JP2001204458A - Microorganism carrier, microorganism counting device and microorganism counting method - Google Patents

Abstract

(57) [Problem] To provide a microbial carrier capable of performing simple and highly sensitive measurement by dielectrophoresis even for microorganisms having a low dielectric constant. SOLUTION: The microorganism carrier of the present invention is composed of a material having a higher dielectric constant than the dielectric constant of the microorganism to be measured,
A microorganism carrier body having a binding group capable of specifically binding to the microorganism on its surface, wherein the microorganisms bound to the binding group are electrophoresed integrally when a dielectrophoretic force acts.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microorganism carrier body provided with a binding group and capable of collecting microorganisms bound to the binding group when dielectrophoretic force acts thereon.
The present invention also relates to a microorganism counting device for measuring the number of microorganisms in a solution using the microorganism carrier, and a method for measuring the number of microorganisms.

[0002]

2. Description of the Related Art Conventionally, a number of techniques for measuring the number of microorganisms in a solution, such as that described in JP-A-57-50652, are known. However, the method of measuring the number of microorganisms according to the conventional technique involves introducing a special agent, for example, an enzyme or a dye, into a sample solution to cause a biochemical reaction, and measuring the progress or result of the reaction by fluorescence or luminescence. Although the measurement sensitivity is relatively high, specialized knowledge in the fields of microorganisms and biochemistry is required, and a dedicated and expensive large-scale measuring device is required, and furthermore, work by a dedicated person is required. It was not possible to measure the number of microorganisms easily and easily.

[0003] Therefore, using only physical means, such as that described in JP-A-59-91900, and using no chemicals, it is small and can be automatically measured by incorporating it into a sample system. , A simple microbial count detection device has been proposed, but the microbial count is 10 8 cells / ml (1
Since the number of microorganisms cannot be detected until the number of microorganisms exceeds 100 million), the range of application has been markedly limited.
As described above, in order to increase the measurement sensitivity in the microorganism counting device according to the conventional technique, it is necessary to use some kind of drug, a dedicated measuring device, and an operation by a dedicated person having specialized knowledge. In addition, with a simple device that does not use drugs, measurement can be performed without the need for a dedicated person.However, if the number of microorganisms is not very large, measurement is difficult, and only a low-sensitivity measuring instrument can be obtained. However, there is a problem that there is no simple and maintenance-free means even if it is desired to improve the sensitivity by locally increasing the density.

[0004] The inventors of the present invention have proposed a method disclosed in
No. 7846 proposes a new measuring method in which a dielectrophoretic force is applied to microorganisms in a measurement sample, and the microorganisms are collected at a location where an electric field is concentrated to measure the concentration. According to this measuring method, no medicine or special device is required,
Simple and highly sensitive measurement was possible, and maintenance-free measurement of microorganisms became possible.

[0005] However, the environment in which microorganisms are measured often contains minerals, organic substances, and the like for the microorganisms to inhabit, which increase the conductivity in the liquid in which the microorganisms inhabit, If this solution is used as a measurement sample to measure the number of microorganisms, there is a problem that the conductivity is high and the concentration of microorganisms cannot be increased by dielectrophoresis. further,
Many microorganisms have a low dielectric constant, and even if the conductivity of the solution is low, there is a problem that the dielectrophoretic force is inherently weak and the concentration cannot be increased similarly. When the conductivity of the measurement sample is high, dielectrophoresis can be performed by performing dialysis or dilution to reduce the conductivity, but if the microorganism itself has a low dielectric constant, the dielectrophoretic force is increased. Since there is no means and the concentration cannot be increased by moving the microorganisms, it has been difficult to measure the number of microorganisms by dielectrophoresis.

[0006]

SUMMARY OF THE INVENTION In order to solve these problems, the present invention provides a carrier for the number of microorganisms, which enables simple and highly sensitive measurement by dielectrophoresis even for microorganisms having a low dielectric constant. The purpose is to:

Further, the present invention enables simple and highly sensitive measurement of microorganisms having a low dielectric constant without requiring a drug or a special device, and enables automatic measurement and maintenance-free microorganism count. It is intended to provide a device.

It is a further object of the present invention to provide a maintenance-free method for measuring the number of microorganisms which enables simple and highly sensitive measurement of microorganisms having a low dielectric constant, enables automatic measurement, and is maintenance-free.

[0009]

In order to achieve the above object, the microorganism carrier of the present invention is made of a material having a higher dielectric constant than that of the microorganism to be measured, and has a surface which is unique to the microorganism. A microorganism carrier body provided with a binding group that can be bound to the substrate, wherein when a dielectrophoretic force acts, the microorganisms bound to the binding group are electrophoresed together.

[0010] Thus, simple and high-sensitivity measurement can be performed by dielectrophoresis even for a microorganism having a low dielectric constant without requiring a drug or a special device.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention described in claim 1 is characterized in that the measurement target is constituted by a material having a higher dielectric constant than the microorganism to be measured, and a surface is provided with a binding group capable of specifically binding to the microorganism. A microbial carrier body, characterized in that when a dielectrophoretic force acts, the microorganisms bonded to the binding group are electrophoresed together, and thus are not easily affected by the dielectrophoretic force. Even for microorganisms with a low dielectric constant, increase the apparent dielectric constant with the microorganism carrier,
This can be collected together with the microorganism carrier in the electric field concentration portion by the dielectrophoretic force.

