Method and device for measuring biomolecule

Abstract

One embodiment of the present disclosure provides a method for measuring a biomolecule. This method comprises: providing a recognition unit that includes a conductive polymer and a molecular recognition pH indicator acting as a dopant for the conductive polymer and recognizing a target biomolecule; introducing a sample containing the target biomolecule into the recognition unit; measuring the electrical characteristics of the conductive polymer, on the basis of a reaction between the target biomolecule and the molecular recognition pH indicator which occurs when the target biomolecule is introduced; and determining the amount of the target biomolecule in the sample, on the basis of the measured electrical characteristics.

Description

Method and apparatus for measuring biomolecules

[0001] The present disclosure relates to methods and devices for measuring biomolecules.

[0002] In biosensing, there is a demand for a technology that can measure biomolecules present in solutions such as body fluids more accurately and more easily.

[0003] For example, GA values ​​have been attracting attention as an important index for blood glucose management. A technology for easily and accurately measuring GA values ​​is needed. GA values ​​are the ratio of the amount of glycated albumin to the total amount of albumin. Biosensing technologies for glycated albumin have been developed. Meanwhile, the total amount of albumin is generally measured using the BCP method, BCG method, HPLC method, immunoturbidimetry, ELISA (enzyme-linked immunosorbent assay), high-performance liquid chromatography (HPLC) precipitation method, etc., but all of these are liquid-phase or solution-based measurement methods. Thus, the development of biosensors for measuring albumin has lagged behind.

[0004] Here, it is recognized that there is a need for a technology that can simply and easily measure, using a biosensor, not only the total amount of albumin but also, for example, but not limited to, various substances (such as proteins) present in body fluids.

[0005] In some embodiments of the present disclosure, a method for measuring a biomolecule is provided. In some embodiments, the method comprises providing a recognition unit including a conductive polymer and a molecular recognition pH indicator that acts as a dopant for the conductive polymer and recognizes a target biomolecule. In some embodiments, the method comprises introducing a sample containing the target biomolecule to the recognition unit. In some embodiments, the method comprises measuring an electrical property of the conductive polymer based on a reaction between the target biomolecule and the molecular recognition pH indicator that occurs when the target biomolecule is introduced. In some embodiments, the method comprises determining the amount of the target biomolecule in the sample based on the measured electrical property.

[0006] According to the above embodiment, for example, the amount of biomolecules in a body fluid can be easily and accurately measured using a biosensor.

[0007]

[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.

[0008]

[0009] The term "conductive polymer" as used herein should be understood as a general scientific term and refers to a polymer that is electrically conductive and whose electrical conductivity can be changed by appropriate doping. Examples of "conductive polymers" include, but are not limited to, poly(3,4-ethylenedioxythiophene) (PEDOT), polyaniline (PANI), polythiophene (PT), polypyrrole (PPy), polyfluorene (PF), poly(paraphenylenevinylene) (PPV), and poly(methyl methacrylate) (PMMA).

[0010] The term "molecular recognition pH indicator" as used herein should be understood as a general scientific term and refers to a substance that has the ability to recognize a target biomolecule and functions as a pH indicator. Generally, pH indicators are small molecules.

[0011] The molecular recognition pH indicator may be selected depending on the target biomolecule. Examples of combinations of target biomolecules and molecular recognition pH indicators that recognize them are shown in Table 1.

[0012] In some embodiments, the target biomolecule is albumin, and the molecular recognition pH indicator may be bromocresol purple (BCP) or bromocresol green (BCG). For example, the molecular recognition pH indicator may be BCP.

[0013] The molecular recognition pH indicator may be disposed inside the conductive polymer network. For example, the molecular recognition pH indicator may be mixed into the monomer at or before the start of polymerization. This allows, for example, the molecular recognition pH indicator to be uniformly disposed inside the conductive polymer network, making it easier for the molecular recognition pH indicator to remain inside the conductive polymer network and / or reducing the probability or speed of desorption from the conductive polymer network.

[0014] In some embodiments, other polymers or dopants may be disposed within the conductive polymer network. Examples of conductive polymer and dopant combinations are shown in Table 2.

[0015] In some embodiments, the conductive polymer may be PEDOT, and polystyrene sulfonate (PSS) may be disposed within the PEDOT network. In some embodiments, the conductive polymer may be PEDOT, and PSS and BCP may be disposed within the PEDOT network.

[0016] The conductive polymer may be prepared by polymerizing a monomer. In some embodiments, a layer including a conductive polymer and a dopant may be formed by providing at least a monomer for the conductive polymer and polymerizing the monomer. The polymerization may be performed by applying a solution including the monomer to a desired position on a substrate surface and leaving it to stand at room temperature. For example, a conductive polymer member or a conductive polymer layer may be formed by applying a polymerization liquid to a predetermined surface such as an electrode or a sensor substrate and polymerizing the monomer contained in the polymerization liquid. For example, the conductive polymer member or conductive polymer layer produced by polymerization may be disposed on a predetermined surface such as an electrode or a sensor substrate.

[0017] The polymerization may be electropolymerization. The polymerization may be electropolymerization. For example, a conductive polymer can be formed on the electrode surface by applying a solution containing a monomer to the electrode surface and applying a potential to the electrode. For example, a conductive polymer can be formed as a channel connecting the drain electrode and the source electrode by applying a solution containing a monomer to the surfaces of a drain electrode, a source electrode, and an insulator therebetween for a transistor and applying an electric field to the drain electrode and the source electrode.

