JP2013257348A - Particle measuring apparatus and particle measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a particle measuring apparatus and a particle measuring method capable of detecting/reducing measurement impedance value fluctuation due to a circuit drift and achieving highly accurate impedance change measurement.SOLUTION: The particle measuring apparatus includes: at least a pair of electrodes 11 immersed inside a cell for introducing particle-containing liquid; a migration power supply part 4 for applying an AC voltage to the electrodes 11 and making dielectrophoretic force act on particles; a measurement part 5 for measuring impedance between the electrodes 11; and a control operation part 6 for calculating the number of particles inside the liquid from the measurement result of the measurement part 5 and measuring the conductance of the liquid. The control operation part 6 refers to a table or a function indicating an electrode impedance fluctuation value for each conductance, and corrects a calculation result on the basis of an electrode impedance fluctuation value according to the measurement result of the conductance.

Description

  The present invention relates to a fine particle measuring apparatus and a fine particle measuring method for measuring the number of fine particles in a sample solution using dielectrophoresis. More specifically, the present invention relates to a fine particle measuring apparatus and a fine particle measuring method for detecting / reducing a measurement impedance value variation due to circuit drift and measuring with high sensitivity and high accuracy.

  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 nanotubes, 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 this application, in particular, measurement of microorganisms is assumed.

  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, in the measurement of the time change of the interelectrode impedance, the measurement is performed after adjusting the measurement range according to the state of the substance to be measured, the state of the electrode, the ambient conditions at the time of measurement, etc. Depending on initial conditions such as circuit temperature to be performed, there is a problem in that the circuit generates heat due to current generated by applying a voltage to the electrodes for dielectrophoresis and impedance measurement, thereby causing undesirable measurement drift. In many cases, the surface state of the electrode changes with time due to adhesion or adsorption of organic substances in the substance to be measured to the electrode of the measuring apparatus. This causes a drift from the desired measurement reference point during measurement.

  In order to cancel fluctuations such as drift, two identical electrode cells are prepared and the difference between the signals of the two is taken. 2. Description of the Related Art An electrical conductivity measuring device that measures changes with high accuracy is known.

  FIG. 16 is a schematic configuration diagram of a conventional electrical conductivity measuring device. This electric conductivity measuring device 71 has electric conductivity measuring cells 62 and 63 having at least two electrodes in contact with a substance to be measured. A multiplier or divider 72 is provided in front of the current supply electrode of one of the conductivity measuring cells 63 to multiply the supplied alternating current value by a predetermined magnification or divide by a predetermined ratio. Therefore, the electric conductivity level of the substance to be detected that is detected in the electric conductivity measuring cell 63 can be made different from that of the electric conductivity measuring cell 62.

  The multiplier or divider 72 has a phase inversion function. That is, the alternating current before being supplied to the current supply electrode is amplified or reduced by a predetermined magnification, and the phase of the supplied alternating current is inverted. As a result, the detection signals themselves from the respective conductivity measuring cells 62 and 63 are substantially subtracted, and the subtracted signal is sent to the amplifier 66 (see, for example, Patent Document 2).

Japanese Patent Laid-Open No. 2000-125846 JP 2001-311710 A

Hywel Morgan, et al .: `` AC Electrokinetics: colloids and nanoparticles '', RESERCH STUDIES PRESS LTD., 2003, pp. 15-63 J. Suehiro, R. Yatsunami, R. Hamada, M, Hara, J. Phys. D: Appl. Phys. 32 (1999) 2814-2820

  As described above, in the measurement circuit that realizes the DEPIM measurement, since it is necessary to measure a slight inclination of the impedance change caused by the fine particles to be measured, the measurement is caused by the temperature variation of the circuit chip, the electrode, the solution, or the like. Value drift is an important issue. In addition, the drift as used in the field of this invention represents the change of the impedance resulting from things other than by the bacteria which are the measuring objects collected by the electrode.

  In the electrical conductivity measuring apparatus described in Patent Document 2, two identical electrode cells are prepared, and fluctuations such as drift are canceled by taking the difference between the signals of both, but due to individual differences of individual electrodes Drift compensation error, and two electrode cells are required, which complicates the system.

  The present invention has been made in view of the above circumstances, and provides a particle measuring apparatus and a particle measuring method capable of detecting / reducing measurement impedance value fluctuation due to circuit drift and realizing highly accurate impedance change measurement. It is intended to provide.

  The fine particle measuring apparatus of the present invention includes at least a pair of electrodes immersed in a cell into which a fine particle-containing liquid is introduced, and an electrophoresis power supply unit that applies a dielectrophoretic force to the fine particles by applying an alternating voltage between the pair of electrodes. An impedance measuring means for measuring the impedance between the pair of electrodes, an arithmetic unit for calculating the number of fine particles in the liquid from the measurement result of the impedance measuring means, and a conductivity measuring means for measuring the conductivity of the liquid And the calculation unit corrects the calculation result based on the electrode impedance fluctuation value according to the measurement result of the conductivity measuring means with reference to a table or function indicating the electrode impedance fluctuation value for each conductivity. To do.

  According to the above configuration, the measurement impedance value fluctuation due to the circuit drift of the liquid having different conductivity can be detected / reduced by performing correction using the table or function indicating the electrode impedance fluctuation value for each conductivity. Impedance measurement can be realized.

  The fine particle measuring apparatus of the present invention further includes a temperature measuring unit that measures the temperature of the liquid, and the calculation unit refers to a table or a function that indicates a conductivity and an electrode impedance variation value for each temperature. The calculation result is corrected based on the electrode impedance fluctuation value corresponding to the conductivity measurement result and the liquid temperature measurement result.

  According to the said structure, the fluctuation | variation of the drift when not only the electrical conductivity of a solution but temperature differs can be correct | amended with high precision.

  The fine particle measuring apparatus of the present invention includes a capacitance connected in parallel with the pair of electrodes, the electrophoretic power supply unit applies an alternating voltage to a parallel circuit including the electrodes and the capacitance, and the impedance measuring means The parallel impedance between the electrodes and the capacitance is measured.

  According to the above configuration, by using the capacitance, it is possible to reduce the fluctuation of the impedance measurement value caused by the phase error of the measurement circuit.

