency is increased.

  A second detection device according to the present invention includes the sample concentrating device of the present invention provided with a liquid feeding electrode, and a sensor disposed outside the region between the concentrating electrodes of the sample concentrating device. It is characterized by that. According to such a detection apparatus, the component substance concentrated by the sample concentration apparatus can be detected by moving it to the position of the sensor. Therefore, detection can be performed without being disturbed by the electrode for concentration or the electric field.

  The means for solving the above-described problems in the present invention has a feature in which the above-described constituent elements are appropriately combined, and the present invention enables many variations by combining such constituent elements. .

  Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings.

(First embodiment)
Hereinafter, the structure of the sample concentrator according to Embodiment 1 will be described with reference to FIGS. 1 (a) and 1 (b). FIG. 1A is a schematic cross-sectional view of a sample concentrating device 11 according to Embodiment 1, and FIG. 1B is a schematic cross-sectional view taken along line XX in FIG. In the first embodiment, a case where particles (component substances) negatively charged are concentrated will be described as an example.

(Description of structure)
The sample concentrator 11 is formed by a plate 12 having a flat plate shape. A pair of reservoirs 13 and 15 (a sample solution supply unit and a recovery unit) are provided in the plate 12, and the reservoirs 13 and 15 are connected to each other by a linear minute channel 14. Further, liquid supply electrodes 16 and 17 are provided on the bottom surfaces of the reservoirs 13 and 15, respectively, and a pair of concentration electrodes 18 and 19 are provided on the bottom surface of the flow channel 14 so as to cross the middle of the flow channel. It has been.

  The plate 12 is formed of a material that is charged to the same polarity as the particles, and at least the inner wall surface of the flow path 14 is previously charged to the same polarity as the particles, that is, negative. In the first embodiment, since the particles are negatively charged, the plate 12 is also formed of a negatively charged material. For example, glass, PDMS (polydimethylsiloxane), or the like can be used, but any other material that is negatively charged can be used. Further, any material can be used for the plate 12 as long as the inner surface of the flow path 14 is negatively charged by surface treatment. The plate 12 may be a transparent material or an opaque material. However, when the particles concentrated in the flow path are measured by an optical sensor or inspection means, at least that portion is made of a transparent material. Need to form.

  The reservoirs 13 and 15 are columnar spaces, and the upper surface of each of the reservoirs 13 and 15 is open at the upper surface of the plate 12. One of the reservoirs 13 is a reservoir on the solution supply side for storing a sample solution to be examined from now on, and the other reservoir 15 is a reservoir on the solution recovery side and includes a flow path 14. The sample solution after passing through is for storing. Each of the reservoirs 13 and 15 has a volume sufficiently larger than the volume of the flow path 14.

  Both ends of the flow path 14 are open at the side surfaces of the lower ends of the reservoir 13 and the reservoir 15, respectively, and communicate with the reservoirs 13 and 15. The cross section of the channel 14 is a rectangular cross section having a height of about 10 μm and a width of about 100 μm.

  The liquid feeding electrodes 16 and 17 and the concentration electrodes 18 and 19 are formed by adhering Au to the plate 12 by a method such as sputtering or chemical plating. In addition to Au, platinum-based metals such as Pt can also be used. It is.

  As shown in FIG. 1B, the liquid feeding electrode 16 is connected to the DC power source 20 by the wiring 23, and the concentration electrode 18 is also connected to the DC power source 20 by the wiring 24 through the switch 21. Further, the liquid feeding electrode 17 is connected to the ground by the wiring 25, and the concentration electrode 19 is also connected to the ground by the wiring 26 through the switch 22. The switch 21 and the switch 22 are continuous throw switches and are opened simultaneously or simultaneously closed. In addition, the DC power supply 20 includes a power switch 27.

(Explanation of concentration operation)
According to the sample concentrating device 11 as described above, particles in the sample solution can be concentrated to a high concentration using electroosmotic flow and electrophoresis. Hereinafter, the concentration operation of the sample solution by the sample concentration device 11 will be described with reference to FIGS. 2 (a) and 2 (b) show the operation of the sample concentrating device 11, and FIGS. 3 and 4 are diagrams schematically showing how particles are concentrated at that time. FIG. 5 is a diagram showing a state where particles are concentrated only by electrophoresis without electroosmotic flow. FIG. 6 is a diagram for explaining electroosmotic flow and electrophoresis.

  When the sample solution is concentrated, the switches 21 and 22 are closed. First, as shown in FIG. 2A, with the power switch 27 turned off, the sample solution 28 is injected and stored in the reservoir 13 on the solution supply side, and the sample solution 28 is also allowed to enter the flow path 14. Keep it. In the state before the start of concentration, as shown in FIG. 3, since the inner wall surface of the channel 14 is negatively charged, the positive ions 30 in the sample solution 28 are attracted to the inner wall surface of the channel 14 and flow. An electric double layer is generated on the inner wall surface of the path 14. However, since there is no electric field in the flow path 14, there is no flow of the sample solution 28 (electroosmotic flow) in the flow path 14, and the particles 29 are evenly dispersed throughout the flow path 14.

  Next, as shown in FIG. 2B, when the power switch 27 is turned on, as shown in FIG. 4, an electroosmotic flow is generated in the flow path 14, and the sample solution 28 moves from the reservoir 13 side to the reservoir 15 side. While flowing and being fed, the particles 29 are attracted to the concentration electrode 18 and concentrated at the location of the concentration electrode 18.

