Microplate, measurement method using same, automatic measurement system, and program

Abstract

Through an embodiment of the present invention, there is provided a microplate comprising an electrochemical sensor and at least one well (or a plurality thereof).

Description

Microplate, measurement method using same, automatic measurement system, and program

[0001] The present disclosure relates to a microplate, a measurement method using the same, an automatic measurement system, and a program.

[0002] Conventionally, microplates have been used in various biochemical analyses, medical diagnoses, and the like. By using a microplate with an automatic pipetting device, it is possible to obtain a large amount of data efficiently and with good reproducibility under the same conditions. A microplate reader can perform optical measurements such as absorbance, fluorescence, and luminescence using the microplate. Microplates equipped with optical measurement cells for lateral incidence of probe light are also known (Patent Document 1).

[0003] Patent Publication No. 2016-24054

[0004] However, optical measurement is not suitable for very small amounts of target solution. Furthermore, when performing electrochemical measurements, the microplate must be removed from the main device and placed in an appropriate sensing device. This can lead to errors in the reaction time from preparation, such as mixing the solutions in the microplate, to electrochemical measurement.

[0005] In some embodiments of the present disclosure, a microplate (cartridge) comprising a sensor is provided.

[0006] In some embodiments, a microplate (cartridge) is provided that includes an optical measurement well and an electrochemical sensor.

[0007] In some embodiments of the present disclosure, an automated measurement system is provided that includes or uses a microplate.

[0008] Some embodiments of the present disclosure provide a measurement method using a microplate equipped with sensors, a control method thereof, and a program or storage medium thereof.

[0009] This allows, for example, relatively accurate or small error measurements to be made for minute amounts of a target substance, for example, when multiple measurements (optical and one or more sensor measurements) are made, measurements with small inter-measurement errors can be made, or multiple measurements can be made in parallel.

[0010]

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0011] FIG. 1 shows a schematic side view illustrating the configuration of a microplate according to one embodiment. FIG. 2 shows a schematic side view illustrating the use of a microplate according to one embodiment. FIG. 3 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. 5 shows a schematic side view illustrating the use of a microplate according to one embodiment. FIG. 6 shows a top view and a cross-sectional view of a microplate according to one embodiment. FIG. 7 shows an exploded view of a sensor according to one embodiment. FIG. 8 shows an exploded view of a sensor according to one embodiment. FIG. 9 shows a perspective view of the appearance of a microplate and a measuring device main body according to one embodiment. FIG. 10 shows a cross-sectional view of a microplate and a measuring device main body according to one embodiment. FIG. 11 shows a perspective view of a microplate according to one embodiment. FIG. 12 shows a top view and a cross-sectional view of a microplate according to one embodiment.

[0012] <Measurement Subject> In some embodiments, the subject (test subject) may include or may be a human. In some embodiments, the subject may include or may be a non-human animal. The subject may include or may be a mammal. The subject may be, for example, but not limited to, a working animal, a livestock animal, a pet, or a wild animal.

[0013] The sample to be measured may be a solution. The "solution" may be a body fluid, a solution derived from a body fluid, or a diluted solution of a body fluid. The solution may be a solution that is not a body fluid (non-body fluid-derived), or a mixture of a body fluid or a body fluid-derived solution and a non-body fluid-derived solution. The solution may be a solution used for sample measurement, or a solution used for calibration measurement. For example, the solution may be a standard solution or a calibration solution. For example, the solution may be a liquid that does not intentionally or deliberately contain the substance to be measured, so that it is used for calibration, etc. The sample to be measured may be a specimen. The solution may be a solution containing a chemical substance.

[0014] The "body fluid" may be lymph, tissue fluid such as interstitial fluid, intercellular fluid, or interstitial fluid, or may be body cavity fluid, serous cavity fluid, pleural fluid, ascites, pericardial fluid, cerebrospinal fluid (spinal fluid), synovial fluid, or aqueous humor (aqueous humor). The body fluid may be digestive fluid such as saliva, gastric juice, bile, pancreatic juice, or intestinal fluid, or may be sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, or milk. The body fluid may be animal or human. The "body fluid" may also be a solution. The solution may contain a physiological buffer solution such as phosphate-buffered saline (PBS) or N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (TES) containing the substance to be measured. The solution is not particularly limited as long as it contains the substance to be measured.

[0015] The solution may contain a target substance. The solution may potentially contain a target substance. In some embodiments, the target substance may be a molecule, ion, polymer, biomolecule, etc. The target substance may comprise a biomolecule. The target substance may be a protein, a glycated protein, etc. For example, the solution may be tears, and the target substance may be albumin, glycated albumin, hemoglobin, and / or glycated hemoglobin contained in tears. Alternatively, the target substance may be albumin, glycated albumin, hemoglobin, or glycated hemoglobin in blood, serum, or plasma, or may be albumin, glycated albumin, hemoglobin, or glycated hemoglobin in interstitial fluid, urine, or saliva. Albumin may be oxidized albumin (HNA) or reduced albumin (HMA). In some embodiments, the substance to be measured may be AGEs (Advanced Glycation End Products). In some embodiments, the substance to be measured may be glycated lipids.

[0016] <Sensor> The sensor may be a chemical sensor, a biosensor, an ion sensor, etc. (hereinafter, these may be referred to as a "sensor," a "biochemical sensor," a "chemical sensor," or an "electrochemical sensor.") The sensor may include a plurality of sensors.

[0017] The sensor may include an electrode. The electrode may be an amperometric electrode. The electrode may include a hydrogen peroxide electrode. The electrode may include an oxygen electrode. The electrode may be a potentiometric electrode. The electrode may be an electrode for detecting ions (such as a pH electrode, a cyanide ion electrode, or an iodide ion electrode).

[0018] In some embodiments, the sensor may output an electrical signal. In some aspects, the sensor may output a current signal. The sensor may output a voltage signal or a charge. The sensor may be electrically connected to an ammeter, voltmeter, or the like.

[0019] <Measurement of Hydrogen Peroxide> In some embodiments, the produced glycated amino acid may be reacted with ketoamine oxidase to generate hydrogen peroxide. In some embodiments, the amount (concentration) of the generated hydrogen peroxide may be measured. In some embodiments, the amount (concentration) of hydrogen peroxide may be measured electrically.

[0020] "Ketoamine oxidase" generally refers to an oxidase that recognizes the ketoamine structure of a glycated amino acid or a peptide or peptide fragment containing a glycated amino acid residue and oxidizes the glycated amino acid to produce an amino acid, glucosone (α-ketoaldehyde), and hydrogen peroxide. Thus, ketoamine oxidase produces hydrogen peroxide at a concentration proportional to or related to the concentration of the glycated amino acid or peptide or peptide fragment containing the glycated amino acid residue that it recognizes.

[0021] The ketoamine oxidase may be a dehydrogenase, a kinase, or an oxidase, and may be fructosyl amino acid oxidase (FAOD), fructosyl peptide oxidase, fructosyl valylhistidine oxidase, fructosyl amine oxidase, amadoriase, fructosyl amine deglycase, or modified forms thereof.

[0022] In some embodiments, the ketoamine oxidase may be an oxidase (Group I ketoamine oxidase) that acts on an amino acid or peptide whose α-amino group is glycated. The amino acid or peptide may be valine, glycine, or valylhistidine. By using an oxidase that acts on an amino acid or peptide whose α-amino group is glycated, a glycated hemoglobin sensor or a glycated hemoglobin A1c (HbA1c) sensor can be constructed.

[0023] In some embodiments, the ketoamine oxidase may be an oxidase that acts on an amino acid or peptide whose ε-amino group is glycated (group II ketoamine oxidase). The amino acid may be lysine. The glycated amino acid may be fructosyl lysine. A glycoalbumin sensor can be constructed using an oxidase that selectively acts on an amino acid or peptide whose ε-amino group is glycated.

[0024] In some embodiments, the ketoamine oxidase may be an oxidase (Group III ketoamine oxidase) that acts on an amino acid or peptide whose α-amino group and / or ε-amino group is glycated. The amino acid or peptide may be lysine, valine, glycine, or valylhistidine. A glycated protein sensor can be constructed using an oxidase that acts on an amino acid or peptide whose α-amino group and / or ε-amino group is glycated.

[0025] In some embodiments, the sensor may include a detection unit. The detection unit may be a hydrogen peroxide detection unit. The "hydrogen peroxide detection unit" (hydrogen peroxide sensor) may be an electrochemical electrode or a hydrogen peroxide electrode. The hydrogen peroxide electrode may have a counter electrode, a reference electrode, and a working electrode. In certain embodiments, the detection unit may detect oxygen. For example, it may detect the amount or concentration of oxygen reduced by an enzyme reaction. Oxygen detection is considered to be relatively insensitive to molecules and ions that are noise sources and resistant to interference. Oxygen consumption may be measured by detecting oxygen. Since the detection unit is saturated with air, it may be used for enzyme sensing. The detection unit may be configured to perform multiple detection methods selectively or in combination.

[0026] Noise Reduction Using Ion Exchange Resin In some embodiments, the sensor may include an ion exchange resin on the detector. The sensor may include an ion exchange resin between the ketoamine oxidase layer and the hydrogen peroxide electrode.

[0027] For example, cation exchange resins such as Nafion (registered trademark) can be used to suppress or prevent ascorbic acid and uric acid, particularly negative ions, present in body fluids from permeating and reaching the detection unit, while anion exchange resins such as polypyrrole can be used to suppress or prevent dopamine, particularly positive ions, from permeating and reaching the detection unit.