[0018] As used herein, the term "polymerization liquid" generally refers to a solution containing the monomers to be polymerized. The polymerization liquid may further contain initiators, catalysts, and other additives.

[0019] In some embodiments, the polymerization liquid may contain a mixture of a dopant or a material that serves as a dopant, in addition to the monomer for the conductive polymer. For example, if the conductive polymer contains PEDOT and the dopant contains BCP or BCG, the polymerization liquid may contain 3,4-ethylenedioxythiophene (EDOT) and BCP or BCG. EDOT is polymerized to form PEDOT. In some embodiments, BCP or BCG may exist as a single molecule within the PEDOT network. In some embodiments, BCP or BCG may be polymerized and exist within the PEDOT network. For example, BCP or BCG may be polymerized simultaneously with or separately from the EDOT polymerization process. Polybromocresol purple or a BCP-containing polymer may be mixed into the conductive polymer network.

[0020] In some embodiments, a conductive polymer may be formed by polymerization, and then a dopant may be introduced into the conductive polymer network, for example, by immersing the formed PEDOT network in a solution of BCP or BCG, thereby impregnating the PEDOT network with BCP or BCG.

[0021] In some embodiments, the target biomolecule may be provided in a liquid (solution). The liquid may be a bodily fluid secreted by the subject, or a liquid other than a bodily fluid. The liquid other than a bodily fluid may be a liquid attached to the subject, or a liquid not attached to the subject. The liquid not attached to the subject may be a liquid contained in the subject.

[0022] The provided liquid may contain or may potentially contain the target biomolecule, or may be a reference solution used to measure the target biomolecule. Hereinafter, these cases will be included and may be referred to as a liquid (or solution) containing the target biomolecule.

[0023] The liquid containing the target biomolecule may be a solution. The liquid may be a body fluid, a solution derived from a body fluid, or a diluted solution of a body fluid. The liquid 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. The sample to be measured may be a specimen.

[0024] 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 be a solution. The solution may contain a physiological buffer solution containing the target substance, such as phosphate-buffered saline (PBS), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid buffer (TES), or hydroxyethylpiperazineethanesulfonic acid buffer (HEPES). The solution is not particularly limited as long as it contains the target substance.

[0025] In some embodiments, the bodily fluid may be blood. In some embodiments, blood may be collected. For example, blood may be collected simultaneously with bleeding from a puncture. For example, blood may be aspirated by inserting a needle. In some embodiments, a puncture device (e.g., a needle, a syringe needle, etc.; the same applies below) may be disposed at the tip of the capillary. In some embodiments, the capillary may be formed as a puncture device.

[0026] In some embodiments, the subject may include or be a human. In some embodiments, the subject may include or be a non-human animal. The non-human animal may include or be a mammal. The non-human animal may be, for example, but not limited to, a working animal, a livestock animal, a pet animal, or a wild animal.

[0027] The term "electrode" as used herein is a broad term and should be given its ordinary and customary meaning to those skilled in the art and should not be construed as being limited to any particular or special meaning. In some embodiments, the electrode may be used in electrochemical measurements. The electrode may be formed of a conductive material. The electrode may be formed of a metal. The electrode may be formed of a metallic material, such as, but not limited to, gold (Au), platinum (Pt), palladium (Pd), etc. The electrode may be formed of a non-metallic material. For example, but not limited to, the electrode may be formed of carbon (C). The electrode may be formed of graphene, carbon nanotubes, etc.

[0028] The electrodes may be connected or configured to be connected to a meter, measuring device or measuring element.

[0029] In some embodiments, the electrodes may be connected to an impedance measuring device. In some embodiments, the electrical signal at the electrodes may be detected or analyzed using electrochemical impedance spectroscopy (EIS), which may generate a Cole-Cole plot or a Nyquist plot. The capacitive component of the measurement system may be determined or analyzed. In some embodiments, the electrical signal at the electrodes may be detected or analyzed using cyclic voltammetry (CV).

[0030] In some embodiments, the current may be measured at a potential corresponding to the redox reaction of the conductive polymer. In this case, a current measuring device may be used. The amount of the target biomolecule may be determined from the peak value of the current at the potential corresponding to the redox reaction or the difference from the background of the peak value.

[0031] In some embodiments, the electrode in the vicinity of the aptamer may be used as the working electrode (WE), and additionally, a counter electrode (CE) and / or a reference electrode (RE) may be placed.

[0032] In some embodiments, the electrodes may be connected to a current meter, ammeter, amperometer, etc. The electrical signal at the electrodes may be measured or analyzed amperometrically.

[0033] In some embodiments, the electrodes may be connected to a voltmeter, potentiometer, transistor (e.g., field effect transistor), etc. The electrical signal at the electrodes may be measured or analyzed potentiometrically.

[0034] In some embodiments, the electrode may be an electrode of a transistor. For example, the electrode may be an electrode of a so-called biotransistor or an electrode of a so-called FET (field effect transistor) biosensor. The electrode may be the drain and / or source of the transistor.

