WO2022158548A1 - マイクロプレート、それを用いた測定方法、自動測定システム、及びプログラム - Google Patents
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Images
Classifications
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- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
- G01N35/1081—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices characterised by the means for relatively moving the transfer device and the containers in an horizontal plane
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/251—Colorimeters; Construction thereof
- G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/27—Association of two or more measuring systems or cells, each measuring a different parameter, where the measurement results may be either used independently, the systems or cells being physically associated, or combined to produce a value for a further parameter
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0663—Whole sensors
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/18—Means for temperature control
- B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
- B01L3/50855—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates using modular assemblies of strips or of individual wells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00346—Heating or cooling arrangements
- G01N2035/00356—Holding samples at elevated temperature (incubation)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
- G01N35/04—Details of the conveyor system
- G01N2035/0401—Sample carriers, cuvettes or reaction vessels
- G01N2035/0418—Plate elements with several rows of samples
Definitions
- the present disclosure relates to a microplate, a measurement method using the same, an automatic measurement system, and a program.
- microplates have been used for various biochemical analyses, medical diagnoses, etc.
- a microplate reader can perform optical measurements, such as absorption, fluorescence, and luminescence, using microplates.
- a microplate provided with an optical measurement cell for incident probe light from the side Patent Document 1.
- optical measurements are not suitable for, for example, trace amounts of target solutions.
- microplates (cartridges) with sensors are provided.
- microplates are provided that are microplates that include optical measurement wells and electrochemical sensors.
- Some embodiments of the present disclosure provide automated measurement systems that include or use microplates.
- Some embodiments of the present disclosure provide a measurement method, a control method thereof, a program thereof, or a storage medium thereof, using a microplate equipped with sensors.
- FIG. 2 shows a schematic side view illustrating the configuration of a microplate according to one embodiment.
- FIG. 4 shows a schematic side view illustrating the use of a microplate according to one embodiment.
- FIG. 4 shows a schematic side view illustrating the use of a microplate according to one embodiment.
- FIG. 4 shows a schematic side view illustrating the use of a microplate according to one embodiment.
- FIG. 4 shows a schematic side view illustrating the use of a microplate according to one embodiment.
- 1 shows a top view and a cross-sectional view of a microplate according to one embodiment.
- FIG. 1 shows an exploded view of a sensor according to one embodiment.
- FIG. 1 shows an exploded view of a sensor according to one embodiment.
- FIG. 2 shows an external perspective view of a microplate and a measuring instrument main body according to one embodiment.
- FIG. 4 shows a cross-sectional view of a microplate and meter body according to one embodiment.
- 1 shows a perspective view of a microplate according to one embodiment.
- FIG. 1 shows a top view and a cross-sectional view of a microplate according to one embodiment.
- the subject may include or be human.
- the subject may include non-human animals and may be non-human animals.
- the subject may include or be a mammal.
- Subjects may be, for example, without limitation, working animals, farm animals, companion animals, and wild animals.
- the sample to be measured may be a solution.
- the “solution” may be a body fluid, a solution derived from the body fluid, or a diluted solution of the body fluid.
- the solution may be a solution that is not a bodily fluid (derived from a non-bodily fluid), or a mixture of a bodily fluid or a bodily fluid-derived solution and a non-bodily fluid-derived solution.
- the solution may be the solution used for sample measurements or the solution used for calibration measurements.
- the solution may be a standard solution or a calibrator solution.
- the solution may be a liquid that does not contain the substance to be measured intentionally or intentionally for use in calibration or the like.
- the sample to be measured may be a specimen.
- the solution may be a solution containing chemicals.
- Body fluid may be lymph fluid, interstitial fluid such as interstitial fluid, intercellular fluid, interstitial fluid, body cavity fluid, serous fluid, pleural fluid, ascites, pericardial fluid, cerebrospinal fluid ( cerebrospinal fluid), synovial fluid (synovial fluid), or aqueous humor (aqueous humor).
- the bodily fluid may be a digestive fluid such as saliva, gastric juice, bile, pancreatic juice, intestinal juice, sweat, tears, runny nose, urine, semen, vaginal fluid, amniotic fluid, milk.
- the bodily fluid may be an animal bodily fluid or a human bodily fluid.
- a "bodily fluid” may be a solution.
- the solution may contain a physiological buffer such as phosphate-buffered saline (PBS) or N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (TES) containing the substance to be measured.
- PBS phosphate-buffered saline
- TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer
- the solution is not particularly limited as long as it contains the substance to be measured.
- the solution may contain the substance to be measured.
- the solution may possibly contain the substance to be measured.
- the substance to be measured may be a molecule, ion, macromolecule, biomolecule, or the like.
- the substance to be measured may comprise a biomolecule.
- the substance to be measured may be protein, glycated protein, or the like.
- the solution may be tears, and the substance to be measured may be albumin, glycoalbumin, hemoglobin, and/or glycated hemoglobin contained in tears.
- the object to be measured may be albumin, glycoalbumin, hemoglobin, or glycohemoglobin in blood, serum, or plasma, or albumin, glycoalbumin, hemoglobin, or glycohemoglobin in interstitial fluid, urine, or saliva.
- Albumin may be oxidized albumin (HNA) or reduced albumin (HMA).
- HNA oxidized albumin
- HMA reduced albumin
- the substance to be measured may be AGE (Advanced Glycation End Products).
- the substance to be measured may be a glycated lipid.
- the sensor may be a chemical sensor, biosensor, ion sensor, etc. (hereinafter sometimes referred to as “sensor”, “biochemical sensor”, “chemical sensor” or “electrochemical sensor”).
- the sensor may comprise multiple sensors.
- the sensor may have electrodes.
- the electrodes may be amperometric electrodes.
- the electrodes may comprise hydrogen peroxide electrodes.
- the electrodes may comprise oxygen electrodes.
- the electrodes may be potentiometric electrodes.
- the electrodes may be electrodes for ion detection (pH electrode, cyanide ion electrode, iodide ion electrode, etc.).
- the senor may output an electrical signal. In some aspects, the sensor may output a current signal. A sensor may output a voltage signal or an electrical charge. The sensor may be electrically connected to an ammeter, voltmeter, or the like.
- the glycated amino acids produced may be reacted with ketoamine oxidase to generate hydrogen peroxide.
- the amount (concentration) of hydrogen peroxide generated may be measured.
- the amount (concentration) of hydrogen peroxide may be measured electronically.
- ketoamine oxidase generally recognizes the ketoamine structure of a peptide or peptide fragment containing glycated amino acids or glycated amino acid residues and oxidizes the glycated amino acids to produce amino acids, glucosone ( ⁇ -ketoaldehyde) and peroxidation.
- An oxidase that produces hydrogen is proportional to or related to the concentration of the peptide or peptide fragment containing the glycated amino acid or glycated amino acid residue that it recognizes.
- a ketoamine oxidase may be a dehydrogenase, a kinase, or an oxidase.
- the ketoamine oxidase may be fructosyl amino acid oxidase (FAOD), fructosyl peptide oxidase, fructosyl valyl histidine oxidase, fructosyl amine oxidase, amadoriase, fructosylamine deglycase and modified forms thereof.
- FOD fructosyl amino acid oxidase
- FOD fructosyl peptide oxidase
- fructosyl valyl histidine oxidase fructosyl amine oxidase
- amadoriase fructosylamine deglycase and modified forms thereof.
- the ketoamine oxidase may be an oxidase (Group I ketoamine oxidase) that acts on amino acids or peptides whose ⁇ -amino groups are glycated.
- the amino acid or peptide may be valine, glycine, valylhistidine.
- a glycated hemoglobin sensor or glycated hemoglobin A1c (HbA1c) sensor can be constructed by using an oxidase that acts on an amino acid or peptide having a glycated ⁇ -amino group.
- the ketoamine oxidase may be an oxidase (group II ketoamine oxidase) that acts on amino acids or peptides in which the ⁇ -amino group is glycated.
- the amino acid may be lysine.
- the glycated amino acid may be fructosyl lysine.
- a glycoalbumin sensor can be constructed by using an oxidase that selectively acts on amino acids or peptides having glycated ⁇ -amino groups.
- the ketoamine oxidase may be an oxidase (group III ketoamine oxidase) that acts on amino acids or peptides whose ⁇ -amino group and/or ⁇ -amino group is glycated.
- the amino acid or peptide may be lysine, valine, glycine, valylhistidine.
- a glycated protein sensor can be constructed by using an oxidase that acts on an amino acid or peptide whose ⁇ -amino group and/or ⁇ -amino group is glycated.
- the senor may include a detector.
- the detector may be a hydrogen peroxide detector.
- the "hydrogen peroxide detector” (hydrogen peroxide sensor) may be an electrochemical electrode or a hydrogen peroxide electrode.
- a hydrogen peroxide electrode may have a counter electrode, a reference electrode, and a working electrode.
- the detector may detect oxygen. For example, the amount or concentration of oxygen that decreases in an enzymatic reaction may be detected.
- Oxygen detection is considered to be relatively insensitive to noise sources such as molecules and ions and tolerant to interference. Oxygen consumption may be measured by oxygen sensing. Since the detector is air saturated, it may be used for enzymatic sensing.
- the detection unit may be configured to be able to selectively or combine a plurality of detection methods.
- a sensor may have an ion exchange resin on the detector.
- the sensor may have an ion exchange resin between the ketoamine oxidase layer and the hydrogen peroxide electrode.
- a cation exchange resin such as Nafion (registered trademark)
- an anion exchange resin such as polypyrrole can be used to suppress or prevent permeation of dopamine and the like, particularly positive ions and the like, from reaching the detection unit.
- the ion exchange resin may contain one, multiple or at least one type of ion exchange resin.
- the ion exchange resin may be configured with one, multiple or at least one type of layer.
- the sensor may comprise a Molecular Imprinted Polymer (MIP) membrane. Changes in charge or potential caused by MIP molecular recognition may be detected (potentiometry type). A change in the current flowing through the electrode (for example, mediator current) caused by MIP molecular recognition may be detected (amperometric type).
- MIP Molecular Imprinted Polymer
- protease is a general term for peptide bond hydrolases that hydrolyze and catabolize proteins and polypeptides.
- a protease may be an enzyme that breaks proteins into peptide fragments.
- the peptide fragments generated by the action of protease may include peptide fragments containing glycosylated amino acid residues and peptide fragments that are not glycosylated at all.
- protease may be an animal-derived protease, a plant-derived protease, or a microorganism-derived protease.
- a protease may be an exopeptidase or an endopeptidase.
- the protease may be an aspartic protease, a metalloprotease, a serine protease, or a thiol protease.
- protease may include multiple types or types of protease, or may include one type or type of protease.
- a protease may contain one or both of an endopeptidase and an exopeptidase. Mixing multiple proteases may increase the efficiency of decomposition.
- Animal-derived proteases include trypsin, chymotrypsin, elastase, bovine pancreatic protease, cathepsin, calpain, protease type-I, protease type-XX, aminopeptidase N, carboxypeptidase, pancreatin (a mixture of multiple enzymes such as proteases and amylases). and so on.
- the plant-derived protease may be papain, bromelain, gingipain, kallikrein, ficin, chymopapain, or the like.
- Microorganism-derived proteases include Bacillus-derived proteases, Aspergillus-derived proteases, Penicillium-derived proteases, Streptomyces-derived proteases, Lysobacter-derived proteases, Yeast-derived proteases, Tritirachium-derived protease, Thermus-derived protease, Pseudomonas-derived protease, Achromobacter-derived protease, and the like may be used.
- the protease may be provided in liquid form or mixed with a solution. In some embodiments, the protease may be added or mixed into the solution in solid form (eg, powder). In some embodiments, the protease may be introduced into solution on a support material. The protease may be supported on a plate, curved surface, sphere, bead, biomolecule such as protein, polymer, or the like.
- optical measurement generally refers to determining optical properties of matter using an optical element or device.
- optical measurements of the substance of interest may be measured.
- a substance that is bound to or related to a target substance hereinafter referred to as a substance (e.g., reagent) that is chemically, biologically or physically bound to or related to a target substance, even if it is not the target substance itself) Properties may be measured. Properties of reagents may be measured.
- the reagent may be referred to as a "target substance”.
- optical measurements may include spectroscopic measurements.
- the absorbance of the target substance may be measured.
- a color changing indicator corresponding to the substance of interest may be introduced.
- a presenting indicator may be detected or measured.
- color changing indicator generally refers to halochromic chemical compounds. Color changes may generally be reversible. Color changing indicators include, for example, without limitation, acid-base indicators (pH indicators), redox indicators, adsorption indicators, TLC color changing indicators, and the like. It changes the color of its solution depending on the pH.
- the pH indicator may be, for example, without limitation, bromocresol purple (BCP), bromocresol green (BCG), and the like.
- BCP or BCG can be used for albumin.