[0028] The ion exchange resin may comprise 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.

[0029] <Molecularly Imprinted Polymer Sensor> The sensor may include a molecularly imprinted polymer (MIP) membrane. The sensor may detect a change in charge or potential caused by molecular recognition by the MIP (potentiometry type). The sensor may also detect a change in current (e.g., mediator current) flowing through an electrode caused by molecular recognition by the MIP (amperometry type).

[0030] <Protease> "Protease" is generally a general term for peptide bond hydrolases that hydrolyze and catabolize proteins and polypeptides. Proteases may also be enzymes that break down proteins into peptide fragments. When a protein contains glycated amino acid residues, the peptide fragments produced by the action of the protease may include peptide fragments containing glycated amino acid residues and peptide fragments that are not glycated at all.

[0031] The "protease" may be an animal-derived protease, a plant-derived protease, or a microbial-derived protease. The protease may be an exopeptidase or an endopeptidase. The protease may be an aspartic protease, a metalloprotease, a serine protease, or a thiol protease.

[0032] The term "protease" may include multiple types or kinds of proteases, or may include a single type or kind of protease. For example, the protease may include either an endopeptidase or an exopeptidase, or may include both. Mixing multiple proteases may increase the decomposition efficiency.

[0033] The animal-derived protease may be 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 protease and amylase), or the like.

[0034] The plant-derived protease may be papain, bromelain, zingipain, kallikrein, ficin, chymopapain, or the like.

[0035] The protease derived from a microorganism may be a protease derived from Bacillus, a protease derived from Aspergillus, a protease derived from Penicillium, a protease derived from Streptomyces, a protease derived from Lysobacter, a protease derived from yeast, a protease derived from Tritirachium, a protease derived from Thermus, a protease derived from Pseudomonas, a protease derived from Achromobacter, or the like.

[0036] In some embodiments, the protease may be provided in liquid form or mixed with a solution. In some embodiments, the protease may be added to or mixed with a solution in solid form (e.g., a powder). In some embodiments, the protease may be supported on a support material and introduced into a solution. The protease may be supported on a flat plate, a curved surface, a sphere, beads, a biomolecule such as a protein, a polymer, etc.

[0037] Optical Measurements: As used herein, "optical measurement" generally refers to determining the optical properties of a substance using an optical element or device. In some embodiments, an optical measurement of a substance of interest may be measured. In some aspects, a property of a substance bound to or associated with the substance of interest (hereinafter, not the substance of interest itself, but a substance (e.g., a reagent) that is chemically, biologically, or physically bound to or associated with the substance of interest) may be measured. A property of a reagent may be measured. The reagent may be referred to as the "substance of interest."

[0038] In some embodiments, the optical measurement may include a spectroscopic measurement. For example, the absorbance of the substance of interest may be measured. In some embodiments, a color-changing indicator corresponding to the substance of interest may be introduced. The indicating indicator may be detected or measured.

[0039] <Color Changing Indicator> As used herein, the term "color changing indicator" generally refers to a halochromic chemical compound. The color change may generally be reversible. Color changing indicators include, but are not limited to, acid-base indicators (pH indicators), redox indicators, adsorption indicators, TLC color indicators, and the like, which change the color of their solution depending on the pH. pH indicators may be, but are not limited to, bromocresol purple (BCP), bromocresol green (BCG), and the like.

[0040] In some embodiments, BCP or BCG can be used for albumin. For example, albumin binds to bromocresol green (BCG) to form an albumin-bromocresol green complex. At around pH 4, this complex exhibits a blue color. Therefore, the albumin concentration can be determined by measuring the absorbance (BCG method). A surfactant may be added to the measurement reagent. A surfactant may not be added to the measurement reagent. For example, a protein denaturant (such as SDS) may or may not be added.

[0041] For example, albumin binds to bromocresol purple (BCP) to form an albumin-bromocresol purple complex. This complex is blue in color. Therefore, the albumin concentration can be determined by measuring absorbance (BCP method). In addition, generally, reduced albumin is converted to oxidized albumin using an oxidizing agent, and then the albumin concentration can be measured by a color reaction with BCP (improved BCP method). A surfactant may be added to the measurement reagent. A surfactant does not have to be added to the measurement reagent. For example, a protein denaturant (such as SDS) may or may not be added.

[0042] In some embodiments, the reaction in which albumin binds to bromocresol purple (BCP) or bromocresol green (BCG) to form a complex may be referred to as the "first reaction."

[0043] In some embodiments, the color changing indicator may be provided in liquid form and mixed with the solution, hi some embodiments, the color changing indicator may be added to or mixed with the solution in solid form (e.g., powder).

[0044] <Measurement of Absorbance> In some embodiments, an absorptiometer (colorimeter) may be used. The absorptiometer has a light source and a light receiver. All or a portion of the light emitted from the light source enters the target solution. All or a portion of the light that has passed through the solution enters the light receiver. The light absorbed by the target substance can be analyzed.

[0045] The wavelength of the light source may be appropriately selected depending on the color indicator used. For example, the absorbance may be measured at a wavelength of 600 to 630 nm for BCP and at 565 to 660 nm for BCG. The measurement wavelength may be changed depending on various conditions such as pH.

[0046] In some embodiments, the change in absorbance of the color indicator over time may be measured. In some aspects, the absorbance may be measured at at least two (or more) times. For example, the total amount of protein at the beginning (before the start of protease degradation, t=0) may be determined based on measurements at two times. In some aspects, the time at which protease degradation begins may be measured, and the total amount of protein may be determined based on measurements corresponding to the time from this start time. In some aspects, the total amount of protein may be determined based on measurements at three or more times. 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.

[0047] In some embodiments, the protein concentration may be determined by referring to a previously prepared calibration curve showing the relationship between absorbance and protein concentration. For example, a calibration curve showing the relationship between absorbance A(t) changing with time (t) and its change over time (ΔA(t) / Δt) is prepared in advance. In an actual measurement, the absorbance A(t1) and its slope (ΔA(t1) / Δt) at a certain time (t=t1) are determined. From this, the absorbance A(t=t0) at the time t=t0 when the protease starts decomposing can be determined.

[0048] In some embodiments, measurement of the glycated protein may be started after a predetermined or specified time has elapsed since the start of protease degradation. In some embodiments, measurement of the glycated protein may be started when the absorbance A becomes sufficiently small. In some embodiments, when the change in absorbance over time (ΔA / Δt) becomes substantially zero, it is determined that sufficient protease degradation has been completed, and measurement of the glycated protein may be started, or the measurement results of the glycated protein at that time may be adopted. In some embodiments, when the change in absorbance over time reaches a predetermined value, it is determined that the predetermined protease degradation has been achieved, and measurement of the glycated protein may be started, or the measurement results of the glycated protein at that time may be adopted.

[0049] In some embodiments, the absorbance measurement may be calibrated. The optical system including the optical measurement cell, the light-emitting element, the light-receiving element, etc. may be calibrated. For example, the absorbance may be measured before the solution is introduced into the optical system (e.g., when the optical measurement cell is empty, in its initial state, etc.) and after the solution is introduced into the optical system (e.g., when the measurement solution is introduced into the optical measurement cell). The difference between these absorbances may be calculated.

[0050] <Microplate> The present disclosure provides a microplate. As used herein, the term "microplate" generally refers to a flat plate having multiple small "wells" that function as test tubes. A microplate is also called a microtiter plate, a microwell plate, a multi-well, a cartridge, etc. (hereinafter, sometimes referred to as a "microplate"). A "microplate" is generally formed in a rectangular shape, but the shape is not limited to this. A "microplate" generally has multiple "wells" regularly arranged in a row or matrix, but the arrangement of the wells is not limited to this.

[0051] At least one or more of the wells and at least one or more of the optical measurement wells may contain a pretreatment solution, a dilution solution, a priming solution, an enzyme solution, a biochemical solution, an indicator solution, or the like.

[0052] In some embodiments, the microplate may comprise an electrochemical sensor and at least one or more wells. In some embodiments, the microplate may comprise an optical measurement well, an electrochemical sensor, and at least one or more wells.

[0053] The present disclosure provides a sensor. In some embodiments of the present disclosure, a microplate may include a sensor. In some embodiments, the microplate may be configured to have a sensor attached or fixed thereto. For example, the sensor may be manufactured separately from the microplate and then attached to the microplate. For example, the microplate and the sensor may each be introduced separately into a measurement device. For example, the microplate and the sensor may be combined within the measurement device. The microplate and the sensor may be physically separate or substantially combined, associated with or cooperating with each other, to perform measurement solution preparation and measurement within the measurement device. For example, one sensor may be used for multiple microplates and may be used repeatedly. In some embodiments, the sensor may be integrally molded with the microplate.

[0054] <Example of Microplate Configuration> In some embodiments, the microplate may include at least one of: (a0) a dilution solution well containing a dilution solution; (b0) a filter well equipped with a filter; (c0) an optical measurement preparation solution well containing a preparation solution for optical measurement; (d0) an indicator solution well containing an indicator solution; (e0) a first stirring well used for stirring the solution; (f0) an optical measurement well into which light can be introduced from the outside and extracted to the outside; (g0) an electrochemical measurement preparation solution well containing a preparation solution (e.g., enzyme) for electrochemical measurement; (h0) a second stirring well used for stirring the solution; (i0) a sensor cleaning solution well containing a sensor cleaning solution; and (j0) a sensor calibration solution well containing a sensor calibration solution; and (s0) an electrochemical sensor.