  In order to explain this reason, electrophoresis and electroosmotic flow will be described. FIG. 6 is a schematic diagram illustrating the reason why electrophoresis and electroosmotic flow occur in the flow path 14. Electrophoresis is a phenomenon in which charged particles are attracted in the direction of an electric field by Coulomb force, and the particles move depending on the amount of charge. Referring to FIG. 6, when a voltage is applied between the electrodes K1 and K2 provided in the flow path 14, an electric field E is generated between the electrodes K1 and K2 in the flow path 14. At this time, if the particle 29 is negatively charged, the particle 29 receives a force by the electric field E and is pulled toward the positive electrode K1.

  The electroosmotic flow is a flow of a solution that occurs when ions of opposite polarities gather on a charged wall surface, the collected ions are attracted in the electric field direction by Coulomb force, and the solution is involved in the movement of ions. Referring to FIG. 6, when the inner wall surface of the flow path 14 is negatively charged, the positive ions 30 in the sample solution 28 are attracted to the inner wall surface of the flow path 14, and the inner wall surface is electrically charged. Multiple layers occur. Moreover, when a voltage is applied between the electrodes K1 and K2 and an electric field E is generated in the flow path 14, the positive ions 30 on the inner wall surface are pulled by the electric field E and pulled toward the negative electrode K2. Moving. At this time, the positive ions 30 moving to the electrode K2 side drag the sample solution 28, so that the sample solution 28 flows toward the K2 side. This phenomenon is called electroosmotic effect, and this flow is called electroosmotic flow.

  Returning to the concentration operation of FIGS. 2B and 4, since the switches 21 and 22 are closed, an electric field E (≠ 0) is generated between the concentration electrode 18 and the concentration electrode 19. However, the electric field E = 0 in the region between the liquid-feeding electrode 16 and the concentration electrode 18 and in the region between the concentration electrode 19 and the liquid-feeding electrode 17. Therefore, the particles 29 in the concentration area between the concentration electrodes 18 and 19 are attracted to the concentration electrode 18 side under the force of the electric field and aggregate in the vicinity of the concentration electrode 18. As a result, the particles 29 dispersed in the sample solution 28 in the concentration area are collected by the concentration electrode 18 and concentrated.

  The sample solution 28 receives a driving force due to the electroosmotic effect because an electric field is present in the region between the concentration electrodes 18 and 19, but the region between the liquid supply electrode 16 and the concentration electrode 18. In addition, since the electric field E = 0 in the region between the concentrating electrode 19 and the liquid feeding electrode 17, no driving force is generated due to the electroosmotic effect. Therefore, the sample solution 28 in the flow channel 14 generates an electroosmotic flow only by the driving force between the concentration electrodes 18 and 19.

  If the inner wall surface of the flow path 14 is not charged and no electroosmotic flow is generated, particles in the concentration area between the concentration electrode 18 and the concentration electrode 19 as shown in FIG. If 29 collects on the concentration electrode 18, the particles 29 are not further concentrated. Therefore, a very large concentration cannot be obtained.

  On the other hand, when the electroosmotic flow exists, even if all the particles 29 in the concentration area gather at the concentration electrode 18 as shown in FIG. 5, the sample solution 28 containing the particles 29 by the electroosmotic flow enters the concentration area. The supplied and newly supplied particles 29 also collect on the concentration electrode 18. By approaching and combining electrophoresis and electroosmotic flow, it is possible to obtain particles 29 gathered locally at a very high concentration.

  However, in the presence of electrophoresis and electroosmotic flow, the particles 29 are caused to flow in the opposite direction to the concentrating electrode 18 by the electroosmotic flow. Therefore, in order to collect the particles 29 on the concentrating electrode 18, The speed attracted to the concentration electrode 18 side must be faster than the flow rate of the electron osmotic flow.

(Description of particle transport operation)
After the concentration of the particles 29 is completed, as shown in FIG. 7, when the switches 21 and 22 are opened so that a voltage is applied only between the liquid feeding electrodes 16 and 17, the electroosmotic effect is generated in the flow path 14. The sample solution 28 is fed, and the particles 29 concentrated on the concentrating electrode 18 are carried on the flow of the sample solution 28. Liquid feeding means moving the sample solution as an electroosmotic flow in the passage, and particle transportation means moving particles together with the sample solution in the same direction as the sample solution.

  When the switches 21 and 22 are opened and a voltage is applied between the liquid feeding electrodes 16 and 17, an electric field E (between the liquid feeding electrode 16 and the liquid feeding electrode 17 as shown in FIG. ≠ 0) occurs and electrophoresis and electroosmotic flow occur. For this purpose, the speed at which the particles 29 are attracted to the concentration electrode 18 side by electrophoresis must be slower than the flow velocity of the electroosmotic flow.

  Therefore, when the switches 21 and 22 are opened, the particles 29 concentrated in the concentration electrode 18 can be moved along the flow path 14. However, since the concentrated particles 29 may be diffused by particle transportation, it is preferable that the flow rate of the electroosmotic flow be an appropriate flow rate in consideration of the diffusion of the particles 29.

  The purpose of moving the particles 29 thus concentrated by the particle conveyance is to move the particles 29 to the inspection position of the sensor or detection device for inspecting the particles 29.

  As understood from the above, according to the sample concentrating device 11, the charged particles 29 contained in the sample solution 28 can be aggregated at a high concentration. Further, since the concentrated particles 29 can be conveyed to a position such as a sensor, the particles 29 can be detected efficiently and with high sensitivity by the sensor or the like. Further, since the particles 29 are conveyed using electroosmotic flow, an external pump or the like is not required, and the sample concentrator 11 can be made compact and the cost can be reduced.