  Further, in the method for measuring fine particles of the present invention, an alternating voltage is applied between a pair of electrodes immersed in a sample solution containing fine particles, the fine particles are arranged at predetermined positions by a dielectrophoretic force, and the number of fine particles in the sample solution is determined. A fine particle measuring method for measuring, a conductivity measuring step for measuring the conductivity of the sample solution, an impedance measuring step for measuring an impedance between the pair of electrodes by applying an AC voltage between the pair of electrodes, and When calculating the number of fine particles in the sample liquid from the measurement result of the impedance, refer to a table or function indicating the electrode impedance fluctuation value for each conductivity, and the electrode impedance corresponding to the measurement result of the conductivity of the sample liquid And a calculation step of correcting the calculation result based on the fluctuation value.

  In addition, the fine particle measurement method of the present invention includes a temperature measurement step of measuring the temperature of the liquid, and the calculation step calculates conductivity and temperature when calculating the number of fine particles in the liquid from the measurement result of the impedance. A calculation result is corrected based on an electrode impedance fluctuation value corresponding to a measurement result of the conductivity of the liquid and a measurement result of the temperature of the liquid with reference to a table or function indicating the electrode impedance fluctuation value for each. .

  Furthermore, in the fine particle measurement method of the present invention, the impedance measurement step applies an AC voltage to a parallel circuit including the pair of electrodes and a capacitance connected in parallel with the pair of electrodes, The parallel impedance is measured.

  According to the present invention, it is possible to detect / reduce a measurement impedance value variation due to a circuit drift and to realize highly accurate impedance change measurement.

Schematic configuration diagram for explaining a particle measuring apparatus according to a first embodiment of the present invention (1) Schematic for demonstrating the electrode tip of the particulate measuring device concerning the embodiment of the present invention. The figure which shows the electric force line 15 produced by the voltage applied between measurement electrode 11a, 11b in embodiment of this invention. Explanatory drawing in which the fine particles 14 are trapped along the lines of electric force at the opposing edge portions of the electrodes 11a and 11b. Schematic configuration diagram (2) for explaining the particle measuring apparatus according to the first embodiment of the present invention. Schematic configuration diagram for explaining the particle measuring apparatus according to the first embodiment of the present invention (3) Graph showing the relationship between solution conductivity (μS / cm) and Re [K] when the frequency of the AC voltage for dielectrophoresis is used as a parameter The graph which shows the relationship between the frequency (Hz) of the alternating voltage for dielectrophoresis, and the real part (Re [K]) of the Clausius Mosotti number The flowchart for demonstrating the fine particle measuring method concerning the 1st Embodiment of this invention. The schematic block diagram for demonstrating the microparticles | fine-particles measuring apparatus concerning the 2nd Embodiment of this invention. The figure for demonstrating the impedance measurement step in the microparticle measuring device concerning the 2nd Embodiment of this invention. The figure for demonstrating the impedance measurement step in the microparticle measuring device concerning the 3rd Embodiment of this invention. The schematic block diagram for demonstrating the microparticles | fine-particles measuring apparatus concerning the 4th Embodiment of this invention. The figure which shows the specific structure of the dummy element in the microparticles | fine-particles measuring apparatus concerning the 8th Embodiment of this invention. The vector diagram which shows that the fluctuation range with respect to an impedance value changes with phase angles in the 8th Embodiment of this invention. The figure which shows the measurement result 1 of the Example of this invention The figure which shows the measurement result 2 of the Example of this invention Schematic configuration diagram of a conventional electrical conductivity measuring device Schematic block diagram for explaining a particle measuring apparatus according to a ninth embodiment of the present invention

(First embodiment)
Hereinafter, a particle 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 particle measuring apparatus according to the present embodiment, and FIG. 2 is a schematic diagram illustrating an electrode chip of the particle measuring apparatus according to the present embodiment.

  In FIG. 1, 1 is a cell for holding a sample solution 2 containing fine particles to be measured, 3 is an electrode chip including an electrode pair for collecting fine particles by dielectrophoresis, 4 is an electrophoretic power supply unit, and 5 is trapped by dielectrophoresis. A measurement unit for measuring an optical or electrical change caused by the fine particles formed, 6 a control calculation unit for controlling the whole fine particle measurement apparatus, performing analysis calculation of the measurement results, input / output processing, and the like, and 7 for the sample liquid 2 Conductivity input means for inputting conductivity.

  In FIG. 2, 10 is a substrate, 11a and 11b are electrodes formed on the substrate 10 to form a pair of electrodes, and 13 is an interelectrode gap between the electrodes 11a and 11b. A pattern of electrodes 11a and 11b is formed on the substrate 10 by a conductive material such as metal. As an example of a preferable material, gold, silver, copper, aluminum, platinum, or the like is desirably sufficient, and silver is used in this embodiment mode.

  FIG. 3 shows electric lines of force 15 caused by the voltage applied between the measuring electrodes 11a and 11b. In the present embodiment, the configuration in the vicinity of the gap 13 between the measurement electrodes 11a and 11b corresponds to the electric field concentration portion, and the gap 13 is most concentrated in the electric field. Therefore, the fine particles are most strongly migrated to the gap 13 portion.

  The electrodes 11a and 11b are desirably thin films that are sufficiently thin with respect to their widths. For example, the thickness is about 1000 mm with respect to a width of 100 μm. As a result, an unequal electric field is formed at the edge portion as viewed in the thickness direction, and the fine particles can be efficiently dielectrophoresed.

  As a method for patterning the electrodes 11a and 11b, a desired pattern may be formed using a selected material. For example, forming a metal thin film by sputtering, vapor deposition, plating, etc., forming a pattern by photolithography, laser processing, etc., forming electrodes, such as a direct pattern formation method such as gravure printing, screen printing, ink jet printing, etc. The general process used to do this can be selected. The most appropriate process should be selected in consideration of productivity and cost. In the present embodiment, a silver thin film is formed by sputtering, and a pattern is formed by photolithography.

  The electrodes 11a and 11b are respectively connected to the migration power supply unit 4, and the migration power supply unit 4 applies an alternating voltage of a specific frequency between the electrodes 11a and 11b. 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.