[0055] In some embodiments, the concentration of the protein of interest in the sample of interest may be measured by optical measurements, hi some embodiments, the concentration of the glycated protein of interest in the sample of interest may be measured by electrical measurements or electrochemical sensors.

[0056] In some embodiments, the microplate may be used for analyzing, detecting, or measuring proteins. One or more wells may contain a solution containing a protease. The protease degrades the protein to be measured into peptide fragments or amino acids. The peptide fragments or amino acids thus generated may be measured optically and / or by an electrochemical sensor.

[0057] In some embodiments, glycated proteins may be measured electrically. Hydrogen peroxide may be generated by the action of ketoamine oxidase, which specifically reacts only with glycated amino acids or peptides among the amino acids or peptides decomposed by proteases, and the hydrogen peroxide may be measured using an electrode. This allows the concentration of glycated proteins in a solution to be determined.

[0058] In the following embodiments 1 to 5, the configuration of a microplate for measuring glycated proteins and steps for using the same will be described as examples.

[0059] <Embodiment 1> Fig. 1 shows a microplate 10 according to an embodiment. The microplate 10 is capable of measuring glycated proteins. The concentration of a target protein (the total amount of glycated and non-glycated proteins) and the concentration of the glycated protein can be measured. The microplate 10 is capable of performing optical and electrical measurements. The microplate 10 includes a plurality of wells (a to j) and an electrochemical sensor (s). By way of example, but not limitation, optical measurement of albumin concentration in blood and measurement of glycated albumin concentration using an electrochemical sensor will be described with reference to the microplate 10 of this embodiment.

[0060] The microplate 10 shown in Fig. 1 includes: (a) a dilution solution well containing a dilution solution; (b) a filter well equipped with a filter; (c) a preparation solution well containing a preparation solution for optical measurement of proteins; (d) an indicator solution well containing an indicator solution; (e) a first stirring well used for stirring the solution; (f) an optical measurement well that allows light to enter from the outside and emit light to the outside; (g) a protease solution well containing a protease solution; (h) a second stirring well used for stirring the solution; (i) a sensor cleaning solution well containing a sensor cleaning solution; (j) a sensor calibration solution well containing a sensor calibration solution; and (s) an electrochemical sensor. Two pipette tips t1 and t2 are attached to the microplate 10. These are used for optical measurement and electrical measurement, respectively.

[0061] The filter in the filter well (b) has the ability to filter blood and extract plasma or serum. For example, the filter may be a plasma or serum separation filter. The filter well (b) is initially empty and receives plasma or serum components that pass through the filter when blood is introduced. The filter may have the ability to remove debris and substances that are unnecessary or adversely affect the measurement.

[0062] The diluent in the diluent well (a) is used to dilute the collected blood, and the diluent is, for example, physiological saline (e.g., phosphate buffer solution, PBS).

[0063] In some embodiments, the diluent may contain ketoamine oxidase. Low-molecular-weight peptides are generally present in plasma or serum. To measure the concentration of glycated albumin, only the glycated peptide fragments produced by protease degradation of glycated albumin should be oxidized with ketoamine oxidase and the generated 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 errors or noise in the measurement results. Adding ketoamine oxidase to plasma or serum components prior to measurement can predegrade low-molecular-weight peptides in the blood, eliminating or reducing these sources of noise.

[0064] In some embodiments, the diluent may contain catalase. Catalase decomposes hydrogen peroxide, thereby eliminating or reducing sources of noise, etc. In some embodiments, sodium azide may be contained in any of the wells, for example, in wells other than the diluent well. Sodium azide inhibits the decomposition reaction of hydrogen peroxide by catalase. Adding sodium azide inhibits catalase, allowing only the glycated peptide fragments generated by decomposition of glycated albumin with protease to be oxidized with ketoamine oxidase, making it possible to measure only the generated hydrogen peroxide.

[0065] The priming solution in priming solution well (c) contains an oxidizing agent. In some embodiments, the priming solution may contain an oxidizing agent. This well may contain other substances, such as buffers, chlorides, surfactants, protein denaturants, pH adjusters, chelating agents / antioxidants (ascorbic acid), preservatives, etc.

[0066] The indicator well (d) contains a BCP solution. In some embodiments, the indicator may be BCP or BCG. This well may contain other substances, such as buffers, chlorides, surfactants, protein denaturants, pH adjusters, chelating agents / antioxidants (ascorbic acid), preservatives, etc.

[0067] The first stirring well (e) is initially empty and receives a predetermined amount of the preparation solution, indicator solution, and dilution mixture. The solution in the first stirring well is thoroughly stirred by pipetting.

[0068] The optical measurement well (f) is initially empty and receives the mixture for optical measurement, and has one or more windows that allow the entrance and exit of light from outside.

[0069] The protease solution well (g) contains the protease solution. This well may contain other substances, such as buffers, chlorides, surfactants, pH adjusters, chelating agents / antioxidants (ascorbic acid), preservatives, mediators, etc.

[0070] The second stirring well (h) is initially empty and receives a predetermined amount of the protease solution and dilution mixture. The solution in the second stirring well is thoroughly stirred by pipetting. The second stirring well is configured to be heated externally.

[0071] The sensor wash wells (i) contain wash solutions for washing the sensor(s), for example, to wash the sensor(s) before use and prepare them for measurement.

[0072] The sensor calibrator well (j) may contain a calibrator, e.g., a substrate for the enzyme of an enzymatic sensor. The calibrator may contain, e.g., fructosyl lysine, a glycated peptide fragment, for measuring glycated albumin.

[0073] In some embodiments, the optical measurement well (f) may have an optical measurement window for the entrance and / or exit of measurement light (probe). The optical measurement window may be used to allow measurement light (probe) to enter the optical measurement mixture and detect the light exiting therefrom. In some embodiments, one optical measurement window may be provided. In some embodiments, one window may allow both incident light and outgoing light to pass through. In some embodiments, two or more optical measurement windows may be provided. Separate windows for incident light and outgoing light may be provided. The incident light window and outgoing light window may be provided at symmetrical positions (surfaces) of the optical measurement well. For example, light windows may be provided on one and the other horizontal faces of the well.

[0074] The use of the microplate 10 will now be described with reference to FIGS.

[0075] <Pretreatment> The collected body fluid (e.g., blood) is introduced into the diluent well (a) (Fig. 2A). The mixture is stirred to prepare a diluted mixture consisting of the introduced amount of body fluid and diluent (Fig. 2B).

[0076] The dilution mixture is taken from the diluent well (a) and passed through the filter of the filter well by pipetting. The filtered dilution mixture is introduced into the filter well (b) (FIG. 2C). This pipetting operation may be performed using a fluid introduction device.

[0077] The filter is removed from the filter well (b) and transferred to the diluent well (a) in Figure 2, allowing a pipette access to the filtered dilution mixture in the filter well (b) (Figure 2D).

[0078] <Optical Measurement> One pipette tip t1 is used to handle the solution for optical measurement (FIG. 3A). In FIGS. 3B to 3D, the pipette tip t1 is used but is not shown in the figures.

[0079] A predetermined amount of preparation liquid (e.g., oxidizing agent) is taken from the preparation liquid well (c) by pipetting and introduced into the first stirring well (e). Furthermore, a predetermined amount of indicator liquid (e.g., BCP) is taken from the indicator liquid well (d) by pipetting and introduced into the first stirring well (e). In this way, an indicator mixture containing the preparation liquid and the indicator is prepared (FIG. 3B).

[0080] A predetermined amount of the filtered diluted mixture is taken from the filter well (b) by pipetting and introduced into the first stirring well (e). After introduction, the solution in the first stirring well (e) is stirred. This prepares the mixture for optical measurement (FIG. 3C).

[0081] A predetermined amount of the optical measurement mixture is taken from the first stirring well (e) by pipetting 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).

[0082] The optical measurement well (f) is configured to allow sufficient introduction of probe light and output of measurement light. Based on the optical properties of the optical measurement mixture, the amount (e.g., concentration) of a target substance (e.g., a protein) in the initial solution can be determined.

[0083] 3C and 3D can be replaced by other methods. For example, the indicator mixture may be first introduced into the optical measurement well (f) and its optical properties may be measured, and then the filtered diluted mixture may be introduced into the optical measurement well (f) and its optical properties may be measured. In this case, the amount of the target substance can be determined from the difference in optical properties before and after the introduction of the measurement sample.

[0084] <Electrical Measurement> The other pipette tip t2 is used to handle the solution for electrical measurement (FIG. 4A). In FIGS. 4B to 4G, the pipette tip t2 is used but is not shown in the figures.

[0085] A predetermined amount of sensor washing solution is taken from the sensor washing solution well (i) by pipetting to wash the sensor (s) (FIG. 4B).

[0086] The sensor calibration solution is taken from the sensor calibration solution well (j) by pipetting and introduced into the sensor (s) (FIG. 4C). The sensor is calibrated using this sensor calibration solution.

[0087] A predetermined amount of sensor washing solution is taken from the sensor washing solution well (i) by pipetting to wash the sensor(s) and remove the calibrator solution from the sensor(s) (FIG. 4D). In some embodiments, the calibrator solution may not be washed after calibration. A measurement solution may be introduced into the sensor(s) with the calibrator solution present, and measurement may begin.