[0035] Example 1 Electrochemical electrodes were fabricated on the end faces of gold (Au) rods (3 mm diameter, BAS). The Au end faces were polished with alumina (0.05 μm particle size) and ultrasonically cleaned in DI water for 1 minute. Galvanostatic deposition was performed in a polymerization solution containing 7 mM EDOT, 1 mM BCP (Fujifilm Wako), and 5-50 mM poly(styrene sulfonate) sodium salt (NaPSS) (Alfa Aesar), and 0.1 M perchloric acid (HClO ４ ) was prepared with deionized water (DI water). This solution was heated under nitrogen (N) with stirring for 30 minutes before use. ２ ) gas is bubbled to completely dissolve the components, and oxygen (O ２ The galvanostatic deposition was performed at 0.10 mA / cm while stirring the solution. ２ The current density was 100 s (Keysight B1500A), which allowed for 375 s. This allowed for a uniform composition within the film. A PEDOT film with a thickness of approximately 100 nm and doped with BCP and PSS was formed on the gold electrode surface.

[0036] All electrochemical measurements were performed using an Ag / AgCl reference electrode and a Pt counter electrode in saturated KCl, and an electrochemical analyzer (ALS 618E, BAS, or B1500A, Keysight). HSA solutions were adjusted to concentrations of 0, 0.17, 0.5, and 1.5 mg / mL using 1x phosphate-buffered saline (PBS).

[0037] The incorporation of BCP into the PEDOT network was confirmed by observing the pH response of the BCP redox couple during cyclic voltammetry (CV) (not shown). A HSA solution was introduced into 1x phosphate-buffered saline (PBS) and incubated at room temperature for 20 minutes. Then, as a pretreatment for the measurement, a voltage of 0.6 V was applied to an Ag / AgCl reference electrode, and a current of 28 μA / cm was applied. ２ A current of was passed.

[0038] FIG. 1 shows the CV characteristics of HSA solutions of various concentrations (horizontal axis: potential (V) applied to the working electrode relative to the Ag / AgCl reference electrode). ＷＥ ), vertical axis: current flowing through the working electrode (I ＷＥ )). The oxidation peak of the redox reaction of BCP incorporated into the PEDOT network was observed at 370 mV. It was observed that the height of this oxidation peak changed with the HSA concentration of the HSA solution.

[0039] Figure 2 shows the working electrode current (peak current I) at 370 mV of the CV characteristics versus HSA concentration. This shows that the peak current at the working electrode monotonically increases with the HSA concentration. This result indicates that the HSA-BCP complex within the PEDOT network can be electrochemically and quantitatively detected.

[0040] One scientific theory is that BCP undergoes a structural change upon binding to albumin, becoming reduced, making the albumin-BCP complex more susceptible to oxidation. The anionic component of BCP may function as a dopant for PEDOT. The change in the oxidation peak height in the CV characteristics of PEDOT containing BCP is thought to reflect changes in the redox state of BCP due to albumin-BCP complex formation or the conductivity of PEDOT. However, this mechanism is merely one of the hypotheses proposed by the inventors at this stage, and the contents of this disclosure should not be interpreted as being limited to this scientific theory. This statement does not exclude the possibility that this disclosure may be interpreted based on other scientific theories or mechanisms.

[0041] BCP is a small molecule compared to polymers. Therefore, it was not known that a sufficient amount of BCP could remain in the vicinity of PEDOT molecules or inside the PEDOT network to allow PEDOT to electrically detect the reaction with the HSA-BCP complex. Furthermore, at least to the inventors' knowledge, it was not known that BCP functions as a dopant for conductive polymers such as PEDOT. The inventors were the first in the world to discover that the HSA-BCP complex can be electrochemically and quantitatively detected by PEDOT.

[0042] Those skilled in the art will recognize that this result does not only apply to the HSA and PEDOT:BCP system, but also applies to a broader concept: the present disclosure also includes the idea that the reaction of a biomolecule with a molecular recognition pH indicator that can recognize the biomolecule and function as a dopant affects the electrical properties of the conductive polymer, and that the biomolecule can be detected or quantified based on the electrical properties.

[0043] The electrical properties of the conductive polymer can be measured by various techniques. In some embodiments, cyclic voltammetry may be used. In some embodiments, a redox reaction near an electrode having a conductive polymer disposed on its surface may be measured. For example, the height (current value) of the redox peak may be measured. In some embodiments, the conductivity of the conductive polymer may be measured. For example, a transistor may be constructed using a conductive polymer containing a molecular recognition pH indicator as its channel. The current (drain current) in the channel generated by the reaction between the target biomolecule and the molecular recognition pH indicator may be measured.

[0044] 3 shows a schematic cross-sectional view of a device 300 for detecting a biomolecule according to an embodiment. The device 300 includes a recognition unit 310 and a transducer 320.

[0045] The recognition unit 310 includes a network of a conductive polymer 311 and a molecular recognition pH indicator 312 that recognizes a target biomolecule 351. The molecular recognition pH indicator 312 is arranged in the network of the conductive polymer 311 so that an electrical signal resulting from recognition of the target biomolecule 351 can be transmitted to the conductive polymer 311.

[0046] 3 and the following figures, the molecular recognition pH indicators 312, 412, and 512 are arranged in the vicinity of the conductive polymers 311, 411, and 511. However, these figures are schematic diagrams, and the location and manner of arrangement of each molecule should not be interpreted as being limited to this.