(Conditions to enable concentration and particle transport)
In order to concentrate the particles while feeding the solution as described above, the particles must be sucked into the concentration electrode 18 without flowing into the sample solution. On the other hand, when carrying the particles by liquid feeding, the force that the sample solution pushes the particles must be superior to the force by which the liquid feeding electrode 16 sucks the particles. Such an action can be realized under the following conditions.

First, a condition for causing particle conveyance by liquid feeding is obtained. In this case, since the switches 21 and 22 are open and the electric field E is generated over the entire length of the flow path 15, the flow velocity ν _EOF of the sample solution 28 flowing due to the electroosmosis effect is It becomes equal to the osmotic flow velocity and is expressed by the following formula (1).
ν _EOF = (εζ / η) E = k _EOF E (1)
ε: Dielectric constant of sample solution
ζ: zeta potential
η: viscosity of the sample solution
E: Potential gradient (electric field) between the liquid feeding electrodes 16 and 17
Here, k _EOF = (εζ / η) is the electroosmotic mobility determined by the flow path 14 and the sample solution 28. The zeta potential ζ is a “slip surface” of the electric double layer (diffusion) generated on the inner wall surface of the flow path 14 (an “ion-fixed layer” formed in the vicinity of the inner wall surface in the sample solution and the outside thereof. It is defined as a potential at a boundary defined for the sake of convenience.

The electrophoretic velocity ν _EP of the particles 29 that move by electrophoresis is expressed by the following formula (2).
ν _EP = [e / (6πηr)] E = k _EP E (2)
e: Charge of particles
η: viscosity of the sample solution
r: radius of the particle
E: Potential gradient (electric field) between the liquid feeding electrodes 16 and 17
Here, k _EP = ([e / (6πηr)] is an electrophoretic mobility determined by the properties of the particles 29.

In order to move the particles 29 in the electroosmotic flow direction when the switches 21 and 22 are opened, it is sufficient that the flow velocity ν _EOF of the sample solution 28 is larger than the electrophoresis velocity ν _EP of the particles 29. Therefore, from the above equations (1) and (2), this condition is
k _EOF E> k _EP E
It is expressed. Further, when the distance between the electrodes 16 and 17 for liquid feeding is L and the voltage applied to the liquid feeding electrode 16 (voltage of the DC power supply 20) is V, E = V / L. The conditions for the particle conveyance are as shown in the following formula (3).
k _EOF (V / L)> k _EP (V / L) (3)

Next, the conditions for concentrating the particles are obtained. In this case, since the switches 21 and 22 are closed and the electric field E is generated between the concentration electrodes 18 and 19, the distance between the electrodes of the concentration electrodes 18 and 19 is x, and the applied voltage to the concentration electrode 18. If (the voltage of the DC power supply 20) is V, E = V / x. Accordingly, the electrophoretic velocity ν _EP of the particles 29 in the concentration area between the concentration electrodes 18 and 19 is expressed by the following equation (4) using the above equation (2).
ν _EP = k _EP (V / x) (4)

On the other hand, since no electric field is generated between the liquid feeding electrode 16 and the concentrating electrode 18 or between the concentrating electrode 19 and the liquid feeding electrode 17 at this time, the sample is out of the area outside these concentrating areas. A driving force is not applied to the solution 28 and a solution resistance is generated. Therefore, the electroosmotic flow is affected by such a solution resistance, and the flow velocity ν _EOF of the sample solution 28 becomes slower than the electroosmotic flow velocity. When the flow rate maintenance rate due to the solution resistance is expressed as a function of the interelectrode distance x of the concentration electrodes 18 and 19, which is f (x), the flow rate of the sample solution is expressed by the following formula (5).
ν _EOF = f (x) · k _EOF (V / x) (5)
The flow rate maintenance rate f (x) is an increasing function of the interelectrode distance x, and f (x) ≦ 1.

In order to move the particles 29 in the direction opposite to the electroosmotic flow direction (electrophoresis direction) when the switches 21 and 22 are closed, the electrophoretic velocity ν _EP of the particles 29 must be larger than the flow velocity ν _{EOF of the} sample solution 28. That's fine. Therefore, the conditions for concentrating the particles 29 by electrophoresis are as shown in the following equation (6) from the equations (4) and (5).
f (x) · k _EOF (V / x) <k _EP (V / x) (6)

Eventually, when the switches 21 and 22 are opened, the particle conveyance by the liquid feeding occurs, and when the switches 21 and 22 are closed, the concentration of the particles 29 by the electrophoresis occurs, the equations (3) and (6) Since it is sufficient to hold simultaneously, this condition is expressed by the following formula (7).
k _EOF > k _EP > f (x) · k _EOF (7)
Alternatively, Equation (7) can also be expressed as follows.
εζ / η> e / (6πηr)> f (x) · (εζ / η) (8)
ε: Dielectric constant of sample solution
ζ: zeta potential
η: viscosity of the sample solution
e: Charge of particles
r: radius of the particle
f (x): Flow rate maintenance rate

  Since the properties of the particles 29 cannot be basically changed, in order to satisfy the inequality on the left side of the equation (8), it is effective to increase the charge amount of the inner wall surface of the flow path 14 and increase the zeta potential ζ. It is. Further, in order to satisfy the inequality on the right side of Expression (8), it is effective to reduce the flow rate maintenance rate f (x) (that is, increase the solution resistance).

  Since the zeta potential ζ can be changed by a surface treatment such as a silane coupling agent or a self-assembled film (SAM), the zeta potential ζ may be increased by these surface treatments.

A pressure loss can be considered as the solution resistance. The pressure loss ΔP is generally expressed by the following equation.
ΔP = (32ηIu) / D ²
η: viscosity of the sample solution
I: Flow representative length
u: Average flow velocity
D: Diameter of the channel Accordingly, if a channel having a small diameter D is used, the pressure loss ΔP increases and the solution resistance increases, so that the flow rate maintenance rate f (x) can be reduced. Therefore, it is obvious that a narrow flow path may be used.