[0088] 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).

[0089] A predetermined amount of the filtered dilution mixture is taken from the filter well (b) by pipetting 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 dilution mixture introduced into the second stirring well (h) may be heated. The second stirring well (h) may be heated before the dilution mixture is introduced. The second stirring well (h) may be heated after the dilution mixture is introduced. The protease mixture may be heated, for example, to the optimal temperature for the protease.

[0090] 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) performs electrical measurements on the protease mixture. The sensor (s) may measure the time change in output based on the time when mixing of the protease solution and the diluted mixture begins.

[0091] For example, the amount of the glycated protein of interest can be determined based on measurements by the sensor(s).

[0092] For example, the degree of glycation 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).

[0093] <Embodiment 2> In some embodiments, the microplate may include at least: (a2) a diluent well containing a diluent; (d2) an indicator well containing an indicator solution and a preparation solution for optical measurement; (e2) a first stirring well used to stir the solution; (f2) an optical measurement well having an optical measurement window; (g2) a protease solution well containing a protease solution; (h2) a second stirring well used to stir the solution; and (s2) an electrochemical sensor.

[0094] That is, the microplate may include at least six wells and one sensor. The microplate may include two agitation wells.

[0095] A collected body fluid (e.g., blood) is first subjected to plasma separation. A sample containing plasma components is introduced into the diluent well (a2). This allows the sample to be mixed with the diluent. The solution may be stirred for mixing.

[0096] A predetermined amount of indicator mixture is taken from the indicator solution well (d2) by pipetting and introduced into the first stirring well (e2).

[0097] 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. This prepares the mixture for optical measurement.

[0098] A predetermined amount of the optical measurement mixture is taken from the first stirring well (e2) by pipetting and introduced into the optical measurement well (f2). The optical properties of the optical measurement mixture in the optical measurement well (f2) are measured.

[0099] A predetermined amount of protease solution is taken from the protease solution well (g2) by pipetting and introduced into the second stirring well (h2).

[0100] A predetermined amount of the dilution mixture is taken from the dilution solution 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.

[0101] A predetermined amount of the protease mixture is taken from the second mixture (h2) by pipetting and introduced into the sensor (s2), which performs electrochemical measurement on the protease mixture.

[0102] In some embodiments, the dilution mixture may be prepared in a diluent well (e.g., a). In this case, a filter well (e.g., b) is not required. In some embodiments, a preparation solution for optical measurement of proteins may already be mixed with an indicator solution in an indicator solution well (e.g., d). In this case, a preparation well (e.g., c) is not required. In some embodiments, a calibrator solution well (e.g., j) is not required. In some embodiments, a sensor (e.g., s) does not require measurement preparation using a sensor wash solution or the like. In that case, a sensor wash solution well (e.g., i) is not required.

[0103] <Embodiment 3> In some embodiments, the microplate may include at least: (a3) ​​a diluent well containing a diluent; (d3) an indicator well containing a protein indicator solution; (f3) an optical measurement well having an optical measurement window; (g3) a protease solution well containing a protease solution; and (j3) an electrochemical sensor. That is, the microplate may include at least four wells and one sensor. The microplate does not need to include a separate stirring well.

[0104] A collected body fluid (e.g., blood) is first subjected to plasma separation. A sample containing plasma components is introduced into the diluent well (a3). This allows the sample to be mixed with the diluent. The solution may be stirred for mixing.

[0105] A predetermined amount of the diluted mixture is taken from the diluent well (a3) ​​by pipetting and introduced into the indicator well (d3). After introduction, the solution in the indicator well (d3) is stirred. This prepares the mixture for optical measurement.

[0106] A predetermined amount of the optical measurement mixture is taken from the indicator solution well (d3) by pipetting and introduced into the optical measurement well (f3). The optical properties of the optical measurement mixture in the optical measurement well (f3) are measured.

[0107] In another embodiment, a predetermined amount of the dilution mixture is taken from the dilution liquid well (a3) ​​and introduced into the optical measurement well (f3) by pipetting, and a predetermined amount of the indicator mixture is taken from the indicator liquid well (d3) and introduced into the optical measurement well (f3). They are mixed in the optical measurement well (f3), and the optical properties of the optical measurement mixture in the optical measurement well (f3) are measured.

[0108] A predetermined amount of the dilution mixture is taken from the dilution mixture well (a3) ​​by pipetting and introduced into the protease mixture well (g3). After introduction, the solution in the protease mixture well (g3) is stirred. This prepares the protease mixture. The protease mixture well (g3) may be heated.

[0109] A predetermined amount of the protease mixture is taken from the protease solution well (g3) by pipetting and introduced into the sensor (s3), which performs electrochemical measurement on the protease mixture.

[0110] In some embodiments, the indicator solution well may contain a metered amount of indicator solution for the protein. In some embodiments, the dilution mixture may be stirred or mixed with the indicator solution for the protein in the indicator solution well. In some embodiments, the dilution mixture may be stirred or mixed with the protease solution in the protease solution well.

[0111] <Embodiment 4> In some embodiments, the microplate may include at least: (a4) a diluent well containing a diluent; (f4) an optical measurement well containing a protein indicator solution and having an optical measurement window; (g4) a protease solution well containing a protease solution; and (s4) an electrochemical sensor. That is, the microplate may include at least three wells and one sensor. The optical measurement well may contain a pre-measured protein indicator solution. The solution may be mixed and stirred in the optical measurement well, and measurements may be performed on the solution.

[0112] The collected body fluid (e.g., blood) is first subjected to plasma separation. The sample containing the plasma component is introduced into the diluent well (a4). This allows the sample to be mixed with the diluent. The solution may be stirred for mixing.

[0113] A predetermined amount of the diluted mixture is taken from the dilution well (a4) by pipetting and introduced into the optical measurement well (f4). After introduction, the solution in the optical measurement well (f4) is stirred. This prepares the optical measurement mixture. The optical properties of the optical measurement mixture in the optical measurement well (f4) are measured.

[0114] A predetermined amount of the dilution mixture is taken from the dilution mixture well (a4) by pipetting and introduced into the protease mixture well (g4). After introduction, the solution in the protease mixture well (g4) is stirred. This prepares the protease mixture. The protease mixture well (g4) may be heated.

[0115] A predetermined amount of the protease mixture is taken from the protease solution well (g4) by pipetting and introduced into the sensor (s4), which performs electrochemical measurement on the protease mixture.

[0116] In some embodiments, the dilution mixture may be stirred or mixed with a mixture containing a protein indicator solution and an optical measurement preparation solution in the optical measurement well, hi some embodiments, the dilution mixture may be stirred or mixed with a protease solution in the protease solution well.

[0117] <Embodiment 5> In some embodiments, the microplate may include 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. That is, the microplate may include at least two wells and one sensor. The optical measurement well may contain a solution containing a quantified protein indicator and a protease. Optical measurement may be performed in the optical measurement well, and the same solution may be introduced to the sensor. In some embodiments, the target protein may be degraded by the protease even in the presence of the optical measurement indicator. In some embodiments, the sensor may contain the indicator or may be capable of measuring peptides degraded by the protease.

[0118] A collected body fluid (e.g., blood) is first subjected to plasma separation. A sample containing plasma components is introduced into the diluent well (a5). This allows the sample to be mixed with the diluent. The solution may be stirred for mixing.

[0119] A predetermined amount of the 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. This prepares a mixture for optical measurement containing protease. The optical properties of the mixture for optical measurement in the optical measurement well (f5) are measured. The optical measurement well (f5) may be heated.

[0120] A predetermined amount of the mixture for optical measurement containing the protease is taken from the optical measurement well (f5) by pipetting and introduced into the sensor (s5). The sensor (s5) performs electrochemical measurement on the mixture for protease.

[0121] In some embodiments, the indicator liquid wells may further contain a preparation liquid for optical measurement. For example, the indicator liquid wells (d2, d3, etc.) may contain a protein indicator and a preparation liquid for optical measurement. For example, the optical measurement wells (f4, etc.) may contain a protein indicator and a preparation liquid for optical measurement. For example, the optical measurement wells (f5, etc.) may contain a protein indicator, a preparation liquid for optical measurement, and a protease.

[0122] In the first to fourth embodiments, an indicator is used for optical measurement and an enzyme is used for electrochemical measurement to measure glycated proteins, but this is merely an example and the present disclosure is not limited thereto. The solution used in the wells may be another solution. In the first to fourth embodiments, the measurement target, solution, number of wells, well names, process, and the like can be changed as appropriate depending on the application.

[0123] In some embodiments, control measurements may be performed before, during, or after the present measurements, for example, introducing standards of known concentration and performing a process similar to that described above, or mutatis mutandis.

[0124] <Waste Tank> In some embodiments, the microplate may further include a waste tank. In some embodiments, the waste tank may be configured to contain a solution discharged from the sensor. In some embodiments, the sensor may include a waste tank. This allows, for example, the liquid used in the microplate to be discarded along with the cartridge. This can improve safety in handling the solution or reduce the risk of infection, for example.

[0125] <Cover> In some embodiments, the microplate may include a cover. The microplate may be configured to be coupled with the cover. The cover can substantially prevent leakage or spillage of the solution remaining in the wells after use. This can, for example, improve the safety of handling the solution or reduce the risk of infection.