[0047] The device 300 may have a fluid chamber (not shown) that can accommodate the recognition unit 310 and receive a fluid. A solution 350 containing a target biomolecule 351 is introduced into the fluid chamber of the device 300 (FIG. 3A). The solution 350 contacts the surface of the recognition unit 310.

[0048] A target biomolecule 351 in a solution 350 flows into the network of the conductive polymer 311. The target biomolecule 351 is captured by the molecular recognition pH indicator 312 to form a complex 330. This complex 330 acts as a dopant, creating a positive charge (indicated by a plus sign in the figure) in the conductive polymer 311, thereby changing the electrical or conductive properties of the conductive polymer 311.

[0049] The transducer 320 is disposed near the recognition unit 310 and is configured to electrochemically detect a chemical reaction in the recognition unit 310. The transducer 320 is configured to electrically detect the electrical properties of the conductive polymer 311 or a change therein.

[0050] The transducer 320 may be connected to electronics or circuitry, such as a computer (not shown), that analyzes the signal output by the transducer 320 .

[0051] A more specific embodiment of this embodiment will be described with reference to FIGS.

[0052] 4 shows a schematic cross-sectional view of a device 400 for detecting biomolecules by cyclic voltammetry (CV method) according to an embodiment. The device 400 includes a recognition unit 410 and an electrode 421 as a transducer.

[0053] The recognition unit 410 includes a network of a conductive polymer 411 and a molecular recognition pH indicator 412 that recognizes a target biomolecule 451. The molecular recognition pH indicator 412 is arranged in the network of the conductive polymer 411 so as to transmit an electrical signal to the conductive polymer 411 by recognizing the target biomolecule 451.

[0054] The device 400 may have a fluid chamber (not shown) that can accommodate the recognition unit 410 and receive a fluid. A solution 450 containing a target biomolecule 451 is introduced into the fluid chamber of the device 400 ( FIG. 4A ). The solution 450 contacts the surface of the recognition unit 410.

[0055] A target biomolecule 451 in a solution 450 flows into the network of the conductive polymer 411. The target biomolecule 451 is captured by the molecular recognition pH indicator 412 to form a complex 430.

[0056] The electrode 420 is disposed near the recognition unit 410 and is configured to electrochemically detect a chemical reaction occurring in the recognition unit 410. In Fig. 4, the electrode 420 has a flat surface on which a conductive polymer 411 is layered. In Fig. 4, a three-electrode method is used. A counter electrode (CE) 423 and a reference electrode (RE) 423 are disposed in contact with the solution 450. The electrode 420 functions as a working electrode (WE).

[0057] This allows the oxidation-reduction reaction of the conductive polymer 411 due to the formation of a complex 430 between the target biomolecule 451 and the molecular recognition pH indicator 412 to be measured by CV using three electrodes.

[0058] 4, device 400 includes an analyzer 440 connected to a working electrode (WE) 421, a counter electrode (CE) 422, and a reference electrode (RE) 423. Analyzer 440 may be a potentiostat, a galvanostat, or a potentiogalvanostat. In some embodiments, analyzer 440 may be included in device 400. In some embodiments, analyzer 440 may be external to device 400 or configured to be connected to device 400.

[0059] In some embodiments, the peak current at the redox potential (redox peak current) may be measured instead of the CV, and a current measuring device may be used for this purpose.

[0060] 5 shows a schematic cross-sectional view of a device 500 for detecting biomolecules using a transistor configuration according to an embodiment. The device 500 includes a recognition unit 510 and a source electrode 521, a drain electrode 522, and a gate electrode 523 as transducers.

[0061] The recognition unit 510 includes a network of a conductive polymer 511 and a molecular recognition pH indicator 512 that recognizes a target biomolecule 551. The molecular recognition pH indicator 512 is arranged in the network of the conductive polymer 511 so as to transmit an electrical signal to the conductive polymer 511 by recognizing the target biomolecule 551.

[0062] The device 500 may have a fluid chamber (not shown) that can receive a fluid and accommodates the recognition unit 510. A solution 550 containing a target biomolecule 551 is introduced into the fluid chamber of the device 500. The solution 550 contacts the surface of the recognition unit 510.

[0063] A target biomolecule 551 in a solution 550 flows into the network of the conductive polymer 511. The target biomolecule 551 is captured by the molecular recognition pH indicator 512 to form a complex 530.

[0064] The source electrode 521 and the drain electrode 522 are formed on the surface of the substrate 520 and are insulated from each other by an insulator 524. The recognition unit 510 is formed on the surface of the insulator 524 between the source electrode 521 and the drain electrode 522, and is connected to the source electrode 521 at one end and to the drain electrode 522 at the other end. This allows the recognition unit 510 to function as a channel between the source electrode 521 and the drain electrode 522. The channel electrode 523 is arranged so as to be in contact with the introduced solution 550.

[0065] This allows the change in conductivity of the conductive polymer 511 due to the formation of a complex 530 between the target biomolecule 551 and the molecular recognition pH indicator 512 to be measured by the biotransistor.

[0066] In Figure 5, device 500 includes an analyzer 540 connected to source electrode 521, drain electrode 522, and gate electrode 523. Figure 5 shows a simplified circuit diagram of analyzer 540 as an example. However, this is merely an example, and other elements, circuits, etc. may be added or substituted for this circuit. Analyzer 540 may be a potentiostat, galvanostat, or potentiogalvanostat. In some embodiments, analyzer 540 may be included in device 500. In some embodiments, analyzer 540 may be external to device 500 or configured to be connected to device 500.

