JP2003000223A - Microorganism measuring device, microbial measuring electrode chip used therefor, and microbial measuring method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microorganism measuring apparatus capable of quantifying a specific microorganism quickly and easily without a specialist in a mixed suspension. I do. SOLUTION: An antibody solution tank 19 for supplying an antibody solution for causing an antigen-antibody reaction to a microorganism concentrated between electrodes 2a and 2b to a measurement chamber 1 is provided. An AC voltage is applied by the unit 3 to concentrate a plurality of types of microorganisms, an antibody solution is supplied from the antibody solution tank 19, specifically reacted with a specific microorganism, and aggregated to separate other microorganisms. The method is characterized in that the number of microorganisms is calculated and output from a change in conductance between the electrodes 2a and 2b measured by the measuring unit 4.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a microorganism measuring apparatus capable of easily and quantitatively detecting a specific microorganism by anyone, an electrode chip used therefor, and a microorganism detecting method.

[0002]

2. Description of the Related Art Recently, microorganisms, especially pathogenic E. coli O-
One of the social problems is the mass infection caused by malignant pathogens such as 157 and the case of bacterial contamination in the food manufacturing process. It is difficult to immediately take the best countermeasures without mistakes due to such mass infections and bacterial contamination cases, and if the countermeasures are delayed, the damage will spread and cause great damage. The biggest reason why the countermeasures are delayed is that the pathogenic bacteria that cause the infection cannot be investigated quickly and with high sensitivity. Therefore,
This is why a new technique for detecting pathogens early and with high sensitivity is desired.

At present, the colony counting method is the most commonly used method for detecting pathogenic bacteria. That is, this method involves spraying a sample of a suspension containing a microorganism on a medium, and quantifying the viable cell count from the number of colonies (communities) formed with the growth of this microorganism. However, this colony counting method requires a surprisingly long time from 1 day to several days until colonies are formed, and although it can be quantified, it can spread the damage in an urgent mass infection or food poisoning. It was something I couldn't stop. In most cases, the test sample liquid contains a plurality of bacteria, and specific bacteria cannot be quantified without auxiliary treatment for selective quantification. In addition, complicated manual work by specialists such as sampling from the test object, concentration and dilution, planting in the medium, and counting the number of colonies (CFU) is required. For these reasons, there is an undeniable possibility that the running cost required for the inspection may increase and that human error may occur.

The term "microorganism" as used herein generally includes all microorganisms such as bacteria, fungi, actinomycetes, rickettsiae, mycoplasmas, viruses, etc., which are the subject of so-called microbiology.

As another method for selective quantification of microorganisms, ATP (Adenosine Tri Phospha) utilizing luminescence is used.
te) There is a law. Since cells such as animals, plants, and bacteria always contain ATP (adenosine triphosphate), ATP contained in bacteria in the test sample is extracted with an ATP extraction reagent, and the extracted ATP is fluorinated. ATP by measuring the amount of luminescence by causing it to emit light by the enzyme (luciferase)
It is to detect the quantity. Since the ATP content in microorganisms has a certain constant value, the number of bacteria contained in the sample is estimated by detecting the ATP amount estimated from the luminescence intensity.

However, the extraction of ATP by an ATP extraction reagent, the action of an enzyme, the measurement of the amount of luminescence, the estimation of the amount of ATP, etc. require a highly sophisticated and complicated work by a specialist, and a very layman can perform a quick measurement. It couldn't be done. Also,
Since all living cells have ATP, and these become background ATP in the measurement, the separation technique may not be perfect and the test becomes uncertain. This method is a method in which bacteria, yeast, fungi, etc. cannot be distinguished as microorganisms, and selective quantitative determination is difficult.

In addition, ELISA (Enzyme-Linked Immun
osorbent Assay) method. The ELISA method is a method for highly sensitively measuring a test target substance by labeling an antibody or an antigen with an enzyme, and is mainly used in clinical tests and the like. The test sample solution is brought into contact with a solid phase such as the surface of plastic, and the test bacteria in the solution are adsorbed through the proteins of the bacteria. After that, the liquid phase is removed to leave only the bacteria adsorbed on the solid phase, and an antibody liquid that specifically reacts only with the protein contained in the target bacteria is added thereto. If the amount of antigen (bacteria) added first and the amount of antibody added later are in the appropriate range, the amount of antibody bound in the antigen-antibody reaction changes depending on the amount of antigen adsorbed on the solid phase. . If the antibody to be used is labeled with an enzyme, a large number of enzymes will be present in proportion to the amount of bound antibody, and the enzymatic reaction will occur strongly. If a chemical substance that develops color by an enzymatic reaction is used, the amount of bound antibody can be determined by measuring the color intensity (absorbance), and thus the amount of the antigen protein (bacteria to be tested) originally present can also be determined.

However, this test method requires complicated experimental procedures, requires a high degree of specialization, and needs to be concentrated to obtain a test object concentration suitable for the measurement of absorbance. The disadvantage is that detection has to take a long time. Further, the ELISA method described above measures the color development by modifying the antibody, and similarly, the method utilizing the antigen-antibody reaction is used, and the agglutination method and the sedimentation method exist as a method of easily confirming with the naked eye. However, this aggregation method,
A high concentration of microorganisms is required to measure by the sedimentation method,
It is necessary to dramatically increase the number of microorganisms by culturing etc. in the previous stage, and it takes a very long time to detect.

Next, PCR (Polymerase Chain Reactio)
The n) method is a technology that uses the polymerase chain reaction to replicate billions of genes in a short time based on the sequence of the specific gene. First, DNA is extracted from an object to be inspected and heated to unravel the double helix structure of the DNA to form a single-stranded state. A gene is composed of four bases, and each base binds to a specific base. This base sequence is called a gene sequence, and a pair of gene fragments (called a primer) is added to this. This primer binds to the target gene sequence with extremely high accuracy, and by adding a thermostable enzyme (Taq enzyme) to this, the original double-helix DNA is extracted from the site where the primer is bound to the single-stranded gene. To play. By repeating this process, the DNA exponentially increases and only the DNA to be inspected is detected by the fluorescence detection device. However, since the DNA of dead bacteria is also amplified, it is necessary to examine the activity of the test subject by taking a culturing procedure in advance. And, a method for simply and quantitatively measuring this microbial activity has not been established. Further, since the DNA explosively increases, it is difficult to estimate the initial number of bacteria, and there is a problem of lack of quantitativeness.

Since the conventional technique has a fatal common drawback that it takes a long time to quantify microorganisms, the present inventor, together with other researchers, combined dielectrophoresis and electrical impedance to determine the number of microorganisms. To measure (DEPI
M method (Dielectrophoretic Impedance Measurement Meth
od)) proposed.

The DEPIM method is characterized in that both the microorganism concentration process and the bacteria concentration detection process are electrically performed. The concentration process is a dielectrophoresis phenomenon (electrical force acts on dielectric particles polarized in an electric field,
The bacteria are concentrated on the integrated microelectrodes by the phenomenon of moving in a certain direction), and the bacteria concentration detection process quantitatively estimates the bacteria concentration from the change in impedance between the electrodes during concentration (Hachinami, Others: "Measurement of Escherichia coli Suspension Concentration by Dielectrophoretic Impedance Measurement (1) -Principle and Device Overview-", Proc.
9); Hamada et al .: "Measurement of Escherichia coli Suspension Concentration by Dielectrophoretic Impedance Measurement (2) -Estimated Model of Suspension Concentration",
Proceedings of the Japan Electrostatic Society '99, pp.341-344 (1999)).

In order to detect microorganisms by the DEPIM method, it is necessary to collect the microorganisms on the microelectrode by the dielectrophoretic force. In other words, under conditions where the dielectrophoretic force does not sufficiently act, the number of bacteria collected on the microelectrodes decreases, so that the detection signal decreases. The dielectrophoretic force F _DEP acting on the microorganism is theoretically given by the following (Equation 1) when expressed as a complex number.

[0013]

[Equation 1] Here, the complex permittivity K is expressed by (Equation 2),

[0014]

[Equation 2] Also, the complex permittivity ε ^* _m of the suspension is expressed by (Equation 3),

[0015]

[Equation 3] ε _m : Permittivity of suspension ε ^* _m : Complex permittivity of suspension ε ^* _i : Complex permittivity of microorganism σ Conductivity of suspension ω: Angular frequency of electric field Also, a: When spherical approximation is performed Radius of the microorganism Re [K]: a parameter E dependent on the complex permittivity of the microorganism and the suspension: electric field strength. As is clear from these (Equation 1) (Equation 2) (Equation 3), if the sizes of the suspension and the microorganism are constant, the dielectrophoretic force F _DEP is proportional to the parameter Re [K]. .

