JPWO2010013757A1 - Somatic cell number measuring device and sensor - Google Patents

Abstract

The somatic cell number measuring apparatus is composed of a sensor and an apparatus main body. The sensor has an examination room (20) for holding raw milk, a working electrode (31) and a counter electrode (33). The working electrode (31) and the counter electrode (33) are provided with a first electrode portion (31a) and a second electrode portion (33a) that face the internal space of the examination room (20), respectively. An enzyme reagent (4) that reacts with active oxygen contained in raw milk is immobilized on the first electrode part (31a). The apparatus body calculates the number of somatic cells contained in the raw milk based on the current that flows when a voltage is applied between the working electrode (31) and the counter electrode (33).

Description

  The present invention relates to an apparatus for measuring the number of somatic cells in raw milk. Moreover, this invention relates to the sensor used for the said apparatus.

  A disease caused by highly pursuing livestock productivity, such as breeding selection for raising the capacity of livestock, breeding with multiple heads, or intensive management, is called production disease. In addition to metabolic disorders, production disorders include reproductive disorders, lactation disorders, and motor organ disorders, as well as gastrointestinal and respiratory diseases in calves and piglets that have the aspect of opportunistic infections. Enter the category. Productive diseases are relatively unfamiliar to general consumers, unlike infectious diseases that are urgent and dangerous in terms of national prevention, such as foot-and-mouth disease, BSE, swine fever, and highly pathogenic influenza in chickens. It is. However, in the livestock field, it is one of the most important illnesses that occur frequently on a daily basis and are putting pressure on the management of producers.

  The main production diseases in question are as follows by breed or animal type. In dairy cows, metabolic disorders (lumen acidosis, liver dysfunction such as fatty liver and ketosis), reproductive disorders (follicular growth disorders, ovarian cysts), motor organ disorders, lactation disorders (clinical mastitis, latent mastitis, chronic breasts) Flame). In beef cattle, there are metabolic disorders (lumen acidosis, liver dysfunction, vitamin deficiencies), chronic respiratory diseases and digestive diseases originating from the barn environment. In pigs and chickens, there are chronic respiratory diseases derived from the barn environment, nutritional disorders due to heat and cold environment, respiratory infections, and the like.

  A latent productive disease is a productive disease in which an abnormality cannot be detected with the naked eye and the progress of the disease state is detected for the first time by examination of body fluid components such as blood and milk. Due to recent advances in nutrition management technology and livestock management in fields related to livestock science, and production veterinary care that tackles these, production diseases with clinical symptoms are decreasing. However, it is not possible to solve the potential production disease, but rather it is in a situation where it is spreading widely and deeply. Latent production disease is often overlooked because it is not accompanied by clinical symptoms. It is said that latent productive diseases have a negative impact on reproductive and immune functions, and the economic damage is several times that of clinical productive diseases.

  One of the latent productive diseases is latent mastitis. Mastitis is the biggest problem among dairy cattle catastrophes. Mastitis is one of the most incurable diseases in the world because it is easy to get ill and difficult to cure. The number of somatic cells in raw milk of dairy cows affected by mastitis is about 300,000 / mL compared with about 300,000 / mL or less for normal dairy cows, including 3 million / mL. There are many cases of chronic mastitis. If it is discovered by the Dairy Inspection Association, etc. that the number of somatic cells in the sampled raw milk exceeds the specified value, the raw milk will be discarded in tank units, and the annual damage amount will be 80 billion yen for Japan as a whole. It is estimated to be ~ 100 billion yen.

  70% to 80% of mastitis damage is said to be due to so-called latent mastitis in which the number of somatic cells in raw milk increases without showing clinical symptoms such as fever, redness, swelling, and pain. The economic damage from latent mastitis is enormous. In particular, the latent mastitis caused by Staphylococcus aureus tends to follow the course of chronicity by forming microabscesses and granulomas in the breast tissue.

  Conventional mastitis diagnosis methods include the somatic cell count method, the CMT (California Mastitis Test) method, and the electrical conductivity method. The somatic cell count method is effective for diagnosis from bacterial invasion to moderate mastitis, and the CMT method and electrical conductivity method are effective for diagnosis from mild mastitis to severe mastitis.

  The somatic cell count method directly measures the number of cells in milk, such as phagocytic cells such as neutrophils and eosinophils, mammary epithelial cells, lymphocytes, monocytes, and plasma cells. It can detect mastitis up to moderate. However, the somatic cell counting method using an automatic cell counting instrument has a problem that it is extremely expensive. In addition, there is a problem in that the measurement of smearing with an optical microscope takes too much time. In particular, colostrum is difficult to smear and fix milk, and when mastitis increases, many leukocytes are adsorbed on bacteria to form a dumpling, which makes measurement impossible.

  The CMT method is a method for estimating the number of somatic cells by utilizing the fact that the degree of aggregation reaction varies depending on the number of somatic cells when a surfactant is added to raw milk. Since BTB reagent is also included for pH measurement, salt such as sodium chloride and potassium chloride in raw milk increases due to increased vascular permeability during mastitis, increased fight between leukocytes and bacteria, etc., and approaches alkalinity Is used to change the color tone from yellow to green to blue to be a mastitis determination index. It can be easily measured by anyone, is characterized by whether or not it is mastitis, and is very cheap. However, the CMT method is difficult to diagnose until after a reaction has occurred, such as after the battle between white blood cells and bacteria, or after increased vascular permeability, and the determination is subject to human subjective judgment. However, there was a problem that it was only a guideline. In particular, when the number of somatic cells in raw milk is 300,000 cells / mL or less, determination is difficult, and there is a problem that is not suitable for diagnosing mastitis at an early stage.

  In the case of mastitis milk, the electrical conductivity method makes it easier for electricity to pass than normal milk because of increased vascular permeability and increased electrolyte components such as sodium and chlorine in the plasma due to the inflammatory reaction of the mammary gland tissue. Is to be used. There is an advantage that a quantitative value can be obtained by quantifying the electrical conductivity. However, the electrical conductivity method increases the number of somatic cells in raw milk and sees changes due to inflammatory reactions after the invasion of bacteria and the fight against white blood cells, so it is necessary to diagnose mastitis at an early stage There was an unsuitable problem. Further, even in normal milk, there are considerable differences in electrolyte components and concentrations depending on each quarter or each cow, so there is a problem that reproducibility is not good and the mastitis determination rate is low.

  As a method to improve these problems, phagocytic leukocytes such as neutrophils, which occupy the body position in somatic cells in raw milk, pay attention to the excellent initial reaction (agility) at the time of infection. It has been proposed to measure phagocytic leukocyte function by luminol chemiluminescence (see Patent Document 1).

  To explain the luminol chemiluminescence method, when the breast begins to invade the bacteria, neutrophils capable of phagocytosis gather in the raw milk in an attempt to stop it. Neutrophils are highly mobile after bacterial invasion and work to stop bacterial invasion by overwhelming numbers. Neutrophils release active oxygen to kill bacteria. The luminol chemiluminescence method is a method in which active oxygen released from neutrophils is excited by a luminol reagent, and the amount of chemiluminescence is detected and amplified and quantified. This is a technique that can detect the number of neutrophils that gather in conjunction with bacterial invasion with high sensitivity over a wide range from the initial stage of bacterial invasion to early mastitis.

  However, the luminol chemiluminescence diagnostic device for mastitis is expensive and requires time to obtain a certain amount of light, so it is excellent for research tests or somatic cell count verification. There was a problem with the speed required in the field. A photomultiplier is used to cause active oxygen released from neutrophils to emit light with a luminol chemiluminescence reagent and detect the weak light quantity. A photomultiplier tube is a high-sensitivity photodetector with a current amplification function based on a photoelectric tube that converts light energy into electrical energy using the photoelectric effect. It can detect faint light but at a high cost. Had problems. Since the size of the photomultiplier tube is large, the apparatus becomes large, and there is a problem in portability when used on the site of a dairy farmer.

  In addition, as described in Patent Document 1, in order to obtain a luminescence peak by a luminol chemiluminescence reagent in a short time, it has been attempted to add zymosan as a neutrophil stimulant to a raw milk sample. However, since the time for obtaining a quantitative emission peak is 5 to 15 minutes, there is a problem in rapidity for use on the site of a dairy farmer.

  As a method for improving this problem, it has been proposed to detect the presence of active oxygen released from neutrophils as an electric current. This proposal focuses on the fact that phagocytic leukocytes such as neutrophils, which occupy the position among somatic cells in raw milk, are excellent in initial reaction (agility) at the time of infection, as in the case of luminol chemiluminescence. We are trying to quantify the amount of active oxygen released from neutrophils. For example, Patent Document 2 discloses a mastitis diagnostic apparatus including a needle-like active oxygen sensor in which a working electrode is formed on the inner peripheral surface of a cylindrical insulating member and a counter electrode is formed on the outer peripheral surface. This mastitis diagnostic apparatus measures the concentration of active oxygen with an active oxygen sensor and determines whether or not the patient has mastitis based on the concentration. The working electrode and the counter electrode are obtained by forming a polymer film of a metal porphyrin complex on the surface of a conductive member, and the principle of measuring active oxygen is as follows.

When the active oxygen sensor is immersed in raw milk in the presence of active oxygen (eg, superoxide anion: O ₂ ⁻ ), the metal in the metal porphyrin complex is reduced by active oxygen (eg, Fe ³⁺ → Fe ²⁺ ). . Therefore, when a certain voltage is applied between the working electrode and the counter electrode, the reduced metal is reoxidized (for example, Fe ²⁺ → Fe ³⁺ ), and a current flows. Since the current value at this time corresponds to the concentration of active oxygen, the concentration of active oxygen can be detected from the current value.