[0067] Application to GA Value Measurement The present disclosure also provides a method and device for determining a GA value. The method or device for measuring the amount of albumin of the present disclosure can also be applied to determining a GA value. In some embodiments, the amount of albumin may be determined using the method or device of the present disclosure, and the amount of glycated albumin may be determined, and the GA value, which is the ratio of the amount of glycated albumin to the total amount of albumin, may be determined from the determined amount of albumin and the amount of glycated albumin.

[0068] An example of a technique for determining the amount of glycoalbumin is given below.

[0069] In some embodiments, the amount of glycated albumin may be measured using an enzymatic method. For example, a sample containing partially glycated albumin is provided, and the albumin is degraded using a protease to generate peptide fragments containing fructosyl lysine. Hydrogen peroxide is generated from the peptide fragments using fructosyl amino acid oxidase (FAOD). The amount of hydrogen peroxide can be quantified to determine the amount of glycated albumin contained in the provided sample.

[0070] In some embodiments, the amount of glycated albumin may be measured using an aptamer. For example, a sample containing partially glycated albumin is provided, and an aptamer that specifically recognizes glycated albumin is used to capture the glycated albumin in the sample. The captured glycated albumin is then quantified to determine the amount of glycated albumin contained in the provided sample. For example, a mediator may be used. An electrode capable of measuring the mediator is provided, and an aptamer is immobilized on the surface of the electrode. The current flowing to the electrode by the mediator decreases depending on the amount of glycated albumin captured by the immobilized aptamer. This current can be measured to determine the amount of glycated albumin contained in the provided sample.

[0071] In some embodiments, the amount of glycoalbumin may be measured using a conductive polymer such as PEDOT. For example, a substance having phenylboronic acid groups that recognize the glucose in glycoalbumin may be mixed with PEDOT. For example, a double-network polymer structure may be provided in which PEDOT and a polymer having phenylboronic acid groups are mixed. A sample containing partially glycated albumin is introduced into this double-network polymer structure, allowing the phenylboronic acid groups to recognize glycoalbumin. The electrical properties of PEDOT change depending on the amount of glycoalbumin recognized by the phenylboronic acid groups. The amount of glycoalbumin contained in the provided sample can be determined by measuring these electrical properties.

[0072] In some embodiments, a device for measuring the total amount of albumin and a device for measuring the amount of glycated albumin may be provided separately, or in some embodiments, a unit for measuring the total amount of albumin and a unit for measuring the amount of glycated albumin may be provided disposed within a single device.

[0073] 6 is a schematic diagram showing a configuration 600 of a fluidic device for determining a GA value according to one embodiment. The fluidic device 600 includes an albumin (Alb) sensor unit 620 using a conductive polymer and a glycoalbumin (GA) sensor unit 630 using an enzymatic method.

[0074] 6 has an inlet 610 for receiving a sample solution. The sample introduced from the inlet 610 is divided and guided to an Alb sensor unit 620 and a GA sensor unit 630, respectively.

[0075] The Alb sensor unit 620 has a first fluid chamber 621. One of the samples diverted from the inlet 610 enters and fills this first fluid chamber 621.

[0076] The GA sensor unit 630 has two fluid chambers fluidly connected in series, a second fluid chamber 631 and a third fluid chamber 633. The other sample diverted from the inlet 610 first enters and fills the second fluid chamber 631, exits the second fluid chamber 631, and then enters and fills the third fluid chamber 633.

[0077] The first fluid chamber 621 accommodates therein an albumin measuring device 622 for measuring albumin as provided herein. The albumin measuring device 622 has a recognition unit in which a conductive polymer and BCP or BCG are mixed. As an example, the albumin measuring device 622 may be an amperometric device. As an example, the albumin measuring device 622 may be a biotransistor. An electrode is disposed near the recognition unit. The electrode may be disposed inside the first fluid chamber 621. An analyzer may be disposed outside the first fluid chamber 621.

[0078] The second fluid chamber 631 contains therein a protease 632. The third fluid chamber 633 contains therein an enzyme electrode 634 including a ketoamine oxidase and a hydrogen peroxide electrode.

[0079] 6 measures albumin and glycoalbumin in the same sample at separate locations, and therefore measurement conditions such as reaction time, temperature, and time profile can be optimized and set independently for each measurement.

[0080] 6, the fluidic device 600 has an outlet 641 for discharging fluid from the first fluid chamber 621 and an outlet 642 for discharging fluid from the third fluid chamber 633. A single outlet may be provided, and fluid from either outlet may be discharged from that outlet. Alternatively, a tank (not shown) may be provided inside the fluidic device 600 to store the discharged solution, rather than providing an outlet.

[0081] 7 is a schematic diagram showing the configuration 700 of a fluidic device for determining a GA value according to one embodiment. The fluidic device 700 includes an albumin (Alb) sensor unit 720 using a conductive polymer and a glycoalbumin (GA) sensor unit 730 using an enzymatic method.

[0082] In the fluidic device 700 of FIG. 7, an inlet 710, an Alb sensor unit 720, and a GA sensor unit 730 are fluidly connected in series in this order.

[0083] The Alb sensor unit 720 has a first fluid chamber 721. The sample introduced from the inlet 710 first enters and fills this first fluid chamber 721.