FIG. 8 shows R with the parameter of cytoplasmic conductivity.
It is a frequency characteristic figure of e [K]. In FIG. 8, the dielectrophoretic force is shown as a function of the cytoplasmic conductivity σ _i with the frequency f of the electric field used for dielectrophoresis as a parameter. Figure 8
According to this, it is understood that the positive dielectrophoretic force works at a frequency of 10 kHz to 1 MHz, and the negative dielectrophoretic force works at other frequencies. It can be seen that a negative dielectrophoretic force works depending on the cytoplasmic conductivity σ _i and the frequency f, and even if the dielectrophoretic force F _DEP acts on the microorganism, it may not be collected.

Therefore, when the frequency f is selected according to the type of the microorganism (cytoplasmic conductivity σ _i ), a positive dielectrophoretic force is applied to collect it, or a negative dielectrophoretic force is applied to eliminate it. Is also possible. However, in the actual dielectrophoresis, it is necessary to consider the influence of the conductivity of the suspension.

However, depending on the type of microorganism, the parameter Re [K] will not be positive and that of all other bacterial species will not be negative, and the distribution of dielectrophoretic force between positive and negative cannot be generalized. That is, by utilizing the dielectric properties (dielectric constant, conductivity) peculiar to microorganisms, a positive dielectrophoretic force is selectively applied only to specific microorganisms in a mixed suspension of various microorganisms, It is difficult to detect only microorganisms.

Further, when the DEPIM method is actually applied, it is expected that it is often required to detect only specific microorganisms from a suspension containing various kinds of unknown microorganisms. However, such a selective microorganism detection is impossible by the conventional DEPIM method.

[0020]

As described above,
The conventional colony counting method sprays a suspension containing a microorganism on a medium and quantifies the viable cell count from the number of colonies formed with the growth of this microorganism. It took a surprisingly long time of one to several days. In the case of a sample solution containing a plurality of bacteria, it is impossible to select and quantify specific bacteria without auxiliary treatment. In addition, a complicated manual work by an expert is required, which may cause an increase in running cost required for the inspection and even a human error.

Further, the ATP method includes the extraction of ATP with an ATP extraction reagent, the action of an enzyme, the measurement of luminescence,
Estimating the amount of ATP, etc. requires a high degree of knowledge from a specialist,
The measurement required a long time, and there was a lot of background in the measurement, which made the inspection uncertain. It is a method that cannot distinguish bacteria, yeast, fungi, etc. as microorganisms,
Selective quantification was difficult.

As with other methods, the ELISA method requires complicated experimental procedures, requires a high degree of specialization, and requires concentration to obtain a concentration of a test object suitable for measurement of absorbance. It has to be done and the detection has to take a long time.

Since the PCR method also amplifies the DNA of dead bacteria, it is necessary to examine the activity of the test subject by carrying out a culturing procedure in advance. Further, since the DNA explosively increases, it is difficult to estimate the initial number of bacteria, and there is a problem of lack of quantitativeness. More expertise is required than any other method.

The DEPIM method proposed by the present inventor together with other researchers conducts both the concentration of microorganisms and the detection of bacterial concentration electrically at the same time, so that the microorganisms can be quantified in a short time without specialists. However, it has been impossible to detect by applying dielectrophoretic force only to specific microorganisms in a suspension in which plural kinds of microorganisms are mixed. The DEPIM method can only solve these problems while other methods for quantifying other microorganisms have to be measured by experts for a long time. However, it is possible to identify them in a sample solution containing multiple types of microorganisms. Was not able to be quantified with high sensitivity. Therefore, there is a need for a new microorganism measuring device capable of rapidly and highly sensitively and selectively quantifying a specific microorganism even in a sample solution containing a plurality of types of microorganisms.

Therefore, in the present invention, in a sample solution containing a plurality of types of microorganisms, specific microorganisms can be rapidly detected with high sensitivity.
It is an object of the present invention to provide a microorganism measuring device that can be easily quantified without depending on an expert.

In addition, the present invention provides an inexpensive electrode chip for measuring microorganisms, which is capable of quantifying specific microorganisms in a sample liquid containing a plurality of kinds of microorganisms with high sensitivity, speed, and easily even without requiring an expert. The purpose is to provide.

The present invention is directed to a specific microorganism in a sample solution containing a plurality of types of microorganisms with high sensitivity and speed,
It is an object of the present invention to provide a method for measuring microorganisms that can be easily quantified without depending on an expert.

[0028]

In order to solve the above problems, the microorganism measuring apparatus of the present invention is an antibody for supplying an antibody solution for causing an antigen-antibody reaction to microorganisms concentrated between electrodes into a measuring chamber. Comprising a liquid supply unit, the arithmetic control unit applies an AC voltage by the dielectrophoresis power supply unit to concentrate multiple types of microorganisms, supplies the antibody liquid from the antibody supply unit, and specifically reacts with specific microorganisms. Then, this is aggregated to separate other microorganisms, and the number of microorganisms is calculated and output from the change in conductance between the electrodes measured by the measurement unit.

This makes it possible to quantify a specific microorganism in a sample solution containing a plurality of kinds of microorganisms with high sensitivity and speed, and easily without requiring a specialist.

In the electrode chip for measuring microorganisms of the present invention, an antibody that specifically reacts with a specific microorganism is immobilized on the substrate between the electrodes, and the reacted microorganisms other than the specific microorganism are separated by fluid force. , A specific number of microorganisms is calculated from the change in conductance between the electrodes and output.

Thus, it is possible to provide an inexpensive microbe-measuring electrode chip capable of quantifying a specific microorganism in a sample solution containing a plurality of types of microorganisms with high sensitivity, speed, and easily without requiring any expert. You can

In the method for measuring a microorganism of the present invention, an AC voltage is applied between a pair of electrodes to concentrate a plurality of types of microorganisms by electrophoresis, and the antibody immobilized between the electrodes specifically binds to a specific microorganism. It is characterized by reacting and separating, and then measuring the impedance between the electrodes to calculate the number of microorganisms of only specific microorganisms.

This makes it possible to quantify a specific microorganism in a sample solution containing a plurality of kinds of microorganisms with high sensitivity and speed, and easily without requiring a specialist.

[0034]

BEST MODE FOR CARRYING OUT THE INVENTION The invention according to claim 1 of the present invention comprises a measurement chamber capable of containing a sample solution,
Immersed in the sample solution in the measurement chamber, generating an unequal electric field and concentrating multiple types of microorganisms contained in the sample solution by dielectrophoresis, leaving only specific microorganisms that have reacted specifically with the antibody. Other microorganisms are separated, a pair of electrodes for measuring the impedance at this time, a power source for dielectrophoresis for applying an AC voltage between the electrodes, and a measuring unit capable of measuring the impedance between the electrodes, Concentrated between the electrodes, the calculation control unit that controls the power supply unit for dielectrophoresis and the measurement unit, the memory unit that stores the measurement table that stores the measurement control data and the calibration curve data for each microorganism to be measured. An antibody liquid supply unit that supplies an antibody liquid for causing an antigen-antibody reaction to microorganisms to the measurement chamber, and the arithmetic and control unit applies an AC voltage by the dielectrophoresis power supply unit to generate a plurality of species. Microorganisms are concentrated, the antibody solution is supplied from the antibody supply section, and they react specifically with specific microorganisms to agglomerate them to separate other microorganisms. Since it is a microorganism measurement device characterized by calculating and outputting the number, multiple types of microorganisms contained in the sample liquid are concentrated by dielectrophoresis by applying an AC voltage between the electrodes, so long time is needed for concentration. No need to spend time, this concentrated microorganism is supplied with an antibody solution that reacts specifically with a specific microorganism, and only specific microorganisms aggregate and separate from other microorganisms. Only specific microorganisms of interest can be selected, and mixed suspension is used to measure the number of microorganisms from the change in conductance by impedance measurement of the specific microorganisms after selection. The particular microorganism in quickly with high sensitivity, without relying on the experts can be quantified in a simple manner.