  In the luminol chemiluminescence method, it is necessary to add zymosan to the raw milk sample as a neutrophil stimulant in order to obtain a luminescence peak in a short time by the luminol chemiluminescence reagent, which is a heavy burden for dairy farmers in the field It was predicted. In a mastitis diagnostic apparatus using an active oxygen sensor, it has been expected that the amount of active oxygen can be measured without adding a neutrophil stimulant to a raw milk sample.

  However, since the mastitis diagnostic apparatus using the active oxygen sensor uses reduction and oxidation of the metal in the metal porphyrin complex, it has been difficult to increase the detection sensitivity of active oxygen.

  In the production of raw milk, the management target value of the number of somatic cells is set to 300 to 500,000 pieces / mL or less. The ratio of neutrophils to the number of somatic cells in raw milk infected with mastitis is reported to be 80% to 90%. When the number of somatic cells is 300 to 500,000 / mL, the number of neutrophils Is estimated to be 27-450,000 / mL. The number of neutrophils in such raw milk is about 1/10 of the number of neutrophils in the blood of healthy cattle, and a low neutrophil count is necessary for early detection of mastitis infection. It is desirable to be able to measure with good reproducibility.

  However, in the case of an active oxygen sensor using a polymer film of a metal porphyrin complex as an electrode, in the initial stage of mastitis where the neutrophil count does not increase so much, the current value obtained by the reaction from the polymer film surface of the metal porphyrin complex The increase was very small and it was difficult to diagnose mastitis at an early stage.

Japanese Patent Laid-Open No. 2000-041696 JP 2005-106490 A

  The conventional somatic cell count method using an optical microscope, which is a diagnostic method for mastitis, has a problem that it takes too much time.

  The CMT method is difficult to diagnose unless the symptoms of dairy cows infected with mastitis are increased, and the judgment is left to the subjective judgment of the person. There was a problem.

  The conductivity method is suitable for diagnosing mastitis at an early stage because the number of somatic cells in raw milk has increased and bacteria have invaded and have seen changes due to the inflammatory response after the fight against leukocytes. There were no problems. Further, even in normal milk, there are considerable differences in electrolyte components and concentrations depending on each quarter or each cow, so there is a problem that reproducibility is not good and the mastitis determination rate is low.

  Luminol chemiluminescence diagnostic device for mastitis is expensive and requires time to obtain a certain amount of light, so it is excellent for research tests or somatic cell count verification. The required speed was a challenge. Moreover, since a photomultiplier tube is used, the apparatus becomes relatively large, and there is a problem in portability when used on the site of a dairy farmer.

  In a mastitis diagnostic apparatus equipped with an active oxygen sensor using a polymer film of a metal porphyrin complex as an electrode, the amount of change in the current value obtained by the reaction from the polymer porphyrin complex surface is very small. It was difficult to diagnose at an early stage.

  The present invention has been made in view of such circumstances, and enables diagnosing mastitis at an early stage in order to prevent the disposal of raw milk in tank units at the production site of raw milk by dairy farmers. An object of the present invention is to provide a somatic cell number device and a sensor used in the somatic cell number measuring device.

  In order to achieve the above object, the present invention has an examination room for holding raw milk, a first electrode part facing the internal space of the examination room, and is included in the raw milk on the first electrode part A voltage is applied between the working electrode and the counter electrode, including a working electrode on which an enzyme reagent that reacts with active oxygen is immobilized, and a counter electrode having a second electrode portion facing the internal space of the examination room. And a device main body for calculating the number of somatic cells contained in the raw milk based on the current flowing when the somatic milk flows.

  The present invention further includes an examination room for holding raw milk and a first electrode part facing the internal space of the examination room, and reacts with active oxygen contained in the raw milk on the first electrode part. A working electrode to which an enzyme reagent is immobilized; a counter electrode having a second electrode portion facing the internal space of the examination room; a reference electrode having a tip facing the internal space of the examination room; the working electrode; the counter electrode; There is provided a sensor comprising: a base plate that supports the reference electrode; and a cover plate that is fixed to the base plate across the working electrode, the counter electrode, and the reference electrode, and forms the examination room together with the base plate.

  According to the present invention, mastitis can be diagnosed at an early stage in a production site of raw milk by a dairy farmer. Thereby, discarding of raw milk in tank units can be effectively prevented.

It is a top view which shows the somatic cell number measuring apparatus which concerns on 1st Embodiment of this invention. It is a perspective view which shows the sensor which comprises the somatic cell number measuring apparatus shown in FIG. It is the III-III sectional view taken on the line of FIG. It is a top view which shows the sensor which abbreviate | omitted the cover plate. It is a perspective view which shows the protrusion formed in the surface of the 1st electrode part. It is a top view which shows the modification of arrangement | positioning of a processus | protrusion. It is an expanded sectional view of a protrusion. It is a perspective view which shows the modification of a sensor. FIG. 9A to FIG. 9C are schematic views showing modifications of the working electrode, reference electrode, and counter electrode patterns. It is a block diagram of the somatic cell number measuring apparatus shown in FIG. It is a top view which shows the somatic cell number measuring apparatus which concerns on 2nd Embodiment of this invention. It is a top view which shows the somatic cell number measuring apparatus which concerns on 3rd Embodiment of this invention. It is a graph (scatter diagram) showing the relationship between the number of somatic cells and the current value.

  The somatic cell number measuring apparatus of the present invention can be reduced in size and cost, and can easily and quickly measure the number of somatic cells with high accuracy. This somatic cell number measuring apparatus is composed of an active oxygen measuring sensor and a main body of the apparatus, and can be mounted on a milker of an automatic milking machine or portable. This feature makes it possible to use dairy farmers at milking sites or for batch measurement by a certifier, etc., so that raw milk with an abnormal somatic cell count can be detected at an early stage, and the disposal of raw milk in tank units can be prevented. In either case of the automatic milking machine mounting type or the portable type, it is preferable that the active oxygen measurement sensor is detachable from the apparatus main body and the active oxygen measurement sensor is made disposable.

  One of the features of the present invention is that attention is paid to active oxygen released from neutrophils in raw milk, and this active oxygen is detected as an electric current by an enzyme. Healthy cows and dairy cows infected with mastitis differ greatly in the components of somatic cells contained in raw milk. In raw milk, mammary epithelial cells, neutrophils, phagocytic lymphocytes such as eosinophils, monocytes, plasma cells and the like are present. When infected with mastitis, neutrophils with phagocytic sterilization ability gather in raw milk in an attempt to stop it, and the ratio of neutrophils increases dramatically.

  In another aspect of the invention, the working electrode and the reference electrode are used to selectively detect active oxygen released from neutrophils to kill bacteria by an enzyme placed on the working electrode, The number of somatic cells in raw milk can be measured without being affected by other components such as potassium. Active oxygen includes superoxide anion radical, hydroxyl radical, hydrogen peroxide, singlet oxygen, nitric oxide, nitrogen dioxide, ozone, lipid peroxide, and the like.

  Another feature of the present invention is that the active oxygen measurement sensor is of a plate type. The plate-type active oxygen measurement sensor includes a laboratory for holding raw milk, a working electrode having a first electrode portion on which an enzyme reagent is immobilized, a counter electrode having a second electrode portion separated from the first electrode portion, and the like. Is provided. By adopting the plate type, it is possible to optimize the layout for increasing the redox current value due to the reaction between active oxygen and the enzyme. For example, the area of the first electrode portion on which the enzyme is disposed can be extremely easily enlarged to obtain the maximum sensitivity. Also in the layout design of the working electrode, the counter electrode, and the insulating material (plate), for example, the design for obtaining the maximum sensitivity is possible such that the second electrode portion surrounds the first electrode portion.

  As a method of immobilizing the enzyme reagent on the first electrode part of the working electrode, the enzyme reagent may be applied as it is on the first electrode part and dried. However, the enzyme reagent may be immobilized by an organic layer having electron transfer properties. preferable. In this way, the enzyme reagent can be firmly held on the first electrode part, and the sensitivity can be stabilized. It also has the effect of suppressing enzyme activity deterioration by maintaining the three-dimensional structure of the enzyme.

  If the organic layer is too thin, it is difficult to maintain the structure of the enzyme. If the organic layer is too thick, the electron propagation property is reduced. Therefore, the thickness of the organic layer is preferably 0.1 nm to 10 μm, and more preferably 0.5 nm to 1 μm.

  Examples of the method for immobilizing an enzyme reagent using an organic layer include a method in which an organic film is formed on the first electrode part, and the enzyme reagent is applied or covalently immobilized thereon, or an organic polymer gel containing the enzyme reagent. And a method of coating the first electrode part on the first electrode part. In particular, it is preferable to use a self-assembled monolayer (SAM) that can easily form a homogeneous organic film and can easily control the film thickness and the amount of immobilized enzyme reagent. Furthermore, the SAM is preferably a SAM formed of a compound having a thiol group or a disulfide group that easily forms a strong film on gold or platinum. Examples of such compounds include benzenethiol, 4-aminothiophenol, 4-mercaptopyridine, 4,4′-dithiobispyridine, methionine, p-methylthiophenol, 2-mercaptopyridine, butanethiol, 2-amino. Ethanethiol, isocysteine, 2- (acetylamino) ethanethiol, N-acetyl-L-cysteine, N-cysteinylglycine, cysteine, L-cysteine, D, L-homocysteine, 3-mercaptopropionic acid, mercapto Examples include undecanoic acid, mercaptosuccinic acid, thiolactic acid, dithiobis (succinimidyl octanate), dithiobis (succinimidyl hexanate), and the like.

