Patent Application
20120250024
The present application claims priority from Japanese patent application JP 2011-076637 filed on Mar. 30, 2011, the content of which is hereby incorporated by reference into this application.
1. Field of the Invention
The present invention relates to a luminescence measuring apparatus for detecting, at high sensitivity and high accuracy, chemoluminescence and bioluminescence of a substance contained in a sample. The present invention also relates to a microbe counting apparatus based on luminescence detection of ATP in a viable microbial cell.
2. Background Art
With the development of genetic engineering, tissue engineering, and basic medical science, cell therapy and regenerative medicine enabling regeneration and reconstruction of organs that make use of biological tissue and cultured cells are being developed. With the development of cell therapy and regenerative medicine, research and development of biopharmaceuticals containing cells in a final product are in progress. The importance of guaranteed sterility in the pharmaceutical field is extremely high. In the food field as well, peace of mind and safety consciousness of consumers rises in response to the enforcement of the Hazard Analysis Critical Control Point (HACCP) system and successive accident reports in recent years. There are an increasing number of food factories that regard sterilization of kitchens and manufacturing lines as important.
In a biological cleanroom, it is important to grasp a microbe contamination state of the indoor environment, i.e., monitor the number of microbes floating in the air (airborne microbes) and the number of microbes present on the surfaces of equipment in a facility (attached microbes).
In the airborne microbe monitoring, the air is brought into collision against the solid surface of an agar culture medium to collect microbes using an air sampler (a collision method). A contamination degree is evaluated using a culturing method by culturing the collected microbes for a few days and counting colonies formed on the agar culture medium. The number of attached microbes is also evaluated by colony count using a culturing method same as that for the evaluation of the airborne microbes. However, a collecting method is different. For example, a method of scraping and collecting a check place with a cotton swab or a gauze and forming a suspension of the check place and then inoculating the suspension into the agar culture medium, or a method of directly closely attaching the agar culture medium to the solid surface, which is the check place, and transferring a contaminated place is used. The airborne and attached microbes to be evaluated are bacteria such as E. coli, staphylococci and Bacillus subtilis, and fungi such as mold and yeast (the bacteria and the fungi are hereinafter collectively referred to as microbe or cell).
In a pharmaceutical manufacturing facility, cleanness management criteria for biological cleanroom indoor environment set by the pharmacopoeia are applied. The pharmaceutical manufacturing facility is required to keep less than one microbial cell (CFU: Colony-Forming Unit) in a safe cabinet per 1 m3 of the air and keep less than 10 CFU in a region around the safe cabinet. CFU is a unit representing the number of viable microbial cells (viable cells). Concerning cleanness management for attached microbes, there are many problems in a testing technique such as a collection efficiency and reproducibility. Therefore, many facilities implement self-imposed controls. A public standard and the like are not established yet. Concerning sterile water (pharmaceutical water) in the pharmaceutical manufacturing facility, there is also a cleanness management standard set by the pharmacopoeia. Standard water for injection needs to be managed at less than 10 CFU/100 mL. The culturing method is used for the testing.
As explained above, the culturing method is the mainstream of the microbe counting method and is a method described in the pharmacopoeia. However, in the culturing method, the agar culture medium is cultured in a constant temperature machine for two to three days or, depending on a type of a microbial cell, for ten or more days and the number of generated colonies is visually counted. Therefore, it takes time to obtain a result.
Because of such a background, development of a quick measuring method for contamination monitoring is desired. There are, for example, a method of detecting metabolism activity in growth of viable cells and a method of detecting a microbe as light using protein in the microbial cell.
As a technique for detecting metabolism activity, there are, for example, an impedance method, a coloring method, and an oxygen electrode method. A change in a culture medium component during a microbial growth process and/or a breathed oxygen amount of microbes are measured. Therefore, culture is necessary for a short time. It takes about a half day or one day to obtain a result. Detection sensitivity is about 105 CFU. The sensitivity is insufficient for application in a cleanness management level set in the pharmacopoeia.
On the other hand, in a method making use of fluorescent staining or fluorescence scattering for detecting light using protein in a microbial cell (a fluorescent method) and an adenosine triphosphate (ATP) bioluminescence method (ATP method), a culturing process is unnecessary. Therefore, a result can be obtained within one hour including time required for sample preparation. If a microbe contamination state can be grasped within one hour, a check and measures can be implemented for a line and a product (including an intermediate) even during a work shift of manufacturing. It is expected that a safety management system and a shipment system are remarkably improved.
In the fluorescent staining method, cell membranes, nucleuses, and the like are stained with a stain having a fluorescent substance and luminescent spots thereof are counted using a fluorescent microscope (see JP Patent Publication (Kokai) No. 2007-60945). It is possible to visualize microbial cells one by one and distinguish viable cells and killed cells. In principle, even a microbial cell that lives in the environment but can not be cultured (Viable but Nonculturable: VBNC) can be detected. Therefore, the fluorescent staining method can also be considered a technique with higher accuracy concerning the cell count than the culturing method. In recent years, not only a function of counting the number of airborne particulates but also a particle counter with a high function that can detect scattering of fluorescence emitted when a laser beam in an ultraviolet region is irradiated on particulates is developed (see WO 2008/105893 A). It is possible to detect the presence of protein used in cellular metabolism, distinguish airborne particulates as abioses and microbes, and count microbial cells one by one in real time. However, in the fluorescent method in general, false luminescence and fluorescence scattering deriving from dusts such as plastic chip, aluminum, soil particles are counted as microbial cells by mistake. Fluorescent substances non-specifically attaching to impurities other than microbial cells in staining are counted. Therefore, there are problems in accuracy and reproducibility.
