JP2009192479A - Particulate measuring device, and electrode used therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a particulate measuring device which is reduced in cost by simplifying an electrode structure that induces nonuniformity in the electric field, and to provide an electrode which is used for the device. <P>SOLUTION: In a microbe measuring device, a chamber 1 is constituted by disposing the electrodes 33 and 34 between substrates 31 and 32 and a structure 35 for generating electric field distortions on one electrode 34. A sample inlet 37 is disposed at one end of a chamber 1, and an outflow port 38 is formed at the other end. The chamber 1 is filled with suspension, the electrodes 33 and 34 are connected to a migration power supply section 4, and the electric field is applied. Thus, the structure 35 is arranged between the pair of electrodes 33 and 34 and induces nonuniformity in the electric field so that the structure of the electrodes 33 and 34 for making dielectrophoresis force acting on the particulates in the suspension is simplified and cost of the particulate measuring device can be lowered. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a fine particle measuring apparatus for measuring the number of fine particles in a suspension using dielectrophoresis and an electrode used therefor. More particularly, the present invention relates to a particle measuring apparatus capable of simplifying an electrode structure for inducing electric field non-uniformity, enabling simple manufacturing, and reducing costs, and an electrode used therefor.

  In recent years, there is a particularly high need for rapid, simple, and highly sensitive quantitative measurement of microorganisms that cause food poisoning, infections, and the like and can cause some harm to the human body. This is because, in a clinic that does not have a food production process or a microbiological examination facility, the microbiological examination can be performed on the spot to prevent or prevent food poisoning or infectious diseases.

  In addition, in so-called biosensors, the number of fine particles in a sample when quantitatively measuring a biochemical substance in a sample using an artificial fine particle such as polystyrene labeled with a substance that specifically binds to an object such as an antibody. Alternatively, it is necessary to quantitatively measure the binding state. Thus, nowadays, there is a high demand for quickly, simply and quantitatively measuring fine particles contained in a liquid.

  Here, the definition of the fine particles in the present application will be described. The microparticles referred to in this application are polystyrene, particles with some coating on them, carbon particles, metal particles such as gold colloids, bacteria, fungi, actinomycetes, rickettsia, mycoplasma, viruses, so-called microorganisms, and protozoa It is a living organism or a fine particle derived from living organisms in a broad sense, including small animals and protozoa, larvae of organisms, animal and plant cells, sperm, blood cells, nucleic acids, proteins, and the like. In addition, the fine particles referred to in the present application mean all particles having a size capable of dielectrophoresis. In the present application, in particular, measurement of microorganisms is assumed, and hereinafter, the measurement target will be described as microorganisms.

  Conventionally, the culture method is the most commonly used method for testing microorganisms. The culture method is a method of quantifying the number of microorganisms by smearing a microorganism sample on a medium, culturing the microorganism under growth conditions, and counting the number of colonies on the formed medium.

  However, since it usually takes 1 to 2 days for colony formation and several weeks depending on the microorganism species, there is a problem that rapid inspection cannot be performed. In addition, since concentration, dilution, smearing on the medium, and the like are necessary, an operation by a specialist is necessary, and a simple test cannot be performed, or there is a problem of a decrease in accuracy due to operational variations.

  In order to solve these conventional problems, the present inventor, together with other inventors, uses a DEPIM (Dielectrophoretic Impedance Measurement Method) method that combines dielectrophoresis and impedance measurement as a rapid, simple, and highly sensitive microbial count measurement method. Proposed (see, for example, Patent Document 1).

  The DEPIM method is a method for quantitatively measuring the number of microorganisms in a sample solution by collecting microorganisms on a microelectrode by dielectrophoretic force and simultaneously measuring the impedance change of the microelectrode. The measurement principle will be outlined below.

  Microorganisms generally have a structure in which cytoplasm and cell walls having a high dielectric constant and conductivity are ion-rich and surrounded by a cell membrane having a relatively low dielectric constant and conductivity, and can be regarded as dielectric particles. In the DEPIM method, a dielectrophoretic force that is a force acting in a certain direction on dielectric particles polarized in an electric field is used to collect microorganisms that are dielectric particles between gaps of microelectrodes.

It is known that the dielectrophoretic force F _DEP acting on the dielectric particles is given by the following ( _Equation 1) (for example, see Non-Patent Document 1). Hereinafter, a case where the dielectric particles are microorganisms will be described as an example.

Here, a: radius of microorganism when approximated by a sphere, ε ₀ : dielectric constant of vacuum, ε _m : relative permittivity of sample liquid, E: electric field strength, and ▽ represents a gradient by an operator . In this case, ▽ E ² is the gradient of the electric field E ² , and means how much E ² has an inclination at that position, that is, how suddenly the electric field E changes spatially. K is called Clausius Mosotti number, expressed by (Expression 2), Re [K]> 0 indicates positive dielectrophoresis, and microorganisms are in the same direction as the electric field gradient, that is, toward the electric field concentration part. Electrophoresed. Re [K] <0 represents negative dielectrophoresis and migrates away from the electrolytic concentration part, that is, toward the weak electric field part.