  The oxidation-reduction current detected from the active oxygen measuring sensor is converted into the number of somatic cells and displayed by, for example, a device body incorporating a current-voltage conversion element or the like. The method for displaying the number of somatic cells may be an oxidation-reduction current value, a DC voltage value, the number of somatic cells, an alarm, LED display, or sound, and is not limited.

  The reaction between the active oxygen and the enzyme reagent varies greatly depending on the measurement temperature environment. Usually, the reactivity is higher at around 37 ° C., which is higher than 23 ° C. near room temperature, and the oxidation-reduction current value shows a high value. If the somatic cell number measuring device is provided with a heater for heating raw milk held in the examination room to a predetermined temperature, it is possible to measure with high accuracy by increasing the reactivity between the active oxygen and the enzyme reagent.

  The actual measurement may be performed simultaneously with milking of a dairy cow, or when an examiner performs batch measurement after collecting raw milk as a sample. It is presumed that the temperature of raw milk for measurement varies depending on the form of measurement. When the standing time has elapsed after collecting raw milk, for example, heating raw milk to 37 ° C. requires time for measurement. For the same raw milk, the somatic cell count calculated based on the redox current detected by the active oxygen measurement sensor is changed according to the raw milk temperature so that the output somatic cell count does not change even if there is a difference in temperature environment. It may be a somatic cell number measuring device to be corrected.

  The plate type active oxygen measurement sensor can easily optimize the arrangement of electrodes and the like, and can provide a plurality of inspection sections (inspection room and desired electrodes) in one sensor. There are four dairy cows' breasts. For example, by providing four inspection units in one sensor, the number of somatic cells by quarter can be measured efficiently. It becomes possible.

  When a plurality of active oxygen measuring sensors are provided, or when a plurality of inspection units are provided in one active oxygen measuring sensor, the apparatus main body has a detection circuit for the number of somatic cells having a plurality of systems. For example, the redox current value of each raw milk may be measured by an automatic changeover switch in one somatic cell number detection circuit. Further, if the data of the measured number of somatic cells in raw milk is automatically managed by a wireless tag built in the apparatus main body, it is possible to minimize the labor of dairymen and examiners.

  The number of somatic cells in raw milk is mainly from inspection institutions that set the upper limit of the management range to 300,000 cells / mL. In order to detect latent mastitis at an early stage, it is desirable to be able to diagnose as early as possible in infection, and it is desirable to have sensitivity to detect whether the number of somatic cells exceeds 50,000 cells / mL.

  It is known that the number of somatic cells in raw milk varies depending on the pre-squeeze, middle squeeze, and post-squeeze, and the post-squeeze that increases the milking time increases the number of somatic cells. Even in healthy dairy cows, the number of somatic cells may exceed 2 million / mL by post-drawing, and may exceed 5 million / mL in dairy cows infected with mastitis. Therefore, the number of somatic cells that can be detected by the somatic cell number measuring apparatus is preferably 50,000 cells / mL or more and 5 million cells / mL or less, more preferably 100,000 cells / mL or more and 3 million cells / mL or less.

  The apparatus body in the somatic cell number measuring apparatus is preferably composed of a general-purpose current-voltage element, voltage amplification element, or the like in order to reduce the manufacturing cost of the apparatus. Due to the recent increase in insulation, resolution, and amplification capability of electronic elements, current / voltage conversion can be performed by a general-purpose element so long as the current value is in units of nA.

  In order to reduce the production cost of the somatic cell number measuring apparatus and to enable measurement of the somatic cell number having measurement accuracy, the current value detected by the active oxygen measurement sensor is preferably in the range of 1 nA to 1000 nA, More preferably, it is in the range of 5 nA to 500 nA.

  In addition, it is desirable that the active oxygen measurement sensor be appropriately stored in an appropriate manner, for example, to be stored in a dark place or in a cold place, or in a packaging bag coated with a metal such as aluminum.

  The somatic cell number measuring apparatus of the present invention can satisfy dimensional accuracy by utilizing an electrode forming method established as an industrial technique. For example, when manufacturing 10 million or more active oxygen measuring sensors per year, satisfying the dimensional accuracy can eliminate a variation between production lots and obtain a reproducible value.

  In addition to the immobilization of enzyme reagent on the working electrode, the detection sensitivity of the active oxygen measurement sensor is dramatically improved by installing a heater, forming a surface layer containing a leukocyte activator, and forming multiple protrusions on the first electrode. Can be increased. With this high sensitivity, the measurement time required for quantifying the number of somatic cells in raw milk can be made within 2 minutes, preferably within 1 minute.

  The ability to quickly measure the number of somatic cells in raw milk at low cost means that abnormalities in the number of somatic cells can be grasped at an early stage in the field use by dairy farmers or batch measurement by a certifier, etc. Can be prevented.

  In the measurement of the number of somatic cells in raw milk, the number of somatic cells contained in the raw milk to be tested can be made the same for the same raw milk by making the amount of raw milk to be examined constant. Depending on the laboratory of the active oxygen measurement sensor, the amount of raw milk used for the measurement becomes constant, and the reproducibility of the somatic cell count measurement value in the same raw milk can be improved. As a method for introducing raw milk into a laboratory, raw milk may be automatically introduced into the laboratory using, for example, capillary action.

  When mastitis pathogens enter the breast through the mouth of the nipple and colonize and proliferate (infection), direct bacterial attack and toxins produced damage the breast cells. As a result, a substance that initiates inflammation is released from damaged mammary gland cells, blood vessel permeability is increased, leukocytes migrate, and leak into the raw milk along with plasma components. Mammary epithelial cells that have fallen into the glandular cavity and leukocytes that have migrated from the blood vessels are discharged out together with raw milk, and these are called somatic cells.

  The type of somatic cells found in raw milk was previously thought to be primarily epithelial cells, but due to the development of laboratory techniques, the type of somatic cells in normal milk and latent mastitis-infected milk It is reported that it is different.

  In normal milk, macrophages are high (60% to 70%), whereas in raw milk of cattle infected with latent mastitis, 90% or more are reported to be neutrophils. When the breast is inflamed, neutrophils migrate from the blood in large quantities to the mammary gland cavity, release active oxygen, kill the phagocytosis, and contribute to the elimination of pathogenic bacteria.

  Moreover, even if it is normal milk, it has been reported that the number of somatic cells in raw milk fluctuates by pre-squeezing, squeezing, and post-squeezing, and the number of somatic cells in post-squeezed raw milk increases. Since the somatic cell counting device calculates the number of somatic cells based on the active oxygen released from neutrophils, the ratio of neutrophils is increased or decreased from the somatic cell count measured by the somatic cell counting device. I can grasp it. For this reason, if the measured number of somatic cells is recorded, the change in the number of somatic cells can be used to determine whether there is mastitis, clinical mastitis, subclinical mastitis, or chronic mastitis in dairy cows from which raw milk has been extracted. Can be diagnosed early.

  The degree of activation of leukocytes, especially neutrophils, can also be confirmed by increasing or decreasing the number, difference in size, and difference in adhesion to the material surface. When the size of neutrophils increases or the neutrophils stick to the material surface, it is predicted that the neutrophils are activated. For example, a simple diagnosis of mastitis, clinical mastitis, subclinical mastitis, and chronic mastitis can be performed by measuring the size of neutrophils and the degree of adhesion using microscopic observation.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. For clarity of explanation, the following description and drawings are omitted and simplified as appropriate.

(First embodiment)
FIG. 1 shows a somatic cell number measuring apparatus 1A according to the first embodiment of the present invention. The somatic cell number measuring apparatus 1A is suitable for continuous measurement in which the somatic cell count is measured simultaneously with milking, and includes four active oxygen measuring sensors (hereinafter simply referred to as “sensors”) corresponding to the number of dairy cows. 2) and the device body (somatic cell number display) 6 connected by these sensors 2 and the cable 71. The apparatus main body 6 is provided with four display units 61 corresponding to each sensor 2 and various push buttons 6a to 6c.

  The somatic cell number measuring apparatus 1A is mounted on a milking machine. In the milking machine, a milk sampling container is attached to the milker or its periphery for each breast, and a sensor 2 is set in each sampling container. The sensor 2 is detachably attached to a connector 7 provided at the tip of the cable 71, so that the sensor 2 can be easily replaced. The sensor 2 outputs an electrical signal when a voltage is applied by the apparatus main body 6 with the tip immersed in raw milk. The electrical signal output from the sensor 2 is transmitted to the apparatus main body 6 via the cable 71. The apparatus body 6 calculates the number of somatic cells in the raw milk based on the electrical signal and displays it on the display unit 61. If it is difficult to manage the calculated number of somatic cells in raw milk each time, the device body 6 is equipped with a wireless tag and sent to a personal computer, etc. It is also possible to manage automatically.

<Sensor>
Next, the configuration of the sensor 2 will be described in detail with reference to FIGS. This sensor 2 is of a substantially rectangular plate type having an examination room 20 for holding raw milk, and raw milk is introduced into the examination room 20 by capillary action. The volume of the examination room 20 is preferably 0.01 to 5 mL, and more preferably 0.05 to 1 mL. A more preferable volume of the examination room 20 is 0.1 to 0.3 mL.