In the invention according to claim 2 of the present invention, the antibody solution supply section comprises an antibody solution tank containing the antibody solution, an antibody solution supply tube for introducing the antibody solution into the measurement chamber, and the antibody solution supply tube. The microbial activity measuring device according to claim 1, wherein the microbial activity measuring device is provided with the provided antibody solution valve, and the antibody solution is supplied to the measurement chamber after concentrating a plurality of types of microorganisms. By doing so, the antibody solution can be automatically supplied to a plurality of types of concentrated microorganisms.

The invention according to claim 3 of the present invention is the microbial activity measuring apparatus according to claim 1 or 2, characterized in that the measuring chamber is provided with a stirring device for stirring the sample solution. Therefore, it is possible to make the microbial concentration uniform and repair the vicinity of the electrode. Furthermore, after the antigen-antibody reaction,
Microorganisms other than the specific microorganisms that have specifically reacted with the antibody can be separated by flow by stirring.

According to a fourth aspect of the present invention, the measurement chamber is provided with a washing pipe for washing liquid, and the washing pipe is provided with a washing valve, and the arithmetic and control unit is based on the timekeeping performed by the clock means. The washing liquid is supplied to the measurement chamber by opening the washing valve, and a specific microorganism is separated and washed away by a fluid force. Therefore, the microorganism activity measuring device according to any one of claims 1 to 3, Microorganisms other than the specifically reacted specific microorganisms can be separated by the fluid force of the cleaning liquid, and the separated unreacted microorganisms can be washed away to enable impedance measurement.

The invention according to claim 5 of the present invention is a measurement chamber capable of containing a sample solution, and the sample solution is immersed in the sample solution in the measurement chamber to generate an unequal electric field and dielectrophoresis the sample solution. In addition to concentrating multiple types of microorganisms contained in, it is separated by leaving only specific microorganisms that have reacted specifically with the antibody, and a microbe measurement electrode chip equipped with a pair of electrodes for measuring the impedance at this time A power source for dielectrophoresis that applies an AC voltage between the electrodes, a measurement unit that can measure the impedance between the electrodes, an operation controller that controls the power source unit for the dielectrophoresis and the measurement unit, and a measurement target. Each of the microorganisms is provided with a memory unit that stores a measurement table that stores measurement control data and calibration curve data.The microorganism measurement electrode chip has a specific microscopic structure on a substrate of a pair of electrodes. The antibody that specifically reacts with is solid-phased, and the arithmetic and control unit applies an AC voltage by the dielectrophoresis power supply unit to concentrate multiple types of microorganisms and separates the solid-phased antibody and the specific microorganisms. Since it is a microorganism measuring device characterized by specifically reacting and separating, and calculating and outputting the number of specific microorganisms from the change in conductance between the electrodes measured by the measuring unit, it is possible to use multiple types of substances contained in the sample liquid. Since it is concentrated by dielectrophoresis by applying an AC voltage between the electrodes, it is not necessary to spend a long time for concentration.
Antibodies that react specifically with specific microorganisms are immobilized, and only specific microorganisms are immobilized on a microbe measuring electrode chip to separate other microorganisms, so only specific microorganisms to be measured are selected from multiple types of microorganisms. By selecting the impedance of a specific microorganism after selection,
Since the number of microorganisms is measured from the change in conductance, specific microorganisms can be quantified in a mixed suspension with high sensitivity and speed, and easily without requiring any expert.

According to the sixth aspect of the present invention, when other microorganisms are separated by leaving only the specific microorganisms that have reacted specifically with the antibody, the aggregate of the reacted specific microorganisms and the other microorganisms are separated. 6. The microorganism measuring device according to any one of claims 1 to 5, wherein an alternating voltage is applied to each of the electrodes to cause a dielectrophoretic force to act on each of them, and the difference is used to perform separation. Therefore, the difference due to the difference in the size of the reacting specific microorganisms and the size of other microorganisms is converted into the difference in the dielectrophoretic force, and the separation can be easily performed by applying the external force such as the fluid force using this difference. Can be done quickly.

The invention according to claim 7 of the present invention is an electrode chip for measuring microorganisms used in the device for measuring microorganisms according to claim 6, wherein a specific microorganism is present on the substrate between the electrodes. An electrode chip for measuring microorganisms, characterized in that the reacting antibody is immobilized and the reacting microorganisms other than the specific microorganisms are separated by fluid force, and the number of the specific microorganisms is calculated and output from the change in conductance between the electrodes. Therefore, it is possible to immobilize the antibody that reacts specifically with a specific microorganism, immobilize only the specific microorganism on the electrode chip for measuring microorganisms, and separate other microorganisms by fluid force. It is possible to inexpensively select only specific microorganisms to be measured from the inside.

According to the eighth aspect of the present invention, an AC voltage is applied between a pair of electrodes to concentrate a plurality of types of microorganisms by electrophoresis, and an antibody solution is supplied to the concentrated microorganisms to produce the antibody. Microorganism measuring method characterized by specifically reacting a liquid with a specific microorganism, separating other unreacted microorganisms, and then measuring the impedance between electrodes to calculate the number of microorganisms of only specific microorganisms Therefore, multiple types of microorganisms contained in the sample solution are concentrated by dielectrophoresis by applying an AC voltage between the electrodes, so it is not necessary to take a long time for concentration, and the microorganisms specific to this concentrated microorganism are Supplying an antibody solution that specifically reacts with and separates other microorganisms by aggregating only specific microorganisms, it is possible to select only specific microorganisms to be measured from among multiple types of microorganisms,
By measuring the impedance of a specific microorganism after selection, the number of microorganisms is measured from the change in conductance.Therefore, a specific microorganism can be quantified in a mixed suspension with high sensitivity and speed, easily and without specialists. can do.

According to a ninth aspect of the present invention, an alternating voltage is applied between a pair of electrodes to concentrate a plurality of types of microorganisms by electrophoresis, and the antibody immobilized between the electrodes is a specific microorganism. It reacts with and separates specifically, and then measures the impedance between the electrodes to calculate the number of microorganisms of only specific microorganisms. It is not necessary to spend a long time for concentration because microorganisms are concentrated by dielectrophoresis by applying an AC voltage between electrodes.Antibodies that react specifically with specific microorganisms are immobilized, and only specific microorganisms are measured. By fixing it to the electrode tip for use and separating other microorganisms, it is possible to select only the specific microorganism to be measured from multiple types of microorganisms, and by measuring the impedance of the specific microorganism after selection. To measure the number of microorganisms from the change in conductance, specific microorganisms in the mixed suspension rapidly with high sensitivity can be determined easily without relying on expert. (Embodiment 1) Hereinafter, a microorganism detecting apparatus and a microorganism detecting method according to Embodiment 1 of the present invention will be described. FIG. 1 is a block diagram of a microorganism measuring apparatus according to Embodiment 1 of the present invention, and FIG. 2A shows an electrode unit of the microorganism measuring apparatus according to Embodiment 1 of the present invention for selectively measuring a specific microorganism. FIG. 2 (b) is a concentration process diagram, FIG. 2 (b) is an antigen-antibody reaction process diagram for selectively measuring a specific microorganism by the electrode unit of the microorganism measuring device according to the first embodiment of the present invention, and FIG. FIG. 3 is a cleaning process diagram for the electrode part of the microorganism measuring device according to Embodiment 1 of the present invention to selectively measure a specific microorganism, and FIG. 3 is a diagram showing a microorganism selectable region of the microorganism measuring device according to Embodiment 1 of the present invention. 4 is a measurement result diagram of a specific microorganism in the microorganism measuring apparatus according to Embodiment 1 of the present invention.

In FIG. 1, 1 is the DEPIM method (Dielec
A measurement chamber containing a sample solution in which a plurality of microorganisms are mixed in a suspension for measuring a specific microorganism by a trophoretic impedance measurement method, 2 is an electrode unit for measuring the number of microorganisms, 2a and 2b are electrodes It is a pair of electrodes provided in the part 2. In the first embodiment, this electrode 2
By the below-described dielectrophoresis performed between a and 2b, a concentration action of microorganisms and a sieving action for separating a specific microorganism aggregated by an antigen-antibody reaction from other microorganisms by a fluid force are performed.