Here, ε _b ^* and ε _m ^* represent the complex permittivity of the microorganism and the solution, respectively, and generally the complex permittivity ε _r ^* is expressed by (Equation 3).

Here, ε _{r represents} the relative dielectric constant of the microorganism or sample solution, σ represents the conductivity of the microorganism or sample solution, and ω represents the angular frequency of the applied electric field.

  From (Equation 1), (Equation 2), and (Equation 3), it can be seen that the dielectrophoretic force depends on the radius of the microorganism, the real part of the Clausius Mosotti number (hereinafter referred to as Re [K]), and the electric field strength. It can also be seen that Re [K] varies depending on the complex permittivity and electric field frequency of the sample solution and the microorganism.

  Therefore, in the DEPIM method, it is necessary to appropriately select these parameters, sufficiently increase the dielectrophoretic force acting on the microorganism, and reliably collect the microorganism in the electrode gap. In addition, the DEPIM method is characterized in that the number of microorganisms in a sample solution is quantitatively measured by performing electrical measurement simultaneously with the collection of microorganisms on the electrode by dielectrophoresis.

  Since the microorganism has the structure described above, it can be considered as a fine particle having an inherent impedance electrically. Therefore, when the number of microorganisms collected between the gaps of the microelectrodes by dielectrophoresis increases, the impedance between the electrodes changes according to the number of collections.

  Therefore, the slope of the interelectrode impedance time change is a value corresponding to the number of microorganisms collected between the electrode gaps per unit time, and the magnitude of the slope corresponds to the microorganism concentration in the sample solution. Therefore, it is possible to measure the microorganism concentration in the sample solution, in other words, the number of microorganisms, by measuring the slope of the interelectrode impedance time change.

  Furthermore, in the DEPIM method, microorganisms are measured in a short time by quantifying the number of microorganisms from the slope of the change in impedance time immediately after the start of dielectrophoresis. The measurement principle of the DEPIM method has been outlined above. For details, refer to Non-Patent Document 2.

  On the other hand, devices and methods are known for the electrical measurement of objects in a medium, in particular cells, liposomes or similar micro objects. FIG. 11 shows an apparatus for measuring the impedance of the liquid 51 filled in the channel. In this apparatus, the electrodes 52 are provided so as to be in electrical contact with the liquid in the channel, respectively, and the electrodes 52 are connected to an electrical measurement device (not shown) for measuring the current and voltage (for example, Patent Document 2). reference).

Japanese Patent Laid-Open No. 2000-125846 Japanese translation of PCT publication No. 2003-527581 Hywel Morgan, et al .: “AC Electronics: colloids and nanoparticulates”, RESERCH STUDIES PRESS LTD. Published in 2003, pp. 15-63 J. Suehiro, R. Yatsunami, R. Hamada, M, Hara, J. Phys. D: Appl. Phys. 32 (1999) 2814-2820

  Conventionally, electrodes used in DEPIM have difficulty in manufacturing because it is necessary to use a fine gap (about 5 μm) in order to trap bacteria by dielectrophoresis using electric field distortion generated between electrode gaps. In addition, since the electric field distortion generated between the electrode gaps is only in a region where the electrode edge is limited, the spatial efficiency of the trap is poor and the measurement sensitivity is limited.

  In addition, although the electrodes are formed in a comb-teeth shape to generate electric field distortion, it is difficult to manufacture the electrodes in a comb-teeth shape, resulting in an increase in cost. In particular, it is desirable to replace the electrode every measurement, and it has been desired to simplify the structure and reduce the cost.

  The present invention has been made in view of the above circumstances, and provides a particle measuring apparatus capable of simplifying an electrode structure for inducing electric field non-uniformity and reducing costs, and an electrode used therefor. The purpose is that.

  The fine particle measuring apparatus of the present invention includes a chamber for introducing a liquid containing fine particles, at least a pair of electrodes provided in the chamber, and a structure that is disposed between the pair of electrodes and induces non-uniformity of an electric field. Calculating the number of fine particles in the chamber by applying an alternating voltage between the pair of electrodes and applying a dielectrophoretic force to the fine particles, and calculating a temporal change in impedance between the pair of electrodes. An impedance measuring device.

  According to the above configuration, since the structure is disposed between the pair of electrodes and induces non-uniformity of the electric field, the structure of the electrode that applies the dielectrophoretic force to the fine particles in the suspension is simplified, and the microorganism is trapped. Since a large amount of space can be taken, it is possible to improve measurement sensitivity while reducing the cost of the particle measuring apparatus.

  In the fine particle measuring apparatus of the present invention, the structure is a filter having fine pores.

  According to the above configuration, it is possible to change the impedance of the suspension by moving the fine particles into the micropores with a simple structure.

  The fine particle measuring apparatus of the present invention includes a plurality of filters having different effective diameters of the micropores, and a plurality of pairs of the pair of electrodes are provided so as to correspond to the plurality of filters.