- albumin combines with bromocresol green (BCG) to produce an albumin-bromocresol green conjugate. Around pH 4, the complex appears blue. Therefore, the albumin concentration can be obtained by measuring the absorbance (BCG method).
- BCG method bromocresol green
- a surfactant may be added to the measurement reagent. Surfactant may not be added to the measurement reagent.
- a protein denaturant such as SDS may or may not be added.
- albumin binds with bromocresol purple (BCP) to generate an albumin-bromocresol purple complex.
- BCP bromocresol purple
- This complex has a blue color. Therefore, the albumin concentration can be obtained by measuring the absorbance (BCP method).
- the albumin concentration can be measured by a color reaction with BCP after converting all of the reduced albumin into oxidized albumin by the action of an oxidizing agent (improved BCP method).
- a surfactant may be added to the measurement reagent. Surfactant may not be added to the measurement reagent.
- a protein denaturant such as SDS may or may not be added.
- the reaction in which albumin combines with bromocresol purple (BCP) or bromocresol green (BCG) to form a conjugate may be referred to as the "first reaction.”
- the color changing indicator may be provided in liquid form or mixed with a solution. In some embodiments, the color changing indicator may be added or mixed into the solution in solid form (eg, powder).
- an absorption photometer (colorimeter) may be used.
- the absorption photometer has a light source and a light receiver. All or part of the light emitted by the light source enters the target solution. All or part of the light that passes through the solution enters the receiver. The light absorbed by the target substance can be analyzed.
- the wavelength of the light source may be appropriately selected according to the color indicator used.
- Absorbance may be measured, for example, at wavelengths such as dominant wavelength 600-630 nm for BCP and 565-660 nm for BCG.
- the wavelength of measurement may be changed according to various conditions such as pH.
- the change in absorbance over time of the color indicator may be measured.
- the time at which protease decomposition starts is measured, and the total amount of protein may be determined based on the measured value corresponding to the time from this start time.
- total protein may be determined based on measurements at three or more time points. From at least three or more measurements, it may be possible to determine the initial total amount of protein even if the time from the start of protease degradation is not clear.
- measurement of glycated proteins may be started after a predetermined or predetermined time has elapsed from the start time of protease degradation.
- glycated protein measurement may begin when the absorbance A is sufficiently small.
- the change in absorbance over time ( ⁇ A/ ⁇ t) becomes substantially zero, it can be determined that sufficient protease degradation has been completed and measurement of glycated proteins can be started, or The measurement result of glycated protein at that time may be adopted.
- the change in absorbance over time reaches a predetermined value, it may be determined that degradation by a predetermined protease has been achieved, and the measurement of glycated protein may be started, or the glycated protein at that time may be measured. Measurement results may be used.
- calibration of absorbance measurements may be performed.
- An optical system including optical measurement cells, light emitters, light receivers, etc. may be calibrated.
- Absorbance may be measured before introduction of the solution into the optical system (e.g., the state in which the optical measurement cell is empty, its initial state, etc.) and after (e.g., the state in which the measurement solution has been introduced into the optical measurement cell).
- Absorbance may be measured before introduction of the solution into the optical system (e.g., the state in which the optical measurement cell is empty, its initial state, etc.) and after (e.g., the state in which the measurement solution has been introduced into the optical measurement cell).
- a difference in absorbance between them may be obtained.
- microplate generally refers to a flat plate having a plurality of small “wells” that function as test tubes. Microplates are also called microtiter plates, microwell plates, multiwells, cartridges, etc. (hereinafter sometimes referred to as “microplates”). A “microplate” is generally rectangular, but the shape is not limited to this. A “microplate” generally has a plurality of "wells” regularly arranged in a row or matrix, but the arrangement of the wells is not limited to this.
- At least one or more wells and at least one or more of the optical measurement wells may contain pretreatment solutions, dilution solutions, preparation solutions, enzyme solutions, biochemical solutions, indicator solutions, and the like.
- a microplate may include an electrochemical sensor and at least one or more wells.
- a microplate may comprise optical measurement wells, electrochemical sensors, and at least one or more wells.
- the microplate may be equipped with sensors.
- the microplate may be configured to have sensors attached or fixed.
- the sensors may be manufactured separately from the microplate and attached to the microplate.
- the microplate and the sensor may each be individually introduced into the measurement device.
- microplates and sensors may be combined in a measurement device.
- the microplate and the sensor may be physically separate, substantially combined, associated with each other, or cooperating within the measurement device to effect the preparation and measurement of the measurement solution.
- one sensor may be used for multiple microplates and may be used repeatedly.
- the sensors may be integrally molded with the microplate.
- the microplate may comprise: (a0) a diluent well containing a diluent; (b0) filter wells fitted with filters; (c0) an optical measurement preparation well containing a preparation for optical measurement; (d0) an indicator liquid well containing an indicator liquid; (e0) a first stirring well used to stir the solution; (f0) an optical measurement well capable of receiving light from the outside and extracting the light to the outside; (g0) an electrochemical assay preparation well containing a preparation (e.g., enzyme) solution for electrochemical assays; (h0) a second stirring well used to stir the solution; (i0) a sensor cleaning solution well containing a sensor cleaning solution; and (j0) a sensor calibrating solution well containing a sensor calibrating solution; at least one of and (s0) an electrochemical sensor.
- a preparation e.g., enzyme
- optical measurements may be used to measure the concentration of the protein of interest in the sample of interest.
- an electrical measurement or an electrochemical sensor may measure the concentration of the glycated protein of interest in the sample of interest.
- microplates may be used for protein analysis, detection or measurement.
- One or more wells may contain a solution containing a protease.
- Protease degrades the protein of the substance to be measured into peptide fragments or amino acids.
- Peptide fragments or amino acids thus generated may be measured optically and/or by electrochemical sensors.
- glycated proteins may be measured electrically.
- ketoamine oxidase which specifically reacts only with saccharified amino acids or peptides, is allowed to act to generate hydrogen peroxide, and the hydrogen peroxide is measured using an electrode.
- the concentration of glycated protein in the solution can be determined.
- Embodiments 1 to 5 the configurations of microplates for measuring glycated proteins and the steps of using them will be described by way of example.
- FIG. 1 shows a microplate 10 according to one embodiment.
- the microplate 10 can measure glycated proteins.
- the concentration of the protein of interest total amount of glycated and non-glycated protein
- the concentration of its glycated protein can be measured.
- Microplate 10 is capable of optical and electrical measurements.
- Microplate 10 comprises a plurality of wells (a-j) and electrochemical sensors (s).
- a-j wells
- electrochemical sensors s
- the microplate 10 shown in FIG. (a) a diluent well containing a diluent; (b) filter wells fitted with filters; (c) a prep well containing a prep for optical measurement of protein; (d) an indicator liquid well containing an indicator liquid; (e) a first stirring well used to stir the solution; (f) an optical measurement well capable of receiving light from the outside and extracting the light to the outside; (g) a protease solution well containing a protease solution; (h) a second stirring well used to stir the solution; (i) a sensor wash well containing sensor wash; (j) a sensor calibrator well containing a sensor calibrator; and (s) an electrochemical sensor; It has A microplate 10 is equipped with two pipette tips t1 and t2. They are used for optical and electrical measurements respectively.
- the filter in the filter well (b) has the ability to filter blood and remove plasma or serum.
- the filter may be a plasma or serum separation filter.
- Filter well (b) is initially empty and receives blood-introduced and filtered plasma or serum components.
- the filter may have the ability to remove dirt, substances that are unwanted or adversely affect the measurement.
- the diluent in the diluent well (a) is used to dilute the collected blood.
- the diluent solution is, for example, physiological saline (eg, phosphate buffer, PBS).
- the diluent may contain ketoamine oxidase.
- Low-molecular-weight peptides are generally present in plasma or serum. In order to measure the concentration of glycated albumin, only glycated peptide fragments produced by degrading glycated albumin with protease should be oxidized with ketoamine oxidase and the resulting hydrogen peroxide should be measured. However, glycated low-molecular-weight peptides present in blood do not undergo protease degradation and contribute to the generation of hydrogen peroxide. This can cause erroneous or noisy measurement results. By adding ketoamine oxidase to plasma or serum components before measurement, low-molecular-weight peptides in blood can be preliminarily degraded, and the causes of noise and the like can be eliminated or reduced.
- the diluent may contain catalase.
- Catalase decomposes hydrogen peroxide. Thereby, causes such as noise can be removed or reduced.
- any well eg, a well other than the diluent, may contain sodium azide.
- Sodium azide inhibits the decomposition reaction of hydrogen peroxide by catalase. By adding sodium azide, it is possible to suppress catalase, oxidize only the glycated peptide fragments produced by degrading glycated albumin with protease with ketoamine oxidase, and measure only the generated hydrogen peroxide. It becomes possible.
- the preparation liquid in the preparation liquid well (c) contains an oxidant.
- the preparation liquid may include an oxidizing agent.
- This well may contain other substances. For example, buffers, chlorides, surfactants, protein denaturants, pH adjusters, chelating agents/antioxidants (ascorbic acid), preservatives and the like may be included.
- the indicator liquid well (d) contains the BCP solution.
- the indicator may be BCP or BCG.
- This well may contain other substances. For example, buffers, chlorides, surfactants, protein denaturants, pH adjusters, chelating agents/antioxidants (ascorbic acid), preservatives and the like may be included.
- the first stirring well (e) is initially empty and receives a predetermined amount of preparation liquid, indicator liquid and dilution mixture.
- the pipetting operation sufficiently agitates the solution in the first agitation well.
- the optical measurement well (f) is initially empty and receives a mixed liquid for optical measurement. It has one or more windows that allow the entrance and exit of light from the outside.
- the protease liquid well (g) contains the protease liquid.
- This well may contain other substances. For example, buffers, chlorides, surfactants, pH adjusters, chelating agents/antioxidants (ascorbic acid), preservatives, mediators and the like may be included.
- the second stirring well (h) is initially empty and receives a predetermined amount of protease liquid and dilution mixture.
- the pipetting operation sufficiently agitates the solution in the second agitation well.
- the second stirring well is configured to be externally heated.
- the sensor cleaning liquid well (i) contains a cleaning liquid for cleaning the sensor (s).
- the sensor(s) can be cleaned and prepared for measurement before use.
- the sensor calibrator well (j) may contain a calibrator, eg a substrate for the enzyme of the enzyme sensor.
- Calibrators may contain fructosyl lysine, glycated peptide fragments, for example, to measure glycated albumin.
- the optical measurement well (f) may have a window for optical measurement for entry and/or extraction of measurement light (probe).
- An optical measurement window may be used to direct the measurement light (probe) into the optical measurement mixture and detect the light emitted therefrom.
- one optical measurement window may be provided.
- a single window may pass both incident and outgoing light.
- two or more optical measurement windows may be provided. Separate windows for incident light and windows for outgoing light may be provided.
- the incident light window and the exit light window may be provided at symmetrical positions (planes) in the optical measurement well. For example, windows for light may be provided on one side and the other side of the horizontal well.
- microplate 10 The use of the microplate 10 will be described with reference to FIGS.
- ⁇ Pretreatment> The collected body fluid (eg blood) is introduced into the diluent well (a) (Fig. 2A). Agitation is carried out to prepare a diluted mixture consisting of the introduced amount of the body fluid and the diluent (Fig. 2B).
- the diluted mixture is taken from the diluent well (a) and passed through the filter of the filter well.
- the filtered diluted mixture is introduced into the filter wells (b) (Fig. 2C). This pipetting operation may be performed with a bodily fluid introduction device.
- a predetermined amount of preparation liquid for example, an oxidizing agent
- a predetermined amount of indicator solution eg, BCP
- an indicator mixed liquid containing the preparatory liquid and the indicator is prepared (FIG. 3B).
- a predetermined amount of the filtered diluted mixture is taken from the filter well (b) by a pipetting operation and introduced into the first stirring well (e). After introduction, the solution in the first stirring well (e) is stirred. This prepares the mixed solution for optical measurement (FIG. 3C).
- a predetermined amount of the mixed liquid for optical measurement is taken from the first stirring well (e) by a pipetting operation and introduced into the optical measurement well (f).
- the optical properties of the optical measurement mixture in the optical measurement well (f) are measured (Fig. 3D).
- the optical measurement well (f) is configured to allow sufficient introduction of probe light and output of measurement light.
- the amount (eg, concentration) of the target substance (eg, protein) in the original solution can be determined based on the optical properties of the optical measurement mixture.
- the steps shown in FIGS. 3C to 3D can be replaced by other methods. For example, first introduce the indicator mixture into the optical measurement well (f) and measure the optical properties, then introduce the filtered diluted mixture into the optical measurement well (f) and measure the optical properties. may In that case, the amount of the target substance can be determined from the difference in optical properties before and after introducing the measurement sample.