  When an AC voltage is applied between the electrodes 11a and 11b in a state where the electrode tip 3 is immersed in the sample liquid 2 and the electrodes 11a and 11b are in contact with the sample liquid 2, fine particles contained in the sample liquid 2 are The gap 13 between the electrodes 11a and 11b is trapped by the dielectrophoretic force.

  When a positive dielectrophoretic force acts on the fine particles, the fine particles 14 follow the lines of electric force along the opposing edge portions of the electrodes 11a and 11b in the region of the gap 13 that is the electric field concentration portion as shown in FIG. Fine particles are trapped in a rosary shape called a pearl chain.

  On the other hand, when negative dielectrophoresis acts on the fine particles 14, as shown in FIG. 4B, in the direction away from the electric field concentration portion, that is, in the region of the gap 13 that is the weak electric field portion, the electrodes 11 a and 11 b are opposed to each other. It is trapped over the part.

  The measurement unit 5 measures the impedance change due to the fine particles trapped in the gap 13 in this way. Specifically, as shown in FIG. 5, a circuit that measures the impedance between the electrodes 11 a and 11 b is configured by the measurement unit 5 between the migration power supply unit 4 and the electrode chip 3.

  In this case, the measurement unit 5 includes a current value flowing between the electrodes 11a and 11b, a circuit for measuring the phase difference between the voltage and current applied by the migration power supply unit 4, and the like. The measuring unit 5 measures changes in the current and the phase difference between the electrodes 11a and 11b due to the movement of the fine particles by dielectrophoresis and the concentration in the vicinity of the electric field concentration portion.

  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 11a and 11b from the information on the current, the phase difference, and the voltage and frequency applied by the electrophoresis power supply unit 4.

  Before the voltage is applied, the region filled only with the sample liquid 2 between the electrodes 11a and 11b is replaced by fine particles having different dielectric constants by trapping by dielectrophoresis, so that the impedance between the electrodes 11a and 11b is the number of trapped fine particles. It changes according to.

  Therefore, it is possible to estimate the number of particles trapped in the gap 13 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 fine particles depends on the concentration of fine particles contained in the sample solution, the number of fine particles in the sample solution can be measured.

  The measuring unit 5 can also be realized by optical measuring means as shown in FIG. In this case, the cells 1 are arranged in such a positional relationship that the gap 13 is included in the optical path between the light source 21 and the light receiving unit 22. The number of fine particles trapped in the gap 13 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 fine particles trapped in the gap 13.

  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 fine particles trapped in the gap 13 thus obtained depends on the concentration of fine particles contained in the sample liquid, the number of fine particles in the sample liquid can be measured.

  As described above, in order to trap the fine particles in the gap 13, it is necessary to induce a sufficiently large dielectrophoretic force against all external forces other than dielectrophoresis, such as viscous force acting on the fine particles, gravity, and Brownian motion. If this is insufficient, the number of fine particles that can be measured by the measurement unit 5 is reduced, so that the measurement sensitivity and accuracy are remarkably lowered. If the measurement unit 5 falls below the magnitude of a signal that can be measured, fine particles cannot be measured.

  Therefore, in the present embodiment, the control arithmetic unit 6 appropriately determines a frequency at which a sufficient dielectrophoretic force acts to trap the fine particles in the gap 13, and the voltage of the frequency determined by the electrophoretic power supply unit 4 is determined. Apply. As a result, a signal that can be sufficiently detected by the measurement unit 5 can be taken out, so that the fine particle concentration can be measured with high accuracy 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 sample solution 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 conductivity value of the sample solution given from the conductivity input means 7. In the frequency selection table, for each conductivity of the sample solution 2, an optimum frequency and applied voltage value at which a sufficient dielectrophoretic force acts on the fine particles are tabulated.

  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 sample solution, the amplitude of the AC voltage to be applied, and the optimum frequency in association with each other. The conductivity of the sample solution 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 sample solution 2 as is clear from (Equation 2) and (Equation 3). FIG. 7 shows how Re [K], that is, the dielectrophoretic force changes when the conductivity of the sample solution 2 changes.

In FIG. 7, 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 sample liquid 2. 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. 7A, for example, when the frequency of the AC voltage for dielectrophoresis is (1) 10 KHz, Re [K] changes from positive to negative near the solution conductivity of 3 μS / cm. 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. 7B 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 minimized with respect to the increase in the conductivity of the sample solution. In order to determine the optimum frequency, it is preferable to perform the following experiment. That is, a plurality of sample solutions having the same fine particle concentration and different conductivity are prepared, and measurement is performed while changing the frequency of the applied voltage for each sample solution. As a result, the frequency with the largest measurement response is the optimum frequency for each sample solution 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. 8 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 sample liquid conductivity of 100 μS / cm, it is optimal if the sufficient dielectrophoretic force to obtain a measurement response is Re [K]> 0.4. The 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, the dielectrophoretic force may be sufficiently obtained at one specific frequency within the range of the sample solution 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 sample solution conductivity that needs to be measured is 0 to 100 μS / cm, if the frequency is 800 KHz, Re [K]> 0.4 for all the conductivity regions. Since the measurement can be performed in the range of the desired sample solution conductivity at the frequency of, the circuit configuration is simple and convenient. In this case, when the sample solution conductivity exceeds 100 μS / cm, data corresponding to the error is written as shown in the frequency selection table.

  FIG. 9 is a flowchart for explaining the fine particle measurement 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 fine particles in the cell 1 will be described. First, in an initial state, a sample solution containing fine particles to be measured is introduced into the cell 1 (step S11).

  Next, the conductivity of the input sample solution is input by the conductivity input means 7. The input conductivity is passed to the control calculation unit 6 (step S12).

  The control calculation unit 6 to which the conductivity of the sample solution is passed refers to the optimum frequency table provided in the memory 6a, and selects a voltage amplitude value and a 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 the fine particles in the gap 13 and is set to 10 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 the particulate trap by 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 11a and 11b to the electrophoresis power supply unit 4 with the voltage amplitude and frequency selected in the optimum frequency table. Is applied (step S15).

  When a predetermined voltage is applied between the electrodes 11a and 11b, the measurement unit 5 immediately measures the impedance between the electrodes 11a and 11b as data in the initial state immediately after the voltage application, and the measurement result is passed to the control calculation unit 6. The initial impedance value is stored in the memory 6a (step S17).