[0084] The GA sensor unit 730 has two fluid chambers fluidly connected in series, a second fluid chamber 731 and a third fluid chamber 732. The sample exits the first fluid chamber 721, enters and fills the second fluid chamber 731, exits the second fluid chamber 731, and then enters and fills the third fluid chamber 733.

[0085] The first fluid chamber 721 accommodates therein an albumin measuring device 722 for measuring albumin as provided herein. The albumin measuring device 722 has a recognition unit in which a conductive polymer and BCP or BCG are mixed. As an example, the albumin measuring device 722 may be an amperometric device. As an example, the albumin measuring device 722 may be a biotransistor. An electrode is disposed near the recognition unit. The electrode may be disposed inside the first fluid chamber 721. An analyzer may be disposed outside the first fluid chamber 721.

[0086] The second fluid chamber 731 contains therein a protease 732. The third fluid chamber 733 contains therein an enzyme electrode 734 including a ketoamine oxidase and a hydrogen peroxide electrode.

[0087] 7 measures albumin and glycoalbumin in separate locations within a single flow path system, which allows measurement conditions such as reaction time, temperature, and time profile to be optimized and set independently for each measurement, and also allows for a compact design of the flow path system.

[0088] The substance contained in the first fluid chamber 721 may leave the first fluid chamber 721 along with the sample flow and reach downstream. In this case, it is important that the substance contained in the first fluid chamber 721, such as BCP used to measure albumin, does not affect the reaction or measurement in the second fluid chamber 731 and / or the third fluid chamber 733 located downstream. Alternatively, the substance contained in the first fluid chamber 721 may be placed or immobilized within the first fluid chamber 721 so as not to leave the first fluid chamber 721. The inventors confirmed that BCP and BCG do not affect the measurement of the protease-mediated degradation of albumin and the reaction of the resulting peptide fragments with the enzyme electrode.

[0089] 7, the fluidic device 700 has an outlet 740 for discharging fluid from the third fluid chamber 733. In other embodiments, an outlet may not be provided, and a tank (not shown) may be provided inside the fluidic device 700 for storing the discharged solution.

[0090] 8 is a schematic diagram showing a configuration 800 of a fluidic device for determining a GA value according to one embodiment. The fluidic device 800 in FIG. 8 has a single fluid chamber 815, in which both an albumin (Alb) sensor unit 820 using a conductive polymer and a glycoalbumin (GA) sensor unit 830 using an enzymatic method are housed.

[0091] A sample introduced through an inlet 810 enters and fills a single fluid chamber 815 .

[0092] The albumin measuring device 822 disposed inside the single fluid chamber 815 has a recognition unit in which a conductive polymer and BCP or BCG are mixed. As an example, the albumin measuring device 822 may be an amperometric device. As an example, the albumin measuring device 822 may be a biotransistor. An electrode is disposed near the recognition unit. The electrode may be disposed inside the single fluid chamber 815. An analyzer may be disposed outside the single fluid chamber 815.

[0093] The GA sensor unit 830 disposed inside the single fluid chamber 815 has a protease 832 and an enzyme electrode 834 including a ketoamine oxidase and a hydrogen peroxide electrode. In Figure 8, the protease 832 and the enzyme electrode 834 are disposed at different locations on a plane. In other embodiments, the protease 832 and the enzyme electrode 834 may be disposed in layers. The arrangement of the GA sensor unit 830, the protease 832, the enzyme electrode 834, etc., inside the single fluid chamber 815 should not be construed as being limited to that shown in Figure 8, and other embodiments are also possible.

[0094] The optimum temperature for operation of the albumin measurement device 822 may differ from the optimum conditions for the protease 832 and the enzyme electrode 834. In such cases, for example, the temperature of the single fluid chamber 815 may be changed to temporally separate the operation of the albumin measurement device 822 from the operation of the protease 832 and the enzyme electrode 834. For example, the albumin measurement device 822 may be first operated at a relatively low temperature (first temperature, T1) to measure the total amount of albumin in the sample, followed by the reaction of the protease 832 at a relatively high temperature (second temperature, T2 > T1), and then the enzyme electrode 834 may be operated at a lower temperature (third temperature, T3 < T2). In this way, multiple reactions or device operations may be performed spatially within a single fluid chamber or may be performed at different times.

[0095] 8 measures albumin and glycoalbumin in a single flow channel chamber, allowing the flow channel system to be designed in an extremely compact manner.

[0096] 8, the fluidic device 800 has an outlet 840 for discharging fluid from the single fluid chamber 815. In other embodiments, there may be no outlet, and a reservoir (not shown) may be provided within the fluidic device 800 to store the discharged solution.

[0097] The above-described configurations of the fluidic device are merely examples, and the present invention should not be construed as being limited thereto. The present disclosure also provides other embodiments of the fluidic device.

[0098] In some embodiments, the glycoalbumin measuring unit or device may be a device including a conductive polymer of the present disclosure and a dopant. For example, the conductive polymer may be PEDOT:PSS, and the dopant may be a polymer including phenylboronic acid groups. The phenylboronic acid groups recognize the glucose in glycoalbumin, thereby changing the electrical properties of the conductive polymer. The amount of glycoalbumin can be measured by measuring the electrical properties.

[0099] Fluid Delivery Mechanism / Liquid Feeder The disclosed device may have a fluid delivery mechanism or fluid feeder that moves introduced solutions to the appropriate fluid chambers. Alternatively, the disclosed device may be configured to move introduced samples to the appropriate fluid chambers by an external fluid delivery mechanism.