[0012] According to a second aspect of the present invention, the microorganism carrier has a representative size that is the same as the representative size of the microorganism or up to several times, and has a surface conductivity lower than the surface conductivity of the microorganism. 2. The method according to claim 1, wherein
Since the microorganism carrier is described, even a microorganism having a low dielectric constant that is not easily subjected to dielectrophoretic force can be collected together with the microorganism carrier in the electric field concentration portion by the dielectrophoretic force. Since one to several microorganisms bind in proportion to the concentration of microorganisms, if the concentration of microorganisms is high, the surface conductivity of both conjugates changes, and the number of microorganisms can be measured by the surface conductivity.

[0013] The invention described in claim 3 is characterized in that the binding group can specifically bind to a functional group of a microorganism,
The microorganism carrier according to claim 1, wherein the microorganism carrier is a compound modified on the surface of the microorganism carrier, even if the microorganism has a low dielectric constant that is not easily affected by dielectrophoretic force. At the same time, it can be stably collected at the electric field concentration portion by the dielectrophoretic force.

[0014] The invention described in claim 4 is that the compound is a compound that specifically binds to an amino acid residue contained in a protein of a microorganism.
The microorganism carrier according to any one of the above, even a microorganism having a low dielectric constant that is less susceptible to the effect of dielectrophoretic force, specifically binds to any living or dead microorganism through amino acid residues, The dielectrophoretic force together with the carrier allows stable collection at the electric field concentration portion.

[0015] The invention described in claim 5 is an antibody having a paratope in which the binding group specifically binds to an epitope of a microorganism which is an antigen, wherein the antibody is modified on the surface of a microorganism carrier. Since the microorganism carrier according to claim 1 or 2, even a microorganism having a low dielectric constant that is less susceptible to the effect of dielectrophoretic force, quickly and specifically binds to the microorganism carrier by an antigen-antibody reaction,
The specific microorganisms can be collected together with the microorganism carrier in the electric field concentration part by the dielectrophoretic force.

According to a sixth aspect of the present invention, a plurality of electrodes are provided therein, and a microorganism-containing liquid is introduced, and the microorganism carrier according to any one of the first to fifth aspects is introduced. A cell, a power supply circuit for applying an AC voltage for generating a dielectrophoretic force in the cell between any of the electrodes, and measuring an electric field with any of the electrodes A measuring unit for measuring the resistance characteristics between the electrodes, a control unit for controlling the power supply circuit and the measuring unit, and a calculating unit for calculating the number of microorganisms by calculating the measurement result of the measuring unit. Since it is a microorganism counting device characterized by having, even in a sample with a small number of microorganisms, after concentrating the microorganisms near the electrode,
Since the number of microorganisms can be measured by the inter-electrode resistance characteristics, simple and highly sensitive measurement can be performed without the need for a drug or a special device.

According to a seventh aspect of the present invention, the measuring section measures a current and a phase difference between the electrodes, and calculates a resistance by measuring a voltage applied between the electrodes. Since the microbial count measuring device described in 6, the microbes are measured by measuring the current, phase difference, and voltage between the electrodes by the measuring unit after the microbes are concentrated near the electrodes even in the sample having a small number of microbes. Therefore, simple and highly sensitive measurement can be performed.

According to the invention described in claim 8, the microorganism-containing liquid and the microorganism carrier described in claims 1 to 5 are introduced into a cell provided with a plurality of electrodes, and any one of the plurality of electrodes is introduced. An AC voltage is applied between the electrodes to generate a dielectrophoretic force in the cell, the microorganisms bound to the microorganism carrier are collected in an electric field concentration portion, and the change in resistance characteristics between the electrodes is measured to determine the number of microorganisms. The method of counting the number of microorganisms is characterized in that the number of microorganisms can be measured by electrical means after the microorganisms are concentrated near the electrodes even in a sample with a small number of microorganisms. Simple and highly sensitive measurement can be performed without the need for a simple device.

Hereinafter, an embodiment of the present invention will be described with reference to FIG.
7 and (Equation 1) to (Equation 5).

(Embodiment) A microorganism counting apparatus according to an embodiment of the present invention will be described in detail with reference to the drawings. FIG. 1 is an overall configuration diagram of an apparatus for measuring the number of microorganisms according to an embodiment of the present invention, FIG. 2 is an explanatory diagram of an electrode according to an embodiment of the present invention, and FIG. Diagram for explaining the state of the electric field of
FIG. 4 is a graph for explaining the relationship between the number of microorganisms, the measurement time, and the resistance R between the electrodes. FIG. 5A is a diagram for explaining a method of calculating the capacitance between the electrodes, and FIG. FIG. 6 is a diagram showing a phase difference between a current and a voltage, FIG. 6 is another diagram for explaining a method of calculating capacitance between electrodes, and FIG. 7 is a schematic diagram showing a molecular structure of a microorganism carrier. .