  According to the said structure, the kind of microbe trapped can be changed with the effective diameter of a micropore, and the dynamic range of a measurement can be improved. In addition, it is possible to selectively detect measurement objects having different sizes.

  The fine particle measurement apparatus of the present invention includes an impedance measurement electrode in the structure or on the surface thereof.

  According to the above configuration, the impedance measurement electrode for measuring the impedance of the suspension can be easily manufactured, and the cost of the particle measuring device can be reduced.

  In the fine particle measuring apparatus of the present invention, the structure is composed of an ion adsorbent or includes an ion adsorbent in part.

  According to the above configuration, since the ion adsorbent can adsorb ions in the suspension and reduce the conductivity of the suspension, the decrease in the dielectrophoretic force due to the increase in the conductivity is prevented, and the high conductivity is achieved. The measurement sample can also be measured with high sensitivity.

  In the fine particle measuring apparatus of the present invention, the filter is on the surface of the pair of electrodes.

  According to the said structure, microparticles can be migrated to the micropore of a filter and the impedance of suspension can be changed with a simple structure.

  In the fine particle measuring apparatus of the present invention, the filter is on the surface of both electrodes of the pair of electrodes.

  According to the said structure, microparticles can be migrated to the micropore of the filter provided in both of a pair of electrodes, and the impedance of suspension can be changed efficiently.

  In the fine particle measuring apparatus of the present invention, the filter is positioned between the pair of electrodes.

  According to the said structure, a filter can be arrange | positioned in the middle of a pair of electrode, a microparticle can be trapped on the both sides of a filter, and the trap efficiency of microparticles | fine-particles can be improved.

  In the fine particle measuring apparatus of the present invention, the impedance measuring device measures the number of fine particles based on an initial change in impedance.

  According to the above configuration, since the number of fine particles is measured by the initial change in impedance, the measurement time can be shortened.

  The fine particle measurement apparatus of the present invention includes a reagent that specifically binds to a detection substance and forms an aggregate.

  According to the above configuration, since it specifically binds to the detection substance and forms an agglomerate, the dielectrophoretic force increases according to the size of the agglomerate. It becomes possible to measure.

  In the particle measuring apparatus of the present invention, the pair of electrodes are transparent, the structure is formed on a transparent substrate, and the number of particles in the chamber is optically measured.

  According to the above configuration, since the number of fine particles in the chamber is optically measured, the number of fine particles can be measured without depending on the conductivity of the suspension.

  Moreover, this invention is an electrode used for said fine particle measuring apparatus.

  According to the above configuration, the structure of the electrode can be simplified and the cost of the particle measuring device can be reduced.

  According to the present invention, since the structure is arranged between the pair of electrodes to induce electric field non-uniformity, the structure of the electrode that causes the dielectrophoretic force to act on the fine particles in the suspension can be simplified. Cost can be reduced.

  Hereinafter, a microorganism measuring apparatus according to an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a configuration diagram of a microorganism measuring apparatus according to the present embodiment.

  In FIG. 1, 1 is a chamber for holding a suspension 2 containing microorganisms to be measured, 3 is an electrode including an electrode pair for collecting microorganisms by dielectrophoresis, 4 is an electrophoretic power supply unit, and 5 is trapped by dielectrophoresis. A measuring unit for measuring an optical or electrical change caused by the microorganisms applied, a control calculating unit for controlling the whole microorganism measuring apparatus, performing an analysis calculation of the measurement result, input / output processing, and the like, and 7 for the sample liquid 2. Conductivity input means for inputting conductivity.

  Fig.2 (a) is a figure for demonstrating the structure of the chamber 1 in the microorganisms measuring apparatus of this embodiment. In the chamber 1 of this embodiment, planar electrodes 33 and 34 are provided between the substrates 31 and 32, and a structure 35 (insulator or the like) for creating an electric field strain is provided on one planar electrode 34. Then, the space between the planar electrode 33 and the structure 35 is filled with the suspension 2, and the electrophoretic power supply unit 4 is connected to the planar electrodes 33 and 34 to apply an electric field.

  Dielectrophoresis is a force that works in places where spatially non-uniform electric fields are formed. Therefore, in normal DEP (dielectrophoresis), the thin film electrodes are opposed to each other, with the sharp edges seen in the cross section facing each other. Create a non-uniform electric field. In the present embodiment, a non-uniform electric field (electric field distortion) is created by disposing a structure 35 such as a membrane filter in the electric field, and a phenomenon (iDEP: insulator-based DiElectroPhoresis) that makes dielectrophoresis work there is used. Any material can be used for the structure 35 as long as a non-uniform electric field can be formed. However, the larger the difference in the dielectric constant from the sample liquid (suspension 2), the larger the electric field distortion can be formed. Such a material is desirable. In this embodiment, since the suspension is assumed to be water having a relative dielectric constant of about 80, it is formed of an insulator such as a resin material having a small relative dielectric constant or a metal that can be regarded as having an infinite relative dielectric constant. Is desirable.