- a predetermined amount of the sensor cleaning liquid is taken from the sensor cleaning liquid well (i) by a pipetting operation to wash the sensor (s) (Fig. 4B).
- the sensor calibration liquid is taken from the sensor calibration liquid well (j) and introduced into the sensor (s) (Fig. 4C). This sensor calibration solution is used to calibrate the sensor.
- a predetermined amount of sensor cleaning solution is taken from the sensor cleaning solution well (i) by pipetting to wash the sensor (s) and the calibrator solution is removed from the sensor (s) (Fig. 4D).
- the calibrator fluid may not be washed after calibration.
- the measurement liquid may be introduced to the sensor(s) in which the calibrator liquid is present and the measurement initiated.
- a predetermined amount of protease solution is taken from the protease solution well (g) by pipetting and introduced into the second stirring well (h) (Fig. 4E).
- a predetermined amount of the filtered diluted mixture is taken from the filter well (b) by a pipetting operation and introduced into the second stirring well (h). After introduction, the solution in the second stirring well (h) is stirred. This prepares the protease mixture (Fig. 4F).
- the diluted mixture introduced into the second stirring well (h) may be heated.
- the second stirring well (h) may be heated even before the diluted mixture is introduced.
- the second stirring well (h) may be heated after the diluted mixture is introduced.
- the protease mixture may be heated, for example, to the optimum temperature for the protease.
- a predetermined amount of the protease mixture is taken from the second stirring well (h) by pipetting and introduced into the sensor (s) (Fig. 4G).
- the sensor(s) make electrical measurements on the protease mixture.
- the sensor(s) may measure the time change of the output based on the time when the protease solution and the diluted mixture start to be mixed.
- the amount of target glycated protein can be determined based on measurement by the sensor(s).
- the degree of saccharification of a protein can be determined from the amount of protein determined by optical measurement in the optical measurement well (f) and the amount of glycated protein determined by the sensor (s).
- the microplate has at least (a2) a diluent well containing a diluent; (d2) an indicator liquid well containing an indicator liquid and an optical measurement preparation liquid; (e2) a first stirring well used to stir the solution; (f2) an optical measurement well having an optical measurement window; (g2) a protease liquid well containing a protease liquid; (h2) a second stirring well used to stir the solution; (s2) an electrochemical sensor; may be provided.
- the microplate may have at least six wells and one sensor.
- the microplate may be equipped with two stirring wells.
- Plasma is separated from the collected body fluid (eg blood) in advance.
- a sample containing plasma components is introduced into the diluent well (a2). This mixes the sample with the diluent. For mixing, the solution may be agitated.
- a predetermined amount of the indicator mixed solution is taken from the indicator solution well (d2) by a pipetting operation and introduced into the first stirring well (e2).
- a predetermined amount of the diluted mixture is taken from the diluent well (a2) by pipetting and introduced into the first stirring well (e2). After introduction, the solution in the first stirring well (e2) is stirred. Thus, a mixed solution for optical measurement is prepared.
- a predetermined amount of the mixed liquid for optical measurement is taken from the first stirring well (e2) by a pipetting operation and introduced into the optical measurement well (f2).
- the optical properties of the mixed solution for optical measurement in the optical measurement well (f2) are measured.
- a predetermined amount of protease solution is taken from the protease solution well (g2) by pipetting and introduced into the second stirring well (h2).
- a predetermined amount of the diluted mixture is taken from the diluent well (a2) by pipetting and introduced into the second stirring well (h2). After introduction, the solution in the second stirring well (h2) is stirred. This prepares the protease mixture.
- a predetermined amount of the protease mixed solution is taken from the second stirring (h2) by a pipetting operation and introduced into the sensor (s2).
- the sensor (s2) performs electrochemical measurements on the protease mixture.
- the diluted mixture may be prepared in a diluent well (such as a). In this case, no filter wells (such as b) are needed.
- the preparatory liquid for the optical measurement of protein may already be mixed with the indicator liquid in the indicator liquid well (eg, d). In this case, the preparation wells (such as c) are unnecessary. In some embodiments, no calibrator wells (such as j) are required.
- the sensor(s, etc.) does not require measurement preparation, such as with sensor cleaning solutions. In that case, sensor wash wells (i, etc.) are not needed.
- the microplate has at least (a3) a diluent well containing a diluent; (d3) an indicator liquid well containing a protein indicator liquid; (f3) an optical measurement well having an optical measurement window; (g3) a protease liquid well containing a protease liquid; and (j3) an electrochemical sensor; may be provided. That is, a microplate may comprise at least four wells and one sensor. The microplate need not have individual stirring wells.
- Plasma is separated from the collected body fluid (eg blood) in advance.
- a sample containing plasma components is introduced into the diluent well (a3). This mixes the sample with the diluent. For mixing, the solution may be agitated.
- a predetermined amount of diluted mixed liquid is taken from the diluent well (a3) by a pipetting operation and introduced into the indicator liquid well (d3). After introduction, the solution in the indicator liquid well (d3) is stirred. Thus, a mixed solution for optical measurement is prepared.
- a predetermined amount of the mixed liquid for optical measurement is taken from the indicator liquid well (d3) by a pipetting operation and introduced into the optical measurement well (f3).
- the optical properties of the mixed solution for optical measurement in the optical measurement well (f3) are measured.
- a predetermined amount of the diluted mixture is taken by pipetting, introduced into the optical measurement well (f3), and a predetermined amount is obtained from the indicator well (d3). and introduce it into the optical measurement well (f3). They are mixed in the optical measurement well (f3), and the optical properties of the mixed solution for optical measurement in the optical measurement well (f3) are measured.
- a predetermined amount of diluted mixture is taken from the diluent well (a3) by pipetting and introduced into the protease liquid well (g3). After introduction, the solution in the protease solution well (g3) is stirred. This prepares the protease mixture.
- the protease liquid well (g3) may be heated.
- a predetermined amount of the protease mixture is taken from the protease liquid well (g3) by pipetting and introduced into the sensor (s3).
- the sensor (s3) performs electrochemical measurements on the protease mixture.
- the indicator liquid well may contain a quantified protein indicator liquid.
- the diluted mixture may be stirred in the indicator liquid well or mixed with the protein indicator liquid.
- the diluted mixture may be stirred in the protease liquid well or mixed with the protease liquid.
- the microplate has at least (a4) a diluent well containing a diluent; (f4) an optical measurement well containing a protein indicator liquid and having an optical measurement window; (g4) a protease liquid well containing a protease liquid; and (s4) an electrochemical sensor; may be provided. That is, a microplate may comprise at least three wells and one sensor. The optical measurement wells may contain a pre-quantitated protein indicator solution. A solution may be mixed and stirred in the optical measurement well, and the solution may be measured.
- Plasma is separated from the collected body fluid (eg blood) in advance.
- a sample containing plasma components is introduced into the diluent well (a4). This mixes the sample with the diluent. For mixing, the solution may be agitated.
- a predetermined amount of diluted mixture is taken from the diluent well (a4) by pipetting and introduced into the optical measurement well (f4). After introduction, the solution in the optical measurement well (f4) is stirred. Thus, a mixed solution for optical measurement is prepared. The optical properties of the mixed solution for optical measurement in the optical measurement well (f4) are measured.
- a predetermined amount of diluted mixture is taken from the diluent well (a4) by pipetting and introduced into the protease liquid well (g4). After introduction, the solution in the protease solution well (g4) is stirred. This prepares the protease mixture.
- the protease liquid well (g4) may be heated.
- a predetermined amount of the protease mixture is taken from the protease liquid well (g4) by pipetting and introduced into the sensor (s4).
- the sensor (s4) performs electrochemical measurements on the protease mixture.
- the diluted mixture may be stirred in the optical measurement well or mixed with a mixture containing the protein indicator liquid and the optical measurement preparation liquid. In some embodiments, the diluted mixture may be stirred in the protease liquid well or mixed with the protease liquid.
- the microplate has at least (a5) a diluent well containing a diluent; (f5) an optical measurement well containing a solution containing a protein indicator and a protease solution and having an optical measurement window; and (s5) an electrochemical sensor; may be provided. That is, a microplate may comprise at least two wells and one sensor.
- the optical measurement wells may contain a solution containing a quantified protein indicator and a protease. In the optical measurement well, optical measurements may be made and the same solution introduced to the sensor.
- the protein of interest may be degraded by a protease, even in the presence of the optical measurement indicator.
- the sensor may include an indicator and be able to measure protease-degraded peptides.
- Plasma is separated from the collected body fluid (eg blood) in advance.
- a sample containing plasma components is introduced into the diluent well (a5). This mixes the sample with the diluent. For mixing, the solution may be agitated.
- a predetermined amount of diluted mixture is taken from the diluent well (a5) by pipetting and introduced into the optical measurement well (f5). After introduction, the solution in the optical measurement well (f5) is stirred. Thus, a mixed solution for optical measurement in which protease is mixed is prepared. The optical properties of the mixed solution for optical measurement in the optical measurement well (f5) are measured.
- the optical measurement well (f5) may be heated.
- a predetermined amount of the mixed liquid for optical measurement mixed with protease is taken from the optical measurement well (f5) by a pipetting operation, and introduced into the sensor (s5).
- the sensor (s5) performs electrochemical measurements on the protease mixture.
- the indicator liquid well may further contain an optical measurement preparation liquid.
- the indicator liquid wells (d2, d3, etc.) may contain a protein indicator and an optical measurement preparation liquid.
- an optical measurement well (such as f4) may contain a protein indicator and an optical measurement preparation liquid.
- an optical measurement well (such as f5) may contain a protein indicator, an optical measurement preparation, and a protease.
- an indicator was used for optical measurement and an enzyme was used for electrochemical measurement in order to measure glycated protein, but this is an example and the present disclosure is not limited to this.
- the solutions used in the wells may be other solutions.
- the object to be measured, the solution, the number of wells, the names of the wells, the steps, etc. can be changed as appropriate according to the application.
- a control measurement may be performed before, during, or after the main measurement.
- a standard solution of known concentration may be introduced and a process similar to or mutated as described above may be performed.
- the microplate may further comprise a waste tank.
- a waste tank may be configured to contain solution drained from the sensor.
- the sensor may include a waste tank. Thereby, for example, the liquid used in the microplate can be discarded together with the cartridge. This can, for example, improve the safety of handling the solution or reduce the risk of infection.
- the microplate may be provided with a cover.
- the microplate may be configured to be coupled with the cover.
- the cover can substantially prevent leakage or spillage of solutions in the wells that remain after use. This can, for example, improve the safety of handling the solution or reduce the risk of infection.
- relatively accurate or small-error measurements can be performed on trace amounts of target substances.
- the coefficient of variation (CV) can be less than 5%, 4%, 3%, 2%, 1%, or any thereof.
- GA value measurements based on glycated albumin concentration can be performed at CV values of 5% to 8% or less.
- the multiple measurements can be performed in parallel, i.e. the measurements can be performed efficiently. .
- FIG. 5 shows a top view (FIG. 5A) of a microplate 20 according to one embodiment and a cross-sectional view (FIG. 5B) taken along line AA in FIG. 5A.
- the microplate 20 has longitudinally arranged wells 20a to 20j and a sensor mounting portion 20s.
- the second stirring well 20h, the optical measurement well 20f, and the sensor mounting portion 20s are arranged on the centerline.
- the wells and positions are arranged in two longitudinal rows symmetrically about the centerline. The wells or structures arranged will be described below in order from the left side of the drawing.
- the filter well 20a and the diluent well 20b are arranged. Walls are provided around the openings of these wells. This wall 21 can receive and stably hold the filter and used blood collection device.
- an open pipette tip holder 22 is provided. These may not be wells, but may be through-holes, for example.
- a second stirring well 20h is arranged next to it.
- the second stirring wells 20h are arranged singly instead of in two rows.
- a lower or lateral space is reserved so that a heater (not shown) can be brought closer.
- the heater may have a concave surface that contacts the outer shape of the second stirring well 20h. Thereby, it is possible to contact the second stirring well 20h over a relatively wide area.
- the heater may be configured to approach the second stirring well 20h from below.
- a heater can control the temperature of the solution in the second stirring well 20h to optimize the reaction of the reagents.
- 6 wells are arranged in 2 rows. In some embodiments, they are used as prep fluid well 20c, indicator fluid well 20d, first stirring well 20e, protease fluid well 20g, sensor wash fluid well 20i, and sensor calibration fluid well 20j.
- optical measurement well 20f is arranged next to it.
- the optical measurement wells 20f are arranged singly instead of in two rows. This allows the probe light to pass through the optical measurement well 20f in a horizontal direction perpendicular to the longitudinal direction.
- a sensor mounting portion 20s for mounting a sensor (not shown) is provided on the far right of the drawing.
- the sensor is manufactured separately and attached here.
- FIG. 6 illustrates an exploded perspective view of sensor 100 according to one embodiment.