That is, the feature of the first embodiment is that the sample liquid containing a plurality of microorganisms is applied to the electrode 2 by dielectrophoresis.
Concentration is performed as a pretreatment between a and 2b, and an antibody solution that specifically reacts with a specific microorganism is introduced into the microorganism that has been concentrated by this pretreatment. After the specific microorganisms are aggregated by the antigen-antibody reaction, a weak dielectrophoretic force is applied between the electrodes 2a and 2b, and the cleaning liquid is introduced and / or stirred on the electrodes 2a and 2b as described later. The sample liquid is caused to flow, and due to the relationship between the fluid force and the dielectrophoretic force, only other unreacted microorganisms are separated and removed, and finally washed with the washing liquid. After that, the number of specific microorganisms is counted between the electrodes 2a and 2b. All of these treatments are performed on the surface of the electrode part 2. In the first embodiment, the difference in size due to aggregation is used, but since this difference in size is reflected in the fluid resistance, dielectrophoretic force, etc., the separation method is It is also possible to separate them by utilizing the fluid force and the fluid force, or by utilizing the relationship between the fluid resistance difference and other external force or the difference between the dielectrophoretic force and other external force.

Here, the antigen-antibody reaction treatment, which is a premise for separating a specific type of microorganism from the above-mentioned plurality of types of microorganisms, will be described. When an antibody solution that specifically reacts only with a unique protein contained in a specific microorganism is added, an antigen-antibody reaction occurs. By utilizing this property, it becomes possible to selectively detect a specific microorganism (antigen). In the case of the first embodiment, after the entire microorganisms are concentrated by dielectrophoresis, specific microorganisms are aggregated by the antigen-antibody reaction, and unreacted microorganisms are not aggregated. Therefore, the difference in dielectrophoretic force caused by the difference in size is caused. Is used to measure only the specific microorganisms left by the fluid force of the cleaning liquid introduction and stirring. Since the microbial concentration necessary for carrying out the antigen-antibody reaction can be obtained by dielectrophoretic force without culturing as in the conventional case, it can be detected in a short time. Since the number of microorganisms is detected as an electrical impedance signal, quantitative measurement is possible.

The electrodes 2a and 2b have a shape in which an unequal electric field is generated in order to perform dielectrophoresis, and a large number of microorganism chains called pearl chains can be bridged between the electrodes 2a and 2b, for example, a castle wall type electrode. A shape such as a comb-shaped electrode is preferable. The castle wall type electrode is one in which electrodes 2a and 2b formed with a large number of rectangular electrode pieces projecting at intervals of 1 pitch (for example, 50 μm to 100 μm) on both sides are displaced by 1 pitch and are arranged at a distance of 5 μm to 10 μm.
The electric field is concentrated between the edges of the rectangular electrode pieces of the electrodes 2a and 2b. Further, in the comb-teeth type electrode, a pair of electrodes having teeth (for example, 30 μm to 100 μm width) formed like a comb are inserted and combined in a groove in a nested manner and face each other with a narrow gap (for example, 5 μm to 10 μm width). It is an electrode, and the electric field is such that an unequal electric field is formed between edges in the thickness direction and a large number of pearl chains are formed. It is desirable to select the most appropriate value for the gap and pitch depending on the size of the microorganism.

The electrodes 2a and 2b are preferably formed as thin film electrodes of chromium, platinum, or the like, formed on a glass substrate or a plastic substrate by sputtering, vapor deposition, plating, or the like, and etched by photolithography or the like. .
It is desirable that the thin film has a thickness of about 50 nm to 200 nm in order to form many pearl chains. The electrode 2
The material of a and 2b is not limited to chromium or platinum, and may be any metal having a small ionization tendency that does not cause electrolysis when an AC voltage is applied.

As shown in FIG. 1, the measurement chamber 1 may have a structure in which the electrode portion 2 is separately housed and the substrate of the electrode portion 2 is covered. For example, electrode 2
A minute measuring chamber 1 can be made compact by disposing a spacer around a and 2b and placing a glass plate, a plastic plate or the like on this spacer and adhering them.
It is effective when the amount of the sample liquid is small, and it is also suitable to flow the microorganisms in the vicinity of the electrodes 2a and 2b by flowing them as a closed circuit instead of stirring, and to make the microorganism concentration uniform.

Next, 3 is a power source for dielectrophoresis that applies an AC voltage to generate dielectrophoresis between the electrodes 2a and 2b, and 4 is a measuring unit that can measure the impedance between the electrodes 2a and 2b. Reference numeral 5 is a microprocessor or the like, which functions by loading a control program and various data into a work area, controls at least the dielectrophoresis power supply unit 3 and the measurement unit 4, and performs an operation control unit 6
Is a clock means. The dielectrophoresis power supply unit 3 includes electrodes 2a,
The voltage and frequency of the AC voltage applied between 2b are controlled by the arithmetic control unit 5. In the first embodiment, 1
You can change the frequency between kHz and 10MHz,
Peak-to-peak voltage (hereinafter referred to as pp) 1Vpp to 20Vp
A function generator that can apply p is used. In the present specification, the alternating voltage is a sine wave, a triangular wave that changes the direction of flow in a substantially constant cycle,
It means a voltage such as a square wave, and has the same average value of the currents on both the positive and negative sides. The frequency changes depending on the type of microorganism and the conductivity of the suspension, and the voltage is selected such that electrolysis does not occur between the electrodes 2a and 2b and the dielectrophoretic force is increased.

The measuring section 4 is provided with a resistance for current detection of about 500Ω, which is inserted in series in the voltage applying circuit shown in FIG. 1. The current flowing through this is measured to measure the electrodes 2a,
The conductance between 2b (the reciprocal of the resistance component) is calculated. That is, the arithmetic control unit 5 controls the frequency and voltage of the dielectrophoresis power supply unit 3 at the timing of, for example, an interval of 10 sec, which is measured by the clock unit 6, and the electrodes 2a and 2b.
At the same time as applying an AC voltage with an optimal frequency, at the same time, let the measuring unit 4 measure the current flowing through the voltage applying circuit, and measure the resistance component due to the concentration of microorganisms in the sample solution. Is calculated to calculate the conductance. The clock means 6 performs not only the timing of this sampling but also the time management for the scheduling for the automatic operation of the microorganism measuring apparatus according to the first embodiment.

Now, 7 is a memory unit for storing programs to be loaded into the arithmetic control unit 5 and various data, and 7a is provided in the memory unit 7 for controlling measurement for collecting and concentrating specific microorganisms. 3 is a measurement table that stores data and calibration curve data for converting the number of microorganisms from the output of the measurement unit 4 indicating the conductance (hereinafter, DEPIM output). The control data for measurement includes, for each microorganism, the suspension conductivity of the sample solution in which the microorganism is suspended and the optimum frequency at which the positive dielectrophoretic force can be applied to the microorganism. Similarly, as shown in (Table 1), the measurement table 7a is separated by utilizing the type of antibody that causes an antigen-antibody reaction for each microorganism, the voltage V _C1 applied for collection, and the fluid resistance. The voltage V _C2 to be applied at this time, the set time t _c1 for aggregating in the antigen-antibody reaction, and the washing time t _{c2 for} washing are stored.

[0052]

[Table 1] Subsequently, in FIG. 1, 8 is a data input section for inputting a microorganism name, antibody type, etc., 9 is a display for data input, and the calculation control section 5 calculates based on the calibration curve data. A display unit such as an LCD for displaying the number of microorganisms. Further, 10 is a sample solution tank in which a sample solution containing microorganisms is stored, and 11 is a supply pipe which connects the sample solution tank 10 and the measurement chamber 1 and guides the sample solution in the sample solution tank 10 to the measurement chamber 1. Reference numeral 12 is a discharge pipe for discharging the sample liquid after measurement from the measurement chamber 1, 13 is a supply valve such as an electromagnetic valve provided in the supply pipe 11, and 14 is a discharge valve provided in the discharge pipe 12.

Reference numeral 15 denotes a stirrer such as a stirrer, which stirs the sample liquid before and after the arithmetic control unit 5 performs dielectrophoresis based on the timekeeping performed by the clock means 6. Thereby, when the sample liquid is introduced from the supply pipe 11 and subjected to dielectrophoresis, the concentration of microorganisms can be made uniform and many microorganisms can be guided to the vicinity of the electrodes 2a and 2b. Further, if necessary, after the antigen-antibody reaction is completed, the stirring device 15 causes a flow to separate the other microorganisms while leaving the specific microorganisms aggregated in the reaction, and the difference in the dielectrophoretic force with respect to the size is generated. It has the action of washing away unnecessary microorganisms.