[0126] By using the microplate or method of the present disclosure, for example and without limitation, measurements with relatively high accuracy or small error can be performed for trace amounts of a target substance. For example, the coefficient of variation (CV) can be 5%, 4%, 3%, 2%, 1%, or less. For example, GA value measurements based on glycated albumin concentration can be performed with a CV value of 5% to 8% or less. For example and without limitation, when multiple measurements (optical measurements and one or more sensor measurements) are performed, the multiple measurements can be performed in parallel, i.e., measurements can be performed efficiently.

[0127] <Example 1: Microplate> Figure 5 shows a top view (Figure 5A) of a microplate 20 according to one example, and a cross-sectional view (Figure 5B) taken along the line A-A in Figure 5A. The microplate 20 has wells 20a-20j arranged in the longitudinal direction 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 center line. The wells and positions are arranged in two longitudinal rows symmetrically about the center line. Below, the wells and structures will be described starting from the left side of the drawing.

[0128] First, a filter well 20a and a diluent well 20b are arranged. A wall 21 is provided around the openings of these wells. This wall 21 can receive and stably hold the filter and the used blood collection device.

[0129] Next, there are provided open pipette tip rests 22. These do not have to be wells, but may for example be through holes.

[0130] Second stirring well 20h is arranged adjacent to it. Second stirring well 20h is arranged singly, not in two rows. Space is secured below or to the side, allowing a heater (not shown) to be placed in close proximity. For example, the heater may have a concave surface that contacts the outer shape of second stirring well 20h. This allows contact over a relatively large area with second stirring well 20h. For example, the heater may be configured to approach second stirring well 20h from below. The heater can control the temperature of the solution in second stirring well 20h and optimize the reaction of the reagent.

[0131] Next, six wells are arranged in two rows, which in some embodiments serve as priming solution well 20c, indicator solution well 20d, first stirring well 20e, protease solution well 20g, sensor cleaning solution well 20i, and sensor calibration solution well 20j.

[0132] The optical measurement well 20f is arranged adjacent to the optical measurement well 20f. The optical measurement well 20f is arranged singly, not in two rows, so that the probe light can pass through the optical measurement well 20f in the horizontal direction, perpendicular to the longitudinal direction.

[0133] A sensor mounting portion 20s for mounting a sensor (not shown) is provided on the far right side of the drawing. The sensor is manufactured separately and mounted here.

[0134] 6 shows an exploded perspective view of a sensor 100 according to one embodiment. The sensor 100 includes a lower body 110, a sensor substrate support 120, a sensor substrate 130, a flow path spacer 140, an upper body 150, an upper sealing 160, and a fixing screw 170.

[0135] The lower body 110 has a recess in which the sensor substrate support 120 is placed. The sensor substrate 130 is usually made of glass or silicon and is therefore relatively fragile. To avoid mechanical damage during assembly or pressing, the sensor substrate support 120 may be made of a relatively soft material such as resin.

[0136] A sensor substrate 130 is placed on top of the substrate. The sensor substrate 130 has, on its surface, a sensing electrode 131 and a sensing electrode terminal 132 electrically connected to the sensing electrode 131. The sensing electrode 131 recognizes chemical reactions occurring on or near its surface and generates a corresponding current or voltage. This electrical signal is transmitted to the outside via the sensing electrode terminal 132.

[0137] A flow channel spacer 140 is placed on the sensor substrate 130. The flow channel spacer 140 has a flow channel through-hole 141. The flow channel spacer 140 is in close contact with the sensing substrate 130 at the bottom around the flow channel through-hole 141 so as to surround the sensing electrode 131, and is in close contact with the upper body 150 at the top. Therefore, the flow channel through-hole 141 substantially determines the shape of the sensing flow channel by its width, thickness, and length. The flow channel spacer 140 further has a terminal through-hole 142 that ensures access to the sensing electrode terminal 132.

[0138] The upper body 150 is placed on top of the upper body 150. The upper body 150 is mechanically fixed to the lower body 110 by the fixing screws 170. This causes the sensor substrate support 120, the sensor substrate 130, and the flow channel spacer 140 to be in close contact with one another. The flow channel is formed as a fluidically sealed structure except for the inlet and outlet.

[0139] The upper body 150 has a waste liquid tank 151. The upper sealing 160 is attached to the upper body 150 so as to close the opening of the waste liquid tank. The upper sealing 160 has an air hole 161.

[0140] The upper body 150 has an inlet 154 for introducing a solution to be measured. The inlet 154 may have a structure that allows easy attachment of a pipette, for example. The solution introduced from the inlet 154 flows through a flow path defined by the flow path through-hole 141. The solution passes over the surface of the sensing electrode 131, where measurement is performed.

[0141] The upper body 150 has an electrode terminal 153. When assembled, the lower end of the electrode terminal 153 passes through the terminal through-hole 142 of the flow path spacer 140 and can be in direct contact with the sensor electrode terminal 132 of the sensor substrate 130. The upper end of the electrode terminal 153 can be connected to an external electric circuit. In this way, the electrical signal obtained by the sensor electrode 131 can be transmitted to the outside of the sensor 100.

[0142] The solution flows up the waste liquid guide 152 provided vertically inside the upper body 150 from the opposite side of the solution inlet 154 of the flow path 141, and flows from the upper end thereof into the waste liquid tank 151. As the solution flows into the waste liquid tank 151, the air inside the tank is discharged to the outside through the air hole 161.

[0143] During normal use, the waste liquid contained in the waste liquid tank 151 does not come out through the air hole 161. Therefore, the waste liquid stored in the waste liquid tank 151 can be disposed of together with the sensor 100 or the microplate (cartridge).

[0144] 7 shows an exploded perspective view of a sensor 200 according to one embodiment. The sensor 200 includes a lower body 210, a sensor substrate support 220, a sensor substrate 230, a flow path spacer 240, an upper body 250, an upper sealing 260, and a fixing screw 270.

[0145] The lower body 210 is provided with a recess, inside which the sensor substrate support 220 is placed. The sensor substrate support 220 may be made of a relatively soft material such as resin. The lower body 210 further includes waste liquid tanks 211 a and 211 b.

[0146] A sensor substrate 230 is placed on top of the substrate. The sensor substrate 230 has, on its surface, a sensing electrode 231 and a sensing electrode terminal 232 electrically connected to the sensing electrode 231. The sensing electrode 231 recognizes chemical reactions occurring on or near its surface and generates a corresponding current or voltage. This electrical signal is transmitted to the outside via the sensing electrode terminal 232.

[0147] A channel spacer 240 is placed on the sensor substrate 230. The channel spacer 240 has a channel through-hole 241. The channel spacer 240 is in close contact with the sensing substrate 230 at the bottom around the channel through-hole 241 so as to surround the sensing electrode 231, and is in close contact with the upper body 250 at the top. Therefore, the channel through-hole 241 substantially determines the shape of the sensing channel by its width, thickness, and length. The channel spacer 240 also has a terminal through-hole 242 that ensures access to the sensing electrode terminal 232. The channel spacer 240 also has a waste liquid through-hole 243 that allows solution to flow between the lower body 210 and the upper body 250.

[0148] The upper body 250 is placed thereon. The upper body 250 is mechanically fixed to the lower body 210 by the fixing screws 270. This causes the sensor substrate support 220, the sensor substrate 230, and the flow channel spacer 240 to be in close contact with one another. The flow channel is formed as a fluidically sealed structure except for the inlet and outlet.

[0149] The upper body 250 has an inlet 254 for introducing a solution to be measured. The solution introduced from the inlet 254 flows through a flow path defined by the flow path through-holes 241. The solution passes over the surface of the sensing electrode 231, where measurement is performed.

[0150] The upper body 250 has an electrode terminal 253. When assembled, the lower end of the electrode terminal 253 passes through the terminal through-hole 242 of the flow path spacer 240 and can be in direct contact with the sensor electrode terminal 232 of the sensor substrate 230. The upper end of the electrode terminal 253 can be connected to an external electric circuit. In this way, the electrical signal obtained by the sensor electrode 231 can be transmitted to the outside of the sensor 200.

[0151] The solution flows up and down the first waste liquid guide 252a arranged vertically within the upper body 250 from the opposite side of the solution inlet 254 of the flow path 241, passes through one waste liquid through hole 243 of the flow path spacer 240, and enters the first waste liquid tank 211a of the lower body 210.

[0152] After the first waste liquid tank 211a becomes full, the supply of waste liquid from the flow path may continue. In this case, the waste liquid flows from the first waste liquid tank 211a through one of the waste liquid through-holes 243 of the flow path spacer 240, up the second waste liquid guide 252b, over the top of the flow path, and into the second waste liquid tank 211b in the lower body 210 located on the opposite side.

[0153] An upper sealing 260 is attached to the top of the upper body 250. The upper flow paths of the waste liquid guides 252a and 252b have an open structure. The upper sealing 260 closes these openings, sealing off the waste liquid guides 252a and 252b.

[0154] The upper sealing 260 further has an air hole 261. The air that was inside the first waste liquid tank 211a and the second waste liquid tank 211 passes through another waste through-hole in the flow path spacer 240, passes through the air guide 252c in the upper body 250, and is finally discharged to the outside through the air hole 261 in the upper sealing 260.

[0155] During normal use, the waste liquid contained in the waste liquid tanks 211a and 211b does not leak out through the air hole 261. Therefore, the waste liquid stored in the waste liquid tanks 211a and 211b can be disposed of together with the sensor 200 or the microplate (cartridge).