Subsequently, in the ATP method, the number of ATPs in a cell can be converted into an amount of light and measured based on a luminescent reaction of a firefly. As the principle of the ATP method, substrate luciferin and ATP molecules are captured into a luciferase enzyme and a luminescence amount at the time when oxidized luciferin (oxyluciferin) transitions from an excited state to a ground state according to the consumption of the ATPs is measured.
At this point, the consumption of one molecule of ATP corresponds to the generation of one photon. Therefore, the number of generated photons is proportional to the number of ATPs. In viable cells, ATP molecules in attomole (amol=10−18 mol) order are present as an energy source. Therefore, it is possible to estimate a total number of viable cells contained in a measurement sample. Further, since quantum efficiency (ΦBL: ≈0.5) of the luminescent reaction is most excellent in bioluminescence and chemoluminescence, it is possible to detect one cell as photons equivalent to several hundred thousands. It is possible in principle to detect light equivalent to one cell in the luminescent reaction. Compared with the fluorescent method, in the ATP method, dust other than a biological substance is not detected as light. Therefore, the ATP method is an effective method with high accuracy.
A standard procedure for measuring standard ATP luminescence in viable cells is briefly explained below. The procedure contains the following three steps:
(1) removal of extraneous ATP molecules by an ATP degrading enzyme;
(2) extraction of ATP molecules from viable cells by a surfactant; and
(3) bioluminescent reaction of the ATP molecules extracted from viable cells and a luminescent reagent.
In the method of removing extraneous ATP molecules of (1), physical processing using a membrane filter having a pore with diameter disallowing microbial cells to pass and allowing ATP molecules to pass may be performed. For example, since the ATP molecules adhere to fibers of the membrane filter, it is difficult to completely remove the ATP molecules. On the other hand, in the enzyme degrading method, since processing has to be only solution operation, a special tool is unnecessary. The degrading enzyme does not react to viable cells present in the measurement sample and acts on ATP contained in killed cells having a weakened cell membrane, organic matters peeled from the human skin, materials deriving from body fluid, and the like (see JP Patent Publication (Kokai) No. 2001-136999). A problem in detection of a very small amount of viable cells using the ATP method is deterioration in measurement sensitivity and reproducibility due to extraneous ATP entering from a worker and an indoor environment. Therefore, a measured value obtained in the normal ATP method is a sum of ATP derived from viable cells (ATP in viable cells) and extraneous ATP. Fewer amount of ATP in viable cells, luminescence deriving from the extraneous ATP more affects a measurement result.
Subsequently, in step (2), the APT degrading enzyme is deactivated. At the same time, ATP is eluted from viable microbe present in the measurement sample. Finally, in step (3), a luciferin-luciferase luminescent reagent and ATP elution liquid are caused to react with each other. An amount of light by a bioluminescent reaction is measured by a photodetector.
In the past, in general, a detection lower limit of the ATP method is about 102 amol (amol=10−18 mol). This is equivalent to one hundred or more viable cells. Since sensitivity corresponding to a cleanness management level is not satisfied, as an example in which the ATP method is used for airborne microbe detection, there is also a report example in which culture for about six hours is performed to enable measurement of an environment in which several to ten viable cells are present (Proceedings of 13th Annual Technological Meeting on Air Cleaning and Contamination Control, pages 331 to 334, 1995). In this way, in the purpose of detecting the level of several viable cells, it is said that time of about a half day to one day is necessary until a result is obtained even if the ATP method is used. In recent years, it is possible to measure an amount of ATP molecule equivalent to 1 amol using a dispensing system comprising a washing function for preventing external contamination and a bioluminescence detecting system in which a high-sensitivity detector is arranged in a space in the same apparatus where light is blocked and contaminants from the outside are suppressed (US 2008/0261294 A1, US 2011/0183371 A1, and US 2008/0241871 A1).
In the ATP method, an ATP amount of 1 amol can be measured through the increase in sensitivity. Therefore, if several microbes are present, in principle, the microbes can be detected. However, in microbe measurement in cleanness management in a pharmaceutical manufacturing facility and a regenerative medicine facility, apparatus performance for guaranteeing without limit that viable cell counts is zero or one is essential. Therefore, in addition to the improvement of sensitivity, it is an object to improve reliability of a photodetector that does not output a misdetection result. This is because, if it is determined by misdetection that “contamination” occurs, disposal of manufactured products and stop of a manufacturing line are requested, leading to a fall in productivity. The misdetection indicates lights in a visible light region deriving from an external factor other than a collected sample (extraneous lights) rather than bioluminescence due to ATP derived from viable cells contained in a sample collected for contamination evaluation. The extraneous lights are roughly divided into four.
First extraneous light is caused by a light blocking failure of an apparatus ideally configured to meet darkroom specifications. Second extraneous light is light temporarily entering into the luminescence measuring apparatus and accumulated in a detection surface of a detector and other members in the luminescence measuring apparatus (accumulated light), so-called stray light. Third light is false positive luminescence caused when a contamination source is present in the luminescence measuring apparatus and a contaminant substance from the contamination source is mixed in microbes and ATP in a collected sample. Fourth extraneous light is false positive luminescence due to contamination of a kit for measurement including consumables such as a so-called reagent and a container. In weak luminescence measurement based on the ATP method, all the extraneous lights may be detected by the photodetector without being distinguished from ATP light in a visible light region of 400 nm to 750 nm. An ATP amount from viable cells in the collected sample and the extraneous lights are detected. Therefore, misdetection occurs in terms of cleanness evaluation.