Further, the measurement chamber 1 is connected to the electrodes 2a, 2
In the case of a minute measurement chamber 1 in which a spacer is arranged around b, and a glass plate or the like is placed on and adhered to the spacer, the discharge tube 12 is replaced by the sample liquid layer 1 instead of the stirring device 15.
It is suitable to use it in combination with a method in which the microorganisms are guided to the vicinity of the electrodes 2a and 2b by being connected to 0 and flowing as a closed circuit, and at the same time, the microorganism concentration is made uniform.

16 is a cleaning liquid tank, 17 is a cleaning pipe, 18
Is a cleaning valve. The specific microorganisms are aggregated by the antigen-antibody reaction, a flow is generated in the stirring device 15, and unreacted microorganisms are applied by a fluid force (a force of a fluid that is applied against a dielectrophoretic force described later, which is based on a viscous force). Separated by force based on fluid resistance) and washed away. Sample liquid tank 10
It is also preferable to provide a pump for the cleaning liquid tank 16 when a head (water head) cannot be secured or when adjustment is difficult. In the time management of the clock means 6, the arithmetic control unit 5 opens the cleaning valve 18 to supply the cleaning liquid to the measurement chamber 1 when a predetermined time tc2 has elapsed, and cleans between the electrodes 2a and 2b. Although a cleaning liquid is used to separate and wash away unreacted microorganisms as described above, in the process of separating unreacted microorganisms, the stirring action of the stirring device 15 should be used in combination instead of the separation by the cleaning liquid. Alternatively, in some cases, it is possible to separate them by the stirring action of the stirring device 15 alone.

Reference numeral 19 is an antibody solution tank containing an antibody solution containing an antibody that specifically reacts with a specific microorganism, 20 is an antibody introduction tube for introducing the antibody solution into the measurement chamber 1, and 21 is an antibody supply valve. The antibody liquid tank 19, the antibody introduction pipe 20, and the antibody supply valve 21 constitute the antibody supply unit of the first embodiment. In the first embodiment, the sample liquid containing a plurality of microorganisms is applied to the electrodes 2a and 2b by dielectrophoresis.
In the meantime, it is concentrated as a pretreatment before the antigen-antibody reaction, and an antibody solution that specifically reacts only with a specific microorganism is introduced into the microorganism concentrated in this pretreatment. The specific microorganisms are aggregated by the antigen-antibody reaction, the microorganisms that are not aggregated by the stirring device 15 and the washing liquid are separated by the fluid force, and finally washed with the washing liquid, and then the number of the specific microorganisms is counted between the electrodes 2a and 2b. All of these treatments are performed on the surface of the electrode part 2.

Furthermore, the microorganism measuring apparatus of the first embodiment is equipped with a measurement self-checking mechanism so that accurate measurement can be performed without much knowledge. That is,
If the microorganism measuring device is not properly prepared, the measurement will be useless. Therefore, the present microorganism measuring device performs a self-check immediately after the start of measurement. Microorganisms are not collected in the state just after starting the measurement. When the impedance between the electrodes 2a and 2b is measured and the DEPIM signal is detected in this state, an initial value that depends only on the conductivity and the dielectric constant of the suspension is shown. If the arithmetic control unit 5
Immediately after starting the measurement, if the DEPIM signal is detected and it is determined that the initial value indicated by the suspension is significantly different from the normal value range, the electrode part 2 may be damaged and short-circuited or contaminated. Therefore, an alarm is sounded or a warning is displayed on the display unit 9 to prompt confirmation or replacement of the electrode or device. The normal value of the initial value indicated by the suspension may be prepared for each suspension in the measurement table 7a, or may be determined by using the stored calibration curve data. By performing such a self-check, even a non-specialist can easily and surely measure the number of microorganisms.

Subsequently, FIGS. 2 (a) (b) (c), FIG.
Based on FIG. 4, a procedure for selectively measuring only specific microorganisms by the microorganism number measuring device and the microorganism number measuring method according to the first embodiment will be described. 2 (a) (b) (c)
For simplicity, the suspended microorganisms are microorganisms A and B.
There are only two types, and it is assumed that the microbial concentrations are almost equal. As shown in FIG. 2A, in step 1, pre-collection by dielectrophoresis is performed. A voltage V _c1 is applied between the electrodes 2a and 2b installed in the sample solution, and all the microorganisms A and B are collected by the dielectrophoretic force. When the DEPIM signal shows a saturation tendency, the pre-collection is terminated. If there is no significant difference in the size and dielectric properties of these microorganisms A and B,
As can be seen from (Equation 1), almost the same dielectrophoretic force acts on both, and both are collected by the electrode. At this time, DEPI
When the impedance is measured by the M method, the microorganisms A and B
Both collections result in approximately the same impedance change, in which case they cannot be distinguished.

In step 2, only a specific microorganism A is selected from a plurality of types of microorganisms A and B by an antigen-antibody reaction. That is, in a state where the pre-collection of the microorganisms A and B is completed, an antibody solution containing an antibody that specifically reacts with the microorganisms A is added. At this time, due to dielectrophoresis, the microorganisms A are in close contact with each other, and when the antibodies act on the microorganisms in such a state, the antibodies crosslink and only the microorganisms A form aggregates. The reaction takes place.
When the clock means 6 measures the predetermined time t _c1 , it is judged that the agglutination reaction has ended.

Then, in step 3, time t _c1
After the passage of time, the arithmetic control unit 5 reduces the voltage to a voltage V _c2 at which the collection of microorganisms by the dielectrophoretic force does not occur any more. However, since a sufficiently high electric field is maintained between the electrodes 2a and 2b, the microorganisms already collected are retained without being diffused. Then, the microorganisms are selected by the fluid force. As is clear from (Equation 1), the dielectrophoretic force F
_DEP is basically proportional to the square of the electric field (voltage V) and the cube of the size of the microorganism (radius a), and (Equation 4) holds.

[0061]

[Equation 4] However, A is a proportional constant. When performing the dielectrophoresis, if the sample liquid in the measurement chamber 1 is agitated to generate a flow,
Fluid resistance due to viscous force is generated in the microorganisms in the sample solution. Since the flow velocity generated by stirring or the flow of the washing liquid is low, from the relationship with the Reynolds number, when the relative velocity between the microorganism and the sample liquid is constant, the normal viscous force F _V is proportional to the size of the microorganism (radius a) (equation 5). ) Is established.

[0062]

[Equation 5] However, B is a proportional constant. From (Equation 4) and (Equation 5), if the ratio of the dielectrophoretic force F _DEP to the viscous force F _V is defined as the relative dielectrophoretic force F * _DEP , F * _DEP is ( _Equation 6)
It is represented by.

[0063]

[Equation 6] The relationship of (Equation 6) is illustrated in FIG. When a microorganism causes an agglutination reaction than when an antibody solution is added, its apparent radius becomes larger, and the rate of increase with respect to the square of the voltage V ² increases. That is, the relative dielectrophoretic force F * _DEP acting on the microorganism before aggregation changes along the straight line OA in FIG. 3, but after the aggregation changes along the straight line OB.

When there is a flow on the surfaces of the electrodes 2a and 2b, in order to overcome the viscous force F _V and collect and retain the microorganisms on the electrodes by the dielectrophoretic force, the relative dielectrophoretic force F * _DEP must be a certain value or more. Needs to be As a guide, when the dielectrophoretic force is larger than the viscous force, that is, the relative dielectrophoretic force F
* _DEP > 1 can be considered as the condition. The threshold value of this relative dielectrophoretic force F * _DEP is represented by a straight line CD in the figure, and in the region above this straight line (hatched portion in the figure), the dielectrophoretic force is relative to the viscous force F _V received from the flow. Is also winning.

[0065] The relative dielectrophoretic force F * _DEP when set to V _C1 voltage in FIG. 3, aggregated microorganisms (radius a _1),
Since it is above the straight line CD with respect to both of the non-aggregated microorganisms (radius a ₂ ), it is possible to overcome the viscous force and collect the microorganisms. On the other hand, when the voltage is set to V _C2 , the relative dielectrophoretic force F * _DEP is point E for the agglutinated microorganisms and is above the straight line CD, so that the microorganisms can be collected, but they are agglutinated. In the case of no microorganism, the point becomes below the straight line CD, and the viscous force F _V is more dominant, so that the microorganism cannot be collected by the dielectrophoretic force.