  Specifically, the sensor 2 includes a base plate 21 having a substantially rectangular shape, and three working electrodes 31, a reference electrode 32, and a counter electrode 33 supported on one surface (one surface in the thickness direction) 21 a of the base plate 21. An electrode and a substantially rectangular cover plate 22 fixed to one surface 21a of the base plate 21 with the electrodes 31 to 33 interposed therebetween are provided. In the following description, for convenience of explanation, one of the longitudinal directions of the base plate 21 (upper right direction in FIG. 2) is referred to as the front, and the other (lower left direction in FIG. 2) is referred to as the rear. (Lower right direction in FIG. 2) is called the right side, and the other (upper left direction in FIG. 2) is called the left side.

[Base plate and cover plate]
The width of the cover plate 22 is the same as the width of the base plate 21, but the length of the cover plate 22 is set shorter than the length of the base plate 21 so as to expose the rear end portion of the one surface 21 a of the base plate 21. Yes. The size of the base plate 21 is, for example, 60 mm long, 30 mm wide, and 1 mm thick. The size of the cover plate 22 is, for example, 50 mm long, 30 mm wide, and 3 mm thick.

  The material of the base plate 21 and the cover plate 22 is not particularly limited as long as it is insulative. For example, acrylic resin, polylactic acid, polyglycolic acid, styrene resin, acrylic-styrene copolymer resin (MS resin), polycarbonate resin, polyester resin such as polyethylene terephthalate, polyvinyl alcohol resin, ethylene-vinyl alcohol copolymer resin, thermoplastic elastomer such as styrene elastomer, vinyl chloride resin, polydimethylsiloxane, etc. Silicone resin, vinyl acetate resin, polyvinyl butyral resin, and the like can be given. The material of the base plate 21 is preferably selected as appropriate in consideration of adhesion with an electrode material described later, and the material of the cover plate 22 is preferably selected as appropriate in consideration of adhesion with the base plate 21. .

  A groove 23 is formed in the cover plate 22 so as to open to the end surface of the cover plate 22 along the base plate 21, and the inspection chamber 20 is configured by the groove 23 and one surface 21 a of the base plate 21. The examination room 20 is automatically filled with raw milk from the opening 23a by capillary action. That is, the opening 23 a of the groove 23 constitutes an introduction port to the examination room 20. In order to utilize the capillary phenomenon, the width of the groove 23 is preferably 1 to 50 mm, the depth is 0.05 to 5 mm, and the length is preferably 2 to 100 mm, more preferably the width of the groove 23 is 3 to 30 mm. The depth is 0.1 to 3 mm and the length is 10 to 70 mm. For example, the width of the groove 23 may be 10 mm, the depth may be 0.5 mm, and the length may be 40 mm.

  The groove 23 can be formed by machining, injection molding, or the like, and the base plate 21 and the cover plate 22 can be joined by heat fusion, laser fusion, solution adhesion technique, or the like. By applying a method established as such an industrial production technique, the accuracy of the volume of the examination room 20, that is, the accuracy of the amount of raw milk introduced into the sensor 2 can be increased. Thereby, if it is the same raw milk, the number of the neutrophils in the laboratory 20 can be made constant, and it becomes possible to improve the measurement accuracy of the number of somatic cells.

  In the present embodiment, since the capillary phenomenon is used, it is possible to fill the examination room 20 with the raw milk in a short time, for example, within 5 seconds just by immersing the end of the sensor 2 on the opening 23a side in the raw milk. .

[electrode]
The working electrode 31, the reference electrode 32, and the counter electrode 33 are arranged in the left-right direction, and each extends linearly in the front-rear direction from the rear end of the base plate 21 to a predetermined position. In the present embodiment, a circular first electrode part (positive electrode part) 31a is provided at the tip of the working electrode 31, and a circular second electrode part (negative electrode part) larger than the first electrode part 31a is provided at the tip of the counter electrode 33. ) 33a is provided. Also, the tip 32a of the reference electrode 32 has a small circular shape. The first electrode portion 31 a and the second electrode portion 33 a and the tip 32 a of the reference electrode 32 all face the internal space of the examination room 20. Moreover, the part which is not covered with the cover plate 22 in each electrode 31-33 comprises the wide terminal parts 31b-33b, and the sensor 2 is inserted in the connector 7 by these terminal parts 31b-33b. When attached, it is electrically connected to a terminal (not shown) of the connector 7.

An enzyme reagent 4 that reacts with active oxygen contained in raw milk is immobilized on the surface 31c of the first electrode portion 31a, and a surface layer 5 containing a leukocyte activator is further laminated thereon. (See FIG. 7). The reference electrode 32 is used as a reference electrode. A voltage of, for example, 1 V DC is applied uniformly between the working electrode 31 and the counter electrode 33 and between the reference electrode 32 and the counter electrode 33. In addition, when an additional voltage of, for example, 0.3 V is applied to the working electrode 31 in a superimposed manner, active oxygen is oxidatively decomposed by the enzyme reagent 4 on the first electrode portion 31a (more precisely, neutrophils in raw milk Oxidative decomposition of the superoxide anion released from (O ₂ ⁻ → O ₂ + e ⁻ ) is generated, and the enzyme dissolved from the first electrode portion 31a is reduced on the second electrode portion 33a. A current flows between 31 and the counter electrode 33. By measuring the amount of current flowing between the working electrode 31 and the counter electrode 33, the amount of active oxygen released from neutrophils, and thus the number of somatic cells in raw milk can be measured. In order to efficiently detect a reduction current for oxidative decomposition on the first electrode portion 31a, the area of the second electrode portion 33a is preferably larger than the area of the first electrode portion 31a. Here, “the area of the second electrode portion 33a” and “the area of the first electrode portion 31a” are the areas when the second electrode portion 33a and the first electrode portion 31a are viewed from a direction orthogonal to the base plate 21. Say. Area of the first electrode portion 31a is preferably 0.7～500Mm ^2, more preferably 4~250mm ^2.

  As a method for forming the electrodes 31 to 33, vapor deposition, sputtering, electrolytic plating, electroless plating, silk screen printing, metal paste injection, or the like can be used. By adopting such an industrially established method, the electrodes 31 to 33 can be formed with high accuracy, and the reproducibility of measured values can be improved in mass production. Examples of the conductive material used for the electrode include gold, silver, silver chloride, platinum, copper, and aluminum. In the present embodiment, superoxide anions released from neutrophils as active oxygen are mainly detected, so that the working electrode 31 is made of gold, the reference electrode 32 is made of silver / silver chloride, and the counter electrode 33 is made of platinum.

  Preferably, each electrode 31 to 33 has a width of 50 to 5000 μm, a length of 5 to 100 mm, and a thickness of 0.003 to 300 μm. More preferably, each electrode 31 to 33 has a width of 100 to 2000 μm and a length. Is 10 to 50 mm, and the thickness is 0.02 to 200 μm.

  In addition, it is preferable that minute unevenness is formed on the one surface 21a of the base plate 21, and the working electrode 31, the reference electrode 32, and the counter electrode 33 are formed on the minute unevenness. If it becomes like this, the adhesiveness of the baseplate 21 and the electrodes 31-33 can be improved by the anchor effect.

  The minute irregularities are preferably such that the arithmetic mean roughness (Ra: JIS B0601) of the one surface 21a falls within the range of 20 nm to 20 μm, and the arithmetic mean roughness of the one surface 21 a is in the range of 100 nm to 2 μm. It is more preferable that it is within the range.

  In order to form the above-described minute unevenness on the one surface 21a of the base plate 21, the one surface 21a of the base plate 21 may be processed by, for example, plasma etching or sand blasting. Alternatively, when forming the base plate 21, minute unevenness may be formed on the one surface 21 a of the base plate 21 by using a mold whose surface is roughened to a predetermined surface roughness.

[Enzyme reagent]
As the enzyme reagent 4 immobilized on the first electrode portion 31a, a plurality of types can be used. For example, the enzyme reagent 4 comprises at least one enzyme selected from the group consisting of SOD (superoxide dismutase), cytochrome c, hemin, horseradish peroxidase, catalase, glutathione peroxidase, polyphenol oxidase, tyrosinase, cholesterol oxidase, and laccase. Including. Among these, it is preferable to use SOD from the viewpoint of long-term stability (weather resistance), industrially stable quality, and cost. Moreover, the enzyme activator may be added to the enzyme reagent 4 as needed.

  The thickness of the enzyme reagent 4 is preferably 0.005 to 1 μm, and more preferably 0.02 to 0.1 μm.

  The enzyme reagent 4 is preferably stable for a period guaranteed by the manufacturer (for example, 6 months) so that the quality is not deteriorated by oxidative deterioration, hydrolysis, photolysis, thermal decomposition or the like. In order to stabilize the quality, an enzyme protecting agent such as trehalose may be added to the enzyme reagent 4.

  As an example of the immobilization method of the enzyme reagent 4, a cysteine self-assembled monolayer 40 (see FIG. 7) is immobilized on the first electrode portion 31a made of gold, and then a dipping method or an ink jet method is formed thereon. It is possible to immobilize by applying the enzyme reagent 4 using an injection method or the like and curing it. Thus, by applying the enzyme reagent 4 to the first electrode portion 31a via the cysteine self-assembled monolayer 40, the electron propagation property from the enzyme reagent 4 to the first electrode portion 31a can be improved. . When the cysteine self-assembled monolayer 40 is not provided, the working electrode 31 may be made of platinum, for example, and the enzyme reagent 4 may be applied directly on the first electrode portion 31a.

  The reason why the enzyme reagent 4 is immobilized only on the first electrode portion 31 a without being immobilized on the second electrode portion 33 a is that the current value flowing between the working electrode 31 and the counter electrode 33 is measured. This is because when a current value is generated in an electrode other than the working electrode 31, this becomes noise and the measurement accuracy is lowered. In addition, the enzyme reagent 4 should just be fix | immobilized on the 1st electrode part 31a so that it may not overlap with the other electrodes 32 and 33, for example, is arrange | positioned in the area one size larger than the 1st electrode part 31a. May be.