In FIG. 1, 1 is a cell, 2 is an electrode on a substrate, 3 is an electrophoresis power supply circuit, 4 is a measuring unit, 5 is an arithmetic unit, 6 is control means, 7 is a sample system pipe, 8 is a solenoid valve, 9 is a display means. In FIG. 2, reference numeral 10 denotes a substrate of the electrode 2;
Reference numeral 1 denotes a thin film electrode, and 12 denotes a minute gap sandwiched between the thin film electrodes 11. In FIG. 3, reference numeral 13 denotes a thin film electrode 11.
These are lines of electric force generated by a voltage applied therebetween. In FIG. 5A, reference numeral 14 denotes one pole of the thin-film electrode 11;
Is the other pole of the thin film electrode 11, and 16 is a CR assumed when a microorganism or a microorganism carrier is placed in the electric flux 13.
In FIG. 5B, reference numeral 17 denotes a capacitance constituting a parallel equivalent circuit, and reference numeral 17 denotes a resistance constituting a CR equivalent circuit when the carrier body is placed in the electric flux lines 13. 18 is the time axis, 1
Reference numeral 9 denotes an axis representing the magnitude of voltage and current, reference numeral 20 denotes a curve representing a change in current, and reference numeral 21 denotes a curve representing a change in voltage. Also,
In FIG. 7, reference numeral 22 denotes a carrier core made of a material having a higher dielectric constant than the microorganism to be measured. In the present embodiment, barium titanate particles are used. A polymer 24 such as polystyrene, which coats 22, is a binding group such as a compound or an antibody which is modified on the surface of the polymer 23 or the core 22 and specifically binds to a microorganism. Core 22 and polymer 2
3 and the binding group 24 as a whole are a microorganism carrier (hereinafter, referred to as a microorganism carrier).
Carrier body). As described later, it is also appropriate that the core 22 itself is made of polymer particles in which fine particles having a high dielectric constant are dispersed (there is no polymer 23 to be coated). Also, this carrier body,
It is preferable to have a representative dimension that is about the same as or several times as large as the representative dimension up to the microorganism to be measured. A typical example of the binding group 24 is a group chemically bonded by a compound. Among them, a carboxyl group or the like binding to an amino acid residue contained in a protein is effective. Paratopes, which are antibodies that bind, are also suitable. In the case of an amino acid residue, it stably and specifically binds to both living and dead microorganisms, and can be collected together with the carrier body at the electric field concentration portion by dielectrophoretic force. The microorganisms can be quickly bound to the carrier body and collected together with the carrier body in the electric field concentration portion.

Here, the dielectrophoretic phenomenon of microorganisms will be described. Due to the action of an AC electric field generated by application of a high-frequency AC voltage, the microorganisms in the cell 1 are migrated to a portion where the electric field is strongest and non-uniform due to its dielectric property, that is, an electric field concentrated portion. On the other hand, the magnitude of the dielectrophoretic force acting on the microorganism generally has a relationship such that it increases as the difference between the dielectric constant of the microorganism and the dielectric constant of the solution around the microorganism increases. Therefore, considering that the dielectric constant of the solution around the microorganism is smaller than the dielectric constant of the microorganism, the acting dielectrophoretic force increases as the microorganism has a higher dielectric constant. As described above, in the present invention, even when the conductivity of the solution becomes relatively high, the conjugate having a higher dielectric constant by the carrier can be migrated. In the present embodiment, the structure near the gap 12 of the electrode 2 corresponds to the electric field concentration portion, and the electric field is most concentrated in the gap 12. Therefore, gap 1
Microorganisms are electrophoresed most strongly in the two parts. The gap 12 shown in FIG. 2 is a portion sandwiched between parallel electrodes, and the electric field distribution is uniform in the direction in which the electrodes extend, that is, in the direction perpendicular to the plane of FIG. However, in the direction perpendicular to the substrate surface, an electric field distribution as shown in FIG. 3 occurs, and the electric field concentrates most on the surface in the length direction connecting the edges of the electrodes. Gap 1
Microorganisms floating near 2 are attracted to the gap 12 by such an electric field effect generated between the electrodes 2 and are aligned along the lines of electric force. At this time, the moving state of the microorganisms near the gap 12 depends on the number of microorganisms present in the sample liquid and the interval between the gaps 12. When the number of microorganisms is sufficiently large, the gap 12 is cross-linked by a chain composed of microorganisms. Will be enough. At this time, the microorganisms floating in the vicinity of the gap 12 from the beginning move to the gap 12 immediately, and the microorganisms floating in the place distant from the gap 12 are removed from the gap 12 after a predetermined time elapse according to the distance.
Therefore, the number of microorganisms that have gathered in a predetermined area near the gap 12 after a certain period of time is proportional to the number of microorganisms in the cell 1. This is, of course, proportional to the number of microorganisms present in the liquid of the measurement sample.

As described above, when the dielectric constant of the microorganism to be measured is sufficiently large, the microorganism can be quickly moved to the position of the measurement electrode by the dielectrophoretic force. When the permittivity is large, the difference between the permittivity of the microorganism and the permittivity of the surrounding solution is reduced, so that the dielectrophoretic force is also reduced, and a sufficient acting force on the microorganism cannot be obtained. Therefore, in the present invention, by combining a plurality of microorganisms with a carrier having a high dielectric constant to form a conjugate having a high dielectric constant, a sufficient acting force can be received. As a result, the microorganisms can be quickly moved to the measurement electrode position as a conjugate with the carrier body, and the concentration of microorganisms in a solution having relatively high conductivity can be measured.