- the sensor 100 comprises a lower body 110 , a sensor substrate support 120 , a sensor substrate 130 , a channel spacer 140 , an upper body 150 , an upper sealing 160 and a set screw 170 .
- a recess is provided in the lower body 110, and the sensor substrate support 120 is laid in this interior.
- Sensor substrate 130 is typically made of glass or silicon and is therefore relatively fragile.
- the sensor substrate support 120 may be made of a relatively soft material such as resin.
- the sensor substrate 130 is placed thereon.
- the sensor substrate 130 has sensing electrodes 131 and sensing electrode terminals 132 electrically connected to the sensing electrodes 131 on its surface.
- Sensing electrode 131 senses a chemical reaction occurring at or near its surface and produces a corresponding current or voltage.
- the electrical signal is transmitted to the outside through the sensing electrode terminal 132 .
- a channel spacer 140 is placed on the sensor substrate 130 .
- the channel spacer 140 has a channel through hole 141 .
- the flow path spacer 140 is closely attached to the sensing substrate 130 so as to surround the sensing electrode 131 on the lower side and to the upper body 150 on the upper side on the surface around the flow path through hole 141 . Therefore, the channel through-hole 141 substantially determines the shape of the sensing channel by its width, thickness and length.
- Channel spacer 140 further includes terminal through-holes 142 to ensure access to sensing electrode terminals 132 .
- the upper body 150 is placed on it. Upper body 150 is mechanically fixed to lower body 110 by fixing screws 170 . As a result, the sensor substrate support 120, the sensor substrate 130, and the channel spacer 140 are in close contact with each other therebetween.
- the channel is formed as a fluidly closed structure, except for the inlet and outlet.
- the upper body 150 has a waste liquid tank 151 .
- the upper sealing 160 is attached to the upper body 150 so as to block the opening of the waste liquid tank.
- the upper ceiling 160 has air holes 161 .
- the upper body 150 has an inlet 154 for introducing the solution to be measured.
- the introduction port 154 may have a structure that facilitates attachment of a pipette, for example.
- a solution introduced from the inlet 154 flows through the channel defined by the channel through-hole 141 .
- the solution passes over the surface of sensing electrode 131 where measurements are taken.
- the upper body 150 has electrode terminals 153 .
- the lower ends of the electrode terminals 153 can pass through the terminal through holes 142 of the channel spacer 140 and come into direct contact with the sensor electrode terminals 132 of the sensor substrate 130 .
- An upper end of the electrode terminal 153 can be connected to an external electric circuit.
- the electric signal obtained by the sensor electrode 131 can be transmitted to the outside of the sensor 100.
- the solution flows up the waste liquid guide 152 vertically provided in the upper body 150 from the side opposite to the solution introduction port 154 of the channel 141 and flows into the waste liquid tank 151 from the upper end thereof. As the solution flows into the waste liquid tank 151 , the air inside it is discharged to the outside through the air holes 161 .
- the waste liquid stored in the waste liquid tank 151 does not come out from the air hole 161 during normal use. Therefore, the waste liquid can be discarded together with the sensor 100 or the microplate (cartridge) while being stored in the waste liquid tank 151 .
- FIG. 7 illustrates an exploded perspective view of sensor 200 according to one embodiment.
- Sensor 200 comprises lower body 210 , sensor substrate support 220 , sensor substrate 230 , flow path spacer 240 , upper body 250 , upper sealing 260 and fixing screw 270 .
- a recess is provided in the lower body 210, and a sensor substrate support 220 is laid inside the recess.
- Sensor substrate support 220 may be formed of a relatively soft material such as resin.
- the lower body 210 further comprises waste liquid tanks 211a, 211b.
- a sensor substrate 230 is placed thereon.
- the sensor substrate 230 has sensing electrodes 231 and sensing electrode terminals 232 electrically connected to the sensing electrodes 231 on its surface.
- Sensing electrode 231 senses chemical reactions occurring at or near its surface and produces a corresponding current or voltage.
- the electrical signal is transmitted to the outside via the sensing electrode terminal 232 .
- a channel spacer 240 is placed on the sensor substrate 230 .
- the channel spacer 240 has a channel through hole 241 .
- the flow path spacer 240 is closely attached to the sensing substrate 230 so as to surround the sensing electrode 231 on the lower side and to the upper body 250 on the upper side on the surface around the flow path through hole 241 . Therefore, the channel through-hole 241 substantially determines the shape of the sensing channel by its width, thickness and length.
- the channel spacer 240 further has terminal through-holes 242 that ensure access to the sensing electrode terminals 232 .
- Channel spacer 240 further includes a waste through-hole 243 that allows solution flow between lower body 210 and upper body 250 .
- the upper body 250 is placed on it. Upper body 250 is mechanically fixed to lower body 210 by fixing screws 270 . As a result, the sensor substrate support 220, the sensor substrate 230, and the channel spacer 240 are in close contact with each other therebetween.
- the channel is formed as a fluidly closed structure, except for the inlet and outlet.
- the upper body 250 has an inlet 254 for introducing the solution to be measured.
- the solution introduced from the inlet 254 flows through the channel defined by the channel through hole 241 .
- the solution passes over the surface of sensing electrode 231 where a measurement is taken.
- the upper body 250 has electrode terminals 253 .
- the lower ends of the electrode terminals 253 can pass through the terminal through holes 242 of the channel spacer 240 and come into direct contact with the sensor electrode terminals 232 of the sensor substrate 230 .
- An upper end of the electrode terminal 253 can be connected to an external electric circuit.
- the electric signal obtained by the sensor electrode 231 can be transmitted to the outside of the sensor 200.
- the solution flows from the side opposite to the solution inlet 254 of the channel 241 up and down the first waste liquid guide 252a provided in the upper body 250 in the vertical direction, and flows through one of the waste liquid through-holes 243 of the channel spacer 240. and into the first waste tank 211 a of the lower body 210 .
- the waste liquid from the channel may continue to be supplied. At that time, the waste liquid flows from the first waste liquid tank 211a through one of the waste liquid through-holes 243 of the channel spacer 240, up the second waste liquid guide 252b, over the flow channel, and then to the opposite side. It flows into the second waste liquid tank 211b of the lower body 210 located there.
- An upper sealing 260 is attached to the top of the upper body 250 .
- Upper channels of the waste liquid guides 252a and 252b have an open structure.
- a top sealing 260 closes them off and seals off the waste guides 252a, 252b.
- the upper sealing 260 further has air holes 261 .
- the air inside the first waste tank 211a and the second waste tank 211 passes through another waste through-hole of the channel spacer 240, passes through the air guide 252c of the upper body 250, and finally reaches the upper sealing 260. is discharged to the outside from the air hole 261 of the .
- waste liquid stored in the waste liquid tanks 211a and 211b does not come out from the air holes 261 during normal use. Therefore, the waste liquid can be discarded together with the sensor 200 or the microplate (cartridge) while being stored in the waste liquid tanks 211a and 211b.
- the well containing the solution in advance may be sealed with a seal.
- the seal may be peeled off prior to use. Movement of the pipette tip may cause its tip to penetrate the seal.
- the electrodes of the sensors of Examples 2 and 3 may be hydrogen peroxide electrodes, oxygen electrodes, other electrodes, or combinations thereof.
- Automatic measurement system> 8 and 9 are perspective and cross-sectional views, respectively, showing the configuration of an automatic measurement system 500 comprising a microplate 510, a measurement body 520, and a pipette 530 according to one embodiment.
- the microplate 510 extends in the longitudinal direction and has a sensor 510s fitted at one end thereof.
- a pipette tip 511 and a filter 512 may be arranged in advance on the microplate 510 .
- a blood collection tube 513 is depicted schematically.
- the measuring body 520 has a linear rail (or guide) 521, which is configured to receive the inserted microplate 510 by sliding in the longitudinal direction (X) direction.
- the measuring body 520 is equipped with a heater system 522 .
- the heater system 522 is configured to contact or approach the bottom and sides of the heating well (e.g., second stirring well) 510i of the microplate 510 and heat it, and move the heater 522a up and down. and a heater moving mechanism 522b that can move the heater.
- Heater system 522 may be connected to a power source (not shown).
- the measurement main body 520 includes an optical measurement system 523 including a light emitting element 523a and a light receiving element 523b.
- the optical path from the light emitting element 523a to the light receiving element 523b is defined perpendicular to the longitudinal direction of the microplate 510 or the rail 521 and passes through the optical measurement wells 510f of the microplate 510. FIG. Thereby, optical properties in the optical measurement well 510f can be measured.
- the measuring body 520 includes an electrical measuring system 524 .
- Electrical measurement system 524 includes probe terminals 524a that can be lowered to contact electrode terminals (not shown) of sensor 510s. Electrical signals output from sensors 510s are received by electrical measurement system 524, analyzed, and/or further transmitted externally.
- the electrical measurement system 524 may be electromagnetically connected, either wirelessly or by wire, to an external computing unit.
- the pipette 530 is configured to move up and down (Z direction) relative to the measurement body 520 or the microplate 510 fixed thereto and in a horizontal direction (Y direction) perpendicular to the longitudinal direction.
- Pipette 530 is coupled to or includes a movement mechanism (pipette movement mechanism, not shown) that allows such movement.
- the measuring body 520 is configured to move in the longitudinal direction (X direction) of the microplate 510 relative to the pipette 530 within the horizontal plane. Measurement body 520 is coupled to a movement mechanism (measurement body movement mechanism, not shown) that enables such movement.
- the movement mechanism, heater system 522, and electrical measurement system 524 may each, or some or all of them collectively, be connected to a control system (not shown).
- the automated measurement device may further comprise a computer or computer system and may be calibrated connectable thereto. From or after attachment of the microplate to the device body, various steps in the method of the present disclosure may be computer controlled.
- the computer may comprise a central processing unit (CPU) and a storage medium storing programs for controlling them.
- the senor may be calibrated before, during, after the measurement, or at multiple times thereof.
- the configuration may use a predetermined calibration liquid.
- the sensor may be pre-calibrated at the time of shipment or manufacture.
- the sensor may have a read code such as a bar code, QR code (registered trademark), character code, image code, or the like.
- the code may be attached or printed on the surface of the sensor body.
- the code may be a magnetic code.
- the cord may be attached inside the sensor.
- the code may be associated with sensor information such as manufacturing information, shipping information, calibration information, and the like. A user or device can obtain relevant information by reading the code. Reading the code may obtain the calibration line for that sensor.
- a microplate may be equipped with multiple sensors. In some embodiments, a microplate may be configured by connecting a plurality of submicroplates. In some embodiments, one well group and one sensor may be provided for one specimen. In some embodiments, multiple well groups and one sensor may be provided for one specimen. In some embodiments, one well group and multiple sensors may be provided for one analyte. In some embodiments, multiple well groups and multiple sensors may be provided for one specimen.
- FIG. 10 shows a perspective view of a microplate 30 according to one embodiment.
- the microplate 30 includes, for example, eight rows of submicroplates 30-1 to 30-8.
- each sub-microplate may be a microplate as described in the examples or embodiments above.
- Each sub-microplate 30-1 to 30-8 in FIG. 10 is provided with sensors 30s-1 to 30s-n, respectively. This allows measurements to be performed in parallel. Measurements can be performed efficiently or with high throughput.
- FIG. 11 shows a top view (FIG. 11A) of a microplate 40 according to one embodiment and a cross-sectional view (FIG. 11B) taken along line AA in FIG. 11A.
- the microplate 40 has longitudinally arranged wells 40a to 40j and a sensor mounting portion 40s.
- One difference from the microplate 20 of Example 1 shown in FIG. 5 is as follows. That is, in the microplate 20 of Example 1 shown in FIG. 5, some of the wells are arranged in two rows in the longitudinal direction symmetrically with respect to the center line. On the other hand, in the microplate 40 shown in FIG. 11, all the wells are arranged longitudinally in a line on the center line. The wells or structures arranged will be described below in order from the left side of the drawing.
- the filter well 40a is arranged.
- a filter (not shown) is press-fitted into this filter well 40a.
- the bottom has a partial bevel that allows the filtered solution (or diluted mixture) to efficiently collect at the deepest point.
- a pipette tip rest 42 has a wall located in the opening. As a result, a pipette tip or the like can be stably held so as not to be displaced.
- the pipette tip rest may have a closed bottom surface, as shown in FIG. 11B. As a result, liquid drips that may occur during and after use can be retained in the cartridge, and the device can be kept clean and free from contamination.
- a sensor washing liquid well 40i and a diluent well 40b are arranged.
- the measuring device approaches the pipette cartridge 40 from below and has a heater surrounding and heating the first stirring well 40e.
- the internal temperature can be controlled to suit the desired mixing or agitation.
- a protease liquid well 40g, a sensor calibration liquid well 40j, a preparation liquid well 40c, and an indicator liquid well 40d are arranged in series.
- An optical measurement well 40f is arranged next to it.