  Here, impedance measurement is described as an example. However, if the measurement unit 5 measures the state of the gap 13 using optical means, the initial state can be measured without applying a voltage. Can be performed before step S15.

  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 11a and 11b, 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 stopping the voltage application, the control calculation unit 6 calculates the fine particle concentration in the sample liquid 2 from the time-dependent change data of the impedance between the electrodes 11a and 11b stored in the memory 6a, and causes the display unit 9 to display the result. (Step S21), a series of measurement operations are terminated (Step S22).

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

  When this conversion formula is stored in the memory 6a of the control calculation unit 6 and a sample having an unknown fine particle concentration is measured, the value of the change in impedance within a predetermined time is substituted to calculate the fine particle concentration in the cell 1. 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 present embodiment, by selecting an optimum applied electric field frequency according to the conductivity of the sample solution, a sufficient dielectrophoretic force for measurement can be applied to the fine particles. Even if the conductivity of the liquid increases, fine particles can be measured without pretreatment.

(Second Embodiment)
FIG. 10a shows a schematic configuration diagram of a particle measuring apparatus according to a second embodiment of the present invention. The particle measuring apparatus of the present embodiment is obtained by adding a new function to the measuring unit 5 of the particle measuring apparatus shown in FIG. 1, and includes a dummy element (impedance element) 40 to reduce or detect drift. The circuit selection means 43 for switching between the dummy element 40 and the electrode 11 is provided, a voltage is applied to the dummy element at least before the DEPIM measurement, and the measurement unit 5 measures the impedance of the dummy element 40.

  That is, the particle measuring apparatus according to the present embodiment applies an AC voltage to the measurement load RL and the measurement load RL including at least a pair of electrodes 11 and a dummy element 40 that are immersed in a cell into which a liquid containing particles is introduced. The number of fine particles in the liquid is calculated from the measurement result of the electrophoretic power supply unit 4 that applies a dielectrophoretic force to the fine particles by a pair of electrodes 11, the measurement unit 5 that measures the impedance of the measurement load RL, and the measurement unit 5. A control calculation unit 6; and a circuit selection unit 43 that switches a measurement load RL connected between the migration power supply unit 4 and the measurement unit 5 to one of the dummy element 40 and the pair of electrodes 11. The power supply unit 4 applies an AC voltage to the measurement load RL selected by the circuit selection unit 43, and the measurement unit 5 selects the measurement load RL selected by the circuit selection unit 43. Impedance is intended to measure.

The dummy element 40 is configured by a parallel circuit or series circuit of a resistance element (R), a capacitance element (C), and a coil element (L), or a combination of parallel and series circuits. In general, since the counter electrode in the solution can express an electrical equivalent circuit by a parallel circuit of R and C, it is desirable that the dummy element 40 is also configured by a parallel circuit of R and C. Furthermore, it is more preferable to configure the combination of the R value and the C value so as to be equivalent to the electrode impedance value in the state of being impregnated in the solution. As will be described later, if the impedance values of the electrode 11 and the dummy element 40 are equivalent, the amount of drift generated in the circuit at the time of measuring the dummy element 40 and the electrode 11 is equivalent, so that highly accurate drift correction can be performed. it can.
Further, depending on the conductivity of the sample solution, in addition to the parallel circuit of R and C, it is necessary to take into account the series solution resistance (Rsol) and the like as an equivalent circuit. For this reason, if necessary, the configuration of the dummy element may be configured in accordance with an assumed equivalent circuit.

  FIG. 10b shows an impedance measurement step in the particle measuring apparatus of the present embodiment. In this measurement step, first, (1) the control calculation unit 6 sets the circuit selection means 43 to the contact (1) side, the migration power supply unit 4 applies a voltage to the dummy element 40, and the measurement unit 5 sets the impedance of the dummy element 40. Measure. Hereinafter, this step is referred to as a first dummy measurement. As shown in FIG. 10b, the impedance of the dummy element 40 causes a drift due to an increase in the temperature of the circuit as indicated by a curve a. This drift has a maximum slope immediately after voltage application and decreases exponentially with time. This is because the cause of the drift is mainly a temperature change.

  Next, (2) when the predetermined time T1 has elapsed, the control calculation unit 6 switches the circuit selection means 43 to the contact (2) side, the electrophoresis power supply unit 4 applies a voltage between the electrodes 11, and the measurement unit 5 The impedance between the electrodes 11 is measured. Hereinafter, this step is referred to as main measurement. The result of this measurement is represented by a straight line b in FIG. 10b and corresponds to a value obtained by adding impedance fluctuations due to particulate collection and drift. The predetermined time T1 is represented by a straight line d, and the fluctuation due to the drift measured when measuring the impedance between the electrodes 11 when the sample liquid 2 does not contain any fine particles affects the measurement accuracy of the fine particles. It is desirable to set a time that is sufficiently small to a certain extent. By doing so, the straight line b may be regarded almost entirely as fluctuation due to particulate collection, and measurement without the influence of impedance fluctuation due to drift becomes possible. In the present embodiment, T1 is set to 1 minute.

  At this time, the amount of drift due to the temperature rise of the circuit depends on the amount of current flowing through the circuit. Therefore, the amount of drift depends on the load impedance connected to the circuit, in other words, the impedance value of the dummy element 40 and the electrode 11. Changes. Therefore, by making the impedance of the dummy element 40 equivalent to the impedance of the electrode 11, the amount of drift between the measurement of the dummy element 40 and the measurement of the electrode 11 can be made constant, so that more accurate measurement is possible. Become.

  Next, (3) the control calculation unit 6 calculates the fine particle concentration from the slope of the straight line b, displays the result, and completes the measurement operation.

  As described above, according to the particle measuring apparatus of this embodiment, before applying a voltage to the electrode for collecting particles, a voltage is applied to the dummy element after the voltage is applied to the dummy element and the drift is sufficiently reduced. Since it is applied, it is possible to measure only the change in impedance due to the collection of fine particles on the electrode, so that it is possible to realize a highly accurate measurement of the number of fine particles.