In this embodiment, as shown in FIG. 7, the carrier body comprises a core 22, a polymer 23, and a bonding group 24.
It has three elements. The core 22 is suitably made of barium titanate particles and has high dielectric constant electrical characteristics. The details of the method for calculating the number of microorganisms will be described later,
Since the number of microorganisms is calculated based on the change in the electrical characteristics of the electrodes, it is necessary to increase the change in the resistance characteristics between the electrodes due to the microorganisms collected in the electric field concentrating part integrally with the carrier body in order to increase the sensitivity The polymer 23 is suitably made of polystyrene or the like having lower conductivity than microorganisms. It is naturally necessary that the carrier body and the microorganisms have a high dielectric constant in an integrated state.However, since the surface conductivity needs to change depending on the number of microorganisms to be bound, the size of the carrier body is It is desirable that the representative dimensions be the same or several times as large. In this embodiment, the carrier body is made of barium titanate particles coated with a polymer, but the core 22 is made of polystyrene particles in which fine particles of barium titanate are dispersed, and the electric characteristics are not changed. Is different, but the original purpose is high dielectric constant,
In order to satisfy the low surface conductivity, it can be used as the core 22 of the carrier body.

The binding group 24 in the present embodiment is a carboxyl group modified on the surface of the core 22 or the polymer 23, and actively binds to an amino group existing at the end as an amino acid residue of a microorganism protein. . In addition, the bonding group 24 made of another compound may be used as long as the bonding group 24 specifically binds to the functional group of the microorganism. Since a large number of binding groups 24 are modified on the surface of the carrier body, one carrier body and a plurality of microorganisms eventually form one combined body. Since both living and dead microorganisms are composed of proteins, the total number of microorganisms can be measured regardless of whether the microorganisms are alive or dead.

Further, the carrier can be bound to the microorganism by using an antigen-antibody reaction as the binding group 24. Generally, the binding between an antigen and an antibody can be regarded as a so-called key-keyhole relationship. The microorganism protein serving as an antigen is a giant macromolecule in which a large number of amino acids composed of carbon, oxygen, hydrogen, nitrogen, sulfur, etc. are bonded, and has a characteristic three-dimensional higher-order structure depending on the arrangement of the amino acids. This higher-order structure is closely related to the function of the protein. In proteins having different functions, some structures are always different from each other, and are one of the antigenic determinants called epitopes. Among such a wide variety of proteins, some proteins are possessed by most microorganisms, while others are possessed only by specific microorganisms. On the other hand, antibodies have a structure that fits well with the characteristic higher-order structure of a specific protein of an antigen, and have an antigen-binding site called a paratope that specifically binds to the epitope described above. I have. Therefore, even if very similar proteins whose binding positions of functional groups are slightly different are present at the same time, if the antibody having a paratope that binds to a specific epitope is modified, it can be identified with high specificity. Causes binding only to proteins. That is,
If the antibody is modified on the surface of the carrier body as the binding group 24, a specific conjugate with the microorganism as an antigen can be obtained promptly, and an antibody that selectively binds only to a specific microorganism can be obtained. If so, a conjugate of the specific microorganism and the carrier can be obtained. This makes it possible to measure the number of specific microorganisms.

Further, as an example of the advanced technique of the antigen-antibody reaction, the extremely high specific binding of a biotin-avidin complex widely used in the field of immunological measurement such as EIA (enzyme immunoassay) and tissue staining is described. Can also be used. Biotin itself can be bonded to various functional groups of a protein by chemically modifying it. Therefore, biotin can be modified in advance on a microorganism serving as an antigen, and avidin can bind to a binding group 24. As a result, a conjugate of a microorganism and a carrier can be obtained through a two-step binding process by using a carrier body modified on the surface.

Here, the microorganism to be detected in the present invention will be described. The microorganisms referred to in the present invention are not only organisms that are subject to so-called microbiology, which are generally classified as bacteria, fungi, actinomycetes, rickettsias, mycoplasmas, viruses, and small protozoa and protozoa. Larvae of living organisms, isolated or cultured animal and plant cells, sperm, blood cells, nucleic acids, proteins, etc. In the present invention, a microorganism in a liquid is assumed as a measurement target.

Next, the electrodes of the microorganism counting apparatus according to the present embodiment will be described. As shown in FIGS. 1 and 2, a thin film electrode 11 on an electrode 2 is provided to face a micro gap 12 in order to move microorganisms in a sample liquid to a predetermined position by dielectrophoresis. In the present embodiment, the thin-film electrode 11 includes two poles 14 and 15, and the comb-shaped thin-film electrodes 11 are arranged so as to be nested in each other as shown in FIGS. Each of the electrodes is made of a conductor that is formed in close contact with the substrate 10 by a method such as sputtering, vapor deposition, or plating, and the portion of the substrate 10 where the thin film electrode 11 is in close contact and the substrate surface are exposed. It is surrounded by the end line that is the boundary of the part. The thin film electrode 11 is formed by two poles 14 and 15.
The gaps 12 formed between the end lines are all at the same interval in the present embodiment. Since the electric field near the gap 12 becomes strongest by the application of the voltage for the dielectrophoresis, the gap 12 becomes the electric field concentration portion in the present embodiment. Therefore, the microorganisms migrate toward the vicinity of the gap 12 where the electric field is most concentrated. The film thickness of the thin film electrode 11 in the embodiment is about 5 nm. The film thickness of 5 nm corresponds to the gap 1 of the gap 12 described later.
Since it is very small as compared to 00 μm and compared to the microorganism to be detected, the thin-film electrode 11 can be considered as an electrode having a substantially negligible two-dimensional spread in thickness. Therefore, in this embodiment, the boundary between the portion where the electrode is in close contact with the substrate and the portion where the substrate is exposed on the substrate 10 is expressed as an end line. At this time, the electric field distribution in the vicinity of the gap 12, which is the electric field concentration portion in the electrode 2, is as shown in FIG. The thickness of the thin film electrode 11 is about 5
As shown in FIG.
The electric field concentrates most on the portion sandwiched between the end lines. The thin-film electrode 11 may be made of any material as long as the resistance is not extremely high. However, when it is assumed that the thin-film electrode 11 is used in a liquid, particularly in water as in the present embodiment, the thin-film electrode 11 has a high ionization tendency. But a low metal is desirable. Since a strong electric field is generated between the electrodes 2 during dielectrophoresis, electrolysis may occur depending on the applied frequency and the electrolyte concentration in water. In the case of an electrode made of a metal having a high ionization tendency when electrolysis occurs, the electrode is dissolved and the shape of the electrode is collapsed, and in extreme cases, the electrode is broken. Therefore, in the present embodiment, platinum is used as the main material of the electrode.