- the optical measurement well 40f is configured to allow the probe light to pass through the optical measurement well 40f in a horizontal direction perpendicular to the longitudinal direction.
- the measuring device has a heater (having a concave surface) that approaches the pipette cartridge 40 from below and contacts these wells from the sides and below in a so-called U-shape. This allows the solutions contained in these wells to be mixed or set to a temperature suitable for measurement within the optical measurement well 40f.
- this heater may be configured to simultaneously heat protease liquid well 40g, sensor calibration liquid well 40j, preparation liquid well 40c, indicator liquid well 40d, and optical measurement well 40f.
- the heater may be configured to simultaneously heat protease liquid well 40g, sensor calibration liquid well 40j, prep liquid well 40c, and indicator liquid well 40d.
- this heater may be configured to heat only the optical measurement wells 40f or to heat the optical measurement wells 40f individually. In some embodiments, the heater is configured to separately heat protease liquid well 40g, sensor calibration liquid well 40j, preparation liquid well 40c, indicator liquid well 40d, and optical measurement well 40f. may
- a sensor mounting portion 40s for mounting a sensor (not shown) is provided on the far right of the drawing.
- the sensor is manufactured separately and attached here.
- ⁇ Application example> Although the above embodiments and examples were described based on the measurement of albumin and glycated albumin, the present disclosure is not limited thereto. In some embodiments, two different measurands can be measured substantially simultaneously or in parallel. Optical measurements may be made on one and electrochemical measurements may be made on the other. In some embodiments, optical measurement is performed on one of multiple forms of one substance due to deformation, modification, difference in group bonding, presence or absence, etc., and electrochemical measurement is performed on the other one. good.
- the present disclosure provides various target substances and various measurement methods. Some applications are given below by way of non-limiting illustration.
- enzymatic sensing may be performed using any of the microplates of the present disclosure.
- predetermined enzymatic reactions may be performed on various substrates, and the products may be measured by a sensor.
- sensing can be performed using a multi-step, two-step enzymatic reaction. Multiple enzymatic reactions may differ in their indicated conditions. Also, one enzymatic reaction may rate-limit the entire enzymatic reaction. By dividing the enzymatic reaction and precisely controlling the amount and time of each by pipetting, accurate and error-free enzymatic sensing can be performed.
- cholesterol may be enzymatically sensed.
- Cholesterol and fatty acids are produced from cholesterol ester and water by the enzymatic reaction of cholesterol esterase.
- Cholestenone and hydrogen peroxide are produced from cholesterol and oxygen by the enzymatic reaction of cholesterol oxidase. By measuring oxygen and hydrogen peroxide at this time, the initial amount of cholesterol ester and/or cholesterol can be obtained.
- the cholesterol esterase solution and the cholesterol oxidase solution may be contained in predetermined wells.
- the sensor may comprise oxygen electrodes, hydrogen peroxide electrodes or other electrodes, or a combination thereof.
- enzymatic sensing may be performed on sucrose.
- ⁇ -D-glucose and fructose are produced from sucrose and water by an enzymatic reaction of invertase.
- ⁇ -D-glucose is produced from ⁇ -D-glucose by the enzymatic reaction of mutarotase.
- Gluconolactone and hydrogen peroxide are produced from ⁇ -D-glucose and oxygen by an enzymatic reaction of glucose oxidase (GOX). By measuring oxygen and hydrogen peroxide at this time, the amount of initial sucrose and/or intermediate products can be obtained.
- GOX glucose oxidase
- the invertase solution and the mutarotase solution may be contained in predetermined wells.
- the sensor may comprise oxygen electrodes, hydrogen peroxide electrodes or other electrodes, or a combination thereof.
- molecules to be measured and enzymes to be used are not limited to the above.
- glucose, phosphatidylcholine, neutral lipid, and other substrates may be subjected to one or more steps of enzymatic reaction in one or more wells, and an intermediate or final product in the enzymatic reaction may be detected with a sensor. good.
- the solution may contain cofactors.
- the solution may contain enzymes and cofactors.
- glucose may be measured using GOX and mediators (ferrocene derivatives, 1,4-benzoquinone, tetrathiafulvalene, etc.).
- mediators may be contained in respective wells.
- the pH of the system in a stable state after adding the mediator. Therefore, the sensitivity was not high.
- the pH can be adjusted to the reducing side and the mediator can be added there. Measurements can be made in that sensitive state. pH changes over time. However, from a state of high sensitivity, it is possible to measure changes in the output of the sensor over time. This allows more accurate and efficient measurements.
- a microplate of any of the present disclosure may be used to measure the titer of an enzyme of interest. For example, enzyme titer may be assessed.
- the enzyme to be evaluated is introduced into the first well. This prepares the enzyme solution. Take an aliquot of the enzyme solution and place it in the optical measurement well. An optical measurement can be performed on this enzyme to determine the total amount of initial enzyme. Take an aliquot of the enzyme solution and introduce it into the second well.
- the oxygen solution is mixed with a known amount or concentration of substrate.
- the substrate may be housed in the second well in advance, or may be introduced into the second well from another well or externally before or after introduction of the oxygen solution. This prepares the substrate mixture.
- Substrates may be, for example, glycated peptides, glycated lysine, and the like. Hydrogen peroxide is thereby produced by an enzymatic reaction.
- this reaction liquid is taken and introduced into the sensor.
- the output as a function of time from the substantial start time of the reaction between the substrate and the enzyme may be measured.
- the amount of enzyme that was active in the enzyme solution can then be determined.
- the titer of the enzyme can be determined from the total amount of enzyme and the amount of active enzyme.
- multiple sensors may be arranged.
- a plurality of sensors may detect or quantify different target substances.
- the ratio of two substances of interest may be determined.
- the albumin/globulin ratio (A/G ratio) may be determined.
- Albumin and globulin may be treated with solutions corresponding to each, and may be measured by methods or sensing adapted to each.
- balance measurements may be made. For example, amino acid balance may be measured. Depending on the functional group of each amino acid, multiple measurement methods (optical measurement, electrochemical measurement) may be used. For example, metallobalance may be measured (MB test). For example, hormone balance may be measured.
- water quality can be measured.
- optical measurements can be used to assess the optical properties of a solution of interest.
- the optical measurements may be spectroscopic measurements. From the properties at each wavelength, the types and/or amounts of each substance in the solution can be determined.
- Electrical measurements can be used to determine pH, amount of electrolytes, and the like. Together, the optical and electrical properties of water can be used to assess the quality of a subject's water.
- microorganisms may be used to evaluate subject solutions. Plant microorganisms may be added to the subject solution. In some embodiments, different microorganisms may be introduced per well. In some embodiments, one or more of the same microorganisms may be introduced into multiple wells, and light irradiation conditions may be set for each well. The mixed solution may be exposed to light of multiple or predetermined wavelengths or intensities. The temperature in the well may be adjusted to the temperature adapted for each microorganism. Thereby, each microorganism generates oxygen by respiration. Alternatively, each plant microorganism generates carbon dioxide through photosynthesis. Their respiration and photosynthesis depend on water quality, an environmental indicator. The solution is introduced into the sensor after a predetermined reaction time. A sensor measures the amount of oxygen and/or carbon dioxide in the solution. Thereby, water quality such as BOD can be measured.
- A001 at least one (or more) wells; and an electrochemical sensor, Microplate with.
- A002 A microplate for optical and electrochemical measurements, comprising at least one (or more) wells; an optical measurement well; and an electrochemical sensor, Microplate with.
- A011 A microplate for optical and electrical measurements, comprising: a diluent well containing a diluent; an optical measurement well containing a solution containing a protein indicator and a protease and having an optical measurement window; and an electrochemical sensor, Microplate with.
- A011b A microplate according to A011, Either one of the dilution well and the optical measurement well contains a preparation solution for optical measurement of protein, microplate.
- A012 A microplate for optical and electrical measurements, comprising: a diluent well containing a diluent; An optical measurement well containing a protein indicator liquid and having an optical measurement window; a protease liquid well containing a protease liquid; and a microplate with an electrochemical sensor.
- A012b A microplate according to A012, Either one of the dilution well and the optical measurement well contains a preparation solution for optical measurement of protein, microplate.
- a microplate for optical and electrical measurements comprising: a diluent well containing a diluent; an indicator liquid well containing a protein indicator liquid; an optical measurement well having an optical measurement window; a protease liquid well containing a protease liquid; and a microplate with an electrochemical sensor.
- A013b A microplate according to A013, any one of the dilution well, the indicator solution well, and the optical measurement well contains a preparation solution for optical measurement of protein; microplate.
- A014 A microplate for optical and electrical measurements, comprising: (a) a diluent well containing a diluent; (d) an indicator liquid well containing an indicator liquid; (e) a first stirring well used to stir the solution; (f) an optical measurement well having an optical measurement window; (g) a protease solution well containing a protease solution; (h) a second stirring well used to stir the solution; and (s) an electrochemical sensor; Microplate with.
- A014b A microplate according to A014, any one of the wells (a) to (h) contains a preparatory liquid for optical measurement of protein; microplate.
- a microplate for optical and electrical measurements comprising: (a) a diluent well containing a diluent; (b) filter wells fitted with filters; (c) a prep well containing a prep for optical measurement of protein; (d) an indicator liquid well containing an indicator liquid; (e) a first stirring well used to stir the solution; (f) an optical measurement well capable of receiving light from the outside and extracting the light to the outside; (g) a protease solution well containing a protease solution; (h) a second stirring well used to stir the solution; (i) a sensor wash well containing a sensor wash; and (s) an electrochemical sensor; Microplate with.
- A016 A microplate for optical and electrical measurements comprising: (a) a diluent well containing a diluent; (b) filter wells fitted with filters; (c) a prep well containing a prep for optical measurement of protein; (d) an indicator liquid well containing an indicator liquid; (e) a first stirring well used to stir the solution; (f) an optical measurement well capable of receiving light from the outside and extracting the light to the outside; (g) a protease solution well containing a protease solution; (h) a second stirring well used to stir the solution; (i) a sensor wash well containing sensor wash; (j) a sensor calibrator well containing a sensor calibrator; and (s) an electrochemical sensor; Microplate with.
- a microplate for optical and electrical measurements comprising: (a) a diluent well containing a diluent; (b) filter wells fitted with filters; (c) a prep well containing a prep for optical measurement of protein; (d) an indicator liquid well containing an indicator liquid; (e) a stirring well used to stir the solution; (f) an optical measurement well capable of receiving light from the outside and extracting the light to the outside; (g) a protease solution well containing a protease solution; (i) a sensor wash well containing sensor wash; (j) a sensor calibrator well containing a sensor calibrator; and (s) an electrochemical sensor; Microplate with.
- A021 The microplate of any one of embodiments A001-A016b, further comprising a waste tank configured to contain effluent from the electrochemical sensor.
- B001 An automatic measurement system for determining the saccharification degree of protein, at least one pipette head; a microplate comprising at least one (or more) wells, optical measurement wells, and electrochemical sensors; a drive system for moving the pipette head and the microplate relative to each other; an electrical measurement unit configured to be connected to the electrochemical sensor;
- a system with B002 The system of embodiment B001, wherein: The system further comprising a temperature control system for controlling the temperature of at least a portion of said microplate.
- the temperature control system comprises a heater configured to move relative to the microplate and proximate at least one well.
- the system further comprising a computer that controls at least one of operation of the drive system, heating by the heater, and operation of the heater.
- C001 A method for electrochemically measuring a target substance, providing a microplate according to any one of embodiments A001 through A021; providing a liquid that may contain the substance of interest; introducing the liquid into any well of the microplate by pipetting; Performing a process necessary for measurement on the liquid or the target substance in the well or another well of the microplate; introducing the treated solution in the well into the electrochemical sensor by a pipetting operation; and detecting or measuring a target substance using the electrochemical sensor; How to prepare.
- a method for measuring the saccharification degree of protein comprising: providing a microplate according to embodiment A016; obtaining body fluids that may have glycated proteins; introducing the collected bodily fluid into a diluent well to prepare a diluent mixture; By pipetting, a mixture of a predetermined amount of the body fluid and the diluent (diluted mixture) is taken from the diluent well and passed through the filter of the filter well, and the filtered diluted mixture is placed in the filter well.
- C012 The method of embodiment C011, wherein: The method wherein preparing the protease mixture comprises heating the solution in the second stirring well to activate the protease reaction.
- C021 A method for measuring the saccharification degree of protein, comprising: providing a microplate according to embodiment A016b; obtaining body fluids that may have glycated proteins; introducing the collected bodily fluid into a diluent well to prepare a diluent mixture; By pipetting, a mixture of a predetermined amount of the body fluid and the diluent (diluted mixture) is taken from the diluent well and passed through the filter of the filter well, and the filtered diluted mixture is placed in the filter well.