  Further, the pressure loss ΔP can be increased and the flow rate maintenance rate f (x) can be decreased by shortening the interelectrode distance x between the concentrating electrodes 18 and 19. FIG. 9 shows a case where the concentration electrodes 18 and 19 are provided at a distance x in the channel 14 having a length of 14 mm, and the sample solution 28 is fed by applying a voltage between the concentration electrodes 18 and 19. This represents the relationship between the interelectrode distance x and the flow rate of the sample solution 28 (per unit voltage). FIG. 9 shows a flow rate obtained by calculation on the assumption that there is no pressure loss, and an actual measurement value of the flow rate of the sample solution 28. According to FIG. 9, the smaller the distance between the electrodes x, the larger the difference between the measured value and the calculated value (theoretical value when there is no pressure loss), and when the distance between the electrodes x is 1 mm, the calculated value and the actually measured value. There is a 6-fold difference between Therefore, it can be seen that the pressure loss increases as the interelectrode distance x between the concentrating electrodes 18 and 19 decreases. Accordingly, if the inter-electrode distance x is reduced, f (x) is reduced, and the value of the right equation of Equation (8) can be reduced.

  Further, since the charge amount e of the particles 29 can be changed by adjusting the pH of the sample solution 28, the value of the middle formula of the formula (8) can be changed. Care must be taken since the zeta potential ζ of the wall surface and the dielectric constant ε of the sample solution also change.

  Although it has been described above that it is preferable to use a thin channel, in the first embodiment, the channel 14 having a height of about 10 μm and a width of about 100 μm is actually used. This is for the following reason. Considering the case where the height of the flow path 14 is large, negative ions are attracted to the inner wall surface of the flow path 14. However, there is a possibility that the flow in the flow path 14 becomes a laminar flow because the flow does not occur in the central portion of the flow path 14 or the flow velocity is slow even if the flow occurs. Experimentally, by making the height of the flow channel 14 smaller than about 10 μm, the entire flow in the flow channel 14 could be made uniform. On the other hand, if the height of the flow path 14 is made too small, the flow rate of the sample solution flowing in the flow path 14 becomes small. Therefore, the height of the flow path 14 is most preferably about 10 μm.

  However, if the channel 14 has a circular cross section with a diameter of about 10 μm, the flow rate of the sample solution flowing in the channel 14 is reduced. For this reason, in the first embodiment, the height of the flow path 14 is set to about 10 μm to uniform the flow rate, and the width of the flow path 14 is increased (about 100 μm) to ensure the flow rate.

  Further, the cross-sectional shape of the flow path 14 can be a vertically long cross-sectional shape having a width of about 10 μm and a height of about 100 μm. However, if the sample has a vertically long cross-sectional shape, there is a possibility that air bubbles may be caught in the flow path 14 when the sample solution in the reservoir 13 becomes small. Therefore, the wide flow path 14 is used in the width direction.

(Particle moving speed and distance between electrodes)
Next, consider the moving speed of the particles 29 during concentration and particle transportation. When the switches 21 and 22 are closed and the concentration is performed, the moving speed of the particles 29 is expressed by the equations (4) and (5).
[K _EP −f (x) · k _EOF ] (V / x)
It becomes. The flow rate of the sample solution 28 at this time is
f (x) · k _EOF (V / x)
It is. Therefore, by reducing the interelectrode distance x between the concentration electrodes 18 and 19, the particle moving speed is increased to improve the concentration efficiency, and the flow rate of the sample solution 28 is increased to efficiently move the particles 29 to the concentration area. We can expect to be able to supply well.

In addition, when the particles 21 are transported with the switches 21 and 22 closed, the moving speed of the particles 29 can be calculated from the equations (1) and (2).
[K _EOF -k _EP ] (V / L)
It becomes. Therefore, in the case of particle conveyance, if the interelectrode distance L between the liquid feeding electrodes 16 and 17 is increased, the moving speed of the particles can be reduced, and the concentrated particles can be conveyed to a predetermined sensing position. It can be stopped with high accuracy, and the measurement sensitivity can be improved by increasing the signal intensity during sensing. Therefore, it is preferable that the interelectrode distance L between the liquid feeding electrodes 16 and 17 be as long as possible within a range where the size of the plate 12 does not increase. In the first embodiment, the liquid feeding electrodes 16 and 17 are connected to the reservoirs 13 and 15. It is provided inside.

  From the above, it is desirable that the ratio L / x of the interelectrode distance L between the liquid feeding electrodes 16 and 17 and the interelectrode distance x between the concentrating electrodes 18 and 19 is as large as possible.

(Production method of sample concentrator)
Next, a method for producing the sample concentrator 11 of Embodiment 1 will be described. 10 (a) to 10 (e), 11 (a) to 11 (d), and 12 (a) to 12 (d) are schematic cross-sectional views illustrating the manufacturing method.

  First, as shown in FIG. 10A, after cleaning the glass substrate 41 (wafer), Au is sputtered to form an Au film 42 having a film thickness of 300 nm on the upper surface of the glass substrate 41. Next, as shown in FIG. 10B, a photoresist film is spin-coated on the Au film 42 to form a resist film 43. Next, as shown in FIG. 10C, the resist film 43 is patterned using a mask. Further, as shown in FIG. 10D, the Au film 42 is etched by an etchant using the patterned resist film 43 as a mask. After that, the resist film 43 on the Au film 42 is removed with acetone. As a result, as shown in FIG. 10E, the liquid feeding electrodes 16 and 17 and the concentration electrodes 18 and 19 are completed on the upper surface of the glass substrate 41 by the Au film 42. Although only one glass substrate 41 is shown in FIG. 10, since the glass substrate 41 is formed with a plurality of electrodes 16 to 19 while maintaining the wafer size, the wafer is cut with a dicing saw, Divide into individual glass substrates 10. The glass substrate thus divided is, for example, 27 mm long, 55 mm wide, and 0.5 mm thick.