The substrate 10 may be made of any material as long as it can hold the thin-film electrode 11 and has a high insulating property. However, a substrate having a low dielectric constant (a low dielectric constant substrate) is desirable. The reason why a substrate having a low dielectric constant is more preferable is that the electric field between the thin-film electrodes 11 is distributed in the inside of the substrate although not shown in detail in FIG. The ratio of the intensity of the electric field inside the substrate to the electric field spreading outside the substrate, ie, the electric field for dielectrophoresis, is determined by the ratio of the dielectric constants. In this embodiment, a borosilicate glass substrate is used as the low dielectric constant substrate because the substrate has high smoothness, is easy to perform fine processing, and has a relatively low dielectric constant. In the present embodiment, the thin film electrode 11 is formed on the substrate 10 by a method using general photolithography. Although the interval between the gaps 12 in the present embodiment is set to 100 μm, the interval between the gaps 12 is adjusted as necessary because it is affected by the size of the microorganism to be measured. For example, large ones such as yeast and isolated cells need to be wide, and small ones such as rickettsia need to be narrow. Also, the wider the gap 12, the larger the amount of microorganisms that can be concentrated and the wider the dynamic range of the measurement, but the longer the time until the measurement, and the larger the power required for dielectrophoresis. . Conversely, if the gap 12 is reduced, the power and the time required for the measurement are reduced, but the dynamic range of the measurement is reduced. For this reason, in the present embodiment, the interval between the gaps 12 is set to 100 μm.
However, it is desirable that this value is appropriately adjusted in the range of 0.2 to 300 μm according to the microorganism to be detected.

The electrophoresis power supply circuit 3 supplies an alternating current for causing dielectrophoresis between the electrodes 2. In the present embodiment, as will be described later, an alternating current for causing dielectrophoresis is temporarily interrupted to measure a change in impedance between the electrodes. A plurality of electrodes 2 may be provided, and two of them may be used as electrodes for measurement. The migration power supply circuit 3 is controlled by the control means 6 together with the electromagnetic valve 8 and the like. The control means 6 includes a microprocessor (not shown), a memory for storing a preset program, a timer, and a signal line between the measurement unit 4 and the like, and opens and closes the electromagnetic valve 8 according to the program. Then, the electrophoresis power supply circuit 3 is controlled to apply an AC voltage having a specific frequency and voltage to the electrode 2. Further, the control means 6 manages the flow of the entire measurement operation by transmitting and receiving signals to and from the measuring unit 4 and the arithmetic unit 5 and performing appropriate control.

In the embodiment, the measuring section 4 is a circuit for applying an AC voltage (hereinafter referred to as a voltage for measurement) for examining the impedance between the electrodes 2. A circuit for measuring the difference between the flowing current and the phase of the voltage and the current, the arithmetic unit 5
And signal lines for transmitting signals to and from the control means 6. This moves the microorganisms by dielectrophoresis,
The concentration is concentrated in the vicinity of the electric field concentration portion, causing a change in the impedance of the electrode 2, which is measured. The voltage applied between the electrodes 2, the current measured by the measuring unit 4, and data indicating the phase difference between the voltage and the current are passed to the calculating unit 5, where the calculation is performed by a procedure described later. In the present embodiment, the measurement unit 4 is controlled by the control unit 6, and a series of measurement operations is smoothly performed in cooperation with each other according to a preset program. The calculation unit 5 includes a microprocessor, a memory, and the like (not shown), and analyzes the impedance of the electrode 2 from the result measured by the measurement unit 4 to calculate a resistance component between the electrodes 2, which will be described later in detail. Then, the number of microorganisms contained in the measurement sample is finally calculated by, for example, storing the calculation result in the memory 14 or reading out the data stored in advance and comparing the data. In addition,
This microprocessor can be shared by the control means 6 and the arithmetic unit 5. The arithmetic unit 5 is also controlled by the control unit 6 in the same manner as the measuring unit 4.

The display means 9 displays the calculated number of microorganisms on the sample 1
Display digitally as the number of microorganisms per mL. The display on the display means 9 is the final output of the microorganism counting device in the embodiment. In this embodiment, the user can directly know the measured number of microorganisms as the number of microorganisms per 1 mL of the sample. However, the display means 9 may be an approximate number such as a large number or a small number, or another number depending on the purpose. The display method may be used. Furthermore, there is no need for the user to know the number of microorganisms directly, such as controlling the sterilization device by checking the number of microorganisms in the sample or controlling the culture conditions such as temperature.
If the number of microorganisms only needs to be ascertained in order to control the apparatus, the display means 9 need not be provided.