Therefore, the pre-collection in step 1 is performed with the voltage V _C1.
Then, both microorganisms A and B are collected by dielectrophoresis. However, when the process proceeds to step 2 and the agglutination reaction is performed, the voltage is reduced to V _{C2 in} step 3 and the sample solution is made to flow, and the microorganisms A that have formed aggregates remain collected on the electrodes 2a and 2b. However, the non-aggregated microorganism B is separated from the electrodes 2a and 2b by the viscous force F _V and is washed away. That is, finally, only the microorganism A to be detected remains in a state of being collected by the electrode, and if the impedance is measured in this state, only the microorganism A can be selectively detected by the DEPIM method.

FIG. 4 shows the measurement results when Escherichia coli was selectively measured from a sample solution in which equal amounts of Escherichia coli and Pseudomonas aeruginosa were mixed according to steps 1 to 3 described above. In FIG. 4, the measurement was performed under the conditions of a suspension conductivity of 0.1 mS / m and a dielectrophoresis frequency of 1 MHz. Since Escherichia coli and Pseudomonas aeruginosa are similar in size and dielectric properties, it is not possible to selectively detect only one of them from the sample solution by the conventional DEPIM method.

In the measurement of FIG. 4, pre-collection by dielectrophoresis is performed at a voltage of 10 V in step 1. The transient increase in conductance observed at this time is a result of the fact that both Escherichia coli and Pseudomonas aeruginosa in the sample solution were trapped in the electrode gap by the dielectrophoretic force as shown in FIG. 2 (a). Next, in step 2, 10 ml of E. coli antibody was added to the sample solution, and E. coli aggregates were added to the electrode 2 as shown in FIG. 2 (b).
It was formed between a and 2b. At this time, the conductance remains constant. After the completion of the agglutination reaction, the voltage is reduced to 1 V in step 3, and the sample solution is stirred as shown in FIG. 2 (c) to introduce the washing solution to separate only the agglutinated Pseudomonas aeruginosa from the electrode by viscous force. Then, the conductance drops sharply by about 50%. The conductance (residual conductance) observed after step 3 corresponds to the concentration of Escherichia coli retained between the electrodes 2a and 2b by aggregation, and the E. coli concentration in the sample solution was quantified by this residual conductance. It will be.

Although the above description is directed to a sample solution in which two kinds of microorganisms are mixed, even in the case of three or more kinds, only a specific microorganism can be selectively detected by the same method. . (Second Embodiment) Hereinafter, a microorganism detecting apparatus and a microorganism detecting method according to a second embodiment of the present invention will be described. FIG. 5 is a configuration diagram of a microorganism measuring device according to the second embodiment of the present invention, and FIG. 6A is an electrode chip diagram before measurement of the microorganism measuring device according to the second embodiment of the present invention.
FIG. 6B is an aggregation process diagram for the electrode part of the microorganism measuring apparatus according to the second embodiment of the present invention to selectively measure a specific microorganism, and FIG. 6C is a microorganism measurement according to the second embodiment of the present invention. FIG. 7 is a cleaning process diagram for the electrode portion of the apparatus to selectively measure specific microorganisms, and FIG. 7 is a measurement result diagram of specific microorganisms in the microorganism measuring apparatus according to the second embodiment of the present invention. The members having the same reference numerals as those of the microorganism measuring device according to the first embodiment indicate the same members also in the microorganism measuring device according to the second embodiment, and the detailed description will be omitted for the microorganism measuring device according to the first embodiment. The description is omitted.

In FIG. 5, reference numeral 1 is a measuring chamber for containing a sample solution in which a plurality of microorganisms are mixed, and 2'is an electrode chip for measuring microorganisms for measuring the number of microorganisms, 2a and 2b.
Is a pair of electrodes provided on the microbe measuring electrode chip 2 ′. In FIGS. 6A, 6B and 6C, 2c is an antibody immobilized between the electrodes 2a and 2b on the microorganism measurement electrode chip 2 '. Here, adsorbing an antibody on the surface of a glass substrate or the like and utilizing it for an antigen-antibody reaction is called immobilization of the antibody. In the second embodiment, a sample solution containing a plurality of microorganisms is concentrated by dielectrophoresis between the electrodes 2a and 2b as a pretreatment for the antigen-antibody reaction, and the microorganism concentrated in this pretreatment is treated as a specific microorganism. The immobilized antibody 2c that reacts specifically is allowed to act. By the antigen-antibody reaction, this specific microorganism is immobilized on the microorganism measuring electrode chip 2'via the antibody 2c. As will be described later, the sample liquid is made to flow by the stirrer 15 to separate unreacted microorganisms, and a washing liquid is introduced to wash away unreacted microorganisms. After that, the number of specific microorganisms is counted between the electrodes 2a and 2b. All of these treatments are performed on the surface of the microbe-measuring electrode tip 2 '.

There are various immobilization methods such as a physical method and a chemical method. In the second embodiment, the antibody 2 is used.
For c, the electrode substrate of the electrode chip 2'for measuring microorganisms is cleaned with acetone, and then the dialyzed antibody solution is dropped on the electrode substrate, kept at 4 ° C in that state, and left to stand for 15 to 18 hours in the solid phase. Turn into. The electrode tip 2'for measuring microorganisms may be a disposable type electrode that is exchanged for each measurement.

3 is a power source for dielectrophoresis, 4 is electrodes 2a, 2
a measuring unit capable of measuring the impedance between b,
Reference numeral 5 is a calculation control unit, and 6 is a clock means. Further, 7 is a memory unit, and 7a is a measurement table which is provided in the memory unit 7 and stores measurement control data for concentrating specific microorganisms and calibration curve data for converting the number of microorganisms. is there. The measurement control data of the second embodiment is as shown in (Table 2), and the measurement table 7a shows in the measurement table 7a the type of antibody that causes an antigen-antibody reaction for each microorganism and the voltage V _C1 applied for collection. , Voltage V _C2 to be applied when separating by fluid force, set time t for aggregation by antigen-antibody reaction
_cl , at least the cleaning time t _{c2 for} cleaning is stored.

[0073]

[Table 2] Reference numeral 8 is a data input unit for inputting a microorganism name and suspension conductivity, 9 is a display unit, 10 is a sample liquid tank, and 11 is a supply pipe for guiding the sample liquid to the measurement chamber 1. Reference numeral 12 denotes a discharge pipe for discharging the sample liquid after measurement from the measurement chamber 1.
3 is a supply valve such as a solenoid valve provided in the supply pipe 11;
Reference numeral 4 is a discharge valve provided in the discharge pipe 12. Reference numeral 15 is a stirrer. 16 is a cleaning liquid tank, 17 is a cleaning pipe,
Reference numeral 18 is a cleaning valve.

Next, based on FIGS. 6 (a), (b), (c) and FIG. 7, a procedure for selectively measuring only specific microorganisms by the microorganism number measuring apparatus and the microorganism number measuring method according to the second embodiment. Will be described. 6A, 6B, and 6C, for simplicity, it is assumed that there are only two kinds of microorganisms suspended, that is, microorganisms A and B, and the microorganism concentrations are almost equal. As shown in FIG. 6A, in step 1,
The antibody 2c is immobilized on the microorganism measurement electrode chip 2 '. Electrode chip 2'for measuring microorganisms used in the DEPIM method
Is a pattern formed by photolithography after forming a thin metal film such as chromium on the surface of the glass substrate, the glass substrate is exposed between the electrodes 2a and 2b, and the antibody 2c is solid-phased in this gap portion. Turn into.

Next, in step 2, the microorganisms are pre-collected by dielectrophoresis and the specific microorganisms are immobilized on the microorganism measuring electrode chip 2'by the immobilized antibody 2c.
That is, the voltage V _c1 is applied to the electrodes installed in the sample solution, and all the microorganisms A and B are first collected by the dielectrophoretic force. When the DEPIM signal shows a saturation tendency, the pre-collection is completed. When there is no significant difference in the size and the dielectric characteristics of the microorganisms A and B, almost the same dielectrophoretic force acts on both of them as expected from (Equation 1), and both are collected by the electrodes 2a and 2b. At this time, if impedance measurement is performed by the DEPIM method, trapping of both microorganisms A and B causes almost the same impedance change, and it is not possible to distinguish them. Part of the dielectrophoresed microorganisms A and B are held in contact with the surface of the glass substrate between the electrodes 2a and 2b. Since the antibody of the microorganism A is immobilized on the glass substrate in step 1, the microorganism A contacting the surface of the glass substrate
Bind to the antibody and are consequently immobilized on the glass surface.