[0100] In some embodiments, the liquid feeder may be a pusher that pushes the deformable fluid chamber from the outside. The liquid feeder may compress the liquid chamber to reduce its volume and feed the liquid contained in the fluid chamber to the outside. In some embodiments, the liquid feeder may be a pump. It may be fluidly connected to the fluid chamber or configured to be fluidly connected to the fluid chamber. In some embodiments, it may be a rotor unit that rotates the fluidic device. The liquid feeder may apply centrifugal force to the fluidic device to move the liquid inside the fluidic device.

[0101] The solution may be moved by positive pressure, negative pressure, or a combination thereof. Positive pressure may be applied to the solution upstream of a predetermined position in the flow path. Positive pressure may be applied to the solution downstream of a predetermined position in the flow path. Positive pressure and negative pressure may be applied by a pump. Positive pressure may be applied by externally compressing a predetermined position in the flow path. Pressure may be applied by centrifugal force. For example, the fluidic device may be configured to be mounted on a rotating disk or a rotating unit. The rotation generates centrifugal force, and the solution in the flow path is moved in the direction of the centrifugal force.

[0102] 6 to 8 are schematic illustrations of fluidic devices 600, 700, and 800, and the dimensions, size, and layout should not be interpreted strictly as being in accordance with the drawings. In the drawings, the flow paths connected to the fluidic devices (solid lines in the drawings) are depicted as straight lines with right-angle bends at the bends, but this should also be understood as being schematic. The flow paths may have curves. A single flow path may be composed of curves, or may be composed of straight and curved lines. A single flow path may have multiple sub-flow paths connected at an angle.

[0103] The fluidic device may have other chambers. The fluidic device may further have elements with other functions. Such elements may be located within or fluidly connected to the channels or chambers.

[0104] In some embodiments, the fluidic device may have a filter or separation unit that removes contaminants from an introduced sample. For example, if the sample is whole blood and albumin in the blood is to be measured, a filter (e.g., a serum filter or plasma filter) that removes blood cell components may be placed upstream of the Alb sensor unit. For example, a filter that removes blood cell components may be placed upstream of the Alb sensor unit and upstream of the GA sensor unit. For example, if the sample is saliva and albumin in the saliva is to be measured, a filter that removes contaminants such as mucin and food particles may be placed upstream of the Alb sensor unit. For example, a filter that removes contaminants such as mucin and food particles may be placed upstream of the Alb sensor unit and upstream of the GA sensor unit.

[0105] [Rule 91 Amendment 09.12.2024] This disclosure includes what is disclosed or described in Figures 9A1 to 9A23.

[0106] [Rule 91 Amendment 09.12.2024] This disclosure includes what is disclosed or described in Figures 9B1 to 9B3.