- BCP BCP from the indicator solution well and introducing it into the optical measurement well to prepare the indicator mixture; pipetting a predetermined amount of the filtered diluted mixture from the filter wells and into the optical measurement wells to prepare the optical measurement mixture; measuring the optical properties of the optical measurement mixture in the optical measurement well; Determining the amount of protein based on the optical properties of the optical measurement mixture; pipetting a predetermined amount of sensor wash from the sensor wash well and onto the sensor; pipetting a predetermined amount of sensor calibrator solution from the sensor calibrator well and flowing it through the sensor; calibrating the sensor with a sensor calibration fluid; pipetting a predetermined amount of protease solution from the protease solution well and introducing it into the first stirring well; pipetting a predetermined amount of the filtered diluted mixture from the filter well and into the first stirring well to provide a protease mixture; A heater surrounding the first stirring well heats the first stirring well at a predetermined temperature for a predetermined time to allow the protease mixture to react.
- D001 Computer software for causing a computer to perform the method of embodiments C001 through C012.
- E001 A storage medium storing the computer software of embodiment D001.
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Abstract
Description
いくつかの実施形態では、対象者(被検者)は、ヒトを含んでいてもよく、ヒトであってもよい。いくつかの実施形態では、対象者は、ヒト以外の動物を含んでいてもよく、ヒト以外の動物であってもよい。対象者は、哺乳類動物を含んでいてもよく、哺乳類動物であってもよい。対象者は、例えば非限定的に、使役動物、家畜動物、愛玩動物、野生動物であってもよい。
センサは、化学センサ、バイオセンサ、イオンセンサなど(以下、「センサ」、「生化学センサ」、「化学センサ」又は「電気化学センサ」と呼ぶ場合がある。)であってもよい。センサは、複数のセンサを備えていてもよい。
いくつかの実施形態では、生成された糖化アミノ酸をケトアミンオキシダーゼと反応させて過酸化水素を発生させてもよい。いくつかの実施形態では、発生した過酸化水素の量(濃度)を測定してもよい。いくつかの実施形態では、過酸化水素の量(濃度)を電気的に測定してもよい。
ある実施形態に係るセンサは検出器上にイオン交換樹脂を有していてもよい。センサは、ケトアミンオキシダーゼ層と過酸化水素電極との間にイオン交換樹脂を有していてもよい。
センサは、分子鋳型ポリマ(Molecular Imprinted Polymer, MIP)膜を備えていてもよい。MIPの分子認識により生じる電荷又は電位の変化を検出してもよい(ポテンシオメトリ型)。MIPの分子認識により生じる電極に流れる電流(例えばメディエータの電流)の変化を検出してもよい(アンペロメトリ型)。
「プロテアーゼ」は一般に、タンパク質やポリペプチドを加水分解して異化する、ペプチド結合加水分解酵素の総称である。プロテアーゼは、タンパク質をペプチド断片に分解する酵素であってもよい。タンパク質が糖化されたアミノ酸残基を含む場合、プロテアーゼの作用に生じたペプチド断片には、糖化されたアミノ酸残基を含むペプチド断片と、全く糖化されていないペプチド断片が存在し得る。
本明細書で用いる「光学測定」は、一般に、光学素子又はデバイスを用いて物質の光学的特性を決定することをさす。いくつかの実施形態では、対象物質の光学測定を測定してもよい。いくつかの態様では、対象物質に結合された又は関連する物質(以下、対象物質そのものでなくとも、対象物質に化学的、生物的又は物理的に結合し又は関連する物質(例えば試薬))の特性を測定してもよい。試薬の特性を測定してもよい。その試薬を、「対象物質」という場合がある。
本明細書で用いられる「呈色指示薬」とは、一般に、ハロクロミックな化学合成物をさす。色の変化は一般に可逆的であってもよい。呈色指示薬は、例えば非限定的に、酸塩基指示薬(pH指示薬)、酸化還元指示薬、吸着指示薬、TLC呈色指示薬などを含む。それはpHに応じてその溶液の色を変化させる。pH指示薬は、例えば非限定的に、ブロモクレゾールパープル(BCP)、ブロモクレゾールグリーン(BCG)などであってもよい。
いくつかの実施形態では、吸光光度計(比色計)を用いてもよい。吸光光度計は、光源と受光器とを有する。光源から出た光の全て又は一部は、対象溶液内に入る。溶液を通った光の全て又は一部は受光器に入る。対象物質が吸収した光を分析することができる。
本開示は、マイクロプレートを提供する。本明細書で用いる「マイクロプレート」は、一般に、複数の、試験管として機能する小型の「ウェル」を有する平板をさす。マイクロプレートは、マイクロタイタープレート、マイクロウェルプレート、マルチウェル、カートリッジなどとも呼ばれる(以下、「マイクロプレート」と呼ぶ場合がある。)。「マイクロプレート」は、一般に、長方形に形成されているが、形状はこれに限定されない。「マイクロプレート」は、一般に、複数の、列状又はマトリックス状に規則的に並べられた「ウェル」を有しているが、ウェルの配列はこれに限定されない。
いつかの実施形態では、マイクロプレートは、以下を備えていてもよい:
(a0) 希釈液を内包する希釈液ウェル;
(b0) フィルタが装着されたフィルタウェル;
(c0) 光学測定のための準備液を内包する光学測定準備液ウェル;
(d0) 指示薬液を内包する指示薬液ウェル;
(e0) 溶液を攪拌するために用いられる第一攪拌ウェル;
(f0) 外部から光を入射し外部に光を取り出すことができる光学測定ウェル;
(g0) 電気化学測定のための準備(例えば酵素)溶液を内包する電気化学測定準備液ウェル;
(h0) 溶液を攪拌するために用いられる第二攪拌ウェル;
(i0) センサ洗浄液を内包するセンサ洗浄液ウェル;及び
(j0) センサ較正液を内包するセンサ較正液ウェル;
の少なくとも一つ、
及び
(s0) 電気化学センサ。
図1に、ある実施形態に係るマイクロプレート10を示す。マイクロプレート10は、糖化タンパク質を測定することができる。対象タンパク質の濃度(糖化及び非糖化タンパク質の合計量)とその糖化タンパク質の濃度とを測定することができる。マイクロプレート10は、光学測定と電気測定とを行うことができる。マイクロプレート10は、複数のウェル(a~j)と電気化学センサ(s)とを備えている。本実施形態のマイクロプレート10を参照して、一例としてしかし非限定的に、血液中のアルブミン濃度の光学測定と、糖化アルブミン濃度の電気化学センサを用いた測定について説明する。
(a) 希釈液を内包する希釈液ウェル;
(b) フィルタが装着されたフィルタウェル;
(c) タンパク質の光学測定のための準備液を内包する準備液ウェル;
(d) 指示薬液を内包する指示薬液ウェル;
(e) 溶液を攪拌するために用いられる第一攪拌ウェル;
(f) 外部から光を入射し外部に光を取り出すことができる光学測定ウェル;
(g) プロテアーゼ液を内包するプロテアーゼ液ウェル;
(h) 溶液を攪拌するために用いられる第二攪拌ウェル;
(i) センサ洗浄液を内容するセンサ洗浄液ウェル;
(j) センサ較正液を内包するセンサ較正液ウェル;及び
(s) 電気化学センサ;
を備えている。マイクロプレート10には、2つのピペットチップt1,t2が装着されている。それぞれ光学測定及び電気測定のために用いられる。
採取された体液(例えば血液)を、希釈液ウェル(a)に導入する(図2A)。攪拌を行い、導入した量の体液と希釈液とからなる希釈混合液を準備する(図2B)。
光学測定のための溶液の取り扱いには、一方のピペットチップt1を用いる(図3A)。図3B~Dでは、ピペットチップt1が用いられるが、図中には示されていない。
電気測定のための溶液の取り扱いには、他方のピペットチップt2を用いる(図4A)。図4B~Gでは、ピペットチップt2が用いられるが、図中には示されていない。
いくつかの実施形態では、マイクロプレートは少なくとも、
(a2) 希釈液を内包する希釈液ウェルと;
(d2) 指示薬液及び光学測定用準備液を内包する指示薬液ウェルと;
(e2) 溶液を攪拌するために用いられる第一攪拌ウェルと;
(f2) 光学測定用窓を有する光学測定ウェルと;
(g2) プロテアーゼ液を内包するプロテアーゼ液ウェルと;
(h2) 溶液を攪拌するために用いられる第二攪拌ウェルと;
(s2) 電気化学センサと;
を備えていてもよい。
いくつかの実施形態では、マイクロプレートは少なくとも、
(a3) 希釈液を内包する希釈液ウェル;
(d3) タンパク質用指示薬液を内包する指示薬液ウェル;
(f3) 光学測定用窓を有する光学測定ウェル;
(g3) プロテアーゼ液を内包するプロテアーゼ液ウェル;及び
(j3) 電気化学センサ;
を備えていてもよい。すなわち、マイクロプレートは、少なくとも4つのウェルと1つのセンサとを備えていてもよい。マイクロプレートは、攪拌ウェルを個別に備えていなくてもよい。
いくつかの実施形態では、マイクロプレートは少なくとも、
(a4) 希釈液を内包する希釈液ウェル;
(f4) タンパク質用指示薬液を内包し、光学測定用窓を有する光学測定ウェル;
(g4) プロテアーゼ液を内包するプロテアーゼ液ウェル;及び
(s4) 電気化学センサ;
を備えていてもよい。すなわち、マイクロプレートは、少なくとも3つのウェルと1つのセンサとを備えていてもよい。光学測定ウェルは、予め定量されたタンパク質用指示薬液を収容していてもよい。光学測定ウェル内で、溶液の混合・攪拌を行い、かつその溶液に対して測定を行ってもよい。
いくつかの実施形態では、マイクロプレートは少なくとも、
(a5) 希釈液を内包する希釈液ウェル;
(f5) タンパク質用指示薬及びプロテアーゼ液を含む溶液を内包し、光学測定用窓を有する光学測定ウェル;及び
(s5) 電気化学センサ;
を備えていてもよい。すなわち、マイクロプレートは、少なくとも2つのウェルと1つのセンサとを備えていてもよい。光学測定ウェルは、定量された、タンパク質用指示薬及びプロテアーゼを含む溶液を収容していてもよい。光学測定ウェル内で、光学測定を行い、同じ溶液をセンサに導入してもよい。いくつかの実施形態では、対象タンパク質は、光学測定用指示薬の存在下でも、プロテアーゼにより分解されてもよい。いくつかの実施形態では、センサは、指示薬を含んでいても、プロテアーゼにより分解されたペプチドを測定することができてもよい。
いくつかの実施形態では、マイクロプレートは、更に廃液タンクを備えていてもよい。いくつかの実施形態では、廃液タンクは、センサから排出される溶液を収容するように構成されていてもよい。いくつかの実施形態では、センサは廃液タンクを備えていてもよい。これにより、例えば、マイクロプレートで使用された液体を、カートリッジごと廃棄することができる。これにより例えば、溶液の取り扱いの安全性を向上させ、又は感染リスクを低減することができる。
いくつかの実施形態では、マイクロプレートは、カバーを備えていてもよい。マイクロプレートは、カバーと結合されるように構成されていてもよい。カバーは、使用後に残るウェル内の溶液が漏れる又はこぼれることを実質的に防ぐことができる。これにより例えば、溶液の取り扱いの安全性を向上させ、又は感染リスクを低減することができる。