It is an object of the present invention to provide an apparatus and a system that can prevent, during luminescence measurement, detection of light due to an external factor and detect only luminescence of interest at high accuracy. More specifically, it is an object of the present invention to provide, in a method for measuring microbes by the ATP method not requiring culture, an apparatus and a system that enable detection of minimum one microbial cell, surely identify only an ATP amount derived from viable cells, measure the ATP amount at high accuracy, and measure a contamination degree.
As a result of extensive researches in order to solve the problems, the inventor performed light measurement at all wavelength regions in a measurement container and light measurement at specific wavelength or in a specific pH based on a characteristic of luminescence whose intensity changes at specific wavelength or in specific pH, checked presence or absence of luminescence due to an external factor from a ratio of luminescence intensities of the luminescence, found that luminescence deriving from a sample can be measured at high sensitivity and high accuracy, and resulted in completing the present invention. Specifically, the present invention is as explained below.
a container for the sample;
a photodetector for detecting luminescence from the container; and
at least one optical filter inserted between the photodetector and the container, and/or a pH modifier added to the container,
wherein the photodetector performs measurement of light emitted from the container at all wavelength regions and spectrometry at a specific wavelength range, and/or measurement of light, the intensity of which is changed by the pH modifier.
a container holder for the container;
an optical filter holder for supporting the optical filter and inserting the optical filter between the photodetector and the container;
an optical filter position control unit that moves the optical filter holder; and
a photodetector position control unit that moves the photodetector relatively to the container.
a container for a sample;
a photodetector for detecting luminescence from the container; and
at least one optical filter inserted between the photodetector and the container, and/or a pH modifier added to the container,
wherein the container contains a solution for processing ATP chemoluminescence introduced therein, the photodetector performs measurement of light emitted from the container at all wavelength regions and spectrometry at a specific wavelength range, and/or measurement of light, the intensity of which is changed by the pH modifier, and
the apparatus measures, from a result of the measurement, the luminescence intensity of ATP from the sample and counts microbes in the sample.
According to the present invention, a luminescence measuring apparatus is provided that can measure luminescence in a sample rapidly and with reliability. The luminescence measuring apparatus has an effect of preventing misdetection of cleanness management monitoring for viable cell count in weak luminescence measurement. Specifically, the luminescence measuring apparatus provides measuring function such as contamination detection for consumables such as reagents and a container, detection of apparatus abnormality, and molecular identification in a measurement sample, and can measure a contamination degree with high reliability. This leads to automation of consumable replacement and early finding of necessity of apparatus maintenance. It is possible to establish a system for cleanness management monitoring that can be operated by an unmanned service.
Problems, configurations, and effects other than those explained above are clarified by the following description of embodiments.
The present invention relates to an apparatus for measuring an amount of luminescence in a sample (a luminescence measuring apparatus). The luminescence measuring apparatus comprises at least one optical filter inserted between a photodetector and a measurement container, and/or a pH modifier added to the measurement container.
There are some types of bioluminescence and chemoluminescence whose intensity changes at a specific rate in different wavelength ranges. Therefore, measurement at all wavelength regions of luminescence in the measurement container and spectrometry at a specific wavelength range using an optical filter are performed. It can be determined based on a ratio of luminescence intensities of the bioluminescence and the chemoluminescence whether the luminescence in the container derives from a sample or light due to an external factor (a light blocking failure, accumulated light, or false positive luminescence) is included. As a result, it is possible to measure luminescence from the sample with high accuracy and reliability.
There are some types of bioluminescence and chemoluminescence whose intensity changes at a specific rate according to a pH change of a sample solution. Therefore, measurement at all wavelength regions of luminescence in a measurement container and measurement of light whose intensity is changed by a pH modifier, are performed. It can be determined based on a ratio of luminescence intensities of the bioluminescence and the chemoluminescence whether the luminescence in the container derives from a sample or light due to an external factor (a light blocking failure, accumulated light, or false positive luminescence) is included. As a result, it is possible to measure luminescence from the sample with high accuracy and reliability.
According to the present invention, luminescence to be measured may be bioluminescence or chemoluminescence, and the intensity of the luminescence changes at a specific rate at different wavelength ranges as explained above and/or the intensity of the luminescence changes at a specific rate according to a pH change. Examples of the luminescence, the luminescence intensity of which changes at a specific rate at different wavelength ranges, include wild-type firefly luciferin-luciferase system (
Therefore, the luminescence measuring apparatus according to the present invention comprises at least
a container for a sample,
a photodetector for detecting luminescence from the container, and
at least one optical filter inserted between the photodetector and the container.
Alternatively, the luminescence measuring apparatus according to the present invention comprises at least
a container for a sample,
a photodetector for detecting luminescence from the container, and
a pH modifier added to the container.
Alternatively, the luminescence measuring apparatus according to the present invention comprises at least
a container for a sample,
a photodetector for detecting luminescence from the container,
at least one optical filter inserted between the photodetector and the container, and
a pH modifier added to the container.
The luminescence measuring apparatus according to the present invention may further comprise at least one component selected from a group consisting of: a container holder for the container, a photodetector position control unit that can change a space between the photodetector and the container, an optical filter holder for inserting the optical filter between the photodetector and the container, and an optical filter holder position control unit that moves the optical filter holder.