  On the other hand, as shown in FIG. 11A, after applying a photoresist on the upper surface of the cleaned silicon substrate 44, the photoresist film 45 is patterned by a photolithography process, and the same pattern as the flow path 14 is formed in the photoresist film 45. The opening 46 is opened. Next, as shown in FIG. 11B, the silicon substrate 44 is etched from the opening 46 of the photoresist film 45 to form a groove 47 for forming the flow path 14, and the photoresist film 45 is peeled off. Thereafter, as shown in FIG. 11 (c), Ni is deposited on the upper surface of the silicon substrate 44 by electroforming, and the grooves 47 are transferred to the Ni forming die 48. As shown in FIG. 11 (d), the Ni forming die is transferred. The ridges 49 having the same cross-sectional size and the same pattern as the flow path 14 are formed on the surface of 48.

  Thereafter, as shown in FIG. 12 (a), the PDMS precursor solution is poured onto the upper surface of the Ni mold 48 and kept at a temperature of 60 ° C. for 2 hours to be cured. By doing so, the flow path substrate 50 as shown in FIG. 12B having the flow path 14 formed on the lower surface is obtained. The flow path substrate 50 thus obtained has a length of 25 mm, a width of 35 mm, and a thickness of 4 mm, for example (may be the same vertical and horizontal size as the glass substrate 41). Next, as shown in FIG. 12C, after the two through holes 51 are opened in the flow path substrate 50, the lower surface of the flow path substrate 50 is bonded to the upper surface of the glass substrate 41, and the flow path shown in FIG. Such a sample concentrator 11 is obtained. In the sample concentrating device 11 thus manufactured, the plate 12 is formed by the glass substrate 41 and the flow path substrate 50, and the lower surfaces of the two opening through holes 51 are closed by the glass substrate 41 to form the reservoirs 13 and 15.

(Example)
The state in which the particles were concentrated and the state in which the particles were transported was observed by an actually produced sample concentrator. 13 (a) to (d) and FIGS. 14 (a) to (c) are obtained by using polystyrene beads (particle diameter: 2 mm) whose surfaces are treated with carboxyl groups as observation particles. ) To (d) show how the particles are concentrated, and FIGS. 14 (a) to (c) show how the particles are conveyed. 15 (a) to (e) and FIGS. 16 (a) to (c) are obtained by using E. coli O-157 as observation particles, and FIGS. 15 (a) to (e) are obtained by using E. coli. FIGS. 16 (a) to 16 (c) show how E. coli is transported.

  The sample concentrator used for this observation had a flow path height of 10 μm and a width of 50 to 200 μm (concentration effect could be confirmed with any width). In the flow path having a height of 40 μm, the flow due to the electroosmotic flow was weak in the vicinity of the center of the flow path, and the particles in the vicinity of the center flowed back toward the positive liquid-feeding electrode side when the particles were conveyed. In the sample concentrator of the present invention, since the balance between electroosmotic flow and electrophoresis is important, the height of the flow path is preferably low. This balance is determined by the charge amount of the particles and the charge amount of the inner wall surface of the flow path as described above.

  The two electrodes for liquid feeding and the two electrodes for concentration used Au electrodes. The liquid feeding electrode was a circular Au electrode, and the concentrating electrode was a rectangular Au electrode arranged so as to cross the flow path. Further, in order to increase the difference between electrophoresis and electroosmotic flow, the concentration electrode was disposed at a shorter distance than the entire length of the flow path (10 mm). Specifically, the distance between the electrodes of the concentration electrode is preferably 1 to 2 mm, but in this example, it was set to 1 mm. Further, the distance between the electrodes of the liquid feeding electrode arranged in the reservoir was 14 mm. The width of the concentration electrode was 0.5 mm. The distance between the plus-side liquid feeding electrode and the plus-side concentration electrode was 5.25 mm, and the distance between the minus-side liquid feeding electrode and the minus-side concentration electrode was 6.75 mm.

  Furthermore, a voltage of 50 volts was applied from the power source to the concentrating electrode and the liquid feeding electrode. The higher the applied voltage, the higher the flow rate and the higher the concentration speed.However, the higher the applied voltage, the more likely the bubbles will be generated in the sample solution. It is a trade-off. Therefore, the applied voltage is 50 volts in consideration of these points.

  Concentration operation and the like were confirmed using a 10 mM baud rate buffer as a buffer solution. With pure water, a larger flow rate was obtained, and the higher the buffer concentration, the slower the flow rate. Therefore, the particle transport speed can be adjusted by using an appropriate buffer solution.

  FIG. 13 shows a concentration operation observed using polystyrene beads having a diameter of 2 mm whose surface is treated with a carboxyl group as an observation particle. FIG. 13 (a) shows a flow path before the start of concentration, and FIG. 13 (b). Shows the state of the flow channel immediately after the start of concentration, FIG. 13 (c) shows the state of the flow channel after 1 minute from the start of concentration, and FIG. 13 (d) shows the flow channel after 3 minutes from the start of concentration. It shows a state. In FIG. 13A before the start of concentration, the particles are uniformly dispersed in the flow path. However, as time passes, the particles are as shown in FIGS. 13B, 13C, and 13D. Gradually, the particles concentrate on the positive concentration electrode. FIG. 13 (d) after 3 minutes can observe that a large amount of particles are aggregated and concentrated in the vicinity of the concentration electrode. In the state after 3 minutes, the particle density was 8.6 times the particle density before concentration.