図5に、一実施例に係るマイクロプレート20の上面図(図5A)と、図5A中のA-A断面図(図5B)を示す。マイクロプレート20は、長手方向に配置されたウェル20a~20jと、センサ取付け部20sとを有している。第二攪拌ウェル20h、光学測定ウェル20f、及びセンサ取付け部20sは、中心線上に配置されている。そのウェル及びポジションは、中心線に対して対称に、長手方向に2列に配置されている。以下、図面の左側から順に、配置されているウェル又は構造について説明する。
図6に、一実施例に係るセンサ100の分解斜視図を示す。センサ100は、下部本体110、センサ基板サポート120、センサ基板130、流路スペーサ140、上部本体150、上部シーリング160、及び固定ネジ170を備えている。
図7に、一実施例に係るセンサ200の分解斜視図を示す。センサ200は、下部本体210、センサ基板サポート220、センサ基板230、流路スペーサ240、上部本体250、上部シーリング260、及び固定ネジ270を備えている。
図8及び図9に、一実施例に係るマイクロプレート510、測定本体520及びピペット530を備える自動測定システム500の構成を示す斜視図及び断面図をそれぞれ示す。
いくつかの実施形態では、センサは、測定の前、途中、後、又はそれらの複数の時点で較正されてもよい。構成には、所定の較正液を用いてもよい。いくつかの実施形態では、センサは、予め、出荷時又は製造時に較正されていてもよい。センサは、バーコード、QRコード(登録商標)、文字コード、画像コードなどの読取りコードを有していてもよい。コードは、センサ本体の表面に取り付け又は印刷されていてもよい。コードは、磁気コードであってもよい。コードは、センサ内部に取り付けられていてもよい。コードは、製造情報、出荷情報、較正情報などのセンサ情報に関連づけられていてもよい。ユーザ又は装置は、コードを読み取ることで、関連情報を取得することができる。コードを読み取ることで、そのセンサの較正線を取得してもよい。
図10に、一実施例に係るマイクロプレート30の斜視図を示す。マイクロプレート30は、一例として、8列のサブマイクロプレート30-1~30-8を備えている。いくつかの実施形態では、各サブマイクロプレートでは、上記の実施例又は実施形態で説明したようなマイクロプレートであってもよい。図10の各サブマイクロプレート30-1~30-8は、それぞれセンサ30s-1~30s-nを備えている。これにより、測定を、並列的に実行することができる。測定を効率的に又は高いスループットで行うことができる。
図11に、一実施例に係るマイクロプレート40の上面図(図11A)と、図11A中のA-A断面図(図11B)を示す。マイクロプレート40は、長手方向に配置されたウェル40a~40jと、センサ取付け部40sとを有している。図5に示す実施例1のマイクロプレート20との一つの相違点は以下のものである。すなわち、図5に示す実施例1のマイクロプレート20では、その一部のウェルが、中心線に対して対称に、長手方向に2列に並んで配列されている。これに対し、図11に示すマイクロプレート40では、すべてのウェルが長手方向に、中心線上に一列に並んで配列されている。以下、図面の左側から順に、配置されているウェル又は構造について説明する。
上記の実施形態及び実施例は、アルブミン及び糖化アルブミンの測定をベースに記載したが、本開示はこれに限られない。いくつかの実施形態では、2つの異なる測定対象物質を実質的に同時に又は並行して測定することができる。一方について光学測定を行い、他方について電気化学測定を行ってもよい。いくつかの実施形態では、ある一つの物質の、変形、修飾、基の結合の相違又は有無などによる複数の形について、一方について光学測定を行い、その他の一つについて電気化学測定を行ってもよい。本開示は、種々の対象物質及び種々の測定方法を提供する。以下、非限定的な例示として、いくつかの応用例を示す。
いくつかの実施形態では、本開示のいずれかのマイクロプレートを用いて、酵素センシングを行ってもよい。例えば、種々の基質に対して、所定の酵素反応を行い、その生産物をセンサにより測定してもよい。
いくつかの実施形態では、コレステロールについて酵素センシングを行ってもよい。コレステロールエステルと水から、コレステロールエステラーゼの酵素反応により、コレステロールと脂肪酸とが生成される。コレステロールと酸素から、コレステロールオキシダーゼの酵素反応により、コレステノンと過酸化水素が生成される。この際の酸素、過酸化水素を測定することで、当初のコレステロールエステル、及び又はコレステロールの量を求めることができる。
いくつかの実施形態では、スクロースについて酵素センシングを行ってもよい。スクロースと水から、インベルターゼの酵素反応により、α―D-グルコースとフルクトースとが生成される。α―D-グルコースから、ムタロターゼの酵素反応により、β―D-グルコースが生成される。β―D-グルコースと酸素から、グルコースオキシダーゼ(GOX)の酵素反応により、グルコノラクトンと過酸化水素が生成される。この際の酸素、過酸化水素を測定することで、当初のスクロース、及び/又は中間生成物の量を求めることができる。
いくつかの実施形態では、グルコースを、GOXとメディエータ(フェロセン誘導体、1,4-ベンゾキノン、テトラチアフルバレンなど)とを用いて測定してもよい。GOXとメディエータとをそれぞれのウェルに収容してもよい。
いくつかの実施形態では、本開示のいずれかのマイクロプレートを用いて、対象酵素の力価を測定してもよい。例えば、酵素力価を評価してもよい。
いくつかの実施形態では、2つの対象物質の比率を求めてよい。例えば、アルブミン/グロブリン比(A/G比)を求めてもよい。アルブミンとグロブリンとは、それぞれに対応した溶液によって処理されてもよく、それぞれに適応した方法又はセンシングで測定されてもよい。
いくつかの実施形態では、バランス測定を行ってもよい。例えば、アミノ酸バランスを測定してもよい。各アミノ酸の官能基に応じて、複数の測定方法(光測定、電気化学測定)を用いてもよい。例えば、メタロバランスを測定してもよい(MB検査)。例えば、ホルモンバランスを測定してもよい。
いくつかの実施形態では、水質を測定することができる。例えば、光測定により対象の溶液の光学的特性を評価することができる。光測定は分光測定であってもよい。各波長での特性から溶液中の物質の種類及び/又は各々の量を求めることができる。電気的測定により、pH、電解質の量などを求めることができる。水の光学的特性と電気的特性とを併せて対象の水の質を評価することができる。
いくつかの実施形態では、微生物を用いて対象溶液を評価してもよい。対象溶液に、植物性の微生物を加えてもよい。いくつかの態様では、ウェルごとに異なる微生物を導入してもよい。いくつかの態様では、一つ又は複数の同じ微生物を複数のウェルに導入し、ウェルごとに、光の照射条件を設定してもよい。その混合溶液に、複数の又は所定の波長又は強度の光を当ててもよい。各微生物に対して適応した温度に、そのウェル内の温度を調節してもよい。それにより、各微生物が呼吸により酸素を発生させる。あるいは、各植物性微生物が、光合成により二酸化炭素を発生させる。それらの、呼吸及び光合成は、環境指標である水質に依存する。所定の反応時間後に溶液をセンサに導入する。センサは溶液内の酸素及び/又は二酸化炭素の量を測定する。それにより、BODなどの水質を測定することができる。
A001
少なくとも1つ(又は複数の)ウェル;及び
電気化学センサ、
を備えるマイクロプレート。
A002
光学測定と電気化学的測定とを行うためのマイクロプレートであって、少なくとも1つ(又は複数の)ウェル;
光学測定ウェル;及び
電気化学センサ、
を備えるマイクロプレート。
A011
光学測定と電気的測定とを行うためのマイクロプレートであって、
希釈液を内包する希釈液ウェル;
タンパク質用指示薬及びプロテアーゼを含む溶液を内包し、光学測定用窓を有する光学測定ウェル;及び
電気化学センサ、
を備えるマイクロプレート。
A011b
A011に記載のマイクロプレートであって、
前記希釈ウェル及び前記光学測定ウェルのいずれか一つに、タンパク質の光学測定のための準備液が内包されている、
マイクロプレート。
A012
光学測定と電気的測定とを行うためのマイクロプレートであって、
希釈液を内包する希釈液ウェル;
タンパク質用指示薬液を内包し、光学測定用窓を有する光学測定ウェル;
プロテアーゼ液を内包するプロテアーゼ液ウェル;及び
電気化学センサ
を備えるマイクロプレート。
A012b
A012に記載のマイクロプレートであって、
前記希釈ウェル及び前記光学測定ウェルのいずれか一つに、タンパク質の光学測定のための準備液が内包されている、
マイクロプレート。
A013
光学測定と電気的測定とを行うためのマイクロプレートであって、
希釈液を内包する希釈液ウェル;
タンパク質用指示薬液を内包する指示薬液ウェル;
光学測定用窓を有する光学測定ウェル;
プロテアーゼ液を内包するプロテアーゼ液ウェル;及び
電気化学センサ
を備えるマイクロプレート。
A013b
A013に記載のマイクロプレートであって、
前記希釈ウェル、前記指示薬液ウェル、及び前記光学測定ウェルのいずれか一つに、タンパク質の光学測定のための準備液が内包されている、
マイクロプレート。
A014
光学測定と電気的測定とを行うためのマイクロプレートであって、
(a) 希釈液を内包する希釈液ウェル;
(d) 指示薬液を内包する指示薬液ウェル;
(e) 溶液を攪拌するために用いられる第一攪拌ウェル;
(f) 光学測定用窓を有する光学測定ウェル;
(g) プロテアーゼ液を内包するプロテアーゼ液ウェル;
(h) 溶液を攪拌するために用いられる第二攪拌ウェル;及び
(s) 電気化学センサ;
を備えるマイクロプレート。
A014b
A014に記載のマイクロプレートであって、
前記ウェル(a)から(h)のいずれか一つに、タンパク質の光学測定のための準備液が内包されている、
マイクロプレート。
A015
光学測定と電気的測定とを行うためのマイクロプレートであって、
(a) 希釈液を内包する希釈液ウェル;
(b) フィルタが装着されたフィルタウェル;
(c) タンパク質の光学測定のための準備液を内包する準備液ウェル;
(d) 指示薬液を内包する指示薬液ウェル;
(e) 溶液を攪拌するために用いられる第一攪拌ウェル;
(f) 外部から光を入射し外部に光を取り出すことができる光学測定ウェル;
(g) プロテアーゼ液を内包するプロテアーゼ液ウェル;
(h) 溶液を攪拌するために用いられる第二攪拌ウェル;
(i) センサ洗浄液を内包するセンサ洗浄液ウェル;及び
(s) 電気化学センサ;
を備えるマイクロプレート。
A015b
A015に記載のマイクロプレートであって、
前記ウェル(a)から(i)のいずれか一つに、タンパク質の光学測定のための準備液が内包されている、
マイクロプレート。
A016
光学測定と電気的測定とを行うためのマイクロプレートであって、
(a) 希釈液を内包する希釈液ウェル;
(b) フィルタが装着されたフィルタウェル;
(c) タンパク質の光学測定のための準備液を内包する準備液ウェル;
(d) 指示薬液を内包する指示薬液ウェル;
(e) 溶液を攪拌するために用いられる第一攪拌ウェル;
(f) 外部から光を入射し外部に光を取り出すことができる光学測定ウェル;
(g) プロテアーゼ液を内包するプロテアーゼ液ウェル;
(h) 溶液を攪拌するために用いられる第二攪拌ウェル;
(i) センサ洗浄液を内包するセンサ洗浄液ウェル;
(j) センサ較正液を内包するセンサ較正液ウェル及び
(s) 電気化学センサ;
を備えるマイクロプレート。
A016b
光学測定と電気的測定とを行うためのマイクロプレートであって、
(a) 希釈液を内包する希釈液ウェル;
(b) フィルタが装着されたフィルタウェル;
(c) タンパク質の光学測定のための準備液を内包する準備液ウェル;
(d) 指示薬液を内包する指示薬液ウェル;
(e) 溶液を攪拌するために用いられる攪拌ウェル;
(f) 外部から光を入射し外部に光を取り出すことができる光学測定ウェル;
(g) プロテアーゼ液を内包するプロテアーゼ液ウェル;
(i) センサ洗浄液を内包するセンサ洗浄液ウェル;
(j) センサ較正液を内包するセンサ較正液ウェル及び
(s) 電気化学センサ;
を備えるマイクロプレート。
A021
実施形態A001からA016bのいずれか一項に記載のマイクロプレートであって、前記電気化学センサからの排出液を収容するように構成された廃液タンクを更に備えるマイクロプレート。
B001
タンパク質の糖化度を求める自動測定システムであって、
少なくとも一つのピペットヘッドと、
少なくとも一つ(又は複数)のウェル、光学測定ウェル、及び電気化学センサを備えるマイクロプレートと、
前記ピペットヘッド及び前記マイクロプレートを相対的に動かすための駆動システムと、
前記電気化学センサに接続されるように構成された電気測定ユニットと、
を備えるシステム。
B002
実施形態B001に記載のシステムであって、
前記マイクロプレートの少なくとも一部の温度を制御する温度制御システムを更に備えるシステム。
B003
実施形態B002に記載のシステムであって、
前記温度制御システムは、前記マイクロプレートに対して相対的に移動し、少なくとも一つのウェルに近接するように構成されたヒータを備えるシステム。
B011
実施形態B003に記載のシステムであって、
前記駆動システムの動作、前記ヒータによる加熱、及び前記ヒータの動作の少なくとも一つを制御するコンピュータを更に備えるシステム。