[0156] In some embodiments, the wells pre-containing the solution may be sealed with a seal, which may be removed prior to use, and movement of the pipette tip may cause the tip to pierce the seal.

[0157] The electrodes of the sensors of Examples 2 and 3 may be hydrogen peroxide electrodes, oxygen electrodes, or other electrodes, or a combination thereof.

[0158] <Embodiment 4: Automatic measurement system> Figs. 8 and 9 are a perspective view and a cross-sectional view, respectively, showing the configuration of an automatic measurement system 500 including a microplate 510, a measurement main body 520, and a pipette 530 according to one embodiment.

[0159] 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 placed in advance on the microplate 510. The blood collection tube 513 is depicted schematically.

[0160] The measurement body 520 has a linear rail (or guide) 521, which is configured to slide in the longitudinal (X) direction to receive the inserted microplate 510.

[0161] The measurement body 520 includes a heater system 522. The heater system 522 includes a heater 522a configured to contact or be in close proximity to the bottom and side of the heating well (e.g., second stirring well) 510i of the microplate 510 and heat it, and a heater moving mechanism 522b capable of moving the heater 522a up and down. The heater system 522 may be connected to a power source (not shown).

[0162] The measurement main body 520 is equipped with an optical measurement system 523 that includes 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 in a direction perpendicular to the longitudinal direction of the microplate 510 or the rail 521, and passes through an optical measurement well 510f of the microplate 510. This makes it possible to measure the optical characteristics in the optical measurement well 510f.

[0163] The measurement body 520 includes an electrical measurement system 524. The electrical measurement system 524 includes a probe terminal 524a that can be lowered to contact an electrode terminal (not shown) of the sensor 510s. The electrical signal output from the sensor 510s is received by the electrical measurement system 524, analyzed, and / or further transmitted to the outside. The electrical measurement system 524 may be electromagnetically connected to an external computing unit wirelessly or via a wire.

[0164] The pipette 530 is configured to move up and down (Z direction) and in a direction perpendicular to the longitudinal direction (Y direction) in a horizontal plane relative to the measurement body 520 or the microplate 510 fixed thereto. The pipette 530 is connected to or includes a movement mechanism (pipette movement mechanism, not shown) that enables such movement.

[0165] The measurement body 520 is configured to move in the longitudinal direction (X direction) of the microplate 510 within a horizontal plane relative to the pipette 530. The measurement body 520 is connected to a movement mechanism (measurement body movement mechanism, not shown) that enables such movement.

[0166] These movement mechanisms allow for the control of the appropriate pipetting action of the pipette 530 relative to the microplate 510 .

[0167] In some embodiments, the movement mechanism, heater system 522, and electrical measurement system 524 may be connected individually, or some or all of them together, to a control system (not shown).

[0168] In some embodiments, the automated measuring device may further include a computer or computer system and may be calibrated to be connectable thereto. Various steps in the disclosed method may be controlled by the computer, from the time when the microplate is loaded into the device body or after the loading. The computer may include a central processing unit (CPU) and a storage medium for storing a program for controlling the CPU and the computer.

[0169] <Calibration> In some embodiments, the sensor may be calibrated before, during, or after measurement, or at multiple points thereof. A predetermined calibration solution may be used for the configuration. In some embodiments, the sensor may be calibrated in advance, at the time of shipment, or at the time of manufacture. The sensor may have a readable code such as a barcode, QR code (registered trademark), character code, or image code. The code may be attached or printed on the surface of the sensor body. The code may be a magnetic code. The code may be attached inside the sensor. The code may be associated with sensor information such as manufacturing information, shipping information, and calibration information. A user or a device can obtain the relevant information by reading the code. A calibration line for the sensor may be obtained by reading the code.

[0170] In some embodiments, the microplate may include multiple sensors. In some embodiments, the microplate may be configured by connecting multiple sub-microplates. In some embodiments, one group of wells and one sensor may be provided for one analyte. In some embodiments, multiple groups of wells and one sensor may be provided for one analyte. In some embodiments, one group of wells and multiple sensors may be provided for one analyte. In some embodiments, multiple groups of wells and multiple sensors may be provided for one analyte.

[0171] Example 5: Multi-Channel Microplate Figure 10 shows a perspective view of a microplate 30 according to one example. As an example, the microplate 30 includes eight columns of sub-microplates 30-1 to 30-8. In some embodiments, each sub-microplate may be a microplate as described in the above examples or embodiments. Each of the sub-microplates 30-1 to 30-8 in Figure 10 includes sensors 30s-1 to 30s-n, respectively. This allows measurements to be performed in parallel. Measurements can be performed efficiently or with high throughput.

[0172] Example 6: A Series of Microplates Figure 11 shows a top view (Figure 11A) of a microplate 40 according to one example, and a cross-sectional view (Figure 11B) taken along the line A-A in Figure 11A. The microplate 40 has wells 40a-40j arranged in the longitudinal direction and a sensor mounting portion 40s. One difference from the microplate 20 of Example 1 shown in Figure 5 is as follows. That is, in the microplate 20 of Example 1 shown in Figure 5, some of the wells are arranged in two longitudinal rows symmetrically about the center line. In contrast, in the microplate 40 shown in Figure 11, all of the wells are arranged in a single longitudinal row on the center line. Below, the arrangement of the wells and their structure will be described starting from the left side of the figure.

[0173] First, a filter well 40a is placed. A filter (not shown) is press-fit into this filter well 40a. As shown in Figure 11B, the bottom has a partial slope, which allows the filtered solution (or diluted mixture) to efficiently collect at the deepest point.

[0174] Next, a pipette tip holder 42 is provided. The pipette tip holder 42 has a wall disposed at the opening. This allows for stable holding of pipette tips and the like to prevent them from shifting out of position. In some embodiments, the pipette tip holder may have a closed bottom, as shown in FIG. 11B. This allows for dripping that may occur during and after use to be contained within the cartridge, keeping the device clean and free of contamination.

[0175] Adjacent to this are a sensor cleaning solution well 40i and a diluent solution well 40b.

[0176] Next, first stirring well 40e is located. The measuring device approaches pipette cartridge 40 from below and has a heater surrounding and heating first stirring well 40e. The internal temperature can be controlled to suit the desired mixing or stirring.

[0177] A protease solution well 40g, a sensor calibration solution well 40j, a preparation solution well 40c, and an indicator solution well 40d are arranged in series.

[0178] An optical measurement well 40f is disposed adjacent to the optical measurement well 40f, which is configured to allow the probe light to pass through the optical measurement well 40f in a horizontal direction perpendicular to the longitudinal direction.

[0179] The measurement device approaches the pipette cartridge 40 from below and has a heater (having a concave surface) that contacts the wells from the sides and below in a so-called U-shape. This allows the solutions contained in the wells to be mixed or set to a temperature suitable for measurement in the optical measurement wells 40f. In some embodiments, the heater may be configured to simultaneously heat the protease solution well 40g, the sensor calibration solution well 40j, the preparation solution well 40c, the indicator solution well 40d, and the optical measurement wells 40f. In some embodiments, the heater may be configured to simultaneously heat the protease solution well 40g, the sensor calibration solution well 40j, the preparation solution well 40c, and the indicator solution well 40d. In some embodiments, the heater may be configured to heat only the optical measurement wells 40f or to individually heat the optical measurement wells 40f. In some embodiments, the heater may be configured to separately heat the protease solution well 40g, the sensor calibration solution well 40j, the priming solution well 40c, the indicator solution well 40d, and the optical measurement well 40f.

[0180] A sensor mounting portion 40s for mounting a sensor (not shown) is provided on the far right side of the drawing. The sensor is manufactured separately and mounted here.

[0181] <Application Examples> The above embodiments and examples have been described based on the measurement of albumin and glycated albumin, but the present disclosure is not limited thereto. In some embodiments, two different target substances can be measured substantially simultaneously or in parallel. Optical measurement may be performed on one and electrochemical measurement on the other. In some embodiments, for multiple forms of a single substance, such as those resulting from deformation, modification, differences in group binding, or the presence or absence of group binding, optical measurement may be performed on one and electrochemical measurement may be performed on the other. The present disclosure provides various target substances and various measurement methods. Below, several application examples are shown as non-limiting examples.

[0182] <Application Example 1: Enzyme Sensing> In some embodiments, any of the microplates disclosed herein may be used for enzyme sensing. For example, a predetermined enzyme reaction may be performed on various substrates, and the resulting product may be measured using a sensor.

[0183] In some embodiments, sensing can be performed using multi-step or two-step enzyme reactions. Multiple enzyme reactions may have different signaling conditions. Furthermore, one enzyme reaction may be rate-limiting for the entire enzyme reaction. By dividing the enzyme reactions and precisely controlling the amount and time of each reaction by pipetting, accurate enzyme sensing with minimal error can be achieved.

[0184] <Cholesterol> In some embodiments, enzyme sensing of cholesterol may be performed. Cholesterol and fatty acids are produced from cholesterol esters and water by an enzymatic reaction of cholesterol esterase. Cholesterol and oxygen are produced from cholesterol oxidase by an enzymatic reaction of cholesterol oxidase. By measuring the oxygen and hydrogen peroxide produced during this process, the initial amounts of cholesterol esters and / or cholesterol can be determined.

[0185] A cholesterol esterase solution and a cholesterol oxidase solution may be contained in respective wells. The sensor may include an oxygen electrode, a hydrogen peroxide electrode, or other electrodes, or a combination thereof.