The luminescence measuring apparatus according to the present invention preferably comprises a container for a sample, a container holder for the container, a photodetector for detecting luminescence from the container, a photodetector position control unit that can change a space between the photodetector and the container, an optical filter holder containing at least one optical filter that can insert the optical filter between the photodetector and the container holder, and an optical filter holder position control unit that moves the optical filter holder. Measurement of light emitted from the container at all wavelength regions that contains the sample and spectrometry at specific wavelength are continuously carried out by the same apparatus using the same sample. The luminescence measuring apparatus preferably further comprises a pH modifier added to the container.
The sample from which luminescence is measured is not particularly limited, and may be a sample from which it is desired to detect or measure bioluminescence or chemoluminescence.
The container for a sample (a measurement container) is not specifically limited as long as the container is made of a material that transmits luminescence from the sample. The container can be a container made of, for example, glass, quartz, or resin. The container is preferably held by the container holder to prevent influence on the container and the sample stored in the container.
The photodetector is not specifically limited as long as the photodetector can detect luminescence. In general, it may be suitable to use a photomultiplier tube (PMT) in terms of sensitivity. The photodetector is provided to be opposed to the container that contains a sample. The optical filter is provided to be opposed to the photodetector. The photodetector is preferably formed to be moved by the photodetector position control unit that can change a space between the photodetector and the container. Consequently, it is possible to prevent operation for taking out the measurement container from the container holder and prevent influence on a measurement result.
At least one optical filter can be used. The optical filter transmits light at certain wavelength or in a certain wavelength band and reflects light at a shorter wavelength and a longer wavelength than the certain wavelength or the wavelength band. Specifically, wavelength or a wavelength band of light to be transmitted and reflected and the number of optical filters to be used may be selected according to a type of luminescence to be measured and a purpose of measurement (luminescence measurement in the sample, microbe counting, etc.). As the optical filter, for example, an interference filter, a dichroic filter, or a combination thereof can be used.
For example, when the optical filter contains at least one filter, an optical filter in a wavelength range suitable for measurement of luminescence to be measured, for example, an optical filter in a wavelength range suitable for measurement of luminescence by luciferin-luciferase can be used. Specific examples of the optical filter include an optical filter having center wavelength in a range of 500 nm to 700 nm, and a dichroic filter having a reflected light region of 500 nm or less on the short wavelength side and 600 nm or more on the long wavelength side. Alternatively, an optical filter in a wavelength range suitable for measurement of luminescence at a luminescence peak of luciferin-luciferase can be used. Specific examples of the optical filter include an interference filter having center wavelength of 550 nm to 570 nm and half width of 10 nm to 60 nm, an interference filter having center wavelength of 600 nm to 630 nm and half width of 10 nm to 60 nm, and an interference filter having center wavelength of 650 nm to 680 nm and half width of 10 nm to 60 nm (
For example, when the optical filter contains two filters, the optical filters exemplified above can be combined as appropriate. The optical filters in a wavelength range suitable for measurement of a luminescence peak by luciferin-luciferase are preferably combined. For example, the interference filter having center wavelength of 550 nm to 570 nm and half width of 10 nm to 60 nm, and the interference filter having center wavelength of 600 nm to 630 nm and half width of 10 nm to 60 nm can be used in combination. For example, when the optical filter contains three filters, for example, the interference filter having center wavelength of 550 nm to 570 nm and half width of 10 nm to 60 nm, the interference filter having center wavelength of 600 nm to 630 nm and half width of 10 nm to 60 nm, and the interference filter having center wavelength of 650 nm to 680 nm and half width is 10 nm to 60 nm can be used in combination. When the optical filter contains four filters, the interference filter having center wavelength of 430 nm to 480 nm and half width of 10 nm to 60 nm, the interference filter having center wavelength of 550 nm to 570 nm and half width of 10 nm to 60 nm, the interference filter having center wavelength of 600 nm to 630 nm and half width of 10 nm to 60 nm, and the interference filter having center wavelength 650 nm to 680 nm and half width 10 nm to 60 nm can be used in combination.
At least one optical filter is preferably inserted into at least one through-hole provided in the optical filter holder. The optical filter may be inserted between the photodetector and the container and retracted by the optical filter holder position control unit. When retracting the optical filter, the photodetector position control unit does not bring the photodetector into contact with the container opposed thereto. The photodetector position control unit moves the photodetector to be arranged in a position as close as possible to the container and set a large solid angle to improve acquisition efficiency for light and detect the light at high sensitivity. On the other hand, when the optical filter is inserted, the photodetector may be moved away from the container but is arranged in a position close to the optical filter inserted between the photodetector and the container. The optical filter holder and the optical filter holder position control unit may be integrated.
As the pH modifier, any reagent that can change pH of a solution of a sample can be used. A change in pH of the sample solution can be selected as appropriate according to a type of luminescence whose intensity changes at a specific rate according to the change. For example, in case of luminescence by wild-type firefly luciferin-luciferase, peak luminescence intensity near 560 nm changes at pH 6.4, pH 7.0, pH 7.2, and pH 8.5 (
The luminescence measuring apparatus according to the present invention preferably contains a solution dispenser. The luminescence measuring apparatus contains, for example, at least one nozzle, at least two reagent solution reservoirs, at least one sample container, at least one piping tube, at least one liquid feeding pump connected to the piping tube, and a nozzle position control unit that moves the nozzle into the container. The nozzle position control unit can move the nozzle to the reagent reservoirs, a buffer reservoir, and an upper part of a container opening position of the sample container and insert the nozzle into the containers.