[Leukocyte activator]
Dairy cattle infected with mastitis increase the phagocytic ability of white blood cells, especially neutrophils, and release active oxygen. If the surface layer 5 laminated on the enzyme reagent 4 contains a leukocyte activator, the phagocytic ability of neutrophils is further increased, and the release amount of active oxygen can be increased to increase the sensitivity. .

  It is known that when the phagocytic ability of neutrophils increases, the amount of released active oxygen increases, and at the same time, a pseudo-scaffold is formed on the surface of neutrophils and adheres to the inside of blood vessels. If the activator for neutrophils is present on the enzyme reagent 4, neutrophils are likely to adhere on the enzyme reagent 4, and higher sensitivity can be expected.

  The leukocyte activator may be expressed as a physiologically active substance. It is known that the leukocyte migration ability varies depending on the presence or absence of a physiologically active substance or a concentration difference. By placing a physiologically active substance on or around the first electrode part 31a and causing the neutrophils to migrate on the first electrode part 31a, the neutrophils are actively attached on the first electrode part 31a, Further sensitivity enhancement is possible.

  Since the quantified leukocyte activator is supported on the enzyme reagent 4 in advance, it is not necessary for a measurer such as a dairy farmer or a tester to measure the leukocyte activator and add it to raw milk. Increases speed.

Examples of activators of leukocytes, especially neutrophils, include chemokines, eicosanoids, leukotrienes, FMLP, zymosan, phorbol myristate acetate (PMA), concanavalin A, free fatty acids, surfactants, metal microparticles, polymer microparticles. Etc. Neutrophils adhered to metal fine particles may be accumulated near the electrode by magnetic force, and neutrophils adhered to polymer fine particles may be accumulated near the electrode by gravity or buoyancy.
Usually, there is no particular limitation, but a suitable amount when using an activator as an activator is 1 ng / mL to 500 μg / mL with respect to the laboratory or the volume of raw milk.

  Formation of the surface layer 5 is preferably carried out by preparing a leukocyte activator by freeze-drying and placing it on the enzyme reagent 4. Freeze-drying is a method in which a food or food material containing water is rapidly frozen at about minus 30 ° C. and further dried under reduced pressure by sublimating the water. It is characterized by little change in form such as shrinkage and cracking due to drying, little change in nutritional components such as vitamins and flavor, and is porous and easily invaded by water and hot water. In this way, since the leukocyte activator dissolves well in the raw milk, the degree of mixing of the leukocyte activator and the raw milk is greatly improved, and an increase in sensitivity can be expected.

In general, in the case of fluid flowing in a circular pipe, the Reynolds number is about 2,000 or less, the laminar flow is about 2,000 to 4,000, and the laminar flow and turbulent flow are changed. The Reynolds number is defined by the ratio of inertial force to frictional force (due to viscosity), and is used for air flow and Karman vortices by airplane wings. The Reynolds number (Re = U × L / ν) depends on the type and state of the fluid (ν: m ² / s), the flow velocity (U: m / s) of the target portion, and the length (L: m) of the target portion. It will be decided.

  In order to efficiently mix the leukocyte activator on the first electrode part 31a (exactly on the enzyme reagent 4) and the raw milk, for example, the raw milk is used rather than dropping the raw milk on the first electrode part 31a. It can be expected that the degree of mixing is improved by coating the leukocyte activator on the groove 23 to be introduced. However, the flow velocity and length in the narrow channel do not become a turbulent region, and the Reynolds number becomes a laminar flow of 2,000 or less, and it is predicted that the mixed state will not be improved significantly.

  The surface layer 5 may be composed of only the leukocyte activator, but may be composed of a mixture of this and a hydrophobic organic material. The amount of active oxygen released from neutrophils in raw milk can be detected with higher sensitivity when the neutrophils are close to or attached to the enzyme reagent 4 on the first electrode portion 31a. It becomes.

  In order for neutrophils in raw milk to approach or adhere to the enzyme reagent 4 on the first electrode portion 31a, water on the surface of the surface layer 5 that contacts the raw milk held in the examination room 20 on the first electrode portion 31a. Is preferably 40 ° or more and 110 ° or less, and more preferably 50 ° or more and 100 ° or less. In order to realize this, the surface layer 5 may contain a hydrophobic organic material. Since active oxygen can pass through the organic material, there is no problem in sensitivity even in this way.

  When the surface layer 5 is not provided, the contact angle of water on the surface of the enzyme reagent 4 that contacts the raw milk held in the examination chamber 20 on the first electrode portion 31a is 40 ° to 110 °. It is preferable.

  Moreover, there exists a method of giving energy to raw milk as a method for obtaining the same effect as an activator. Therefore, the somatic cell number measurement apparatus 1 </ b> A may include a means for applying energy to the raw milk held in the examination room 20. The energy is not particularly limited as long as it has an action of activating NADPH oxidase, which is an active oxygen producing enzyme on the cell membrane of neutrophils. For example, electromagnetic waves, shear stress, pressure, ultrasonic waves, electric fields, magnetic fields, microwaves, infrared rays, visible light, and one of these may be used as the energy, or any combination thereof may be used as the energy. It may be energy. That is, the energy may be at least one of electromagnetic waves, shear stress, pressure, ultrasonic waves, electric fields, magnetic fields, microwaves, infrared rays, and visible rays.

The activation mechanism is described. First, neutrophils sense extracellular stimuli and the intracellular signal transduction system is started. As a result, the intracellular calcium ion concentration increases. And if the component of NADPH oxidase moves from the cytosol to the cell membrane and the association occurs, NADPH oxidase is activated and the amount of active oxygen production increases.
In other words, in order to activate neutrophils and increase the amount of active oxygen produced, neutrophils sense extracellular stimuli by applying energy to raw milk, or soften the cell membrane structure by applying energy to raw milk. This makes it possible to easily increase the intracellular calcium ion concentration, or to make it easier for components of NADPH oxidase to migrate from the cytosol onto the cell membrane.

  According to the method for applying energy to raw milk, it is possible to supply energy with low cost and high accuracy using an element established in industrial technology, so that it is possible to reduce the manufacturing cost of a somatic cell measuring device. . In addition, the method of applying energy to raw milk has long-term stability compared to drug stimulation, and is less susceptible to the environmental temperature and humidity in which the somatic cell measuring device is installed. High measurement values can be reproduced.

From the viewpoint of causing neutrophils to sense extracellular stimuli, there is a method using electromagnetic waves as the energy. In this case, it is preferable to irradiate raw milk with ultraviolet rays having a wavelength of 0.01 to 0.4 μm which acts on cell nuclei. A more preferable wavelength of ultraviolet rays is 0.1 to 0.3 μm.
Similarly, from the viewpoint of causing neutrophils to sense extracellular stimuli, there is a method of applying shear stress to raw milk. In this case, the shear stress is preferably 10~10000dynes / cm ^2, more preferably 50~5000dynes / cm ^2.

From the viewpoint of softening the cell membrane structure, there is a method using pressure as the energy.
To soften the cell membrane structure using pressure, neutrophils may be swollen using osmotic pressure. In this case, the range of osmotic pressure is preferably 10 to 250 mOsm, and more preferably 20 to 100 mOsm.
Further, any of air pressure, water pressure, and vacuum pressure may be used as the pressure. When air pressure, water pressure, or vacuum pressure is used as an activator, pressurization and decompression should be performed to such an extent that leukocytes are not destroyed, and the pressure is not particularly limited, but should be in the range of 0.01 Pa to 10 MPa. Is preferable, and the range of 0.1 Pa to 1 MPa is more preferable.

In addition, the method of softening the cell membrane structure by a method other than pressure includes a method of vibrating the cell membrane constituent molecules to soften the cell membrane, a method using ultrasonic waves, an electric field, or a magnetic field, or a molecular motion of intracellular water. And a method using microwaves, infrared rays, or visible rays, etc., to increase the cell membrane flexibility.
Usually, there is no particular limitation, but the ultrasonic range is preferably 10 to 200 kHz, more preferably 20 to 100 kHz. In the electric field, a range of 0.001 to 10 V / cm is preferable, and a range of 0.01 to 5 V / cm is more preferable. In the magnetic field, a range of 0.05 to 30 mT is preferable, and a range of 0.1 to 20 mT is more preferable. Furthermore, you may apply the said electric field and magnetic field in the pulse form of 20 Hz-10 MHz.
In the microwave, a range of 1 to 10 GHz is preferable, and a range of 2 to 4 GHz is more preferable. In infrared rays, the range of 0.7-1000 micrometers is preferable, and the range of 2.0-500 micrometers is more preferable. In visible light, the range of 380-750 nm is preferable, and the range of 430-600 nm is more preferable.

[First electrode section]
Next, the first electrode part 31a will be described in more detail.

  The surface of the first electrode portion 31a may be flat, but it is preferable that the first electrode portion 31a has a concavo-convex shape by a plurality of point-like protrusions or a plurality of point-like depressions. Usually, when the area of the first electrode portion 31a is increased, the amount of current flowing from the working electrode 31 to the counter electrode 33 is increased due to an increase in the attachment area of somatic cells, and detection sensitivity is improved. On the other hand, if the area of the first electrode portion 31a becomes too large, the current on the first electrode portion 31a diffuses into the raw milk, and the amount of current flowing from the working electrode 31 to the counter electrode 33 decreases, and is detected. The result is a decrease in sensitivity.