C001
対象物質を電気化学的に測定する方法であって、
実施形態A001からA021のいずれか一項に記載のマイクロプレートを提供すること;
対象物質を含む可能性を有する液体を提供すること;
ピペッティング操作により、前記液体を前記マイクロプレートのいずれかのウェルに導入すること;
前記ウェル内又は前記マイクロプレートの他のウェル内で前記液体又は前記対象物質に対して、測定に必要な処理を行うこと;
ピペッティング操作により、前記処理された前記ウェル内の溶液を電気化学センサに導入すること;及び
前記電気化学センサを用いて、対象物質を検出し又は測定すること;
を備える方法。
C011
タンパク質の糖化度を測定する方法であって、
実施形態A016に記載のマイクロプレートを提供すること;
糖化タンパク質を有する可能性がある体液を取得すること;
採取された体液を、希釈液ウェルに導入し、希釈混合液を準備すること;
ピペッティング操作により、所定の量の体液と希釈液との混合液(希釈混合液)を、希釈液ウェルから取り、フィルタウェルのフィルタを通過させ、フィルタされた希釈混合液は、フィルタウェル内に導入すること;
フィルタをフィルタウェルから取り外すこと;
ピペッティング操作により、所定の量の準備液ウェル内の溶液(例えば酸化剤)を、準備液フィルタウェルから取り、第一攪拌ウェルに導入すること;
ピペッティング操作により、所定の量の指示薬液(例えばBCP)を指示薬液ウェルから取り、第一攪拌ウェルに導入し、指示薬混合液を準備すること;
ピペッティング操作により、所定の量のフィルタされた混合液を、フィルタウェルから取り、第一攪拌ウェルに導入し、光学測定用混合液を準備すること;
ピペッティング操作により、所定の量の光学測定用混合液を、第一攪拌ウェルから取り出し、光学測定ウェルに導入すること;
光学測定ウェル内にある光学測定用混合液の光学特性を測定すること;
光学測定用混合液の光学特性に基づいて、タンパク質の量を求めること;
ピペッティング操作により、所定の量のセンサ洗浄液を、センサ洗浄液ウェルからとり、センサに流すこと;
ピペッティング操作により、所定の量のセンサ較正液を、センサ較正液ウェルからとり、センサに流すこと;
センサ較正液を用いてセンサを較正すること;
ピペッティング操作により、所定の量のプロテアーゼ溶液を、プロテアーゼ液ウェルから取り、第二攪拌ウェルに導入すること;
ピペッティング操作により、所定の量のフィルタされた混合液を、フィルタウェルから取り、第二攪拌ウェルに導入し、プロテアーゼ混合液を準備すること;
ピペッティング操作により、所定の量のプロテアーゼ混合液を、プロテアーゼ導入から所定に時刻に、第二攪拌ウェルから取り、センサに導入すること;
センサにより、プロテアーゼ混合液に対して、電気測定を行うこと;
電気測定に基づいて、対象となる糖化タンパク質の量を求めること;及び
求められたタンパク質の量と、求められた糖化タンパク質の量とから、タンパク質の糖化度を求めること、
を備える方法。
C012
実施形態C011に記載の方法であって、
前記プロテアーゼ混合液を準備することは、前記第二攪拌ウェル内の溶液を加熱し、プロテアーゼ反応を活性化させることを備える方法。
C021
タンパク質の糖化度を測定する方法であって、
実施形態A016bに記載のマイクロプレートを提供すること;
糖化タンパク質を有する可能性がある体液を取得すること;
採取された体液を、希釈液ウェルに導入し、希釈混合液を準備すること;
ピペッティング操作により、所定の量の体液と希釈液との混合液(希釈混合液)を、希釈液ウェルから取り、フィルタウェルのフィルタを通過させ、フィルタされた希釈混合液は、フィルタウェル内に導入すること;
フィルタをフィルタウェルから取り外すこと;
ピペッティング操作により、所定の量の準備液(例えば酸化剤)を準備液ウェルから取り、光学測定ウェルに導入すること;
ピペッティング操作により、所定の量の指示薬液(例えばBCP)を指示薬液ウェルから取り、光学測定ウェルに導入し、指示薬混合液を準備すること;
ピペッティング操作により、所定の量のフィルタされた希釈混合液を、フィルタウェルから取り、光学測定ウェルに導入し、光学測定用混合液準備すること;
光学測定ウェル内にある光学測定用混合液の光学特性を測定すること;
光学測定用混合液の光学特性に基づいて、タンパク質の量を求めること;
ピペッティング操作により、所定の量のセンサ洗浄液を、センサ洗浄液ウェルからとり、センサに流すこと;
ピペッティング操作により、所定の量のセンサ較正液を、センサ較正液ウェルからとり、センサに流すこと;
センサ較正液を用いてセンサを較正すること;
ピペッティング操作により、所定の量のプロテアーゼ溶液を、プロテアーゼ液ウェルから取り、第一攪拌ウェルに導入すること;
ピペッティング操作により、所定の量のフィルタされた希釈混合液を、フィルタウェルから取り、第一攪拌ウェルに導入し、プロテアーゼ混合液を準備すること;
第一撹拌ウェルの周囲のヒータにより、所定温度で、所定時間、第一撹拌ウェルを加熱し、プロテアーゼ混合液を反応させること
ピペッティング操作により、所定の量のプロテアーゼ混合液を、プロテアーゼ導入から所定に時刻に、第一攪拌ウェルから取り、センサに導入すること;
センサにより、プロテアーゼ混合液に対して、電気測定を行うこと;
電気測定に基づいて、対象となる糖化タンパク質の量を求めること;及び
求められたタンパク質の量と、求められた糖化タンパク質の量とから、タンパク質の糖化度を求めること、
を備える方法。
D001
実施形態C001からC012に記載の方法を、コンピュータに実行させるためのコンピュータソフトウェア。
E001
実施形態D001のコンピュータソフトウェアを格納する記憶媒体。
Claims (17)
- 少なくとも1つ又は複数のウェル;及び
電気化学センサ、
を備えるマイクロプレート。 - 光学測定と電気化学的測定とを行うためのマイクロプレートであって、少なくとも1つ(又は複数の)ウェル;
光学測定ウェル;及び
電気化学センサ、
を備えるマイクロプレート。 - 光学測定と電気的測定とを行うためのマイクロプレートであって、
希釈液を内包する希釈液ウェル;
タンパク質用指示薬及びプロテアーゼを含む溶液を内包し、光学測定用窓を有する光学測定ウェル;及び
電気化学センサ、
を備えるマイクロプレート。 - 光学測定と電気的測定とを行うためのマイクロプレートであって、
希釈液を内包する希釈液ウェル;
タンパク質用指示薬液を内包し、光学測定用窓を有する光学測定ウェル;プロテアーゼ液を内包するプロテアーゼ液ウェル;及び
電気化学センサ
を備えるマイクロプレート。 - 光学測定と電気的測定とを行うためのマイクロプレートであって、
希釈液を内包する希釈液ウェル;
タンパク質用指示薬液を内包する指示薬液ウェル;
光学測定用窓を有する光学測定ウェル;
プロテアーゼ液を内包するプロテアーゼ液ウェル;及び
電気化学センサ
を備えるマイクロプレート。 - 光学測定と電気的測定とを行うためのマイクロプレートであって、
(a) 希釈液を内包する希釈液ウェル;
(d) 指示薬液を内包する指示薬液ウェル;
(e) 溶液を攪拌するために用いられる第一攪拌ウェル;
(f) 光学測定用窓を有する光学測定ウェル;
(g) プロテアーゼ液を内包するプロテアーゼ液ウェル;
(h) 溶液を攪拌するために用いられる第二攪拌ウェル;及び
(s) 電気化学センサ;
を備えるマイクロプレート。 - 光学測定と電気的測定とを行うためのマイクロプレートであって、
(a) 希釈液を内包する希釈液ウェル;
(b) フィルタが装着されたフィルタウェル;
(c) タンパク質の光学測定のための準備液を内包する準備液ウェル;
(d) 指示薬液を内包する指示薬液ウェル;
(e) 溶液を攪拌するために用いられる第一攪拌ウェル;
(f) 外部から光を入射し外部に光を取り出すことができる光学測定ウェル;
(g) プロテアーゼ液を内包するプロテアーゼ液ウェル;
(h) 溶液を攪拌するために用いられる第二攪拌ウェル;
(i) センサ洗浄液を内容するセンサ洗浄液ウェル;及び
(s) 電気化学センサ;
を備えるマイクロプレート。 - 光学測定と電気的測定とを行うためのマイクロプレートであって、
(a) 希釈液を内包する希釈液ウェル;
(b) フィルタが装着されたフィルタウェル;
(c) タンパク質の光学測定のための準備液を内包する準備液ウェル;
(d) 指示薬液を内包する指示薬液ウェル;
(e) 溶液を攪拌するために用いられる第一攪拌ウェル;
(f) 外部から光を入射し外部に光を取り出すことができる光学測定ウェル;
(g) プロテアーゼ液を内包するプロテアーゼ液ウェル;
(h) 溶液を攪拌するために用いられる第二攪拌ウェル;
(i) センサ洗浄液を内包するセンサ洗浄液ウェル;
(j) センサ較正液を内包するセンサ較正液ウェル及び
(s) 電気化学センサ;
を備えるマイクロプレート。 - 請求項1から8のいずれか一項に記載のマイクロプレートであって、
前記電気化学センサからの排出液を収容するように構成された廃液タンクを更に備えるマイクロプレート。 - タンパク質の糖化度を求める自動測定システムであって、
少なくとも一つのピペットヘッドと、
少なくとも一つ(又は複数)のウェル、光学測定ウェル、及び電気化学センサを備えるマイクロプレートと、
前記ピペットヘッド及び前記マイクロプレートを相対的に動かすための駆動システムと、
前記電気化学センサに接続されるように構成された電気測定ユニットと、
を備えるシステム。 - 請求項10に記載のシステムであって、
前記マイクロプレートの少なくとも一部の温度を制御する温度制御システムを更に備えるシステム。 - 請求項11に記載のシステムであって、
前記温度制御システムは、前記マイクロプレートに対して相対的に移動し、少なくとも一つのウェルに近接するように構成されたヒータを備えるシステム。 - 請求項12に記載のシステムであって、
前記駆動システムの動作、前記ヒータによる加熱、及び前記ヒータの動作の少なくとも一つを制御するコンピュータを更に備えるシステム。 - 対象物質を電気化学的に測定する方法であって、
請求項1から9のいずれか一項に記載のマイクロプレートを提供すること;
対象物質を含む可能性を有する液体を提供すること;
ピペッティング操作により、前記液体を前記マイクロプレートのいずれかのウェルに導入すること;
前記ウェル内又は前記マイクロプレートの他のウェル内で前記液体又は前記対象物質に対して、測定に必要な処理を行うこと;
ピペッティング操作により、前記処理された前記ウェル内の溶液を電気化学センサに導入すること;及び
前記電気化学センサを用いて、対象物質を検出し又は測定すること;
を備える方法。 - タンパク質の糖化度を測定する方法であって、
請求項8に記載のマイクロプレートを提供すること;
糖化タンパク質を有する可能性がある体液を取得すること;
採取された体液を、希釈液ウェルに導入し、希釈混合液を準備すること;
ピペッティング操作により、所定の量の体液と希釈液との混合液(希釈混合液)を、希釈液ウェルから取り、フィルタウェルのフィルタを通過させ、フィルタされた希釈混合液は、フィルタウェル内に導入すること;
フィルタをフィルタウェルから取り外すこと;
ピペッティング操作により、所定の量の準備液ウェル内の溶液(例えば酸化剤)を、準備液フィルタウェルから取り、第一攪拌ウェルに導入すること;
ピペッティング操作により、所定の量の指示薬液(例えばBCP)を指示薬液ウェルから取り、第一攪拌ウェルに導入し、指示薬混合液を準備すること;
ピペッティング操作により、所定の量のフィルタされた混合液を、フィルタウェルから取り、第一攪拌ウェルに導入し、光学測定用混合液を準備すること;
ピペッティング操作により、所定の量の光学測定用混合液を、第一攪拌ウェルから取り出し、光学測定ウェルに導入すること;
光学測定ウェル内にある光学測定用混合液の光学特性を測定すること;
光学測定用混合液の光学特性に基づいて、タンパク質の量を求めること;
ピペッティング操作により、所定の量のセンサ洗浄液を、センサ洗浄液ウェルからとり、センサに流すこと;
ピペッティング操作により、所定の量のセンサ較正液を、センサ較正液ウェルからとり、センサに流すこと;
センサ較正液を用いてセンサを較正すること;
ピペッティング操作により、所定の量のプロテアーゼ溶液を、プロテアーゼ液ウェルから取り、第二攪拌ウェルに導入すること;
ピペッティング操作により、所定の量のフィルタされた混合液を、フィルタウェルから取り、第二攪拌ウェルに導入し、プロテアーゼ混合液を準備すること;
ピペッティング操作により、所定の量のプロテアーゼ混合液を、プロテアーゼ導入から所定に時刻に、第二攪拌ウェルから取り、センサに導入すること;
センサにより、プロテアーゼ混合液に対して、電気測定を行うこと;
電気測定に基づいて、対象となる糖化タンパク質の量を求めること;及び
求められたタンパク質の量と、求められた糖化タンパク質の量とから、タンパク質の糖化度を求めること、
を備える方法。 - 請求項15に記載の方法であって、
前記プロテアーゼ混合液を準備することは、前記第二攪拌ウェル内の溶液を加熱し、プロテアーゼ反応を活性化させることを備える方法。 - 請求項14から16に記載の方法を、コンピュータに実行させるためのコンピュータソフトウェア。
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