The luminescence measuring apparatus according to the present invention preferably provides a function of counting microbes in a sample. In other words, in the microbe counting apparatus, the nozzle position control unit controls the nozzle to be inserted into the container. The microbe counting apparatus sequentially suctions a solution for processing ATP chemoluminescence from distal ends of nozzles, leads the solution into a collected sample container, and extracts ATP from viable cells. Thereafter, the microbe counting apparatus mixes the solution in the collected sample container with a luciferin-luciferase luminescent reagent, calculates an ATP amount based on the luminescence intensity of the viable cells, and calculates the number of microbes on the basis of the ATP amount. The solution for processing the ATP chemoluminescence includes an ATP eliminating solution (ATP degrading enzyme, etc.) for removing unnecessary ATP molecules, ATP extraction solution (a surfactant, etc.) for extracting ATP molecules from viable cells, and a luminescent reagent that performs luminescent reaction using ATP molecules (a luciferin-luciferase reagent). Those skilled in the art can appropriately select the solution according to, for example, a type of luminescence to be measured, a measurement purpose of luminescence, and the like.
A sample to be subjected to microbe counting is not limited as long as the sample is a sample suspected to contain microbes. Examples of the sample include industrial products such as foods and beverages, pharmaceuticals, and cosmetics and raw materials of the industrial products; and environmental samples such as sea water, river water, industrial water, sewage, soil, and the air. A method of preparing these samples is known in the art.
In the luminescence measurement, first, for several seconds to several tens seconds, the luminescence measuring apparatus measures light emitted from the measurement container at all wavelength regions along a sensitivity region of the photodetector in use without dispersing light and converts the intensity of the light into data. Subsequently, the luminescence measuring apparatus inserts the optical filter between the measurement container and the photodetector, measures the light dispersed through the optical filter with the photodetector, and converts the intensity of the light into data. The luminescence measuring apparatus continuously measures light emitted from the measurement container at all wavelength regions and spectrometry at a specific wavelength range to measure an amount of light in the measurement container and distinguish an amount of luminescence due to a specific substance in the light amount and other wrong factors. In other words, quantitative information of luminescence in the container may be obtained from the light intensity at all the wavelength regions in the container. Qualitative information of luminescence, for example, information concerning presence of luminescence due to an external factor is obtained from a ratio of the light intensity at all the wavelength regions in the container and light intensity obtained by the spectrometry. In the light measurement at all the wavelength regions or the spectrometry, the luminescence measuring apparatus dispenses the pH modifier (a buffer for changing pH, etc.) and converts an intensity change with respect to pH into data to determine whether a contaminant substance is present. Further, the luminescence measuring apparatus derives only a target luminescence amount and improves reliability of a result. It is also possible to separately perform inspection related to reagent consumables using the optical filter and/or the pH modifier.
As explained above, the luminescence measuring apparatus and the microbe counting apparatus according to the present invention can determine presence or absence of luminescence due to an external factor, for example, luminescence deriving from a light blocking failure, accumulated light, or a contamination source contained in the luminescence measuring apparatus or reagents and prevent a false positive result. As a result, the luminescence measuring apparatuses can measure target luminescence with reliability and count even very few microbes in a sample with high sensitivity and high accuracy.
Specific examples of embodiments of the present invention are explained below with reference to the drawings. However, it should be noted that the embodiments are only examples for realizing the present invention and does not limit the present invention. In the figures, common components are denoted by the same reference numerals.
As a high-sensitivity detector that captures weak luminescence, a photomultiplier tube has been conventionally used. In the case of higher-sensitivity specifications, a single photon counting method for subjecting a signal of the photomultiplier tube to digital processing is adopted.
The internal configuration of the luminescence measuring apparatus is as shown in
The measurement container holder 6 has a structure for efficiently causing the photoelectric surface 11 to receive luminescence in the measurement container 5. The measurement container holder 6 reflects, in the direction of the photodetector 10, light diverging in a direction different from a direction in which the light enters into the photoelectric surface 11, and leads the light to the photoelectric surface 11. As a method for reflecting and leading the light, the use of mirror reflection is suitable. A member obtained by machining a metal material or forming a metal film on the inner surface of the measurement container holder 6 is used for the measurement container holder 6. As the metal film material, it may be preferably to use silver or aluminum with which reflection efficiency equal to or higher than 80% can be stably obtained. An internal shape of the measurement container holder 6 is preferably a taper shape or a semispherical shape.
The measurement container 5 is inserted from a columnar opening with a small diameter in an upper part of the measurement container holder 6. To fix the measurement container 5, the measurement container 5 is mounted using a brim 12 in an upper part of the measurement container 5 in a state in which the measurement container 5 hangs down in the measurement container holder 6. When the measurement container 5 not having the brim 12 is used, an exclusive stopper or the like (not shown) attached to the measurement container 5 can be prepared. The bottom of the measurement container 5 may be supported by a transparent plate. A substrate with high light transmissibility, in which transparency of a visible light region is about 100%, and made of quartz glass or resin having thickness of 0.5 mm or less may be provided in a lower part of the measurement container holder 6.
In
The photodetector 10 can be moved in a z-axis direction by a second actuator 19. This has a function for reducing and increasing a distance between the photoelectric surface 11 of the photodetector 10 and the measurement container 5. The photoelectric surface 11 is brought close to the measurement container holder 6 to set a large solid angle during measurement. To achieve this, the photodetector 10 is inserted into the top plate 8 through-hole 9 of the light blocking box 7 or moves beyond the through-hole 9.