  If the current value due to active oxygen released from somatic cells is S (signal) and the base current value flowing from the working electrode 31 to the counter electrode 33 is N (noise), the detection sensitivity is increased by increasing the S / N ratio. In order to improve the S / N ratio, a method of arranging a plurality of small electrodes can be considered in order to suppress the diffusion of the current value into the raw milk as much as possible and to reduce the current value flowing from the working electrode 31 to the counter electrode 33. Having a plurality of electrodes requires shaping accuracy of the electrodes and decreases productivity due to a decrease in yield.

  By disposing a plurality of protrusions or depressions on the surface of the first electrode part 31a, diffusion of current into raw milk can be suppressed, and an improvement in S / N ratio due to an increase in current density can be expected. By having a plurality of protrusions or depressions on the first electrode portion 31a, the current generated on the concavo-convex tip surface (the top surface of the protrusion in the case of a protrusion or the surface other than the depression in the case of a depression) is diffused in the raw milk. However, the current generated on the surface other than the concavo-convex tip surface can be passed from the working electrode 31 to the counter electrode 33 without diffusing into the raw milk, and the S / N ratio can be dramatically increased. It becomes possible.

Preferred density of protrusions or depressions is preferably 200 to 15000 pieces / mm ^2, and more preferably 500 to 10,000 pieces / mm ^2. The protrusion or the depression has, for example, a column shape, and the cross-sectional shape may be a circle or a polygon. When the density of protrusions or depressions is less than 200 pieces / mm ² , current tends to diffuse, and it may be difficult to increase the S / N ratio. On the other hand, if it exceeds 15000 pieces / mm ² , the difficulty in industrial production may increase, such as an increase in the manufacturing tact time in order to maintain the modeling accuracy, and the cost may increase. “Density” refers to the number of protrusions or depressions per area when viewed from a direction orthogonal to the base plate.

In raw milk, lipid particles, which are solids, are present at least 100 times the neutrophil count. The particle size of the lipid particles varies depending on the breeding of dairy cows, the content of the feed, etc., and is in a wide range of 2 μm to 30 μm. The distance between adjacent protrusions or the width of the dent is preferably 4 to 80 μm and more preferably 10 to 60 μm in consideration of blockage by lipid particles in raw milk.
The height of the protrusion or the depth of the recess is preferably 0.5 to 1000 μm, more preferably 2 to 500 μm. When the height of the protrusion or the depth of the depression is less than 2 μm, there is a case where it is difficult to improve the S / N ratio without changing from a flat working electrode. On the other hand, when the thickness exceeds 500 μm, the difficulty in industrial production may increase, such as an increase in manufacturing tact time in order to maintain modeling accuracy, and the cost may increase. A particularly preferable height or depth of the protrusion is 5 to 500 μm.

  On the other hand, the aspect ratio of the height to the width of the protrusion or the aspect ratio of the depth to the width of the depression increases the possibility that neutrophils adhere, but at the same time, the degree of difficulty increases in terms of industrial technology, Since productivity may fall, it is preferable to select from the range of 0.2-10, and it is more preferable to select from the range of 0.5-5.

  In the present embodiment, as shown in FIG. 5, the surface 31c of the first electrode portion 31a has an uneven shape by a plurality of protrusions 31d. The reaction between the active oxygen and the enzyme reagent 4 is higher when the neutrophil is close to or attached to the first electrode portion 31a than when the neutrophil is floating in the raw milk. It becomes possible to detect the amount of active oxygen released with high sensitivity. By forming the protrusion 31d on the surface 31c of the first electrode portion 31a, a structure in which neutrophils are easily attached can be obtained. Furthermore, the protrusion 31d can increase the area of the first electrode portion 31a, for example, by a factor of two or more without changing the size of the first electrode portion 31a and thus the outer dimensions of the sensor 2, thereby increasing the sensitivity and reducing the cost. It becomes possible to make it compatible.

  The width (thickness) of the protrusion 31d is preferably 1 to 1000 μm, more preferably 5 to 500 μm.

  Specifically, the uneven shape of the surface 31c of the first electrode portion 31a is formed by the first electrode portion 31a being along the uneven structure 21b formed on the base plate 21, as shown in FIG. Further, the above-described cysteine self-assembled monolayer 40, enzyme reagent 4, and surface layer 5 are laminated in this order.

  Although the formation method of the uneven structure 21b is not specifically limited, For example, the roll transfer method by injection molding, press molding, monomer cast molding, solvent cast molding, hot emboss molding, extrusion molding, etc. can be mentioned. Of these, injection molding is preferably used from the viewpoint of productivity and mold transferability.

The protrusions 31d may be arranged in a matrix as shown in FIG. 5, or may be arranged in a staggered manner as shown in FIG. For example, protrusions 31d having a height of 10 μm and a diameter of 5 μm may be arranged at a pitch of 20 μm vertically and horizontally (arranged in a matrix at about 2400 pieces / mm ² ), and protrusions 31d having a height of 30 μm and a diameter of 10 μm are vertically and horizontally. They may be arranged at a pitch of 30 μm and shifted by half a pitch in either the vertical or horizontal direction (arranged in a staggered manner at about 1000 / mm ² ). Further, the protrusions 31d do not need to be regularly arranged, and may be arranged at random.

  The distance L between the adjacent protrusions 31d is preferably 4 to 80 μm and more preferably 6 to 50 μm from the viewpoint of efficiently attaching neutrophils in raw milk. That is, the pitch and thickness of the protrusions 31d may be determined so that the distance L between the adjacent protrusions 31d is within a preferable range. Here, the distance L between the adjacent protrusions 31d is the distance between the protrusions 31d in the direction in which the protrusions 31d are arranged, and the arrangement directions are the vertical direction and the horizontal direction in the case of the matrix form shown in FIG. In the case of the zigzag pattern shown in FIG. 6, the horizontal direction (or vertical direction) and the diagonal direction are used.

<Modified example of sensor>
Note that the sensor 2 does not necessarily have to use the capillary phenomenon, and may have a structure as shown in FIG. 8, for example. The sensor 2 ′ shown in FIG. 8 corresponds to the raw milk spotting method. In the sensor 2 ′, a substantially rectangular through hole 24 that penetrates the cover plate 22 in the thickness direction is provided in the cover plate 22, and the through hole 24 is blocked by the one surface 21 a of the base plate 21 from below. Thus, an examination room 20 is formed that opens largely upward. As an example, the external dimensions of the sensor 2 ′ can be 40 mm in length, 30 mm in width, and 4 mm in height. As an example, the examination room 20 can have a length of 20 mm, a width of 20 mm, and a depth of 3 mm.

  In the case of this configuration, a plurality of examination chambers 20 are provided in one sensor 2 ′, and the same number of electrodes 31 to 33 as the number of examination chambers 20 are provided so that raw milk can be examined at a plurality of locations. Also good. In this case, the number of somatic cells can be measured for a large amount of raw milk at once by using a plurality of pipettes and spotting the raw milk in each examination room 20.

  The patterns of the electrodes 31 to 33 can be appropriately selected so as to have an optimal layout according to the introduction route of raw milk, the arrangement region of the enzyme reagent 4 and the like. For example, as shown in FIG. 9A, the first electrode portion 31a of the working electrode 31 may be rectangular, and the second electrode portion 33a of the counter electrode 33 may be disposed so as to surround the first electrode portion 31a. By doing so, it is possible to more efficiently detect the current due to the reduction of the enzyme with respect to the oxidative degradation on the first electrode portion 31a.

  Alternatively, as shown in FIG. 9B, the first electrode portion 31a of the working electrode 31 is configured by linear electrode fingers 31e arranged in parallel to each other, and a reference current amount flowing between the working electrode 31 and the counter electrode 33 is set. It may be suppressed. In this case, the enzyme reagent 4 may be disposed so as to straddle all the electrode fingers 31e, as indicated by the alternate long and short dash line in FIG. 9B.

  Between the working electrode 31 and the counter electrode 33, not only an oxidation-reduction current flows, but also a reference current corresponding to the additional voltage applied to the working electrode 31. If this reference current amount becomes excessive and becomes larger than the oxidation-reduction current amount, the S / N (oxidation-reduction current amount serving as a signal / reference current amount serving as noise) ratio is lowered, and the detection sensitivity may be lowered. is there.

  Since the amount of current (I) is inversely proportional to the resistance value (R) (I = V / R), in order to reduce the amount of current flowing through the working electrode 31, the pattern of the working electrode 31 is made finer. This can be achieved by increasing the resistance value. Specifically, the electrode finger 31e preferably has a width of 2 to 100 μm, a length of 100 to 5000 μm, and a thickness of 0.003 to 1 μm, a width of 5 to 50 μm, a length of 500 to 3000 μm, and a thickness of 0.01 to More preferably, it is 0.1 μm.

  Alternatively, as shown in FIG. 9C, the second electrode portion 33a is also composed of linear electrode fingers 33c arranged in parallel to each other, and the electrode finger 31e of the first electrode portion 33a and the electrode finger 33c of the second electrode portion 33a are connected to each other. It may be arranged alternately.

<Main unit>
Next, the configuration of the apparatus main body 6 will be described in detail with reference to FIG. The apparatus main body 6 calculates the number of somatic cells contained in the raw milk based on the current that flows when a voltage is applied between the working electrode 31 and the counter electrode 33. Specifically, the apparatus main body 6 applies a voltage between the power source 65, the working electrode 31 and the counter electrode 33, and between the reference electrode 32 and the counter electrode 33, and detects a current flowing between them. A circuit 62, a current-voltage conversion circuit 63 that converts the detected current into a voltage, and a calculation unit 64 that calculates the number of somatic cells from the voltage value are displayed. The number of somatic cells calculated by the calculation unit 64 is displayed. Displayed on the part 61.