Then, in step 3, the cleaning liquid is sorted by the fluid resistance (viscous force). When the time t _c1 set in advance by the clock means 6 has elapsed, it is judged that the antigen-antibody reaction has ended, and the voltage is lowered to V _c2 to reduce the dielectrophoretic force, and the sample solution is made to flow by stirring or introducing a washing solution. Apply a fluid force (viscous force) to microorganisms. If the viscous force has an appropriate value, the microorganism A immobilized on the glass surface by the antibody 2c immobilized in step 2 is retained between the electrodes 2a and 2b even if the voltage is lowered. On the other hand, the non-immobilized microorganism B is caused by the viscous force of the flowing liquid to cause the electrodes 2a,
After being separated from 2b, it is finally washed away with the cleaning liquid. That is, finally, only the microorganism A to be detected remains in a state of being collected by the electrode. If the impedance is measured in this state, only the microorganism A is selectively detected by the microorganism measuring device according to the second embodiment.

FIG. 7 shows the measurement results when Escherichia coli was selectively measured from the sample solution in which equal amounts of Escherichia coli and Pseudomonas aeruginosa were mixed according to steps 1 to 3 described above. In FIG. 7, the measurement was performed under the conditions of a suspension conductivity of 0.1 mS / m and a dielectrophoresis frequency of 1 MHz. Since Escherichia coli and Pseudomonas aeruginosa are similar in size and dielectric properties, it is not possible to selectively detect either one from the mixed suspension by the conventional DEPIM method.

First, in step 1, the antibody of Escherichia coli is immobilized on the surface of the glass substrate of the electrode by the physical adsorption method. In step 2, pre-collection is performed by dielectrophoresis at a voltage of 10V. The transient increase in conductance observed at this time was caused as a result of both Escherichia coli and Pseudomonas aeruginosa in the suspension being trapped between the electrode gaps by the dielectrophoretic force. In the DEPIM method, the bacterial concentration can be quantitatively estimated from this initial increase rate of conductance, but in the mixed suspension, a signal corresponding to the mixed concentration of both bacteria is obtained. Further, a part of the Escherichia coli collected at this time binds to the antibody 2c immobilized on the surface of the glass substrate.

After the completion of the pre-collection, the voltage was lowered to 1 V in step 3, and as shown in FIG. 6 (c), a flow was caused by stirring or the introduction of a washing solution, and the antibody 2c immobilized by the viscous force was bound. When only Pseudomonas aeruginosa, which was not present, was detached from the electrode, the conductance rapidly decreased by about 50%. The conductance (residual conductance) observed after step 3 corresponds to the concentration of Escherichia coli held between the electrodes 2a and 2b by the immobilized antibody 2c. The E. coli concentration of can be quantified.

Although the above description is directed to sample liquids of two types of microorganisms, even in the case of three or more types, only specific microorganisms can be selectively detected by the same method.

[0081]

The microorganism measuring device according to claim 1 of the present invention concentrates a plurality of types of microorganisms, supplies an antibody solution from the antibody supply unit, and causes a specific reaction with a specific microorganism to agglutinate the same. Other microorganisms are separated and the number of microorganisms is calculated from the change in conductance between the electrodes and output. Therefore, multiple types of microorganisms contained in the sample solution are concentrated by dielectrophoresis by applying an AC voltage between the electrodes. Therefore, it is not necessary to take a long time for concentration, and this concentrated microorganism is supplied with an antibody solution that specifically reacts with a specific microorganism, and only specific microorganisms are aggregated and separated from other microorganisms. Only the specific microorganisms to be measured can be selected from among the microorganisms in the above, and the impedance of the selected specific microorganisms is measured to measure the number of microorganisms from the change in conductance. Quickly with high sensitivity to the particular microorganism in Nigoeki, without relying on the experts it can be quantified in a simple manner.

In the microorganism measuring apparatus according to the second aspect of the present invention, the antibody solution supply section includes an antibody solution tank, an antibody solution supply pipe,
Since the antibody solution valve is provided, the antibody solution can be automatically supplied to a plurality of types of concentrated microorganisms by operating the antibody solution valve.

The microorganism measuring device according to claim 3 of the present invention is a sample for separating a microorganism other than a specific microorganism after the measuring chamber is provided with an agitator and the arithmetic and control unit performs dielectrophoresis. Since the liquid is stirred, the concentration of microorganisms can be made uniform and repair can be performed near the electrodes. Furthermore, after the antigen-antibody reaction, microorganisms other than the specific microorganisms that have specifically reacted with the antibody can be separated by flow by stirring.

According to a fourth aspect of the present invention, in the microorganism measuring apparatus, the arithmetic control unit opens the cleaning valve to supply the cleaning liquid to the measurement chamber and discharge the sample liquid after the chamber is opened based on the timekeeping performed by the clock means. Since the inside is washed, microorganisms other than the specific microorganisms that have reacted specifically with the antibody can be separated by the fluid force of the washing liquid, and the separated unreacted microorganisms can be washed away to enable impedance measurement.

In the microorganism measuring device according to claim 5 of the present invention, the microorganism measuring electrode chip has an antibody that specifically reacts with a specific microorganism immobilized on a substrate of a pair of electrodes,
Multiple types of microorganisms are concentrated by applying an AC voltage, and the immobilized antibody and specific microorganisms are caused to react specifically and separated, and the number of specific microorganisms is calculated and output from the change in conductance between the electrodes. Therefore, multiple types of microorganisms contained in the sample solution are concentrated by dielectrophoresis by applying an AC voltage between the electrodes, so that it is not necessary to take a long time for concentration, and they react specifically with specific microorganisms. Since the antibody is immobilized and only specific microorganisms are immobilized on the electrode tip for measuring microorganisms and other microorganisms are separated, it is possible to select only specific microorganisms to be measured from multiple types of microorganisms. By measuring the impedance of a specific microorganism, the number of microorganisms is measured from the change in conductance. Therefore, it is possible to detect a specific microorganism in a mixed suspension with high sensitivity and speed, easily and without the need of an expert. It is possible to amount.

According to the sixth aspect of the present invention, the microorganism measuring apparatus applies an alternating voltage to a group of specific microorganisms that have reacted and another microorganism to cause a dielectrophoretic force to act on each of the microorganisms, and Since separation is performed using the difference, the difference due to the difference in the size of the reacting specific microorganisms and the size of other microorganisms is converted into the difference in the electrophoretic force, and this difference is used to apply the external force such as fluid force. Separation can be performed easily and quickly by adding.

According to the seventh aspect of the present invention, there is provided a microbe-measuring electrode chip, in which an antibody which specifically reacts with a particular microbe is immobilized on a substrate between electrodes. It is possible to immobilize the reacting antibody, immobilize only specific microorganisms on the electrode tip for measuring microorganisms, and wash other microorganisms.It is possible to select only specific microorganisms to be measured from multiple types of microorganisms. It can be realized at low cost.

The method for measuring a microorganism according to claim 8 of the present invention is a method of concentrating a plurality of types of microorganisms by electrophoresis, supplying an antibody solution to the concentrated microorganisms, and specifically targeting the antibody solution and a specific microorganism. After reacting, other unreacted microorganisms are separated, and then the impedance between the electrodes is measured to calculate the number of microorganisms of only specific microorganisms. It is not necessary to take a long time for concentration because it is applied to
An antibody solution that reacts specifically with a specific microorganism is supplied to this concentrated microorganism, and only the specific microorganism aggregates to separate the other microorganisms. By selecting the impedance of a specific microorganism after selection, the number of microorganisms is measured from the change in conductance, so that a specific microorganism in a mixed suspension can be sensitively and quickly provided to an expert. It can be easily quantified without having to read it.