  In the present embodiment, a filter that can be easily obtained from a general material is used as the structure 35. The filter is a structure having fine holes in a direction perpendicular to the surface, and generally a membrane filter or the like is easily available. There are various materials for the membrane filter, and there are resin materials such as polycarbonate. Since the relative dielectric constant of the resin material is about 2 to 3, it is suitable for forming an electric field strain. Due to availability and physical characteristics, a polycarbonate membrane filter is used in this embodiment.

  FIG. 2B shows a micrograph in which bacteria are trapped by p-DEP (positive DiElectroPhoresis) acting on the edge of the hole of the structure 35. There are two types of dielectrophoresis, positive dielectrophoresis (p-DEP) and negative dielectrophoresis (n-DEP; negative DiElectroPhoresis), depending on the conditions of the electric field frequency and particle / solution physical properties (dielectric constant, conductivity). The phenomenon is seen. In the positive dielectrophoresis, the particles move toward the location where the electric field concentrates, and in the negative dielectrophoresis, the particles move in the direction away from the location where the electric field concentrates (that is, the direction opposite to the positive dielectrophoresis). In this case, the edge of the hole becomes an electric field concentration portion, and the dielectrophoretic force acts on the edge to trap bacteria.

で表わされる。懸濁液２のインピーダンスＺｍおよび構造体３５のインピーダンスＺｓは略一定なので、チャンバ１のインピーダンスの時間変化は、菌のインピーダンスＺｂの増分に依存する。 FIG. 2C shows an impedance equivalent circuit of the chamber 1. When the impedance of the suspension 2 is Zm, the impedance of the structure 35 is Zs, and the impedance of the fungus is Zb, the impedance Zc of the chamber 1 is

It is represented by Since the impedance Zm of the suspension 2 and the impedance Zs of the structure 35 are substantially constant, the time change of the impedance of the chamber 1 depends on the increment of the bacteria impedance Zb.

  Moreover, in the microorganism measuring apparatus of this embodiment, the structure 35 can also be comprised with an ion adsorption body. As described above, the dielectrophoretic force is a force that varies depending on the conductivity of the suspension 2, and generally the dielectrophoretic force tends to decrease as the conductivity increases. Therefore, according to this configuration, the ion adsorbent can adsorb ions in the suspension 2 and reduce the conductivity of the suspension 2, thereby preventing a decrease in dielectrophoretic force due to an increase in conductivity, Since sufficient microorganisms necessary for the measurement can be trapped by a dielectrophoretic force having a sufficient strength, a measurement sample having a high conductivity can be measured with high sensitivity.

  FIG. 3 is a diagram for explaining a specific configuration of the chamber 1 in the microorganism measuring apparatus of the present embodiment. In the microorganism measuring apparatus of the present embodiment, the chambers 1 are configured by providing electrodes 33 and 34 between the substrates 31 and 32 and providing a structure 35 for generating electric field strain on one electrode 34. A sample introduction port 37 is provided at one end of the chamber 1 and an outflow port 38 is provided at the other end. Then, the chamber 1 is filled with the suspension, and the electrodes 33 and 34 are connected to the electrophoresis power supply unit 4 to apply an electric field.

  Fig.4 (a) shows the variation (1) of the chamber 1 in the microorganisms measuring apparatus of this embodiment. In variation (1), the filter 41 (structure) is disposed in a hollow state in the suspension 36 to further improve the trap efficiency. By making the filter 41 hollow, it becomes possible to trap microorganisms on both the upper side and the lower side of the filter 41 in FIG.

  FIG.4 (b) shows the variation (2) of the chamber 1 in the microorganisms measuring apparatus of this embodiment. In the variation (2), the electrodes 33 and 34 are disposed on the substrates 31 and 32, the filters 42 and 43 are disposed on both the electrodes 33 and 34, and the suspension 36 is filled between the filters 42 and 43. . According to this configuration, fine particles can be migrated into the micropores of the filters 42 and 43 provided in both the pair of electrodes 33 and 34, and the impedance of the suspension can be efficiently changed.

  FIG.4 (c) shows the variation (3) of the chamber 1 in the microorganisms measuring apparatus of this embodiment. In the variation (3), filters 41a and 41b having different eye roughness are provided between the plurality of electrode pairs 33a, 34a, 33b, and 34b, and the electrode pairs 33a and 34a, the filter 41a, and the electrode pairs 33b and 34b are provided. The filter 41b is divided by partition plates 45, 46 and 47.

  According to the variation (3), the type of bacteria to be trapped can be changed depending on the diameters of the filters 41a and 41b, and the dynamic range of measurement can be improved. Moreover, measurement objects having different sizes can be selectively detected, and immunoaggregated particles can be detected.