  The power source 65 may be an internal power source such as a battery or a battery, or may be an external power source such as a household power source. The current detection circuit 62 is configured by a switching element or the like. The current-voltage conversion circuit 63 includes a current-voltage conversion element and an operational amplifier that amplifies the voltage. The calculation unit 64 includes a CPU, a storage unit (ROM and RAM), and the like. The storage unit stores a calibration curve that associates the voltage value with the number of somatic cells, and the calculation unit 64 calculates the number of somatic cells according to the voltage value sent from the current-voltage conversion circuit 63. When a plurality of types of sensors 2 having different sensitivities are used, a calibration curve may be stored in the storage unit for each sensor 2.

  In the present embodiment, since the reference electrode 32 is provided, the voltage value when calculating the number of somatic cells is a value considering the reference electrode 32. That is, the apparatus main body 6 converts the current that flows when a voltage is applied between the working electrode 31 and the counter electrode 33 into a voltage to obtain a working voltage value, and generates a voltage between the reference electrode 32 and the counter electrode 33. A reference voltage value is obtained by converting a current flowing when applied to a voltage. Then, when calculating the number of somatic cells, the apparatus body 6 calculates the number of somatic cells from a voltage value obtained by subtracting the reference voltage value from the working voltage value. In this way, the voltage value based on the reaction between the active oxygen contained in the raw milk and the enzyme reagent 4 can be obtained accurately.

<How to use>
Next, a method for measuring the number of somatic cells using the somatic cell number measuring apparatus 1A will be described. The sensor 2 is attached to each connector 7 arranged in the sampling container for raw milk. Next, the on button 6a is pressed to turn on the power switch. When milking is started, the measurement button 6c is pushed to make it measurable. When raw milk is filled in the sampling container, raw milk is filled into the examination room 20 from the opening 23a of the sensor 2 by capillary action. As a result, somatic cells in raw milk held in the laboratory 20, particularly active oxygen released by neutrophils, react with the enzyme reagent 4, and the oxidation-reduction current is sent to the apparatus body 6, so that the number of somatic cells is increased. Calculated. The calculated number of somatic cells is displayed on the display unit 61 for each quarter. It is also possible to input a reference somatic cell number into the apparatus main body 6 in advance, and when the calculated somatic cell number exceeds the reference somatic cell number, the lamp can be turned on or notified by voice.

  When milking and the measurement of the number of somatic cells are completed, the milker and the raw milk sampling container are washed, and the sensor 2 used for the measurement is replaced with a new sensor 2 in preparation for the next measurement.

  In the somatic cell number measuring apparatus 1A of the present embodiment, the size and cost can be reduced, and the number of somatic cells can be measured easily and quickly with high accuracy. Therefore, mastitis can be diagnosed at an early stage in the production site of raw milk by a dairy farmer. Thereby, discarding of raw milk in tank units can be effectively prevented.

(Second Embodiment)
FIG. 11 shows a somatic cell number measuring apparatus 1B according to the second embodiment of the present invention. In the second embodiment and the third embodiment to be described later, the same components as those in the first embodiment are denoted by the same reference numerals, and the description thereof is omitted.

  The somatic cell number measuring apparatus 1B according to the second embodiment is suitable for batch measurement, and uses a raw milk sampled by a dairy farmer, a raw milk collected and distributed to a certification association, a raw milk sampled in tank units, etc. Etc. are effective when measuring quickly. The somatic cell number measuring apparatus 1B can have outer dimensions of, for example, a height of 120 mm, a width of 80 mm, and a weight of about 300 g so as to be excellent in portability. The connector 7 to which the sensor 2 is attached is provided directly on the apparatus main body 6, and the cable 71 as in the first embodiment is not necessary.

  In addition, the somatic cell count apparatus 1B is provided with a heater 8 for heating the raw milk held in the examination room 20 to a predetermined temperature. Since the raw milk during milking becomes a measuring object, the continuous measurement somatic cell number measuring apparatus 1A of the first embodiment has a high raw milk temperature of 30 ° C. to 37 ° C. and is released from neutrophils in the raw milk. It is predicted that oxygen and the enzyme reagent 4 of the sensor 2 have high reactivity and sufficient detection sensitivity is obtained. In the batch measurement type somatic cell count measuring apparatus 1B, it is predicted that the temperature of the sampled raw milk is lowered to around room temperature, for example, about 23 ° C. Thus, when the temperature of raw milk is low, there is a possibility that sufficient detection sensitivity cannot be obtained if the number of somatic cells is as small as 300,000 cells / mL or less. In such a case, the heater 8 is provided, and the temperature of the raw milk is increased to, for example, around 37 ° C., thereby increasing the reactivity between the active oxygen released by the neutrophils in the raw milk and the enzyme reagent 4, and sufficient detection sensitivity. Can be obtained.

  In the batch measurement type somatic cell number measuring apparatus 1B having the heater 8, the amount of raw milk held in the examination room 20 of the sensor 2 is preferably 1 mL or less. Since the sensor 2 is a plate type, if a plate-type heater 8 is provided, the temperature can be raised and measured within 1 minute. The amount of power consumed can also be suppressed, and can be used without impairing portability.

  For example, the heater 8 may be heated to a set temperature, for example, 37 ° C. at the same time as the power switch is turned on by pressing the on button 6 a of the apparatus body 6.

(Third embodiment)
FIG. 12 shows a somatic cell number measuring apparatus 1C according to the third embodiment of the present invention. This somatic cell number measuring device 1C is of a batch measurement type capable of measuring the somatic cell count of four raw milk samples, and is attached to each connector 7 with four connectors 7 connected to the device body 6 by cables 71. The four heaters 8 and a connecting plate 9 for connecting the heaters 8 are provided.

  With this configuration, the number of somatic cells can be measured for a plurality of raw milk at a time in batch measurement.

  Examples will be described below. The somatic cell number measuring apparatus shown in this embodiment is an example, and the present invention is not limited to these embodiments.

[Test method for somatic cell count in raw milk samples]
A raw milk sample is smeared onto a fixed area on a slide glass, dried, stained, and microscopically examined. The number of cells is counted, and the area of the field of view of the microscope (observation of oil immersion lens) is related to the raw milk sample. The number of somatic cells present in was calculated. In addition, the representative visual field measured 16 visual fields or more, and the number of nucleated somatic cells in the entire visual field was measured. An inspection microscope (model: BX41) manufactured by Olympus Optical Co., Ltd. was used for the somatic cell number inspection.

  When raw milk was sampled from two healthy cattle (a, b) and five cattle infected with mastitis (c, d, e, f, g), the number of somatic cells in the raw milk was a: 90,000 pieces / mL, b: 220,000 pieces / mL, c: 380,000 pieces / mL, d: 620,000 pieces / mL, e: 880,000 pieces / mL, f: 1,650,000 pieces / mL, g: 2.1 million Pieces / mL.

[Manufacture of active oxygen measurement sensor]
[Example 1]
A base plate having a length of 60 mm, a width of 30 mm, and a thickness of 1 mm was formed by an injection molding method using an acrylic resin (manufactured by Kuraray Co., Ltd., Parapet GH-S). Next, the base plate is masked, and a working electrode (gold), a reference electrode (silver / silver chloride), and a counter electrode (platinum) are shown in FIG. 4 by using a vapor deposition apparatus (manufactured by ULVAC, Inc., model: UEP). Formed with a pattern. Specifically, the diameter of the first electrode part is 4 mm, and the diameter of the second electrode part is 8 mm.

  Next, a cysteine solution was immersed in the working electrode (gold) to form a self-assembled monolayer, and then an enzyme reagent (SOD) was applied and fixed by an immersion method. The thickness of the enzyme reagent is 0.02 μm.

  Next, acrylic resin (Kuraray Co., Ltd., Parapet GH-S) is used, and has a length of 40 mm, a width of 10 mm, a depth of 0.5 mm, and a length of 50 mm, a width of 30 mm, and a thickness of 3 mm by injection molding. The cover plate was formed.

  Next, using a laser resin welding machine (Miyachi Technos, model: ML-5220B), the base plate and the cover plate were welded to obtain an active oxygen measurement sensor.

[Example 2]
When forming a base plate 60 mm long, 30 mm wide and 1 mm thick by an injection molding method using acrylic resin (Kuraray Co., Ltd., Parapet GH-S), a first electrode on one side of the base plate is used using a stamper. In the region where the portion is to be formed, except that convex portions having a height of 10 μm and a diameter of 5 μm are formed at a pitch of 20 μm vertically and horizontally, and a plurality of protrusions are formed on the surface of the first electrode portion by forming a working electrode thereon. Obtained an active oxygen measurement sensor in the same manner as in Example 1.

[Example 3]
A reactive oxygen measurement sensor was obtained in the same manner as in Example 2 except that a leukocyte activator (FMLP) was further immobilized on the enzyme reagent to form a surface layer.

[Example 4]
An active oxygen measurement sensor was obtained in the same manner as in Example 2 except that a leukocyte activator (PMA) was further immobilized on the enzyme reagent to form a surface layer.

[Example 5]
When a base plate having a length of 60 mm, a width of 30 mm, and a thickness of 1 mm is formed by injection molding using acrylic resin (Kuraray Co., Ltd., Parapet GH-S), the surface is roughened to a predetermined surface roughness. An active oxygen measurement sensor was obtained in the same manner as in Example 1 except that a micro unevenness was formed on one surface of the base plate using a mold, and a working electrode, a reference electrode and a counter electrode were formed thereon. In addition, the arithmetic mean roughness of one surface of the base plate on which minute irregularities were formed was 110 nm.