(Third embodiment)
FIG. 10c shows an impedance measurement step in the particle measuring apparatus of the present embodiment. In this measurement step, first, (1) the control calculation unit 6 sets the circuit selection means 43 to the contact (1) side, and the measurement unit 5 measures the impedance of the dummy element 40 (first dummy measurement). This measurement result is represented by a curve a in FIG. 10c, and the impedance of the dummy element 40 after the predetermined time T1 becomes a value a ′, and fluctuation from the initial value occurs due to drift. a ′ corresponds to the state before the drift of the circuit immediately before the main measurement (impedance at the start of measurement).

  Next, (2) the control calculation unit 6 switches the circuit selection means 43 to the contact (2) side, and the measurement unit 5 measures the impedance between the electrodes 11 (main measurement). This measurement result is represented by a straight line b in FIG. 10c and corresponds to a value obtained by adding impedance fluctuations due to particulate collection and drift.

  Next, (3) the control calculation unit 6 switches the circuit selection means 43 to the contact (1) side, and the measurement unit 5 measures the impedance of the dummy element 40 (second dummy measurement). The impedance of the dummy element 40 at this time is represented by a value d ′ in FIG. 10 c and corresponds to a state after the drift of the circuit immediately after the main measurement (impedance at the end of measurement). Next, (4) the slope of the straight line d within a predetermined measurement time (T2−T1) obtained from the value a ′ at the measurement time T1 and the value d ′ at the measurement time T2 is subtracted from the straight line b to obtain the drift amount. And the straight line e (final result) is obtained. Next, (5) the control calculation unit 6 calculates the fine particle concentration from the slope of the straight line b, displays the result, etc., and completes the measurement operation.

  As described above, according to the microparticle measuring apparatus of the present embodiment, the drift amount (straight line d) of the measurement circuit is obtained from the measurement start impedance (value a ′) and the measurement end impedance (value d ′), and between the electrodes 11. Is corrected by the drift of the measurement circuit (straight line e), so that a sufficient time cannot be taken for the time (T1) for measuring the dummy element 40 before this measurement, and the influence of drift Even if the change in impedance due to the collection of fine particles on the electrode is a relatively small change compared to the change in impedance due to drift, the measurement impedance value fluctuation due to circuit drift is appropriate. Therefore, highly accurate particle measurement can be realized.

(Fourth embodiment)
FIG. 11 is a diagram showing a particle measuring apparatus according to the fourth embodiment of the present invention. In the particle measuring apparatus of the present embodiment, a dummy element array 44 including a plurality of dummy elements, and the number N + 1 of switching elements included in the dummy element array 44 ((1), (2), (3)... Multistage circuit selection means 45 having a contact point indicated by N). The control calculation unit 6 selects a predetermined dummy element N from the dummy element array 44 in accordance with the conductance or conductivity of the solution. As described above, the impedance of the dummy element is preferably equivalent to the impedance of the electrode 11, but the impedance of the electrode 11 varies depending on the conductivity of the sample solution 2. Therefore, the value of the dummy element can be corrected with higher accuracy by selecting an optimum value according to the conductivity of the sample solution 2.

  First, (1) the control calculation unit 6 sets the multistage circuit selection means 45 to the contact E side, the migration power supply unit 4 applies a voltage to the electrode 11, and the measurement unit 5 performs impedance (hereinafter referred to as the impedance between the electrodes 11). , Called electrode impedance) (electrode impedance measurement step). At this time, the voltage to be applied and the measurement time are set to a low voltage and a short time so that fine particles are not collected on the electrode. As a result, the electrode impedance can be measured with higher accuracy, and the measurement error during the actual measurement can be reduced. The control calculation unit 6 calculates a conductance value or a conductivity value from the measured impedance value. Thus, the apparatus can be simplified by the electrode also serving as the conductivity measuring means.

  Next, (2) the control calculation unit 6 switches the multistage circuit selection means 45 in accordance with the impedance, conductance, or conductivity measured in step (1), and from the dummy element array 44, Select a value close to the value of the electrode impedance. Hereinafter, since it is the same as the above-mentioned measurement step, description is abbreviate | omitted.

  According to this, it is possible to select an optimum dummy element according to the conductance or conductivity of the solution, and to correct the fluctuation of the drift when the conductivity of the solution is different with high accuracy.

(Fifth embodiment)
The fine particle measurement apparatus according to the present embodiment is realized using the apparatus described with reference to FIG. 10a or FIG. In the following, for simplicity of explanation, a case where the apparatus of FIG. 10A is used will be described. The fine particle measuring apparatus according to the present embodiment estimates and corrects the drift amount generated during the main measurement according to the conductivity of the sample liquid 2. If correction of drift is to be performed with a single dummy element 40 in order to avoid complication of the apparatus, as described above, the amount of drift changes depending on the conductivity of the sample solution 2, and thus accurate correction cannot be performed. Problems arise. Therefore, in the present embodiment, when a dummy element 40 having a certain impedance value is used, a drift amount (corresponding to the straight line d in FIGS. 10b to 10c) caused by the conductivity of the sample liquid 2 is estimated in advance. By subtracting the estimated drift amount from the inclination measurement result b, high-accuracy correction can be performed even with a single dummy element.

In this case, the structure of the dummy element and a resistor R _D and a parallel circuit of a capacitance C _D, when regarded impedance of the electrode 11 and the parallel circuit of the resistor R _E and capacitance C _E, C _D and C _E of equivalents It is desirable to choose.

Specifically, the drift amount is estimated by measuring a solution having a different conductivity and containing no fine particles in a predetermined dummy element 40 and electrode 11 (referred to as a blank measurement experiment). The slope of the straight line d measured at this time corresponds to a drift in each conductivity. A table in which the drift slope corresponding to each conductivity and the solution conductivity obtained in this way are associated with each other is stored in the memory 6a in the control calculation unit 6, and the impedance obtained by the electrode impedance measurement step. The drift is estimated by subtracting the estimated drift value from the slope of the straight line d with reference to the estimated drift value from the conductance or conductivity calculated from the equation (1). Alternatively, the relationship between the solution conductivity and the drift obtained from the blank measurement experiment may be expressed as a function, and an estimated value of the drift may be obtained from the solution conductivity and corrected.
At this time, the electrode impedance measurement step is not necessarily required, and the conductance or conductivity value can be estimated from the initial value of the electrode impedance measured in this measurement. Since correction is performed after the main measurement, the electrode impedance need not be measured first. Thus, the measurement time can be shortened by omitting the electrode impedance measurement step.
According to this, when measuring sample liquids having different electrical conductivities, it is possible to perform highly accurate correction with a single dummy element, so that the measuring apparatus can be simplified.