[0186] <Sucrose> In some embodiments, enzymatic sensing of sucrose may be performed. α-D-glucose and fructose are produced from sucrose and water by the enzymatic reaction of invertase. β-D-glucose is produced from α-D-glucose by the enzymatic reaction of mutarotase. Gluconolactone and hydrogen peroxide are produced from β-D-glucose and oxygen by the enzymatic reaction of glucose oxidase (GOX). By measuring the oxygen and hydrogen peroxide during this process, the amount of initial sucrose and / or intermediate products can be determined.

[0187] The invertase solution and the mutarotase solution may be contained in respective predetermined wells. The sensor may include an oxygen electrode, a hydrogen peroxide electrode, or other electrodes, or a combination thereof.

[0188] In addition to the substrates described above, the molecules to be measured and the enzymes used are not limited to those described above. For example, a single or multiple-step enzymatic reaction may be performed on glucose, phosphatidylcholine, neutral lipids, or other substrates in one or more wells, and an intermediate or final product of the enzymatic reaction may be detected with a sensor. In some embodiments, the solution may contain a cofactor. In some embodiments, the solution may contain an enzyme and a cofactor.

[0189] <Application Example 2: Mediator-Type Sensing> In some embodiments, glucose may be measured using GOX and a mediator (such as a ferrocene derivative, 1,4-benzoquinone, or tetrathiafulvalene). The GOX and the mediator may be contained in respective wells.

[0190] In conventional methods, once a mediator is added, the pH of the system must be stabilized before measurement. This results in a low sensitivity. In this application example, the pH can be adjusted to the reducing side, and then a mediator can be added. Measurement can be performed in this highly sensitive state. pH changes over time. However, from this highly sensitive state, the change in sensor output over time can be measured. This allows for more accurate and efficient measurements.

[0191] Application Example 3: Evaluation of Enzyme Titer In some embodiments, any of the microplates disclosed herein may be used to measure the titer of an enzyme of interest. For example, enzyme titer may be evaluated.

[0192] In some embodiments, the enzyme to be evaluated is introduced into a first well, thereby preparing an enzyme solution. A portion of the enzyme solution is taken and placed in an optical measurement well. Optical measurement of this enzyme can be performed to determine the initial total amount of enzyme. A portion of the enzyme solution is taken and introduced into a second well. In the second well, an oxygen solution is mixed with a known amount or concentration of substrate. The substrate may be contained in the second well in advance, or may be introduced into the second well from another well or externally before or after the introduction of the oxygen solution. This prepares a substrate mixture. The substrate may be, for example, a glycated peptide, glycated lysine, etc. This generates hydrogen peroxide through an enzymatic reaction. After a predetermined time, this reaction solution is taken and introduced into a sensor. The output may be measured as a function of time from the actual start time of the reaction between the substrate and the enzyme. The amount of active enzyme in the enzyme solution can then be determined. The enzyme titer can be determined from the total amount of enzyme and the amount of active enzyme.

[0193] In some embodiments, multiple sensors may be deployed, each of which may detect or quantify a different substance of interest.

[0194] Application Example 4: Ratio Measurement In some embodiments, the ratio of two target substances may be determined. For example, the albumin / globulin ratio (A / G ratio) may be determined. Albumin and globulin may be treated with their respective corresponding solutions and measured using methods or sensing techniques suited to each.

[0195] <Application Example 5: Balance Measurement> In some embodiments, balance measurement may be performed. 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.

[0196] Application Example 6: Water Quality Measurement In some embodiments, water quality can be measured. For example, the optical properties of a target solution can be evaluated by optical measurement. The optical measurement can be spectroscopic measurement. The type and / or amount of each substance in the solution can be determined from the properties at each wavelength. Electrical measurement can determine pH, the amount of electrolytes, etc. The quality of the target water can be evaluated by combining the optical and electrical properties of the water.

[0197] <Application Example 7: Microbial Sensing> In some embodiments, a target solution may be evaluated using microorganisms. Plant-based microorganisms may be added to the target solution. In some aspects, different microorganisms may be introduced into each well. In some aspects, 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 irradiated with light of multiple or predetermined wavelengths or intensities. The temperature in the well may be adjusted to a temperature suitable for each microorganism. As a result, each microorganism generates oxygen through respiration. Alternatively, each plant-based microorganism generates carbon dioxide through photosynthesis. These respiration and photosynthesis depend on water quality, which is an environmental indicator. After a predetermined reaction time, the solution is introduced into a sensor. The sensor measures the amount of oxygen and / or carbon dioxide in the solution. This makes it possible to measure water quality, such as BOD.

[0198] The present disclosure also provides the following embodiments. A001: A microplate comprising at least one (or more) wells; and an electrochemical sensor. A002: A microplate for performing optical measurements and electrochemical measurements, comprising at least one (or more) wells; an optical measurement well; and an electrochemical sensor. A011: A microplate for performing optical measurements and electrical measurements, comprising: a dilution solution well containing a dilution solution; an optical measurement well containing a solution containing a protein indicator and a protease, and having an optical measurement window; and an electrochemical sensor. A011b: The microplate according to A011, wherein a preparation solution for optical measurement of protein is contained in either the dilution well or the optical measurement well. A012 A microplate for performing optical measurements and electrical measurements, comprising: a dilution solution well containing a dilution solution; an optical measurement well containing a protein indicator solution and having an optical measurement window; a protease solution well containing a protease solution; and an electrochemical sensor. A012b The microplate according to A012, wherein a preparation solution for optical measurement of proteins is contained in any one of the dilution well and the optical measurement well. A013 A microplate for performing optical measurements and electrical measurements, comprising: a dilution solution well containing a dilution solution; an indicator solution well containing a protein indicator solution; an optical measurement well having an optical measurement window; a protease solution well containing a protease solution; and an electrochemical sensor. A013b The microplate according to A013, wherein a preparation solution for optical measurement of proteins is contained in any one of the dilution well, the indicator solution well, and the optical measurement well. A014 A microplate for performing optical and electrical measurements, comprising: (a) a diluent well containing a diluent; (d) an indicator well containing an indicator solution; (e) a first stirring well used for stirring the solution;(f) an optical measurement well having a window for optical measurement; (g) a protease solution well containing a protease solution; (h) a second stirring well used to stir the solution; and (s) an electrochemical sensor. A014b The microplate according to A014, wherein any one of the wells (a) to (h) contains a preparation solution for optical measurement of proteins. A015 A microplate for performing optical measurements and electrical measurements, comprising: (a) a dilution solution well containing a dilution solution; (b) a filter well equipped with a filter; (c) a preparation solution well containing a preparation solution for optical measurement of proteins; (d) an indicator solution well containing an indicator solution; (e) a first stirring well used for stirring the solution; (f) an optical measurement well capable of admitting light from the outside and extracting light to the outside; (g) a protease solution well containing a protease solution; (h) a second stirring well used for stirring the solution; (i) a sensor washing solution well containing a sensor washing solution; and (s) an electrochemical sensor. A015b The microplate according to A015, wherein any one of the wells (a) to (i) contains a preparation solution for optical measurement of proteins. A016 A microplate for performing optical measurements and electrical measurements, comprising: (a) a dilution well containing a dilution solution; (b) a filter well fitted with a filter; (c) a preparation well containing a preparation solution for optical measurement of proteins; (d) an indicator solution well containing an indicator solution; (e) a first stirring well used for stirring the solution; (f) an optical measurement well into which light can be introduced from the outside and extracted to the outside; (g) a protease solution well containing a protease solution; (h) a second stirring well used for stirring the solution; (i) a sensor cleaning solution well containing a sensor cleaning solution; (j) a sensor calibration solution well containing a sensor calibration solution; and (s) an electrochemical sensor;A microplate comprising: A016b A microplate for performing optical measurements and electrical measurements, comprising: (a) a dilution solution well containing a dilution solution; (b) a filter well equipped with a filter; (c) a preparation solution well containing a preparation solution for optical measurement of proteins; (d) an indicator solution well containing an indicator solution; (e) a stirring well used for stirring the solution; (f) an optical measurement well into which light can be introduced from the outside and extracted to the outside; (g) a protease solution well containing a protease solution; (i) a sensor cleaning solution well containing a sensor cleaning solution; (j) a sensor calibration solution well containing a sensor calibration solution; and (s) an electrochemical sensor. A021 The microplate according to any one of embodiments A001 to A016b, further comprising a waste tank configured to receive a liquid discharged from the electrochemical sensor. B001 An automated measurement system for determining the glycosylation degree of a protein, comprising: at least one pipette head; a microplate comprising at least one well (or a plurality of wells), an optical measurement well, and an electrochemical sensor; a drive system for moving the pipette head and the microplate relative to one another; and an electrical measurement unit configured to be connected to the electrochemical sensor. B002 The system of embodiment B001, further comprising a temperature control system for controlling the temperature of at least a portion of the microplate. B003 The system of embodiment B002, wherein the temperature control system comprises a heater configured to move relative to the microplate and to be in proximity to at least one well. B011 The system of embodiment B003, further comprising a computer for controlling at least one of the operation of the drive system, the heating by the heater, and the operation of the heater. C001 A method for electrochemically measuring a substance of interest, comprising: providing a microplate of any one of embodiments A001 to A021; providing a liquid possibly containing the substance of interest;A method comprising: introducing the liquid into any well of the microplate by a pipetting operation; performing a process required 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 by a pipetting operation into an electrochemical sensor; and detecting or measuring the target substance using the electrochemical sensor. C011 A method for measuring the glycosylation degree of a protein, comprising: providing the microplate according to embodiment A016; obtaining a body fluid that may have a glycated protein; introducing the obtained body fluid into a diluent well to prepare a dilution mixture; pipetting a predetermined amount of a mixture of the body fluid and the diluent (dilution mixture) from the diluent well and passing it through the filter of the filter well, and introducing the filtered dilution mixture into the filter well; removing the filter from the filter well; pipetting a predetermined amount of a solution (e.g., an oxidizing agent) in a preparation well from the preparation filter well and introducing it into a first stirring well; pipetting a predetermined amount of an indicator solution (e.g., BCP) from the indicator solution well and introducing it into the first stirring well to prepare an indicator mixture; pipetting a predetermined amount of the filtered mixture from the filter well and introducing it into the first stirring well to prepare a mixture for optical measurement; pipetting a predetermined amount of the optical measurement mixture from the first stirring well and introducing it into the optical measurement well; 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 the sensor cleaning solution from the sensor cleaning solution well and flowing it onto the sensor; pipetting a predetermined amount of the sensor calibration solution from the sensor calibration solution well and flowing it onto the sensor; calibrating the sensor using the sensor calibration solution; pipetting a predetermined amount of the protease solution from the protease solution well and introducing it into the second stirring well;A method comprising: pipetting a predetermined amount of the filtered mixture from the filter well and introducing it into a second stirring well to prepare a protease mixture; pipetting a predetermined amount of the protease mixture from the second stirring well at a predetermined time after introducing the protease and introducing it into a sensor; performing an electrical measurement on the protease mixture with the sensor; determining an amount of the glycated protein of interest based on the electrical measurement; and determining the degree of glycosylation of the protein from the determined amount of protein and the determined amount of glycated protein. C012 The method of embodiment C011, wherein preparing the protease mixture comprises heating the solution in the second stirring well to activate a protease reaction. C021 A method for measuring the glycosylation degree of a protein, comprising: providing a microplate according to embodiment A016b; obtaining a body fluid that may have a glycated protein; introducing the obtained body fluid into a diluent well to prepare a dilution mixture; pipetting a predetermined amount of a mixture of the body fluid and the diluent (dilution mixture) from the diluent well and passing it through the filter of the filter well, and introducing the filtered dilution mixture into the filter well; removing the filter from the filter well; pipetting a predetermined amount of a preparation solution (e.g., an oxidizing agent) from the preparation solution well and introducing it into an optical measurement well; pipetting a predetermined amount of an indicator solution (e.g., BCP) from the indicator solution well and introducing it into the optical measurement well to prepare an indicator mixture; pipetting a predetermined amount of the filtered dilution mixture from the filter well and introducing it into the optical measurement well to prepare an 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 cleaning solution from the sensor cleaning solution well and applying it to the sensor; pipetting a predetermined amount of sensor calibration solution from the sensor calibration solution well and applying it to the sensor; calibrating the sensor using the sensor calibration solution;A method comprising: pipetting a predetermined amount of protease solution from the protease solution well and introducing it into a first stirring well; pipetting a predetermined amount of filtered diluted mixture from the filter well and introducing it into a first stirring well to prepare a protease mixture; heating the first stirring well at a predetermined temperature for a predetermined time using a heater surrounding the first stirring well to react the protease mixture; pipetting a predetermined amount of protease mixture from the first stirring well at a predetermined time after introducing the protease and introducing it into a sensor; performing electrical measurements on the protease mixture using the sensor; determining the amount of glycated protein of interest based on the electrical measurements; and determining the glycosylation degree of the protein from the determined amount of protein and the determined amount of glycated protein. D001: Computer software for causing a computer to execute the methods of embodiments C001 to C012. E001: A storage medium storing the computer software of embodiment D001.