Subsequently, a series of flows from the introduction of the liquid of the measurement sample to the concentration, measurement, and washing of the microorganisms in the cell 1 will be described. In the initial state, the electromagnetic valve 8 for shutting off the sample system pipe 7 and the cell 1 is in an open state, and the liquid of the measurement sample passes freely through the cell 1. Here, the liquid of the measurement sample is a mixture of a solution containing a microorganism to be measured and a carrier. At a predetermined timing, when a measurement operation set in advance by a program is started, the control means 6 closes the solenoid valve 8, shuts off the cell 1 from the sample pipe 7, and forms a closed system only in the cell 1. afterwards,
The control unit 6 sends a measurement start signal to the measuring unit 4 to start the measurement after a predetermined period of time, which is expected to stop the flow of the liquid in the cell 1, has elapsed. Upon receiving the measurement start command, the measurement unit 4 immediately applies a sine wave AC voltage having a frequency of 100 kHz and a voltage of 1 V between the electrodes 2 as a voltage for measurement, and outputs the voltage, current, and phase of the voltage and current at that time. Measure the difference. Here, the applied AC frequency and voltage for measurement may be optimally selected at the time of calibration with a sample containing a microorganism whose concentration is apparent in advance, as will be described later. It is not necessary to be bound by the values exemplified in the above. The AC voltage here is not only a sine wave,
A voltage that changes the direction of flow at a substantially constant cycle, and has an equal average value of currents in both directions.

The measurement result is sent to the arithmetic section 5. Arithmetic unit 5
Is the impedance between the electrodes 2 from the obtained measurement results,
A resistance value is calculated when a microorganism, a carrier, a solution, and the like existing between the electrodes 2 are regarded as a CR parallel equivalent circuit including resistance and capacitance. Although the details will be described below, the impedance is obtained by dividing the applied voltage and the current, and the resistance value R
Is the reactance and resistance calculated using the value representing the difference between the impedance, the phase of the voltage, and the phase of the current as the angular difference of the angular frequency (hereinafter referred to as the phase angle). And by solving the simultaneous equations, it can be calculated together with C.

5 and 6, the impedance is Z, the capacitance is C, the reactance is x, and the resistance is r.
And (Equation 1) to (Equation 5) will be described in detail.
(Equation 1) is an equation representing the combined impedance of the CR parallel equivalent circuit, (Equation 2) is an equation representing the resistance of the CR parallel equivalent circuit, (Equation 3) is an equation representing the reactance of the CR parallel equivalent circuit, and (Equation 4) is An expression representing the resistance value of the CR parallel equivalent circuit,
(Equation 5) is an expression representing the capacitance value of the CR parallel equivalent circuit.

[0037]

(Equation 1)

[0038]

(Equation 2)

[0039]

(Equation 3)

[0040]

(Equation 4)

[0041]

(Equation 5)

Water containing microorganisms is present between the poles 14 and 15 of the thin-film electrode 11, and before the microorganisms move to the gap between the electrodes together with the carrier by dielectrophoresis, the water is dissipated between the electrodes. Capacitance C16 configured as a body
It is considered that the resistor R17 and the water connect the two poles 14 and 15 in parallel. Further, even after the microorganisms move as a conjugate by dielectrophoresis, as will be described later, it can be considered that the connection form of the equivalent circuit does not change even if the absolute values of the capacitance C16 and the resistance R17 change. When an AC voltage is applied to such a CR parallel circuit, a phase difference between the current 21 flowing in the circuit and the applied voltage 20 appears as shown in FIG.

FIG. 5B is a diagram showing the phase difference between the current and the voltage. When the frequency of the voltage to which the phase difference is applied is expressed in polar coordinates on a complex plane using the angle difference θ when the frequency is expressed by the angular frequency ω, there is a relationship shown in FIG. 6 between the voltage, the current, and the phase angle. The impedance Z is obtained by dividing the measured applied voltage and current, and corresponds to the absolute value of the vector shown in FIG. At this time, the impedance Z is Z = r + j
x (j is an imaginary unit), the resistance value r is r = Zsinθ, the resistance component of the combined impedance of the CR parallel circuit shown in FIG. 5A, and the reactance x is x = Zcosθ. It is related to the reciprocal of the capacitance component of the circuit. On the other hand, the combined impedance of the CR equivalent circuit in FIG. 5A is expressed by (Equation 1), and the equation of (Equation 1) is expressed by Z
= R + jx to obtain the resistance r and the reactance x to obtain (Equation 2) and (Equation 3). When (Equation 2) and (Equation 3) are simultaneously deformed, (Equation 4) and (Equation 5) are obtained. (Equation 4) and (Equation 5) show the voltage value for measurement, the current value at that time, r, x, calculated from the measured value of the phase angle of voltage and current.
By substituting ω, the resistance R17 and the capacitance C16 can be known.

The arithmetic unit 5 ends a series of arithmetic operations instantaneously by a microprocessor (not shown). Arithmetic unit 5
Stores the calculated value of the resistor R in the memory as an initial value, and notifies the control means 6 by sending a signal that the measurement of the initial value has been completed. If calculations corresponding to the measured values are performed in advance and stored in a table as a memory, the calculations can be converted into microorganisms simply by referring to the table, instead of performing the calculation each time the measurement is performed. That is, the measurement is performed after the microorganisms are concentrated by dielectrophoresis at a preset time, and the voltage value for the measurement, the current value at that time, and the phase difference between the voltage and the current are measured. If a table on the memory is referred to, the number of microorganisms calculated in advance is stored in the table. According to such a configuration, it is possible to provide a microorganism counting apparatus having a simpler structure and capable of quick measurement without the provision of the arithmetic section 5.