(Sixth embodiment)
The fine particle measuring apparatus of this embodiment includes temperature measuring means (not shown) for measuring the temperature of the sample liquid 2 in the apparatus shown in FIG. 11, and the control calculation unit 6 responds to the temperature of the sample liquid 2. To select a predetermined dummy element. As the temperature measuring means, a known measuring means such as a thermocouple or a thermistor can be used. According to this, it is possible to select an optimum dummy element according to the temperature of the sample solution 2 and correct the drift fluctuation when the solution temperature is different with high accuracy.

(Seventh embodiment)
The particle measuring apparatus according to the seventh embodiment is realized by a configuration excluding the apparatus shown in FIG. 5, that is, the dummy element 40 in FIG. A table in which the slope of drift corresponding to each conductivity and the solution conductivity are associated with each other in the memory 6a in the control calculation unit 6 or the relationship between the solution conductivity and drift. The function that represents is stored.

First, (1) the control calculation unit 6 measures the electrode impedance and calculates a conductance or conductivity value in the electrode impedance measurement step.
Next, (2) the control calculation unit 6 performs the main measurement, and obtains a slope line b of impedance change to which impedance fluctuation due to particulate collection and drift is added.
Next, (3) the control calculation unit 6 calculates the estimated drift value corresponding to the conductance or conductivity obtained in step (1) using the table or function stored in the memory 6a, and calculates the estimated Correction is performed by subtracting the drift value from the slope value of the straight line b to obtain the final slope (straight line e). Here, the conductance or the conductivity can also be obtained from the initial value of the impedance of the main measurement in step (2). Thereby, step (1) can be omitted and the measurement time can be shortened.
Next, (4) the control calculation unit 6 calculates the fine particle concentration from the slope of the straight line e, displays the result, and completes the measurement operation.

  According to this, even if there is no dummy element, it is possible to estimate the drift of sample liquids having different conductivities and perform highly accurate correction. Therefore, the measurement time can be reduced by simplifying the measuring device and reducing the dummy element measurement time. Can be shortened.

(Eighth embodiment)
FIG. 12 shows a specific configuration of the dummy element in the particle measuring apparatus according to the eighth embodiment of the present invention. As shown in FIG. 12A, a detection resistor 51 is connected to the particle measuring apparatus to measure the impedance between the electrodes 11, but in this embodiment, as shown in FIG. The capacitance 52 as a dummy element is inserted between the electrodes 11 to reduce the influence of phase fluctuation.

  As shown in FIG. 12B, the electrode 11 is represented by a parallel circuit of a resistance component 54 and a capacitance component 53, and the resistance component 54 varies depending on the solution conductivity. In the present embodiment, by inserting the capacitance 52 in parallel with the capacitance component 53 of the electrode 11, fluctuations in the impedance measurement value due to the phase error of the measurement circuit can be reduced.

  The fact that the fluctuation range for the impedance value varies depending on the phase angle even with the same phase error will be described with reference to FIG. 13 which is a vector diagram of impedance including the detection resistor.

  A vector (1) in FIG. 13A is an impedance vector of the electrode 11 and the detection resistor 51. The total 22KΩ of the resistance component 54 of the electrode 11 and the detection resistor 51 (560Ω) and a capacitance component 53 of the electrode 11 are shown. The phase angle is −64 ° when measured at a frequency of 800 kHz.

  When the solution conductivity increases, the impedance vector of the electrode 11 and the detection resistor 51 changes to the vector (2), the sum of the resistance component 54 of the electrode 11 and the detection resistor 51 is 1 KΩ, and the capacitance component 53 of the electrode 11 is 150 pF. The phase angle becomes −22 °. Note that the increase in conductivity occurs due to the increase in the concentration of ions (Na +, Cl-, etc.) in the solution, and if this level is increased, the dielectric constant of the solution hardly changes, so that the capacitance component 53 of the electrode 11 changes. do not do.

  In the vector (3), the capacitance 52 (200 pF) is added to the electrode 11, the sum of the resistance component 54 and the detection resistor 51 of the electrode 11 is 1 KΩ, the sum of the capacitance 52 and the capacitance component 53 of the electrode 11 is 350 pF, and the phase angle Shows the state of −28 °.

  FIG. 13B shows capacitance variation when the phase angle of the impedance vectors (1), (2), and (3) shown in FIG. 13A varies by 0.02 °. That is, the capacitance variation of the vector (1) is 0.018 pF, the capacitance variation of the vector (2) is 0.086 pF, and the capacitance variation of the vector (3) is 0.012 pF.

  Thus, the capacitance fluctuation of the vector (2) when the solution conductivity is increased was 0.086 pF, whereas the capacitance fluctuation of the vector (3) added with the capacitance 52 (200 pF) is 0.012 pF. It is reduced to about 1/7 to 1/8.

  If the impedance measurement circuit is ideally stable, the phase fluctuation in the measurement circuit becomes zero, but actually a phase fluctuation of about 0.02 ° occurs. According to this embodiment, the phase is shifted in advance by adding a capacitance so that the phase fluctuation caused by the measurement error of the measurement circuit does not affect the true measurement value, and the measurement error due to the phase fluctuation can be suppressed. .

(Ninth embodiment)
FIG. 17 shows a schematic diagram of the particle measuring apparatus according to the present embodiment. The present embodiment is characterized by having a plurality of electrode pairs on the electrode tip 3 of the fine particle measuring apparatus in FIG. 5 described above, and the description of the overlapping portions is omitted. In FIG. 17, a first electrode pair 60 and a second electrode pair 61 are formed on the electrode chip 3, and are connected to the migration power supply unit 4 and the measurement unit 5, respectively. As will be described in detail later, the impedance change is measured while performing dielectrophoresis on one electrode, and the other electrode does not perform dielectrophoresis, but only the impedance change is measured and the drift variation is detected and offset. The sizes are preferably the same.