[0199] Several embodiments and examples of the present disclosure have been described above, but these embodiments and examples exemplify the present disclosure. For example, the above embodiments have been described in detail to clearly explain the present disclosure, and additional changes in dimensions, configurations, materials, and circuits may be made as necessary. Note that embodiments that combine any one or more features of the present disclosure described above are also within the scope of the present disclosure. The claims encompass numerous modifications to the embodiments without departing from the technical spirit of the present disclosure. Therefore, the embodiments and examples disclosed herein are provided for illustrative purposes and should not be considered to limit the scope of the present disclosure.

Claims

1. A microplate for determining the degree of glycation of a protein, comprising: (f) an optical measurement well; and (s) an electrochemical sensor; A microplate comprising:

2. The microplate according to claim 1, The (f) optical measurement well contains a protein indicator; Microplates.

3. The microplate according to claim 2, The optical measurement well further contains a protease. Microplates.

4. The microplate according to claim 1, (d) an indicator well containing a protein indicator; and (g) a protease liquid well containing a protease; The microplate further comprises:

5. The microplate according to claim 4, (a) a diluent well containing a diluent; (b) a filter well with a filter attached; (c) a preparation well containing a preparation solution for optical measurement of proteins; (e) a first stirring well used to stir the solution; (h) a second stirring well used to stir the solution; (i) a sensor wash well containing a sensor wash solution; and (j) a sensor calibrator well containing a sensor calibrator; The microplate further comprises any one or more of:

6. The microplate according to claim 4, (a) a diluent well containing a diluent; (b) a filter well with a filter attached; (c) a preparation well containing a preparation solution for optical measurement of proteins; (e) a first stirring well used to stir the solution; (h) a second stirring well used to stir the solution; (i) a sensor wash well containing a sensor wash solution; and (j) a sensor calibrator well containing a sensor calibrator; A microplate comprising:

7. 7. The microplate according to claim 1, a waste tank configured to receive effluent from the electrochemical sensor; The microplate further comprises:

8. An automated measurement system for determining the glycation degree of a protein, comprising: At least one pipette head; a drive system for moving the pipette head and the microplate relative to one another; an electrical measurement unit configured to be connected to the electrochemical sensor; Equipped with configured to receive a microplate having optical measurement wells and electrochemical sensors; system.

9. 9. The system of claim 8, A temperature control system for controlling the temperature of at least a portion of the microplate. Further systems.

10. 10. The system of claim 9, the temperature control system comprising a heater configured to move relative to the microplate and proximate to at least one well; system.

11. 11. The system of claim 10, a computer that controls at least one of the operation of the drive system, the heating by the heater, and the operation of the heater; Further systems.

12. A method for measuring the degree of glycation of a protein, comprising the steps of: Providing a microplate according to any one of claims 1 to 6; Providing a liquid potentially containing glycated proteins; introducing said liquid into any well of said microplate by a pipetting operation; subjecting the liquid in said well or in other wells of said microplate to any treatment required for the measurement of said protein; introducing said treated solution into said optical measurement well (f) by a pipetting operation; optically detecting or measuring proteins in said optical measurement well (f); subjecting said liquid and / or said protein in said well or in other wells of said microplate to any treatment required for the determination of said glycated protein; introducing the treated solution in the well to the electrochemical sensor(s) by a pipetting operation; and detecting or measuring glycated proteins using said electrochemical sensor(s); A method for providing the above.

13. A method for measuring a glycosylation degree of a protein, comprising the steps of: Providing a microplate according to claim 6; Obtaining a body fluid that may have glycated proteins; introducing the collected bodily fluid into said diluent well (a) to prepare a diluent mixture; pipetting a predetermined amount of a mixture of the body fluid and a diluent (dilution mixture) from the diluent well (a) through the filter of the filter well (b), and introducing the filtered dilution mixture into the filter well (b); removing the filter from said filter well (b); removing a predetermined amount of the solution in said priming well (c) from said priming well (c) and introducing it into said first stirring well (e) by a pipetting operation; removing a predetermined amount of indicator liquid from said indicator liquid well (d) by pipetting and introducing it into said first mixing well (e) to prepare an indicator mixture; removing a predetermined amount of the filtered mixture from the filter well (b) by pipetting and introducing it into the first stirring well (e) to prepare the mixture for optical measurement; removing a predetermined amount of the optical measurement mixture from the first stirring well (e) and introducing it into the optical measurement well (f) by a pipetting operation; Measuring an optical property of the optical measurement mixture in the optical measurement well (f); determining the amount of protein based on the optical property of the optical measurement mixture; removing a predetermined amount of sensor wash solution from said sensor wash solution well (i) and introducing it onto said sensor (s) by a pipetting operation; removing a predetermined amount of sensor calibrant from said sensor calibrant well (j) and introducing it into said sensor (s) by a pipetting operation; calibrating the (s) sensor with the sensor calibration solution; removing a predetermined amount of protease solution from said protease solution well (g) and introducing it into said second stirring well (h) by pipetting; removing a predetermined amount of the filtered mixture from the filter well (b) by pipetting and introducing it into the second mixing well (h) to prepare a protease mixture; removing a predetermined amount of the protease mixture from the second stirring well (h) at a predetermined time from protease introduction and introducing it to the sensor (s) by a pipetting operation; (s) performing an electrical measurement on the protease mixture with a sensor; determining the amount of the glycated protein of interest based on the electrical measurement; and determining the degree of glycation of the protein from the determined amount of protein and the determined amount of glycated protein; A method for providing the above.

14. 14. The method of claim 13, preparing the protease mixture comprises heating the solution in the second stirring well (h) to activate the protease reaction. method.

15. Computer software for causing a computer to carry out the method according to claims 12 to 14.