In
In a state shown in
The center of the measurement container 5, the center of the measurement container holder 6, the center of the top plate through-hole 9, the center of the photoelectric surface 11 of the photodetector 10, and the center position of the optical filter interposed between the measurement container 5 and the photodetector 10 are aligned to be present on the same axis in the z-axis direction. This alignment is preferably executed when the luminescence measuring apparatus is assembled. As the first actuator 14 and the second actuator 19, for example, an actuator controlled by power supply or air supply can be used. The consumable kit 3 for measurement shown in
In
In
The top plate 8 of the light blocking box 7 has structure for enabling the tabular optical filter setting holder 13 to be inserted into the inside of the top plate 8. The inserted tabular optical filter setting holder 13 can be moved in a y-axis direction in the top plate 8 by the first actuator 14.
The photodetector 10 is stored in the light blocking box 7. The photodetector 10 can be moved in the z-axis direction by the second actuator 19.
The solution dispenser is composed of a first dispensing nozzle 33, a second dispensing nozzle 34, a first liquid feeding pump 35, a second liquid feeding pump 36, and a first liquid conveying pipe 37 and a second liquid conveying pipe 38 that respectively connect the first and second dispensing nozzles 33 and 34 and the first and second liquid feeding pumps 35 and 36. The solution dispenser drives the respective liquid feeding pumps 35 and 36 to suck or discharge liquid. In the example shown in
As the first liquid feeding pump 35 and the second liquid feeding pump 36, a pump of any form can be applied. Specifically, a syringe pump or a peristaltic pump is suitable. 41 denotes a buffer solution tank. The buffer solution tank 41 is connected to the second liquid feeding pump 36 via a third liquid conveying pipe 88. If electromagnetic valves having three-way valves are separately prepared and a pump having a combination of the electromagnetic valves is used, a buffer solution can be automatically supplied into the pump. The pump can be used as an automatic supply system for pipe filling water for performing washing of the liquid conveying pipes and the dispensing nozzles and improving suctioning accuracy and dispensing accuracy. This is an example of a configuration for simplifying work for buffer replacement in the pump.
The first actuator 14, the second actuator 19, the third actuator 29, the fourth actuator 30, the fifth actuator 31, the sixth actuator 32, the first liquid feeding pump 35, and the second liquid feeding pump 36 are driven on the basis of an operation sequence set by the control apparatus 2. Further, a signal from the photodetector 10 is also imported into the control apparatus 2.
A normal operation sequence in performing viable cell count using the ATP method is explained below.
As the nozzle washing solutions, ATP-free diluted and sterilized water of a standard for injection, an ATP-free HEPES buffer solution, an ATP-free PBS buffer solution, an ATP-free Tris buffer solution, and the like are suitable. To consume ATP, a luciferin-luciferase luminescent reagent may be used as the nozzle washing solution. The measurement container 5 is set in the measurement container holder 6. Thereafter, the opening and closing stage 4 is closed (S303). Subsequently, HV (High Voltage) is applied to the PMT functioning as the photodetector 10 (S304). It is possible to confirm that a dark count value is a usual value, and detect the presence or absence of a light blocking failure or accumulated light. The first actuator 14 is driven such that the passing through-hole 21 explained with reference to
Further, if the sensitivity of a measuring system is sufficient because of a sufficient ATP amount, movement of the photodetector 10 is unnecessary. The background light measurement and the ATP luminescence measurement may be carried out with the position during the dark count measurement fixed. In S307, it is confirmed that a background light value is a usual value and that there is no false positive luminescence due to contamination of consumables such as a reagent and a container.
Subsequently, the ATP eliminating solution is suctioned from the first solution container 23, dispensed into the third solution container 25, and mixed and reacted with a collected microbial sample (S308). The ATP eliminating solution is caused to react for about 10 to 30 minutes, whereby killed cells other than viable cells and free ATP are eliminated. Subsequently, the ATP extraction solution is suctioned from the second solution container 24 and dispensed into the third solution container 25. A reaction for extracting ATP from viable cells occurs (S309). Since the ATP extraction solution has a function of deactivating an enzyme reaction of the ATP eliminating solution, the ATP extracted from the viable cells is not eliminated by the ATP eliminating solution added in the preceding process. Time required for the extraction is several minutes. The ATP eliminating solution is an ATP degrading enzyme and contains an enzyme of apyrase or deaminase as a main component. The ATP extraction solution is a solution reagent for a cell membrane and contains a surfactant such as benzalkonium chloride as a main component. The ATP eliminating solution and the ATP extraction solution are commercially available as a kit by plural reagent manufacturers and can be readily available as commercial products.
Subsequently, the microbial sample in the third solution container 25 in which the ATP is extracted from the viable cells (S310) is suctioned and dispensed into the measurement container 5 (S311). The PMT functioning as the photodetector 10 is already in an ON state in S304 and continues measurement of a light amount and obtains data as spectra of continuous dark count, background light, and followed by ATP luminescence (
It is preferable that the flow of
In this example, an operation flow is explained in which spectrometry via an optical filter is added when viable cell count is performed using the ATP method.