  As described above, the electrophoretic power supply unit 4 is configured by an AC power supply circuit, and the measurement unit 5 is configured by an impedance measurement circuit, but the AC power supply circuit and the second electrode pair 61 are connected to the first electrode pair 60 and the second electrode pair 61. The impedance measurement circuit may be provided individually, or the AC power supply circuit and the impedance measurement circuit may be shared as one system. As will be described in detail later, if the voltage application is stopped in order to measure the impedance of the other electrode during the dielectrophoresis, fine particles trapped between the electrodes are dispersed in the sample liquid 1, and the AC power supply circuit It is desirable to provide the electrode pair 60 and the second electrode pair 61 separately. In addition, the impedance measurement circuit is desirable because it simplifies the circuit configuration by measuring the impedance changes of each of the first electrode pair 60 and the second electrode pair 61 by sequentially switching one system to be shared.

  In the following, an apparatus configuration and method for detecting and compensating for an undesirable measurement drift amount in the present embodiment will be described. First, measurement is started with the sample solution 2 introduced into the cell 1. The electrode tip 3 is provided in the cell 1 at a position where both the first electrode pair 60 and the second electrode pair 61 impregnate the sample solution 2. That is, the first electrode pair 60 and the second electrode pair 61 are impregnated with a liquid having the same conductivity.

  Next, the control calculation unit 6 instructs the migration power supply unit 4 to apply a voltage for performing measurement. Upon receiving the instruction, the migration power supply unit 4 applies an AC voltage to the first electrode pair 60 and the second electrode pair 61, respectively. Specifically, the voltage to be applied is a voltage for inducing dielectrophoresis and impedance measurement for one electrode pair, and only for impedance measurement without inducing dielectrophoresis for the other electrode pair. Apply the voltage to be performed. Either electrode may be used for dielectrophoresis, but for the sake of simplicity of explanation, the first electrode pair 60 performs the dielectrophoresis of the first electrode pair, and the second electrode pair 61 performs the impedance measurement only. The electrode will be described.

  The dielectrophoresis applied to one electrode and the voltage for impedance measurement are as described above. About the voltage which performs only the impedance measurement applied to the other electrode, a voltage that does not cause an impedance change due to the dielectrophoresis of the detection target fine particles between the electrodes is applied. Specifically, any amplitude that does not induce dielectrophoresis and can perform impedance measurement with high accuracy may be used. More specifically, an amplitude of about 0.001 to 1 Vpp is desirable. Further, for the reason described later, it is desirable that the frequency for impedance measurement of one electrode pair and the other electrode pair is the same. At this time, since one electrode pair needs to continuously perform dielectrophoresis of the fine particles in the sample liquid 1 and detect the impedance in which the fine particles are trapped between the electrodes as a signal, the electrophoretic power supply unit 4 It is desirable to provide a separate circuit system for the electrode pair and apply the AC voltage independently.

  When voltage application is started, the control calculation unit 6 instructs the measurement unit 5 to measure the impedance of each electrode pair. This impedance measurement is performed at predetermined time intervals. As described above, the impedance measurement may be performed at a minimum time interval in order to obtain a necessary resolution. That is, since the measurement is intermittent, the measurement circuit is set to 1 to simplify the measurement circuit. It is good to make it a system | strain and to switch sequentially according to the timing of the impedance measurement with respect to each electrode pair. Further, the impedance measurement result is transferred to the memory 6a and used for calculation after the measurement is completed. This measurement operation is smoothly performed in cooperation with the control calculation unit 6 and the measurement unit 5.

  When the preset measurement time elapses, the control calculation unit 6 instructs the migration power supply unit 4 to stop the voltage application and the measurement unit 5 to stop the impedance measurement. When the measurement operation is completed, the control calculation unit 6 calculates the number of fine particles in the sample liquid 1 from the impedance measurement results of the two pairs of electrodes accumulated in the memory 6a. The impedance change of one electrode includes a drift due to an electrode surface state and a circuit state in addition to an impedance change caused by trapping fine particles between the electrodes. The change in impedance of the other electrode includes a change only in drift. Therefore, by calculating the difference between the two impedance changes, it is possible to substantially obtain an impedance change caused by trapping the fine particles, that is, an impedance change only for the signal amount. Therefore, it is possible to measure the number of fine particles with high accuracy.

  At this time, since it is necessary for the other electrode to reproduce the drift caused by the one electrode, the shape and size are preferably the same as those of the one electrode. When the shape and size of the electrode chip 3 is unavoidable due to restrictions on the size of the electrode chip 3, sample liquids (hereinafter referred to as blank liquids) having different conductivity and not containing fine particles to be detected are measured. What is necessary is just to preserve | save in the memory 6a what acquired the correlation of the drift amount in an electrode and the other electrode beforehand, and to correct | amend it when calculating a difference.

  In addition, the impedance measurement circuit in the measurement unit 5 may be different in the amount of drift generated in each circuit due to variations in parts constituting the circuit when a measurement circuit system is individually provided for each electrode pair. It is done. For this reason, it is possible to cancel the drift amount resulting from the circuit state with higher accuracy by using the same circuit system for each electrode pair. The main cause of the drift amount generated by the measurement circuit is the heat generation of the parts constituting the circuit. For this reason, if the amount of current generated for impedance measurement is different, the drift amount is also different. Therefore, it is desirable that the two pairs of electrodes are impregnated in the same sample solution 1 to have the same electrode impedance depending on the solution conductivity and the same amount of current generated for impedance measurement. When imbalance occurs in the amount of current flowing between both electrodes due to the application of voltage for performing dielectrophoresis, a memory that has previously obtained the correlation of the drift amount generated in each electrode using a blank solution having different conductivity It may be stored in 6a and corrected when the difference is calculated.

  As described above, according to the present embodiment, the actual drift amount caused by the electrode and the circuit can be directly measured and subtracted, so that it is possible to provide a more accurate particle measuring apparatus and particle measuring method. It is.

  Examples of the present invention are shown below.

(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 collected with a rod and suspended in a 0.1 M D-mannitol solution (conductivity, about 5 μS / cm) is used as a standard sample, and appropriately diluted to 1.4 × 10 ^ 5 to 1.4 × 10 ^ 7 cfu. A three-stage dilution series was prepared to achieve a suspension concentration of / ml.