  FIG. 14 shows the state when the voltage is applied between the liquid-feeding electrodes after the concentration step of FIG. 13 and the operation is switched to the particle transport operation, and FIG. 14 (a) starts the particle transport. 14 (b) shows the state of the flow path after 3 seconds from the start of particle transportation, and FIG. 14 (c) shows the state of the flow path after 5 seconds. The state of the subsequent flow path is shown. In FIG. 14A, one second after the start of particle conveyance, the particles concentrated by the positive concentration electrode are conveyed to the negative concentration electrode side. 14B after 3 seconds from the start of particle conveyance, the concentrated particles are conveyed to the negative concentration electrode side in the concentration area. Furthermore, in FIG. 14C, 5 seconds after the start of particle conveyance, the concentrated particles are conveyed outside the concentration area beyond the minus concentration electrode. When the integrated light amount was measured by image processing at 3 seconds after the start of conveyance, it was confirmed that the integrated light amount was 8.6 times that before concentration.

  FIG. 15 shows the concentration operation observed using Escherichia coli O-157 as an observation particle. FIG. 15 (a) shows the flow path before the start of concentration, and FIG. 15 (b) shows the flow path immediately after the start of concentration. FIG. 15C shows the state of the flow channel after 1 minute has elapsed since the start of concentration, FIG. 15D shows the state of the flow channel when 3 minutes have elapsed since the start of concentration, and FIG. ) Shows the flow path after 5 minutes from the start of concentration. In FIG. 15 (a) before the start of concentration, E. coli seems to be uniformly dispersed in the flow path, but as time passes, FIG. 15 (b), FIG. 15 (c), FIG. 15 (d), FIG. As shown in FIG. 15 (e), Escherichia coli gradually aggregates on the positive concentration electrode, and in FIG. 15 (e) after 5 minutes, many E. coli are aggregated and concentrated near the concentration electrode. Although the concentration rate is slower than when polystyrene beads are used, it can be observed that even E. coli can be sufficiently concentrated.

  FIG. 16 shows a state in which, after the concentration step in FIG. 15, a voltage is applied between the liquid-feeding electrodes to switch to the particle transport operation, FIG. 16 (a) starts the particle transport. 16 (b) shows the state of the flow path after 3 seconds from the start of particle transportation, and FIG. 16 (c) shows the state of the flow path after 5 seconds from the start of particle transportation. The state of the subsequent flow path is shown. In the case of E. coli as well, it can be observed that E. coli concentrated at a slow rate is transported toward the negative concentration electrode.

  Therefore, the effectiveness of the sample concentrator of Embodiment 1 could be confirmed by observation in such an example.

(Other)
In the above description, the case where the particles are negatively charged has been described, but the sample concentrator 11 can also be used when the particles are positively charged. In this case, a positively chargeable plate 12 is used to positively charge the inner wall surface of the flow path 14, the DC power supply 20 is connected to the liquid supply electrode 16 and the concentration electrode 18, and the liquid supply If the electrode 17 and the concentration electrode 19 side are connected to the ground, the particles are concentrated at the same place (negative concentration electrode).

  In addition, the target to be concentrated is not limited to charged particles, but can also concentrate charged ions, biomolecules, etc., or transport the concentrated particles.

  Also, a switch may be provided on the liquid-feeding electrode, and at the time of concentration, the liquid-feeding electrode switch may be opened and disconnected from the power source.

(Second Embodiment)
FIG. 17 is a schematic diagram showing a sample concentrator 61 according to Embodiment 2 of the present invention. The sample concentrator 61 is configured by three electrodes, that is, two concentrating electrodes 18 and 19 and one liquid-feeding electrode 16, omitting the negative-side liquid-feeding electrode. In the sample concentrating device 61, the liquid feeding electrode 16 is directly connected to the DC power source 20 by a wiring 64. The concentrating electrode 18 can be connected to the DC power source 20 by wirings 65 and 66, can be connected to the ground by wirings 66 and 67, and can be switched between the power source side and the ground side by the switch 62. It has become. The concentrating electrode 19 can be connected to the ground by wirings 68 and 69, and can be separated from the ground by the switch 62.

  Thus, as shown in FIG. 17A, the switch 62 is switched to the power supply side, the concentration electrode 18 is connected to the DC power supply 20, and the switch 63 is closed, and the concentration electrode 19 is connected to the ground. Then, the negative particles in the concentration area between the concentration electrodes 18 and 19 are attracted to the concentration electrode 18 on the plus side and concentrated at the location where the concentration electrode 18 is located. At the same time, an electroosmotic flow is generated in the flow path 14 due to the electroosmotic effect between the concentration electrodes 18 and 19, and the sample solution is sent from the reservoir 13 to the reservoir 15.

  Further, as shown in FIG. 17B, in the state where the switch 62 is switched to the ground side and the concentrating electrode 18 is connected to the ground, and the switch 63 is opened and the concentrating electrode 19 is open, Due to the electroosmotic effect between the electrode 16 and the concentrating electrode 18, an electroosmotic flow is generated in the flow path 14, and the particles concentrated by the concentrating electrode 18 are conveyed to the concentrating electrode 19 side.

  As can be seen from the operation as described above, in the sample concentrating device 61, the concentrating electrode 18 also functions as a negative liquid feeding electrode. Further, since the particles are concentrated by the concentrating electrodes 18 and 19 and the particles are transported by the liquid sending electrode 16 and the concentrating electrode 18, the distance between the liquid sending electrode 16 and the concentrating electrode 18 is the concentration distance. It is longer than the distance between the electrodes 18 and 19.