The method for measuring microorganisms according to claim 9 of the present invention is a method in which a plurality of types of microorganisms are concentrated by electrophoresis, and the antibody immobilized between the electrodes specifically reacts with specific microorganisms to separate them. After that, since the impedance between the electrodes is measured to calculate the number of microorganisms of only specific microorganisms, multiple types of microorganisms contained in the sample solution are concentrated by dielectrophoresis by applying an alternating voltage between the electrodes. Therefore, it is not necessary to take a long time, and the antibody that specifically reacts with a specific microorganism is immobilized on the solid phase, and only the specific microorganism is immobilized on the electrode tip for measuring microorganisms to separate other microorganisms. You can select only specific microorganisms to be measured from the
By measuring the impedance of a specific microorganism after selection, the number of microorganisms is measured from the change in conductance.Therefore, a specific microorganism can be quantified in a mixed suspension with high sensitivity and speed, easily and without specialists. can do.

[Brief description of drawings]

FIG. 1 is a configuration diagram of a microorganism measuring device according to a first embodiment of the present invention.

FIG. 2 (a) is a concentration process diagram for selectively measuring a specific microorganism by the electrode part of the microorganism measuring apparatus according to the first embodiment of the present invention. (B) Microorganism measuring apparatus according to the first embodiment of the present invention (C) is an antigen-antibody reaction process diagram for selectively measuring a specific microorganism by the electrode section of FIG. 1 shows the electrode section of the microorganism measuring apparatus according to the first embodiment of the present invention for selectively measuring a specific microorganism. Cleaning process diagram

FIG. 3 is a diagram showing a microorganism selectable area of the microbiological analyzer according to the first embodiment of the present invention.

FIG. 4 is a measurement result diagram of a specific microorganism by the microorganism measuring apparatus according to the first embodiment of the present invention.

FIG. 5 is a configuration diagram of a microorganism measuring device according to a second embodiment of the present invention.

FIG. 6 (a) is an electrode chip diagram of the microorganism measuring apparatus according to the second embodiment of the present invention when it is not processed. (B) The electrode portion of the microorganism measuring apparatus according to the second embodiment of the present invention selectively selects a specific microorganism. (C) Cleaning process diagram for selectively measuring specific microorganisms by the electrode unit of the microorganism measuring device according to the second embodiment of the present invention

FIG. 7 is a measurement result diagram of a specific microorganism by the microorganism measuring apparatus according to the second embodiment of the present invention.

FIG. 8 is a frequency characteristic diagram of Re [K] with a parameter of cytoplasmic conductivity.

[Explanation of symbols]

1 measurement chamber 2 electrodes 2'Electrode tip for measuring microorganisms 2a, 2b electrodes 2c antibody 3 Power source for dielectrophoresis 4 measuring section 5 Operation control section 6 clock means 7 Memory section 7a Measuring table 8 Data input section 9 Display 10 sample tank 11 supply pipe 12 discharge pipe 13 Supply valve 14 Discharge valve 15 Stirrer 16 Cleaning solution tank 17 Washing pipe 18 Wash valve 19 Antibody solution tank 20 Antibody introduction tube 21 Antibody supply valve

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 27/447 G01N 33/02 33/02 33/48 M 33/48 33/483 E 33/483 33/53 Y 33 / 53 33/543 521 33/543 521 33/569 B 33/569 27/26 301Z 331Z F term (reference) 2G045 AA28 BB14 CB21 CB30 FA04 FA34 FB03 FB05 FB15 GC22 JA01 JA07 2G060 AA16 AD08 AE40 AF03 AF06 AF08 AF20 AG11 GA01 HA02 HC13 HC19 HC22 HD01 HD02 KA09 4B029 AA07 BB01 CC01 FA10 4B063 QA01 QQ05 QS16 QS33 QX05

Claims (9)

[Claims] 1. A measurement chamber capable of containing a sample solution, and a plurality of types of microorganisms contained in the sample solution which are immersed in the sample solution in the measurement chamber and generate an unequal electric field to cause dielectrophoresis. While concentrating, other microorganisms are separated leaving only specific microorganisms that have reacted specifically with the antibody, and a pair of electrodes for measuring the impedance at this time, and dielectrophoresis for applying an AC voltage between the electrodes. Power supply unit, a measurement unit capable of measuring the impedance between the electrodes, an operation control unit for controlling the dielectrophoresis power supply unit and the measurement unit, measurement control data and calibration for each microorganism to be measured. A memory unit that stores a measurement table that stores line data, and an antibody solution for causing an antigen-antibody reaction to the microorganisms concentrated between the electrodes are supplied to the measurement chamber. And an antibody solution supply section for supplying the antibody solution, wherein the arithmetic control section applies an AC voltage by the dielectrophoresis power supply section to concentrate a plurality of types of microorganisms, and supplies the antibody solution from the antibody supply section. The microorganism measuring device characterized in that it specifically reacts with the above microorganisms to aggregate them to separate other microorganisms, and calculates and outputs the number of microorganisms from the change in conductance between the electrodes measured by the measuring unit. . 2. The antibody solution supply section comprises an antibody solution tank containing the antibody solution, an antibody solution supply pipe for introducing the antibody solution into the measurement chamber, and an antibody solution valve provided in the antibody supply pipe. The microorganism measuring apparatus according to claim 1, wherein the antibody solution is supplied to the measurement chamber after concentrating a plurality of types of microorganisms. 3. The microorganism measuring device according to claim 1, wherein the measuring chamber is provided with a stirring device to stir the sample liquid. 4. A washing pipe for washing liquid is provided in the measurement chamber, and a washing valve is provided in the washing pipe, and the arithmetic control unit is based on timekeeping performed by clock means.
The microorganism measuring device according to any one of claims 1 to 3, wherein the cleaning valve is opened to supply a cleaning liquid to the measurement chamber, and a specific microorganism is separated and washed away by a fluid force. 5. A measurement chamber capable of containing a sample solution, and a plurality of types of microorganisms contained in the sample solution immersed in the sample solution in the measurement chamber to generate an unequal electric field and dielectrophoresis. While concentrating, it is separated leaving only specific microorganisms that have reacted specifically with the antibody, and an electrode tip for microorganisms provided with a pair of electrodes for measuring the impedance at this time, and an alternating voltage between the electrodes. A dielectrophoresis power supply unit to be applied, a measurement unit capable of measuring the impedance between the electrodes, an operation control unit for controlling the dielectrophoresis power supply unit and the measurement unit, and a measurement for each microorganism to be measured. A memory unit in which a measurement table storing control data and calibration curve data is stored, and the microorganism measurement electrode chip has a specific microscopic structure on a substrate of the pair of electrodes. An antibody that specifically reacts with a substance is immobilized, and the arithmetic control unit applies an AC voltage by the dielectrophoresis power source unit to concentrate a plurality of types of microorganisms, and the immobilized antibody and the identification The microorganism measuring apparatus characterized by specifically reacting and separating the microorganism, and calculating and outputting the specific number of microorganisms from the change in conductance between the electrodes measured by the measuring unit. 6. When other microorganisms are separated while leaving only the specific microorganisms that have specifically reacted with the antibody, an alternating voltage is applied to the aggregates of the reacted specific microorganisms and the other microorganisms, respectively. The microorganism measuring device according to any one of claims 1 to 5, wherein a dielectrophoretic force is applied to perform separation by utilizing a difference in the dielectrophoretic force. 7. An electrode chip for microbe measurement used in the microbe measurement apparatus according to claim 6, wherein an antibody that specifically reacts with a particular microbe is immobilized on a substrate between the electrodes, A microorganism measuring electrode chip, wherein microorganisms other than the reacted specific microorganisms are separated by a fluid force larger than dielectrophoretic force, and the specific microorganism number is calculated and output from the change in conductance between the electrodes. 8. An AC voltage is applied between a pair of electrodes to concentrate a plurality of types of microorganisms by electrophoresis, an antibody solution is supplied to the concentrated microorganisms, and the antibody solution specifically reacts with the specific microorganisms. A method for measuring microorganisms, characterized in that the other unreacted microorganisms are separated and then the impedance between the electrodes is measured to calculate the number of microorganisms of only specific microorganisms. 9. An AC voltage is applied between a pair of electrodes to concentrate a plurality of types of microorganisms by electrophoresis, and the antibody immobilized between the electrodes specifically reacts with a specific microorganism to separate it. ,
Then, the impedance between the electrodes is measured to calculate the number of microorganisms of only specific microorganisms.