During the background light measurement, an eliminating reaction of the free ATP and the ATP deriving from killed cells in the microbial sample and an ATP extraction reaction from the viable cells are carried out. The ATP eliminating solution is suctioned from the first solution container 23, dispensed into the third solution container 25, and mixed and reacted with the microbial sample (S717). The ATP eliminating solution is caused to react for about 10 minutes to 30 minutes, whereby ATP from the killed cells other than the viable cells and the free ATP are eliminated. Subsequently, the ATP extraction solution is suctioned from the second solution container 24 and dispensed into the third solution container 25, and a reaction for extracting ATP from the viable cells is performed (S718). The microbial sample in the third solution container 25 in which the ATP is extracted from the viable cells is suctioned (S719), and dispensed into the measurement container 5 (S720). Simultaneously with the dispensing, the ATP in the sample after the ATP extraction reaction reacts with the luminescent reagent. Biochemical luminescence of the luminescent reagent appears as an optical signal amount depending on an ATP amount (S721). Subsequently, while continuing the measurement of the ATP luminescence, the second actuator 19 is driven such that the PMT photodetector 10 is moved downward (S722), and the first optical filter 15 is inserted between the photoelectric surface 11 and the measurement container 5 or, when the measurement container 5 is held by the quartz glass thin plate 42, the first optical filter 15 is inserted between the measurement container 5 and the quartz glass thin plate 42. After performing the measurement for a certain time, the tabular optical filter setting holder 13 is further driven such that each of the second optical filter 16, the third optical filter 17, and the fourth optical filter 18 is inserted between the photoelectric surface 11 and the measurement container 5 in order or, when the measurement container 5 is held by the quartz glass thin plate 42, each of the second optical filter 16, the third optical filter 17, and the fourth optical filter 18 is inserted between the measurement container 5 and the quartz glass thin plate 42 in order, to carry out measurement for a certain time (S723 to S726). After the spectrometry via the four optical filters ends, the optical filters are returned to the position of the passing through-hole 21 and measurement are performed (S727). Finally, the HV of the PMT is turned off (S728 to S729). An ATP amount is calculated based on a light measurement value and viable cell counts are obtained (S730). After the measurement ends, the PMT photodetector 10 is moved downward with the second actuator 19, pulled out from the passing through-hole 21, and returned to the state during the operation sequence start. In this state, the operation sequence ends. The opening and closing stage 4 is opened and the first solution container 23, the second solution container 24, the third solution container 25, the fourth solution container 26, the fifth solution container 27, the sixth solution container 28, and the measurement container 5 used for the measurement are collected. The control apparatus 2 shifts to measurement of the next sample.
It is preferable that the flows of
The first optical filter 15, the second optical filter 16, the third optical filter 17, and the fourth optical filter 18 are respectively a band-pass filter (a first optical filter) having center wavelength of 562 nm (69 in
As characteristics of the ATP luminescence spectrometry spectrum 68 of firefly luciferin-luciferase, the ATP luminescence spectrometry spectrum 68 includes three center wavelengths of 560 nm, 620 nm, and 670 nm and a sum of peaks A, B, and C that can be fit by a Gaussian curve. The intensities of the three center wavelengths change according to pH of a solution (Table 1 and
In the step where the fourth optical filter 18 is used, since light detection in a region not present in an ATP luminescence spectrometry spectrum of firefly luciferin-luciferase is performed, intensity falls to photon counts equal to the dark count signal 53. In other words, the third output data 60 shown in
Photon counts 82 via the fourth optical filter 18 is wavelength unrelated to luciferin-luciferase luminescence based on ATP. Therefore, the photon counts 82 is photon counts same as the dark count signal 44. When photon counts larger than the dark count signal 44 is observed, this indicates that impurities are apparently present, stray light or accumulated light occurs, or light blocking is insufficient. For example, indoor illumination including a fluorescent tube emits light having center wavelength of 430 nm to 450 nm. Therefore, the fourth optical filter 18 is an effective filter that detects abnormality of the luminescence measuring apparatus. When the dark count signal 44 and photon counts of the fourth optical filter 18 are the same but does not reflect a peak ratio for pH in Table 1, not only ATP luminescence of firefly luciferin-luciferase but also luminescence mixed with light due to other factors is detected. Collected data is not based on accurate viable cell count. In this case, contamination that emits fluorescence of used reagents and light enter reflecting broad indoor illumination light at 500 nm to 700 nm are likely to occur. It is possible to determine that, for example, light blocking properties of the luminescence measuring apparatus, stray light entering into the luminescence measuring apparatus, and accumulated light on the photoelectric surface 11 of the photodetector 10 are suspected and re-inspection can be carried out.
When it is confirmed that viable cell counts is accurately measured, as an amount of viable cell counts itself, photon counts (1) 90 of white light of the first ATP luminescence signal 61 is converted into an ATP amount to obtain viable cell counts. The photon counts (1) 90 of white light can be a value obtained by calculating a difference of photon counts of the sixth background light signal 59. This is because, since signal intensity is high without the use of a filter, it is possible to analyze an ATP amount and convert the ATP amount into viable cell counts on the basis of data having a high SN ratio. Reliability of the data is improved. From a result obtained by performing the process according to the flowchart shown in
With respect to the qualitative information of the measurement result, (photon counts (2) 92 of white light) is represented as A, (photon counts (1) 75 via the first optical filter) is represented as B, (photon counts (2) 93 via the first optical filter) is represented as C, (photon counts (1) 76 via the second optical filter) is represented as D, (photon counts (2) 89 via the second optical filter) is represented as E, (photon counts 77 via the third optical filter) is represented as F, (photon counts 82 via the fourth optical filter) is represented as G, and (photon counts 94 of dark count) is represented as H. Then, the following expressions are obtained.
A/B=K1 (Expression 1)
C/D=K2 (Expression 2)
E/F=K3 (Expression 3)
G/H=K4 (Expression 4)
Further, when Expressions 1 to 4 are combined, the following expressions are obtained.