Next, the control means 6 controls the electrophoresis power supply circuit 3 to apply a sine-wave AC voltage (hereinafter, a voltage for electrophoresis) between the electrodes 2 at a frequency of 1 MHz and a peak voltage of 100 V. The frequency of the alternating current applied at this time can be arbitrarily selected as long as the frequency range in which dielectrophoresis occurs,
If the frequency is too low, undesirable electrolysis occurs between the electrodes 2, and if the frequency is too high, the power supply circuit becomes complicated. Therefore, in the present embodiment, the frequency of 1 MHz is selected because dielectrophoresis can be sufficiently caused and the migration power supply circuit 3 can be relatively simple. Although the voltage for electrophoresis is 100 V in the embodiment, when the conductivity of the sample is large, it is appropriate to select a lower voltage so that electrolysis does not occur. Depending on the type of microorganism to be measured, the voltage for measurement and the voltage for electrophoresis do not need to be different voltages and different frequencies, and the same frequency may be used. In other words, the AC frequency to be applied for electrophoresis of any microorganisms most efficiently is not always the same as the AC frequency to be applied to measure them most efficiently, but depending on the type of microorganism, the frequency of electrophoresis and measurement may not be the same. The optimum frequency will be the same. In this case, since the migration and measurement can be performed at a single frequency, the migration power supply circuit 3 can be simplified, and further, the measurement can be performed continuously while performing the migration.
More precise results can be obtained.

Thereafter, at predetermined time intervals, the control means 6, the measuring unit 4, and the calculating unit 5 cooperate to repeat the electrophoresis and measurement, and the calculating unit 5 stores the calculated resistance R in the memory each time. Thus, gap 1 of microorganisms by dielectrophoresis
The time change of the resistance R between the electrodes 2 can be examined by repeating the movement to the vicinity of the electrode 2 and the measurement of the impedance of the electrode 2. After the start of AC voltage application for dielectrophoresis,
When the impedance of the electrode 2 is measured a predetermined number of times in advance, the calculation unit 5 calculates the resistance R between the electrodes 2 up to that point as shown in FIG. The rate of change of the resistance R with time is calculated, and the number of microorganisms in the measurement sample is calculated according to a conversion formula described later.

The reason that the number of microorganisms can be calculated by measuring the rate of change of the resistance R with time will be described. The microorganisms to be measured consist of cell walls and phospholipids, which are ion-rich and have relatively large conductivity, are surrounded by cell membranes on the outside, and are small dielectric particles. It has a high dielectric constant and a low surface conductivity. The combined body after combining the carrier body with the plurality of microorganisms has a high dielectric constant and a high surface conductivity. Therefore, considering that the carrier alone and the binder move at a certain ratio near the gap 12 by dielectrophoresis, as the number of binders increases as compared to the case where only the carrier moves. As a result, the resistance R near the gap 12 decreases. Therefore, if the change in resistance R between the electrodes 2 is measured, the value is the number of conjugates that have moved near the gap 12,
As a result, a measurement result correlated with the number of microorganisms present in the measurement sample can be obtained. FIG. 4 shows an example of such a change in the resistance R with time. And as can be seen from FIG. 4, the rate of change (gradient) of the time change of the resistance R at the initial stage of the measurement.
It can also be seen that, like the temporal change of the absolute value of the resistance R, the resistance R decreases in accordance with the number of microorganisms. When calculating the number of microorganisms by the absolute value of the resistance R, it is more accurate to measure after the transient state, so it takes a long time, but the number of microorganisms is determined by the change rate (gradient) of the resistance R at the initial stage of the measurement. When calculating, the number of microorganisms can be calculated in a relatively short time. Now, in order to associate the change in resistance R with the number of microorganisms in the measurement sample, a conversion formula between the resistance R and the number of microorganisms is required. This conversion formula measures a calibration sample with a clear microbial count in advance using the measurement system of the microbial count measuring apparatus described in the present embodiment, and calculates the variation from the correlation between the microbial count and resistance R at that time. A function that represents a curve obtained by regression analysis is used. When this conversion formula is stored in the memory of the arithmetic unit 5 and a sample whose number of microorganisms is unknown is measured, the number of microorganisms in the measurement sample can be calculated by substituting the rate of change of the resistance R within a predetermined time. In the case of using the conversion table, the calculation result by the conversion formula is stored in advance. Here, as the sample of the embodiment, for example, a single microbial system such as a culture solution of yeast is assumed, but even in a mixed microbial system, the specificity of the binding group modifying the carrier is utilized. By binding only a specific microorganism to a carrier and subjecting the conjugate to dielectrophoresis, only the specific microorganism can be collected and measured.

As described above, after calculating the number of microorganisms,
After a lapse of a predetermined time programmed in advance, the calculation unit 5 sends a notification of the end of the measurement to the control unit 6. In response,
The control means 6 stops energization of the electrode 2 and opens the solenoid valve 8 to start cleaning. The combined body gathered near the gap 12 is supplied to the sample piping 7 by the opening of the solenoid valve 8.
And the series of measurement operations is completed.