(Third embodiment)
FIG. 18 is a schematic diagram showing a sample concentrator 71 according to Embodiment 3 of the present invention. The sample concentrating device 71 is configured by branching a concentration channel 72 from the channel 14 and forming the channel in a T shape. Liquid supply electrodes 16 and 17 are provided in reservoirs 13 and 15 provided at both ends of the flow path 14, respectively. A reservoir 80 is also formed at the end of the concentration channel 72, a liquid feeding electrode 73 is provided in the reservoir 80, and a concentration electrode 74 is provided in the middle of the concentration channel 72. The liquid feeding electrode 16 is directly connected to the DC power source 20 by a wiring 75, and the liquid feeding electrode 73 is also directly connected to the DC power source 20 by a wiring 76. The liquid feeding electrode 17 is connected to the ground by a wiring 77. The concentrating electrode 74 is connected to the DC power supply 20 through a switch 78 by a wiring 79. Further, the interelectrode distance along the flow path between the concentrating electrode 74 and the liquid feeding electrode 17 is sufficiently shorter than the interelectrode distance between the liquid feeding electrode 16 and the liquid feeding electrode 17.

  Thus, when the switch 78 is closed and a voltage is applied to the concentration electrode 74, the particles 29 in the concentration area between the concentration electrode 74 and the liquid feeding electrode 17 are concentrated as shown in FIG. The particles 29 are attracted to the electrode 74 (electrophoresis), and the particles 29 are aggregated and concentrated at the location of the concentration electrode 74. At this time, an electroosmotic flow is generated from the liquid feeding electrode 16 toward the liquid feeding electrode 17. In addition, an electroosmotic flow is generated from the concentration electrode 74 toward the liquid feeding electrode 17, whereby the sample solution 28 flows also from the reservoir 80 toward the concentration electrode 74, and the sample including the particles 29 is generated. The solution 28 is supplied from the reservoir 80 to the concentration area.

  After the concentration of the particles 29 is finished, when the switch 78 is opened to open the concentration electrode 74, an electroosmotic flow is generated from the liquid supply electrode 16 toward the liquid supply electrode 17, as shown in FIG. Further, an electroosmotic flow is also generated from the liquid-feeding electrode 73 toward the liquid-feeding electrode 17, whereby the particles 29 concentrated at the location of the concentration electrode 74 are sent along the concentration flow path 72 and the flow path 14. It is conveyed toward the liquid electrode 17 side (particle conveyance).

(Fourth embodiment)
FIG. 21 is a schematic plan view showing a detection device 81 according to Embodiment 4 of the present invention. This detection device 81 uses the sample concentration device 11 of Embodiment 1, and is provided with a sensor 82 on the lower surface of the plate 12 at a position corresponding to the concentration area between the concentration electrodes 18 and 19. is there. The sensor 82 may be a biosensor such as an SPR (surface plasmon resonance) sensor, a QCM (quartz crystal microbalance), an enzyme sensor, or an electrochemical sensor, or a fluorescence detection sensor or a spectroscopic device.

  In this detection device 81, the sensor 82 is arranged in the concentration area, particularly in the vicinity of the concentration electrode 18, so that the particles 29 are concentrated by the concentration electrode 18 or immediately after the concentration of the particles 29 is completed (particle conveyance). The particles 29 can be detected by the sensor 82. Therefore, since the concentrated particles 29 can be detected as they are, the diffusion of the concentrated particles 29 due to particle transportation can be avoided, and the detection by the sensor 82 can be made highly sensitive. In addition, since such an arrangement is an in-capillary system, the detection operation can be performed efficiently. In addition, in such a form, since it is not necessary to convey the concentrated particle | grains 29, the electrodes 16 and 18 for liquid feeding can be abbreviate | omitted.

  However, with the configuration as shown in FIG. 21, the sensor 82 may not be usable due to the influence of the electric field E. In such a case, like the detection device 83 shown in FIG. 22, the sensor 82 may be disposed outside the concentration area and at a position downstream of the particle conveyance direction. In this case, after the particles 29 are concentrated at the location of the concentration electrode 18, the particles 29 concentrated by the particle transport are moved to the position of the sensor 82 and detection is performed. With such a structure, the concentrated particles 29 may be somewhat diffused by particle transportation, but can be applied to almost all sensors.

  The sample concentrator according to the present invention can be used when a minute chemical analysis system called micro TAS (Total Analysis Systems) is constructed. For example, collected in order to improve sensor sensitivity in the development of biochips for detecting bacteria, viruses, DNA, proteins, etc., and in the development of high-sensitivity sensors (SPR sensors, chemical sensors, biosensors, etc.) It can be used in the step of pretreating the sample. Compared to bacterial culture, the concentration of component substances can be increased easily.

  Moreover, application as a nanopillar chip for the purpose of separating components is also conceivable.