  The electrodes 33 and 34 are respectively connected to the migration power supply unit 4, and the migration power supply unit 4 applies an AC voltage having a specific frequency between the electrodes 33 and 34. Here, the AC voltage is not only a sine wave but also a voltage that changes the direction of the flow at a substantially constant period, and the average value of the currents in both directions is equal. As will be described later, the frequency applied by the electrophoresis power supply unit 4 is appropriately determined by the control calculation unit 6 (see FIG. 1).

  When an alternating voltage is applied between the electrodes 33 and 34 in a state where the electrodes 33 and 34 are in contact with the suspension 36, a structure in which microorganisms contained in the suspension 36 are sandwiched between the electrodes 33 and 34. It is trapped by the dielectrophoretic force in the fine holes such as 41.

When a sufficiently large positive dielectrophoretic force acts on the microorganism, the microorganism is trapped at the edge portion of the hole which is the electric field concentration portion as shown in FIG.

  The measurement unit 5 measures the impedance change caused by the microorganisms trapped in the structure 35 in this way. Specifically, as shown in FIG. 6, a circuit for measuring the impedance between the electrodes 3 is configured between the migration power supply unit 4 and the electrodes 3.

  In this case, the measurement unit 5 includes a current value flowing between the electrodes 3 and a circuit for measuring a phase difference between the voltage and current applied by the migration power supply unit 4. The measuring unit 5 measures changes in the current and phase difference between the electrodes 3 caused by microorganisms moving by dielectrophoresis and concentrated near the electric field concentration part.

  The current value and phase difference measured by the measurement unit 5 are passed to the control calculation unit 6. The control calculation unit 6 calculates the impedance value between the electrodes 3 from the information on the current, the phase difference, and the voltage and frequency applied by the migration power supply unit 4.

  Before the voltage application, the region filled only with the suspension 36 between the electrodes 3 is replaced by microorganisms having different dielectric constants by trapping by dielectrophoresis, so that the impedance between the electrodes 3 depends on the number of trapped microorganisms. Change.

  Therefore, the number of microorganisms trapped in the micropores can be estimated from the difference between the impedance value at a certain time and the initial impedance value immediately after the voltage application, in other words, the amount of change. Since the number of trapped microorganisms depends on the concentration of microorganisms contained in the suspension, the number of microorganisms in the suspension can be measured.

  The measuring unit 5 can also be realized by optical measuring means as shown in FIG. In this case, the chamber 1 is arranged in such a positional relationship that a gap is included in the optical path between the light source 21 and the light receiving unit 22. The number of microorganisms trapped in the gap can be estimated by utilizing the fact that the amount of light incident on the light receiving unit 21 varies depending on the number of microorganisms trapped in the gap.

  Alternatively, the information of the light receiving unit 22 may be transferred to the control calculation unit 6 to be imaged, and the control calculation unit 6 may directly count the number of particles using a particle determination algorithm or the like, or obtain the fine particle area with respect to the visual field area. You may convert into the number of fine particles. Since the number of microorganisms trapped in the gap thus obtained depends on the concentration of microorganisms contained in the suspension, the number of microorganisms in the suspension can be measured.

  As described above, in order to trap microorganisms in the gap, it is necessary to induce a sufficiently large dielectrophoretic force against all external forces other than dielectrophoresis, such as viscous force acting on the microorganism, gravity, and Brownian motion. If this is insufficient, the number of microorganisms that can be measured by the measurement unit 5 is reduced, so that the measurement sensitivity and accuracy are remarkably reduced. If the measurement unit 5 falls below the magnitude of a signal that can be measured, microorganisms cannot be measured.

  Therefore, in the present embodiment, the control arithmetic unit 6 appropriately determines a frequency at which a sufficient dielectrophoretic force is exerted to trap microorganisms in the gap, and the voltage of the frequency determined by the electrophoresis power supply unit 4 is applied. To do. Thereby, since the measurement part 5 can take out the signal which can fully be detected, the measurement of microorganism concentration can be performed with high precision and high sensitivity.

  The control calculation unit 6 includes a CPU (not shown) and a circuit such as a memory 6a in which a program for defining a series of operations and various data are stored, and controls a series of measurement operations. The conductivity input means 7 can input the conductivity of the suspension before measurement. For example, it can be realized by a method of inputting a numerical value with a numeric keypad or a method of pressing a switch corresponding to a plurality of conductivity ranges such as “0 to 50 μS / cm” and “50 to 100 μS / cm”.

  The memory 6 a has a frequency selection table for selecting an appropriate frequency of the voltage applied by the electrophoresis power supply unit 4 from the value of the conductivity of the suspension given from the conductivity input means 7. In the frequency selection table, the optimum frequency and applied voltage value at which a sufficient dielectrophoretic force acts on microorganisms are tabulated for each conductivity of the suspension.

  Here, the frequency selection table stored in the memory 6a will be described in detail. As shown in Table 1, the frequency selection table stores at least the conductivity of the suspension, the amplitude of the alternating voltage to be applied, and the optimum frequency. The conductivity of the suspension may be a specific numerical value, or a table may be created by setting a certain range. The control calculation unit 6 selects the amplitude and optimum frequency of the AC voltage corresponding to the given conductivity. Note that “E” of the frequency corresponding to the conductivity of 300 μS / cm˜ indicates an error, and indicates that measurement cannot be performed when the conductivity is too high.