  The suspension concentration was defined by smearing an appropriately diluted standard sample into a standard agar medium and counting the number of colonies grown as a result of aerobic culture at 37 ° C. for 16 hours. A NaCl solution was appropriately added to the standard sample, and the sample solution conductivity was adjusted to 5, 50, and 100 μS / cm. The sample solution conductivity was measured with a conductivity meter (B-173, Horiba, Ltd.).

(2) Measuring device The measuring device of Fig. 10a was used. The applied voltage amplitude was 10 Vp-p, and the frequency was 800 KHz. The dummy element was a parallel circuit of a 5.2 KΩ resistor and a 150 pF capacitor.

(3) Result 1 (blank measurement experiment)
The amount of drift was measured when the sample liquid conductivity was 5, 50, 100, 250 μS / cm. The first dummy element measurement time T1 was 30 seconds, and the main measurement time was 20 seconds. FIG. 14 shows a plot of the drift amount at this time normalized by the drift amount in the case of 5 μS / cm. It can be seen that the drift amount increases as the conductivity increases.

(4) Result 2 (microbe measurement experiment)
From the drift amount measurement result of Result 1, a function was obtained from the quadratic approximation formula for the relationship between the solution conductivity and the drift amount, and was used as the drift correction amount. The conductivity was 5, 50, 100 μS / cm, the Escherichia coli concentration was 1.4 × 10 5 to 1.4 × 10 7 cfu / ml, and the same measurement as in Result 1 was performed. Table 3 shows measurement results that do not include drift correction.

  The values shown in Table 3 are values normalized by the slope when the conductivity is 5 μS / cm and the Escherichia coli concentration is 1.4 × 10 ^ 5 cfu / ml. When the conductivity is 50 μS / cm and the Escherichia coli concentration is 1.4 × 10 ^ 5 cfu / ml, and the conductivity is 100 μS / cm and the Escherichia coli concentration is less than 1.4 × 10 ^ 7 cfu / ml, the slope of the impedance change becomes a negative value. Measurement becomes impossible.

  On the other hand, FIG. 15 shows the slope of the impedance (capacitance) with respect to the Escherichia coli concentration corrected by the obtained drift correction amount. A positive slope was obtained for all the conductivities of Escherichia coli concentration of 1.4 × 10 ^ 5 cfu / ml or more, and the linearity of the slope of the capacitance with respect to the Escherichia coli concentration was good, and the effect of the correction was proved. .

  INDUSTRIAL APPLICABILITY The present invention can be used as a particle measuring apparatus and a particle measuring method capable of detecting / reducing measurement impedance value fluctuation due to temperature rise of an electrode or a sample solution and circuit drift and realizing high-precision impedance change measurement. is there.

DESCRIPTION OF SYMBOLS 1 Cell 2 Sample solution 3 Electrode chip 4 Electrophoresis power supply part 5 Measurement part 6 Control operation part 6a Memory 7 Conductivity input means 9 Display means 10 Substrate 11,11a, 11b Electrode 13 Gap 14 Fine particle 15 Electric force line 17 Stirring means 21 Light source 22 Photodetector 40 Dummy element 43 Circuit selection means 44 Dummy element array 45 Multi-stage circuit selection means 51 Detection resistance 52 Capacitance 53 Capacitance component 54 Resistance component RL Measurement load 60 First electrode pair 61 Second electrode pair

Claims (6)

At least a pair of electrodes immersed in a cell for introducing a liquid containing fine particles;
An electrophoretic power supply unit that applies an alternating voltage between the pair of electrodes and applies a dielectrophoretic force to the fine particles;
Impedance measuring means for measuring impedance between the pair of electrodes;
An arithmetic unit for calculating the number of fine particles in the liquid from the measurement result of the impedance measuring means;
Conductivity measuring means for measuring the conductivity of the liquid;
With
The calculation unit refers to a table or function indicating the electrode impedance fluctuation value for each conductivity, and corrects the calculation result based on the electrode impedance fluctuation value according to the measurement result of the conductivity measuring unit.
Fine particle measuring device. The fine particle measuring device according to claim 1,
Comprising temperature measuring means for measuring the temperature of the liquid;
The computing unit refers to a table or function indicating the electrode impedance variation value for each conductivity and temperature, and based on the measurement result of the conductivity of the liquid and the electrode impedance variation value corresponding to the measurement result of the temperature of the liquid. , Correct the calculation results,
Fine particle measuring device. The fine particle measuring device according to claim 1,
A capacitance connected in parallel with the pair of electrodes;
The electrophoretic power supply unit applies an alternating voltage to a parallel circuit composed of the electrodes and the capacitance, and the impedance measuring means measures a parallel impedance between the electrodes and the capacitance.
Fine particle measuring device. A method for measuring fine particles in which an alternating voltage is applied between a pair of electrodes immersed in a sample solution containing fine particles, the fine particles are arranged at predetermined positions by a dielectrophoretic force, and the number of fine particles in the sample solution is measured.
A conductivity measuring step for measuring the conductivity of the sample solution;
An impedance measurement step of applying an alternating voltage between the pair of electrodes and measuring an impedance between the pair of electrodes;
When calculating the number of fine particles in the sample liquid from the measurement result of the impedance, refer to a table or function indicating the electrode impedance fluctuation value for each conductivity, and the electrode impedance fluctuation according to the measurement result of the conductivity of the sample liquid A calculation step for correcting the calculation result based on the value;
A method for measuring fine particles. The fine particle measurement method according to claim 4,
A temperature measuring step for measuring the temperature of the liquid;
When calculating the number of fine particles in the liquid from the measurement result of the impedance, the calculation step refers to a table or function indicating an electrode impedance variation value for each conductivity and temperature, and the measurement result of the conductivity of the liquid and Based on the electrode impedance fluctuation value according to the measurement result of the temperature of the liquid, the calculation result is corrected.
Fine particle measurement method. The fine particle measurement method according to claim 4,
In the impedance measuring step, an AC voltage is applied to a parallel circuit including the pair of electrodes and a capacitance connected in parallel with the pair of electrodes, and a parallel impedance between the electrodes and the capacitance is measured.
Fine particle measurement method.

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