FIG. 1A is a schematic cross-sectional view of a sample concentrating device according to Embodiment 1, and FIG. 1B is a schematic cross-sectional view taken along line XX of FIG. FIGS. 2A and 2B are cross-sectional views illustrating a method for concentrating particles using the sample concentration apparatus of the first embodiment. FIG. 3 is a diagram schematically showing the distribution of particles in a state before starting the concentration of particles. FIG. 4 is a diagram schematically showing the distribution of particles in a state where the particles are concentrated. FIG. 5 is a diagram schematically showing a state where particles are concentrated only by electrophoresis without an electroosmotic flow in the flow path. FIG. 6 is a schematic diagram for explaining the reason why electrophoresis and electroosmotic flow occur in the flow path. FIG. 7 is a cross-sectional view showing a state in which particles are transported by the sample concentrating device of the first embodiment. FIG. 8 is a diagram schematically showing the distribution of particles in a state where particle conveyance is performed. FIG. 9 shows a distance x between electrodes when a pair of concentrating electrodes is provided in a flow path having a length of 14 mm and a sample solution is fed by applying a voltage between the concentrating electrodes. It is a figure showing the relationship with the flow rate (per unit voltage) of a sample solution. FIGS. 10A to 10E are diagrams showing a manufacturing process of the sample concentrating device of Embodiment 1, and show a process until an electrode is formed on a glass substrate. FIGS. 11A to 11D are diagrams showing a manufacturing process of the sample concentrating device of the first embodiment and showing a process until a Ni mold is manufactured. 12 (a) to 12 (d) are diagrams showing manufacturing steps of the sample concentrating device of Embodiment 1, in which a flow path substrate is manufactured using a Ni mold, and the flow path substrate is placed on the upper surface of the glass substrate. It shows the process until the sample concentration device is manufactured by pasting together. FIGS. 13A to 13D show the observation of the state in which particles are concentrated using polystyrene beads (particle diameter: 2 mm) whose surfaces are treated with carboxyl groups as observation particles. FIGS. 14A to 14C show the observation of how the particles are conveyed using polystyrene beads (particle diameter: 2 mm) whose surfaces are treated with carboxyl groups as observation particles. FIGS. 15 (a) to 15 (e) are obtained by observing how E. coli is concentrated using E. coli O-157 as observation particles. FIGS. 16 (a) to (c) are observations of E. coli being transported using E. coli O-157 as observation particles. 17 (a) and 17 (b) are schematic views showing a sample concentrating device according to Embodiment 2 of the present invention. FIG. 18 is a schematic diagram showing a sample concentrator according to Embodiment 3 of the present invention. FIG. 19 is a schematic view schematically illustrating a state in which particles are concentrated on the concentration electrode in the sample concentration apparatus of the third embodiment. FIG. 20 is a schematic view schematically illustrating how concentrated particles are conveyed in the sample concentration apparatus of the third embodiment. FIG. 21 is a schematic plan view showing a detection device according to Embodiment 4 of the present invention. FIG. 22 is a schematic plan view showing another detection apparatus according to Embodiment 4 of the present invention.

Explanation of symbols

11, 61, 71 Sample concentrator 13, 15 Reservoir 14 Flow path 16, 17, 73 Electrode for feeding 18, 19, 74 Electrode for concentrating 20 DC power source 21, 22 Switch 28 Sample solution 29 Particle 30 Plus ion 62 Switch 63 Switch 72 Concentration flow path 78 Switch 80 Reservoir 81, 83 Detector 82 Sensor

Claims (11)

  It has a flow path connecting the sample solution supply section and the recovery section, and is equipped with at least two electrodes for concentrating the charged component substance of the sample solution to be detected, with the same polarity as the component substance. A sample concentrating device, wherein an inner wall surface of the flow path is charged.   The sample concentrator according to claim 1, wherein the cross section of the flow path has a flat shape whose height is smaller than its width. The charged charge of the component material to be detected is e, the viscosity of the sample solution is η, the average radius of the component material to be detected is r, the dielectric constant of the sample solution is ε, the zeta potential is ζ, and the electric field is applied to the entire flow path. A sample driven by the electric field generated between the concentrating electrodes when the concentrating electrodes are arranged at a distance x with respect to the flow rate of the sample solution driven by the electric field when When the ratio of the solution flow rates is f (x),
e / (6πηr)> f (x) · (εζ / η)
The sample concentrator according to claim 1, wherein the following relationship is established.   The at least 2 electrode for sending the said component substance to the same direction with the sample solution in the said flow path is provided in the said flow path or the vicinity of the said flow path, The said flow path is characterized by the above-mentioned. Sample concentrator.   The sample concentrator according to claim 4, wherein a distance between the liquid feeding electrodes is longer than a distance between the concentrating electrodes.   The sample concentration apparatus according to claim 4, wherein the liquid feeding electrode is provided in the supply unit and the recovery unit.   The sample concentrator according to claim 4, wherein a part of the concentrating electrode also serves as the liquid feeding electrode. When the dielectric constant of the sample solution is ε, the zeta potential is ζ, the viscosity of the sample solution is η, the charged charge of the component substance to be detected is e, and the average radius of the component substance to be detected is r,
εζ / η> e / (6πηr)
The sample concentrator according to claim 4, wherein the following relationship is established. A flow path connecting the supply section and the collection section of the sample solution, the flow path having at least two electrodes for concentrating the component substances of the target sample solution in the same direction as the sample solution in the flow path At least two electrodes for feeding the component substance to the inside of the channel or in the vicinity of the channel, and the inner wall surface of the channel with the opposite polarity to the charged component substance of the sample solution to be detected Is a sample concentration method using a charged sample concentration device,
Generating an electric field between the electrodes for concentration, concentrating the component substance on any of the electrodes, and causing a flow of the sample solution in a direction opposite to the suction direction of the component substance; ,
Generating a flow of the sample solution in the flow path by generating an electric field between the electrodes for liquid feeding, and moving the component substances in the same direction as the flow of the sample solution;
A method for concentrating a sample, comprising:   A detection apparatus comprising: the sample concentration apparatus according to claim 1; and a sensor disposed in a region between the concentration electrodes of the sample concentration apparatus.   5. A detection apparatus comprising: the sample concentrating device according to claim 4; and a sensor disposed outside a region between the concentrating electrodes of the sample concentrating device.

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