Next, the optimum frequency will be described. In (Equation 1), the dielectrophoretic force F _DEP is proportional to the real part of the Clausius Mosotti number K, that is, Re [K]. Re [K] depends on the conductivity of the suspension, as is clear from (Equation 2) and (Equation 3). FIG. 8 shows how Re [K], that is, the dielectrophoretic force changes when the conductivity of the suspension changes.

In FIG. 8, the electric field used for dielectrophoresis, in other words, the frequency of the applied voltage is used as a parameter, and is shown as a function of the conductivity of the suspension. Re [K] corresponds to the dielectrophoretic force F _DEP , and the sign thereof corresponds to whether the dielectrophoretic force acts as an attractive force or a repulsive force.

As shown in FIG. 8A, for example, when the frequency of the alternating voltage for dielectrophoresis is (1) 10 KHz, Re [K] changes from positive to negative near the solution conductivity of 3 μS / cm, and the fine particles The dielectrophoretic force F _DEP acting on the surface changes from attractive to repulsive.

On the other hand, when the frequency of the AC voltage for dielectrophoresis is (2) 100 KHz, Re [K] changes from positive to negative near the solution conductivity of 30 μS / cm, and the dielectrophoretic force F _DEP acting on the fine particles is attractive. It changes from repulsive force.

FIG. 8B shows the dielectrophoretic force when the solution conductivity is changed from 1 μS / cm to 1000 μS / cm when the frequency of the AC voltage for dielectrophoresis is (2) 100 KHz and (3) 800 KHz. The change in F _DEP is shown. When the frequency is (2) 100 KHz, Re [K] <0 at about 20 μS / cm or more, but at 800 KHz, the decrease in the dielectrophoretic force against the increase in conductivity is suppressed, and Re [K]> up to about 250 μS / cm. It becomes 0 and can be trapped at the edge of the gap 13 by attractive force.

  This indicates that there exists an optimum frequency at which the decrease in the dielectrophoretic force is the smallest with respect to the increase in the conductivity of the suspension. In order to determine the optimum frequency, it is preferable to perform the following experiment. That is, a plurality of suspensions having the same fine particle concentration and varying conductivity are prepared, and measurement is performed while changing the frequency of the applied voltage for each suspension. As a result, the frequency at which the measurement response is maximized is the optimum frequency for each suspension conductivity. The optimum frequency shown in Table 1 is determined in this way.

  However, if the frequency is too high, it is difficult to realize a measurement circuit. If the frequency is too low, convection due to Joule heat or, in an extreme case, bubble generation due to electrolysis adversely affects the measurement. For this reason, the optimum frequency is not optimum for dielectrophoresis, but there is an acceptable frequency range that can cause sufficient dielectrophoresis to perform fine particle measurement.

  FIG. 9 is a graph showing the relationship between the frequency (Hz) of the AC voltage for dielectrophoresis and the real part (Re [K]) of the Clausius Mosotti number when the solution conductivity (μS / cm) is used as a parameter. . When measuring fine particles using positive dielectrophoresis at a suspension conductivity of 100 μS / cm, assuming that the sufficient dielectrophoretic force to obtain a measurement response is Re [K]> 0.4, The optimum frequency is about 700 KHz to 4 MHz. In this case, in order to avoid complication of the measurement circuit due to high frequency, it is possible to adopt 700 KHz which is the lower limit frequency as the optimum frequency.

  In addition, a sufficient dielectrophoretic force may be obtained at one specific frequency within the range of the suspension conductivity that needs to be measured. In that case, a frequency selection table as shown in Table 2 is obtained.

  For example, when the range of the suspension conductivity that needs to be measured is 0 to 100 μS / cm, if the frequency is 800 KHz, Re [K]> 0.4 for all conductivity regions. Since measurement can be performed within a desired suspension conductivity range at one frequency, the circuit configuration is simple and convenient. In this case, when the suspension conductivity exceeds 100 μS / cm, data corresponding to the error is written as shown in the frequency selection table.

  FIG. 10 is a flowchart for explaining the microorganism measuring method according to the present embodiment. Hereinafter, with reference to a flowchart, a series of flow from introduction of a sample to concentration, measurement, and result presentation of microorganisms in the chamber 1 will be described. First, in the initial state, a suspension containing the microorganism to be measured is put into the chamber 1 (step S11).

  Next, the conductivity input means 7 inputs the conductivity of the added suspension. The input conductivity is passed to the control calculation unit 6 (step S12).

  The control calculation unit 6 to which the conductivity of the suspension is passed refers to the optimum frequency table provided in the memory 6a, and selects the voltage amplitude value and the frequency to be applied to the electrode (step S13). The voltage amplitude value at this time (hereinafter referred to as “voltage for dielectrophoresis”) may be selected to be a value sufficient to trap microorganisms in the micropores, and is set to 5 Vp-p in this embodiment. .