K1/K2/K3=fixed (Expression 5)
K2/K3=fixed (Expression 6)
K4=1 (Expression 7)
K1 to K4 are constants that change according to pH. Since a difference of reagent lots affects the K values, a slight difference occurs in a peak intensity ratio. Therefore, it is important to perform spectrometry spectrum measurement for each reagent lot, compile results of the spectrometry spectrum measurement as a database, and store the database in the control apparatus 2. Further, since a slight error is included in the K values, it is preferable to give likelihood of ±5 to 10% to the respective K values.
It may be appreciated that the optical filters are not limited with their types and the number of thereof used in this example. It is important to select optical filters according to a characteristic of wavelength of a luminescent reagent in use.
On the other hand, it is possible to check contamination of the measurement container 5 and a state of a firefly luciferin-luciferase luminescent reagent itself from the second output data 52 shown in
With respect to the qualitative information of the measurement result, (photon counts 96 of a background light signal of white light) is represented as a, (photon counts 78 of background light via the first optical filter) is represented as b, (photon counts 79 of background light via the second optical filter) is represented as c, (photon counts 80 of background light via the third optical filter) is represented as d, (photon counts 83 of background light via the fourth optical filter) is represented as e, and (photon counts 94 of dark count) is represented as f. Then, the following expressions are obtained.
a/b=k1 (Expression 8)
b/c=k2 (Expression 9)
c/d=k3 (Expression 10)
e/f=k4 (Expression 11)
Further, when Expressions 8 to 11 are combined, the following expressions are obtained.
k1/k2/k3=fixed (Expression 12)
k2/k3=fixed (Expression 13)
k4=1 (Expression 14)
Since a difference of reagent lots affects the respective k values, a slight difference occurs in a peak intensity ratio. Therefore, it is important to perform spectrometry spectrum measurement for each reagent lot, compile results of the spectrometry spectrum measurement as a database, and store the database in the control apparatus 2. Further, since a slight error is included in the k values, it is preferable to give likelihood of ±5 to 10% to the respective k values.
In this example, a representative example concerning an operation flow of microbe contamination monitoring is explained. For simplification of explanation, a luminescent consumable kit in which a luminescent reagent is filled in advance is prepared in the measurement container 5. A reagent kit containing the ATP eliminating solution, the ATP extraction solution, and the washing solutions is prepared.
A measurement sample and consumables (the luminescent consumable kit and the reagent kit) are set (S1001), measurement is started (S1002), dark count measurement (S1003) and first to sixth background light measurements (S1004 to S1009) are sequentially performed, and data analysis is carried out (S1010). When quantitative information of intrinsic luminescence and qualitative information of intrinsic fluorescence based on a luminescence spectrometry spectrum coincide with the database based on a data analysis result, the control apparatus 2 shifts to first ATP luminescence measurement (S1011). However, when the quantitative information and the qualitative information do not coincide with the database and a fifth background light measurement result is the same as a dark count, the control apparatus 2 shifts to replacement of the luminescent consumable kit (S1012) and performs measurement again (
If a loading mechanism for continuously automatically leading the luminescent consumable kit, the reagent kit, and/or the measurement sample into the luminescence measuring apparatus 1 is provided, a full automatic operation is possible except a case of a shift to apparatus maintenance.
In
As shown in
On the other hand, when it is determined as NG in the Comparison 1, Comparison 3 (K2/K3) is performed (S1310). When the output calculation value data and the spectrometry spectrum data coincide with each other, subsequently, the Comparison 2 (K4 value) is performed (S1311). When K4(data)=K4(ref) holds, a warning message instructing to carry out replacement of the consumable kit is to be announced (S1313). When the output calculation value data and the spectrometry spectrum data do not coincide with each other, a warning message instructing to carry out maintenance of the luminescence measuring apparatus (S1314) is to be announced (S1314). When it is determined as NG in the Comparison 3 (S1310), the Comparison 2 is performed (S1312). In the case of OK in a result of the Comparison 2, the warning message instructing to replace the consumable kit (S1313) is to be announced. On the other hand, a result of NG is obtained in (S1312), a warning for apparatus maintenance (S1314) is to be announced. In the case of OK in the Comparison 1 (S1304) but NG in the next Comparison 2 (S1305), the warning for apparatus maintenance (S1314) is to be announced. When the operation sequence reaches (S1313), if a replacement system for consumables is established, an automatic replacement function works and it is possible to cause the luminescence measuring apparatus to continuously operate. However, when the operation reaches (S1314), the luminescence measuring apparatus is stopped and maintenance of the luminescence measuring apparatus is performed with the intervention of a person.
It is possible to determine whether a signal is based on ATP luminescence by checking pH dependency of an ATP luminescence signal in a measurement solution. For example, when both a background signal and an ATP luminescence signal are extremely weak, for example, photon counts (1) 75 via a first optical filter equivalent to the intensity of a peak A shown in
In this example, both the optical filter and the pH modifier are used in combination. However, it is also possible to independently use the pH modifier without using the optical filter. In that case, a ratio of light intensity at all wavelength regions measured without the addition of the pH modifier and light intensity at all wavelength regions measured with the addition of the pH modifier is calculated and qualitative information of luminescence measurement can be obtained.
The present invention is not limited to the embodiments and examples explained above and includes various modifications. For example, the embodiments and examples are explained in detail in order to clearly explain the present invention. The embodiments and examples are not always limited to specific embodiments or examples including all the components explained above. A part of the components of a certain embodiment or example can be replaced with the components of another embodiment or example. The component of another embodiment or example can be added to the components of a certain embodiment or example. Concerning a part of the components of the embodiments or examples, addition of other components or deletion or replacement of the components can be performed.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011-076637 | Mar 2011 | JP | national |