In the present embodiment, the case where a single set of thin film electrodes 11 is used has been described, but this does not prevent the use of a plurality of thin film electrodes 11. That is, a plurality of electrodes having the same shape as the electrode 2, that is, having the same impedance under the same conditions, may be provided in the cell 1. In that case, it is possible to obtain a more accurate measurement result by calculating the resistance R after performing the statistical processing such as independently measuring the impedance of each electrode 2 and averaging the values. become. For example, even if a value that is not related to the number of microorganisms is measured due to the influence of undesired impurities or dust among the plurality of electrodes 2, the influence can be reduced by averaging the measured values, Other electrodes at altitude 2
It is also possible to perform processing such as truncation as an abnormal value with reference to the measurement result in the above. It can be said that performing measurement using a plurality of pairs of electrodes 2 having the same shape as described above is rather desirable from the viewpoint of improving accuracy, except that the structure is slightly complicated.

As described above, the microorganism carrier of the present embodiment forms a conjugate with a microorganism by a binding group, and enables measurement of a microorganism having a low dielectric constant and difficult to measure.
Since an alternating voltage for dielectrophoresis and a voltage for measurement are alternately applied, the impedance of the electrode 2 by the measuring unit 4 can be periodically measured while concentrating the microorganisms by dielectrophoresis. Since the time change of the resistance R between the electrodes 2 can be detected by the unit 5, the measurement sensitivity is high in a relatively short time and with a simple structure, and the measurement can be performed automatically. An apparatus can be provided.

[0051]

According to the microorganism carrier of the present invention, simple and highly sensitive measurement can be carried out by dielectrophoresis even for microorganisms having a low dielectric constant.

Further, according to the microorganism counting device of the present invention, simple and highly sensitive measurement of microorganisms having a low dielectric constant can be performed without the need for drugs or special devices, and automatic measurement can be performed. Possible and maintenance-free.

Further, the method for measuring the number of microorganisms of the present invention can perform simple and highly sensitive measurement even for microorganisms having a low dielectric constant, can perform automatic measurement, and can achieve maintenance-free operation.

[Brief description of the drawings]

FIG. 1 is an overall configuration diagram of a microorganism count measuring apparatus according to an embodiment of the present invention.

FIG. 2 is an explanatory diagram of an electrode according to an embodiment of the present invention.

FIG. 3 is a diagram illustrating a cross section of an electrode and a state of an electric field between the electrodes according to the embodiment of the present invention.

FIG. 4 is a graph for explaining the relationship between the number of microorganisms, measurement time, and resistance R between electrodes.

5A is a diagram for explaining a method of calculating a capacitance between electrodes; FIG. 5B is a diagram showing a phase difference between current and voltage;

FIG. 6 is another diagram for explaining a method of calculating capacitance between electrodes;

FIG. 7 is a schematic diagram showing the molecular structure of a microorganism carrier.

[Explanation of symbols]

 Reference Signs List 1 cell 2 electrode 3 migration power supply circuit 4 measurement unit 5 calculation unit 6 control means 7 sample system piping 8 solenoid valve 9 display means 10 substrate 11 thin film electrode 12 gap 13 line of electric force 14, 15 poles 16 capacitance 17 resistance 18 hours Axis 19 Voltage and current magnitude axis 20 Current variation curve 21 Voltage variation curve 22 Core 23 Polymer 24 Linking group

 ──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Yuichi Nakajima 1006 Kadoma Kadoma, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. F term (reference) 2G060 AA16 AD08 AE20 AF07 AG06 AG08 AG10 GA01 HA02 HC10 KA09 4B029 AA07 BB01 CC03 CC08 FA10 FA11

Claims (8)

[Claims] 1. A microorganism carrier comprising a material having a higher dielectric constant than a microorganism to be measured, the surface of which is provided with a binding group capable of specifically binding to the microorganism,
A microorganism carrier body, wherein when a dielectrophoretic force acts, microorganisms bound to the binding group are electrophoresed together. 2. The microorganism carrier body has a representative size of about the same as the representative size of the microorganism or up to several times,
2. The microorganism carrier according to claim 1, wherein the microorganism carrier has a surface conductivity lower than the surface conductivity of the microorganism. 3. The microorganism carrier according to claim 1, wherein the binding group is a compound capable of specifically chemically bonding to a functional group of the microorganism and being modified on the surface of the microorganism carrier. 4. The microorganism carrier according to claim 1, wherein the compound specifically binds to an amino acid residue contained in a microorganism protein. 5. The microorganism according to claim 1, wherein the binding group is an antibody having a paratope that specifically binds to an epitope of a microorganism serving as an antigen, and is modified on the surface of the microorganism carrier. Microorganism carrier body. 6. A cell having a plurality of electrodes therein, into which a microorganism-containing liquid is introduced, and into which the microorganism carrier according to any one of claims 1 to 5 can be introduced. A power supply circuit for applying an AC voltage for generating a dielectrophoretic force between any of the electrodes, and a resistance characteristic between the electrodes for measuring an electric field by any of the electrodes. A measuring unit for measuring, a control unit for controlling the power supply circuit and the measuring unit,
Calculating means for calculating the number of microorganisms by calculating the measurement result of the measuring section. 7. An apparatus according to claim 6, wherein said measuring section measures a current and a phase difference between the electrodes and calculates a resistance by measuring an applied voltage between the electrodes. 8. A microorganism-containing liquid and the microorganism carrier according to claim 1 are introduced into a cell provided with a plurality of electrodes,
An AC voltage is applied between any of the plurality of electrodes to generate a dielectrophoretic force in the cell, and the microorganisms bound to the microorganism carrier are collected in an electric field concentration portion, and the resistance between the electrodes is increased. A method for measuring the number of microorganisms, comprising measuring a change in characteristics to calculate the number of microorganisms.