  In Tables 1 and 2, the voltage for dielectrophoresis is a constant value with respect to the conductivity, but an optimum value can be selected for each conductivity. For example, if the conductivity is high, Joule heat is generated if the voltage is too high, and this affects microbial traps due to dielectrophoresis, so the voltage for dielectrophoresis is lowered as the conductivity increases. And

  Next, the control calculation unit 6 determines whether or not the frequency stored in the memory and corresponding to the input conductivity is the error code (E) (step S14). If it is an error code (E), the process proceeds to step S16, and the control calculation unit 6 instructs the display means 9 to display that the input conductivity is out of the measurement range, and ends the measurement. (Step S22).

  In step S14, when the selected frequency is not the error code (E), the control calculation unit 6 applies a voltage between the electrodes 3 to the electrophoresis power source unit 4 with the voltage amplitude and frequency selected in the optimum frequency table. (Step S15).

  When a predetermined voltage is applied between the electrodes 3, the measurement unit 5 immediately measures the impedance between the electrodes 3 as data in an initial state immediately after the voltage application, and the measurement result is passed to the control calculation unit 6 and the memory 6a. Is stored as an initial impedance value (step S17).

  Here, impedance measurement is described as an example. However, if the measurement unit 5 measures the gap state using optical means, the initial state can be measured without applying a voltage. It is also possible to carry out before step S15. In addition, when the measurement part 5 performs an optical measurement, a transparent electrode and a transparent substrate are used.

  Next, the control calculation unit 6 waits until a predetermined time elapses by a clock means (not shown). At this time, the electrophoretic power supply unit 4 keeps voltage application (step S18).

  When the predetermined time has elapsed, the control calculation unit 6 determines whether the predetermined number of measurements has expired (step S19), and if not, returns to step S17. Returning to step S17, the control calculation unit 6 instructs the measurement unit 5 to measure the impedance between the electrodes 3, and stores the result in the memory 6a as a result after a predetermined time has elapsed.

  When the predetermined number of measurements has expired, the control calculation unit 6 instructs the electrophoresis power supply unit 4 to stop the voltage application (step S20).

  After the voltage application is stopped, the control calculation unit 6 calculates the concentration of fine particles in the suspension 2 from the time-dependent change data of the impedance between the electrodes 3 stored in the memory 6a, and causes the display means 9 to display the result ( Step S21) and a series of measurement operations are terminated (Step S22).

  The calculation of the microorganism concentration can be obtained from a calibration curve stored in advance in the memory 6a. This calibration curve uses a measurement system of the microbe measurement apparatus described in this embodiment to measure a calibration sample with a clear microbial concentration in advance, and performs regression analysis of the variation from the correlation between the number of microbes and the impedance change at that time. A function that represents the curve obtained as described above is used.

  When this conversion equation is stored in the memory 6a of the control calculation unit 6 and a sample having an unknown microorganism concentration is measured, the microorganism concentration in the cell 1 is calculated by substituting the value of the impedance change within a predetermined time. it can. When a conversion table is used, the calculation result based on the conversion formula is stored in advance.

  As described above, according to the particle measuring apparatus of the present embodiment, the chamber 1 for introducing the liquid containing the particles, the pair of electrodes 3 provided in the chamber 1, and the pair of electrodes 3 are disposed. Changes in impedance between the pair of electrodes 3 and the structure 35 that induces non-uniformity of the electric field and the pair of electrodes 3 by applying an alternating voltage between the pair of electrodes 3 and applying a dielectrophoretic force to the fine particles. And a measurement unit 5 for calculating the number of particles in the chamber 1, simplifying the structure of the electrode 3 that applies a dielectrophoretic force to the particles in the suspension, thereby reducing the cost of the particle measuring device. It becomes possible.

  A liquid containing a reagent that specifically binds to the detection substance and forms an aggregate may be introduced into the chamber 1. As described above, the dielectrophoretic force is a force that varies depending on the size of the object to be migrated, and the effective dielectrophoretic force increases as the effective particle size increases. For this reason, when the detection substance which is a measurement object and the foreign substance which is not a measurement object coexist in the suspension 2, the effective particle diameter of the detection substance which formed the aggregate is large. Thus, a large dielectrophoretic force is exerted compared to the interstitial material. If the voltage value to be applied is adjusted, only a dielectrophoretic force that is insufficient to trap the interstitial material acts, and a sufficient dielectrophoretic force can be exerted on the aggregate to be measured. Under this condition, the measured result is considered to be a reaction due to the substance to be measured only. Therefore, according to this configuration, it is possible to specifically measure the detection substance even when other substances (contaminants) are mixed.