[Example 6]
When forming a base plate 60 mm long, 30 mm wide and 1 mm thick by an injection molding method using acrylic resin (Kuraray Co., Ltd., Parapet GH-S), a first electrode on one side of the base plate is used using a stamper. In the region for forming the portion, convex portions having a height of 40 μm and a diameter of 20 μm are formed at a pitch of 35 μm vertically and horizontally, and a plurality of protrusions are formed on the surface of the first electrode portion by forming a working electrode thereon, And the active oxygen measurement sensor was obtained like Example 1 except the diameter of the 1st electrode part having been 6 mm.

[Example 7]
An active oxygen measurement sensor was obtained in the same manner as in Example 6 except that the height of the convex portion formed on the base plate was changed from 40 μm to 80 μm.

[Example 8]
An active oxygen measurement sensor was obtained in the same manner as in Example 6 except that the height of the convex portion formed on the base plate was changed from 40 μm to 1 μm and the diameter was changed from 20 μm to 2 μm.

[Example 9]
An active oxygen measurement sensor was obtained in the same manner as in Example 6 except that the height of the convex portion formed on the base plate was changed from 40 μm to 2 μm.

[Example 10]
An active oxygen measurement sensor was obtained in the same manner as in Example 6 except that the pitch of the protrusions formed on the base plate was changed from 35 μm to 200 μm.

[Example 11]
When forming a base plate 60 mm long, 30 mm wide and 1 mm thick by an injection molding method using acrylic resin (Kuraray Co., Ltd., Parapet GH-S), a first electrode on one side of the base plate is used using a stamper. Forming recesses having a depth of 60 μm and a diameter of 20 μm in a vertical and horizontal pitch in the region where the portion is to be formed, and forming a working electrode thereon to form a plurality of depressions on the surface of the first electrode portion; and An active oxygen measurement sensor was obtained in the same manner as in Example 1 except that the diameter of the first electrode part was 6 mm.

[Comparative Example 1]
A reactive oxygen measurement sensor was obtained in the same manner as in Example 1 except that the cysteine monomolecular film and the enzyme reagent were not provided, that is, the first electrode part was exposed.

[Comparative Example 2]
A reactive oxygen measurement sensor was obtained in the same manner as in Example 2 except that the cysteine monomolecular film and the enzyme reagent were not provided, that is, the first electrode part was exposed.

[Comparative Example 3]
An active oxygen measurement sensor was obtained in the same manner as in Example 1, except that a polymer film of a metal porphyrin complex was formed on the first electrode part instead of the cysteine monomolecular film and the enzyme reagent.

[Measurement of current value detected by active oxygen sensor]
About the raw milk extracted from each cow ag mentioned above, the electric current was detected using the active oxygen measuring sensor of an Example and a comparative example, and the electric current value was measured. For the measurement of the current value, a potentiostat / galvanostat (Hokuto Denko Co., Ltd., model: HA-151) was used.

  After applying 1V DC to the working electrode and the reference electrode, an additional voltage of 0.3V was applied to the working electrode. At the same time as oxidative degradation occurred on the working electrode, enzyme reduction occurred on the counter electrode, and current flowed between the working electrode and the counter electrode. The current value was measured. The current value was measured by fixing the active oxygen measurement sensor on a plate heater set at 37 ° C.

  The measurement results were as shown in Tables 1 and 2 and FIG.

  From Table 1 and Table 2, in Comparative Examples 1 to 3, there is a difference in current value between cattle c suffering from mastitis but having a low somatic cell count of 380,000 / mL in raw milk and cattle b of a healthy cow. Almost no mastitis is found to be difficult to diagnose between early stages. In contrast, in Examples 1 to 11, there is a large difference in current value between cow c and cow b, and mastitis can be diagnosed between the initial stages.

  From the graph of FIG. 13, in Examples 1 to 11, as the number of somatic cells in raw milk increases, the current value detected by the active oxygen measurement sensor greatly increases, and the number of somatic cells and current value in raw milk It can be confirmed that the correlation is good. Especially, when the 1st electrode part of Example 2 had several protrusion of a predetermined magnitude | size, it has confirmed that an electric current value became high. It can be seen that when the leukocyte activator is provided on the enzyme reagents of Example 3 and Example 4, the current value is further increased. Furthermore, in Examples 6 and 7 in which the size (width and height) of the protrusions was larger than that in Example 2, the current value was significantly higher.

  Table 3 summarizes the dimensions of the protrusions in Example 2 and Examples 6 to 10.

  When Table 3 is combined with Table 1 and Table 2, in Example 8 in which the height of the protrusion is less than 2 μm and Example 9 in which the aspect ratio is less than 0.2, the same current as in Example 1 without protrusions. It turns out that only the value can be obtained. Further, when Example 6 and Example 10 are compared, it can be seen that a higher current value can be obtained even if the protrusions have the same size and the interval between the protrusions is set appropriately.

Claims (20)

An examination room for holding raw milk, a first electrode part facing the internal space of the examination room, and an enzyme reagent that reacts with active oxygen contained in the raw milk is immobilized on the first electrode part A sensor including a working electrode and a counter electrode having a second electrode portion facing the internal space of the examination room;
An apparatus main body for calculating the number of somatic cells contained in the raw milk based on a current flowing when a voltage is applied between the working electrode and the counter electrode;
A somatic cell number measuring apparatus.   The somatic cell number measuring apparatus according to claim 1, wherein the sensor further includes a reference electrode that has a tip that faces the internal space of the examination room and is used as a reference electrode.   The sensor includes a base plate that supports the working electrode, the counter electrode, and the reference electrode, and is fixed to the base plate across the working electrode, the counter electrode, and the reference electrode, and forms the inspection chamber together with the base plate. The somatic cell number measuring apparatus according to claim 2, further comprising a cover plate. The somatic cell number measuring apparatus according to claim 3, wherein an area of the first electrode portion when viewed from a direction orthogonal to the base plate is 0.7 to 500 mm ² .   The somatic cell number measuring apparatus according to claim 4, wherein the base plate has a surface on which minute irregularities are formed, and the working electrode, the counter electrode, and the reference electrode are formed on the minute irregularities. The surface of the first electrode part has a concavo-convex shape by a plurality of protrusions or a plurality of depressions,
The density of the plurality of protrusions or the plurality of depressions is 200 to 15000 pieces / mm ² , the height of the plurality of protrusions or the depth of the plurality of depressions is 2 to 500 μm, and the height with respect to the width of the protrusions The somatic cell number measuring device according to claim 4, wherein the aspect ratio of the depth or the aspect ratio of the depth to the width of the depression is 0.2 to 10.   The somatic cell number measuring apparatus according to claim 6, wherein the uneven shape is formed by the first electrode portion being placed along an uneven structure formed on the base plate.   The somatic cell number measuring apparatus according to claim 6, wherein a distance between adjacent protrusions in the plurality of protrusions or a width of the dent is 4 to 80 µm.   The body according to claim 3, wherein a groove is formed in the cover plate along the base plate so as to open to an end surface of the cover plate, and the inspection chamber is configured by the groove and the surface of the base plate. Cell number measuring device.   The somatic cell number measuring apparatus according to claim 9, wherein the groove has a width of 1 to 50 mm, a depth of 0.05 to 5 mm, and a length of 2 to 100 mm.   The somatic cell number measuring apparatus according to claim 1, wherein the active oxygen is a superoxide anion released from neutrophils in the raw milk.   The somatic cell number measurement apparatus according to claim 1, further comprising a heater for heating raw milk held in the examination room to a predetermined temperature.   The enzyme reagent includes at least one enzyme selected from the group consisting of SOD, cytochrome c, hemin, horseradish peroxidase, catalase, glutathione peroxidase, polyphenol oxidase, tyrosinase, cholesterol oxidase, and laccase. Somatic cell counting device.   The somatic cell number measuring apparatus according to claim 1, wherein the enzyme reagent includes an enzyme protecting agent.   The somatic cell number measuring apparatus according to claim 1, wherein the enzyme reagent is immobilized on the first electrode portion by an organic layer having a thickness of 0.1 nm to 10 µm.   The somatic cell number measuring apparatus according to claim 1, wherein a surface layer containing a leukocyte activator is laminated on the enzyme reagent.   The somatic cell number measuring apparatus according to claim 1, further comprising means for applying energy to raw milk held in the examination room.   The somatic cell number measuring apparatus according to claim 17, wherein the energy is at least one of electromagnetic waves, shear stress, pressure, ultrasonic waves, electric fields, magnetic fields, microwaves, infrared rays, and visible rays. A laboratory to hold raw milk,
A working electrode in which an enzyme reagent that reacts with active oxygen contained in the raw milk is immobilized on the first electrode part, the first electrode part facing the internal space of the examination room;
A counter electrode having a second electrode portion facing the internal space of the examination room;
A reference electrode whose tip faces the internal space of the examination room;
A base plate that supports the working electrode, the counter electrode, and the reference electrode;
A cover plate that is fixed to the base plate across the working electrode, the counter electrode, and the reference electrode, and forms the inspection chamber together with the base plate;
Comprising a sensor. The surface of the first electrode part has a concavo-convex shape by a plurality of protrusions or a plurality of depressions,
The density of the plurality of protrusions or the plurality of depressions is 200 to 15000 pieces / mm ² , the height of the plurality of protrusions or the depth of the plurality of depressions is 2 to 500 μm, and the height with respect to the width of the protrusions The sensor according to claim 19, wherein the aspect ratio of the depth or the aspect ratio of the depth to the width of the depression is 0.2 to 10.

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