Examples of the present invention are shown below.
[Example 1]
(1) Preparation of sample solution Congealed Escherichia coli K-12 (NBRC3301, Product Evaluation Technology Infrastructure) that was aerobically cultured at 37 ° C for 16 hours on a standard agar medium (MB0010, Eiken Equipment Co., Ltd.) A sample solution used for measurement was prepared by using a standard sample that was collected with a stick and suspended in a 0.1 M D-mannitol solution (conductivity, approximately 1 μS / cm).
(2) Measuring apparatus The measuring apparatus of FIG. 3 was used. As the membrane filter as a structure, a polycarbonate membrane filter (K020N025A, ADVANTEC) having an effective diameter of 0.2 μm was used. The applied voltage amplitude was 5 Vp-p, and the frequency was 100 KHz. First, after measuring using a blank solution that does not contain E. coli in the sample solution, measure the sample solution containing E. coli, and record the impedance value of each measurement, specifically the capacitance value calculated from the impedance. did.
(3) Results FIG. 9 shows the measurement results. In FIG. 9, the horizontal axis represents the measurement time (seconds), and the vertical axis represents the capacitance change CT (pF) obtained by subtracting the capacitance change of the blank from the capacitance increase of the sample solution containing E. coli. As voltage was applied and E. coli was trapped on the edge of the filter, the capacitance value between the electrodes increased, indicating that this measurement device can measure E. coli in the sample solution. .

  INDUSTRIAL APPLICABILITY The present invention can be used as a particle measuring apparatus capable of simplifying an electrode structure for inducing electric field inhomogeneity and reducing costs and an electrode used therefor.

Schematic configuration diagram for explaining a particle measuring apparatus according to an embodiment of the present invention (1) The figure for demonstrating the structure of the chamber 1 of the particulate measuring device concerning embodiment of this invention. The figure for demonstrating the specific structure of the chamber 1 of the microparticles | fine-particles measuring apparatus concerning embodiment of this invention. The figure which shows the variation of the chamber 1 of the microparticle measuring apparatus concerning embodiment of this invention. Schematic configuration diagram (2) for explaining a particle measuring apparatus according to an embodiment of the present invention Schematic configuration diagram (3) for explaining a particle measuring apparatus according to an embodiment of the present invention Graph showing the relationship between solution conductivity (μS / cm) and Re [K] when the frequency of AC voltage for dielectrophoresis is used as a parameter A graph showing the relationship between the frequency (Hz) of the AC voltage for dielectrophoresis and the real part (Re [K]) of the Clausius Mosotti number The flowchart for demonstrating the fine particle measuring method concerning embodiment of this invention. The graph which shows the microparticle measurement result concerning Example 1 of this invention The figure which shows the conventional apparatus which measures the impedance of the liquid 51 with which it filled in the channel

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Chamber 2,36 Suspension 3,11a, 11b Electrode 4 Electrophoresis power supply part 5 Measuring part 6 Control calculating part 6a Memory 7 Conductivity input means 9 Display means 13 Gap 14 Fine particle 17 Agitation means 21 Light source 22 Light-receiving part 31, 32 Substrate 33, 34 Planar electrode 35, 41, 42, 43 Structure 37 Sample inlet 38 Outlet 45, 46, 47 Partition plate

Claims (12)

A chamber for introducing a liquid containing fine particles;
At least a pair of electrodes provided in the chamber;
A structure disposed between the pair of electrodes and inducing non-uniformity of the electric field;
An electrophoretic power supply unit that applies an alternating voltage between the pair of electrodes and applies a dielectrophoretic force to the fine particles;
A particle measuring device comprising: an impedance measuring device that calculates a time change in impedance between the pair of electrodes and calculates the number of particles in the chamber. The fine particle measuring device according to claim 1,
A fine particle measuring apparatus, wherein the structure is a filter having fine pores. The fine particle measuring apparatus according to claim 2,
A plurality of filters having different effective diameters of the micropores,
A fine particle measuring apparatus provided with a plurality of pairs of the pair of electrodes in correspondence with the plurality of filters. The fine particle measuring apparatus according to claim 2,
A fine particle measuring apparatus including an impedance measuring electrode in the structure or on the surface thereof. The fine particle measuring apparatus according to claim 2,
A fine particle measuring apparatus in which the structure is composed of an ion adsorbent or includes a part of the ion adsorbent. The fine particle measuring apparatus according to claim 2,
A fine particle measuring apparatus in which the filter is on the surface of the pair of electrodes. The fine particle measuring apparatus according to claim 2,
A fine particle measuring apparatus in which the filter is on the surface of both electrodes of the pair of electrodes. The fine particle measuring apparatus according to claim 2,
A fine particle measuring apparatus in which the filter is positioned between the pair of electrodes. The fine particle measuring device according to claim 1,
The impedance measurement device is a fine particle measurement device that measures the number of fine particles by an initial change in impedance. The fine particle measuring device according to claim 1,
A fine particle measuring apparatus containing a reagent that specifically binds to a detection substance and forms an aggregate. The fine particle measuring device according to claim 1,
The pair of electrodes is transparent;
The structure is configured on a transparent substrate,
A fine particle measuring apparatus for optically measuring the number of fine particles in the chamber.   The electrode used for the particulate measuring device according to any one of claims 1 to 11.

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