[0107] The present disclosure includes the following embodiments: A001. A method for measuring a biomolecule, comprising: providing a recognition unit including a conductive polymer and a molecular recognition pH indicator that acts as a dopant for the conductive polymer and recognizes a target biomolecule; introducing a sample containing the target biomolecule to the recognition unit; measuring an electrical property of the conductive polymer based on a reaction between the target biomolecule and the molecular recognition pH indicator that occurs when the target biomolecule is introduced; and determining the amount of the target biomolecule in the sample based on the measured electrical property. A002. The method of A001 or any embodiment, wherein the molecular recognition pH indicator is disposed within a network of the conductive polymer. A011. The method of A001 or any embodiment, wherein the recognition unit further comprises a second polymer disposed within the network of the conductive polymer. A012. The method of A011 or any embodiment, wherein the second polymer has the ability to act as a dopant for the conductive polymer. A021. A001. The method according to any one of the preceding embodiments, wherein the electrical property is a CV property associated with the reaction between the target biomolecule and the molecular recognition pH indicator. A022. The method according to any one of the preceding embodiments, wherein the electrical property is a conductive property of the conductive polymer. A101a. The method according to any one of the preceding embodiments, wherein the target biomolecule is albumin, and the molecular recognition pH indicator is BCP or BCG. A101b. A method for measuring albumin, comprising: providing a recognition unit including a conductive polymer and BCP or BCG; introducing a sample containing albumin to the recognition unit; measuring the electrical property of the conductive polymer based on the reaction between the albumin and the BCP or BCG that occurs when the albumin is introduced; and determining the amount of albumin in the sample based on the measured electrical property.A105. The method according to A101a or A101b, or any of the embodiments, wherein the conductive polymer comprises PEDOT. A106. The method according to A105, or any of the embodiments, wherein the recognition unit further comprises a PSS disposed in the network of the conductive polymer. A201. A method for determining a GA value, comprising: providing a sample containing albumin; determining the amount of albumin in the sample using the method according to any of A101a to A106, or any of the embodiments; determining the amount of glycated albumin in the sample; and determining the GA value of the sample from the amount of albumin and the amount of glycated albumin. A211. A201. The method according to any one of the preceding embodiments, wherein determining the amount of glycated albumin comprises: introducing a protease into the sample and causing the protease to degrade albumin to generate peptide fragments; introducing ketoamine oxidase to the peptide fragments and generating hydrogen peroxide; measuring the amount of hydrogen peroxide; and determining the amount of glycated albumin in the sample from the measured amount of hydrogen peroxide. B001. A device for measuring a biomolecule, comprising: a recognition unit including a conductive polymer and a molecular recognition pH indicator that acts as a dopant for the conductive polymer and recognizes a target biomolecule; and a transducer that converts a reaction between the molecular recognition pH indicator and the target biomolecule, which occurs when the target biomolecule is introduced, into an electrical signal. B005. The device according to any one of the preceding embodiments, further comprising a circuit connected to the transducer and configured to process the electrical signal based on the reaction between the molecular recognition pH indicator and the target biomolecule.B011. The device of B001 or any embodiment, wherein the transducer comprises an electrode, wherein the conductive polymer and the molecular recognition pH indicator are disposed on a surface of the electrode, and wherein the device further comprises a circuit connected to the electrode and configured to evaluate a redox reaction of the conductive polymer associated with a reaction between the molecular recognition pH indicator and the target biomolecule upon introduction of the target biomolecule. B021. The device of B001 or any embodiment, comprising a drain electrode and a source electrode; the conductive polymer is disposed so as to connect the drain electrode and the source electrode, and wherein the device comprises a circuit connected to the drain electrode and the source electrode and configured to measure a transistor characteristic between the drain electrode and the source electrode associated with a reaction between the molecular recognition pH indicator and the target biomolecule upon introduction of the target biomolecule. B101. C001. A device (unit) for measuring albumin, comprising: a recognition unit containing PEDOT and BCP or BCG; and a transducer for detecting a reaction between the BCP or BCG and the albumin when albumin is introduced. C001. A device for measuring GA value, comprising: a unit for measuring albumin according to B101 or any of the embodiments; and a unit for measuring glycoalbumin. C101. A fluidic device for measuring GA value, comprising: an albumin measurement chamber having therein the unit for measuring albumin according to B101 or any of the embodiments; and a glycoalbumin measurement chamber having therein a unit for measuring glycoalbumin. C111. The fluidic device according to C101 or any of the embodiments, further comprising a liquid delivery mechanism configured to introduce a target sample into the albumin measurement chamber and the glycoalbumin measurement chamber.C121. The fluidic device according to C101 or C111, or any of the embodiments, wherein the albumin measurement chamber and the glycoalbumin measurement chamber are fluidly connected, and the glycoalbumin measurement chamber is disposed downstream of the albumin measurement chamber. C131. The fluidic device according to C101 or C111, or any of the embodiments, wherein a sample introduced into the fluidic device is divided and introduced into each of the albumin measurement chamber and the glycoalbumin measurement chamber. C141. The fluidic device according to C101 or C111, or any of the embodiments, further comprising a measurement fluid chamber, and the measurement fluid chamber houses both a unit for measuring albumin and a unit for measuring glycoalbumin therein.

[0108] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided within the specification. While the present invention has been described with reference to the foregoing specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the invention. It is therefore intended that the present invention cover all such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A method for measuring a biomolecule, comprising: providing a recognition unit including a conductive polymer and a molecular recognition pH indicator that acts as a dopant for the conductive polymer and recognizes a target biomolecule; introducing a sample containing the target biomolecule to the recognition unit; measuring an electrical characteristic of the conductive polymer based on a reaction between the target biomolecule and the molecular recognition pH indicator that occurs when the target biomolecule is introduced; and determining the amount of the target biomolecule in the sample based on the measured electrical characteristic.

2. The method of claim 1, wherein said molecular recognition pH indicator is disposed within a network of said conductive polymer.

3. The method of claim 1, wherein the recognition unit further comprises a second polymer disposed within the network of the conductive polymer.

4. The method of claim 3, wherein said second polymer is capable of acting as a dopant for said conductive polymer.

5. The method of claim 1, wherein the target biomolecule is albumin, the molecular recognition pH indicator is BCP or BCG, and the conductive polymer comprises PEDOT.

6. The method of claim 5, wherein the recognition unit further comprises a PSS disposed within the conductive polymer network.

7. A method for determining a GA value, comprising: providing a sample containing albumin; determining the amount of albumin in the sample using the method of claim 5 or 6; determining the amount of glycated albumin in the sample; and determining the GA value of the sample from the amount of albumin and the amount of glycated albumin.

8. A device for measuring a biomolecule, comprising: a recognition unit including a conductive polymer and a molecular recognition pH indicator that acts as a dopant for the conductive polymer and recognizes a target biomolecule; and a transducer that converts a reaction between the molecular recognition pH indicator and the target biomolecule that occurs when the target biomolecule is introduced into an electrical signal.

9. The device of claim 8, further comprising a circuit connected to said transducer and configured to process said electrical signal based on a reaction between said molecular recognition pH indicator and said target biomolecule.

10. The device of claim 8, wherein the transducer comprises an electrode, the conductive polymer and the molecular recognition pH indicator are disposed on a surface of the electrode, and the device further comprises a circuit connected to the electrode and configured to evaluate a redox reaction of the conductive polymer associated with a reaction between the molecular recognition pH indicator and the target biomolecule upon introduction of the target biomolecule.

11. The device of claim 8, comprising a drain electrode and a source electrode; the conductive polymer is arranged to connect the drain electrode and the source electrode, the device comprising a circuit connected to the drain electrode and the source electrode, the circuit configured to measure a transistor characteristic between the drain electrode and the source electrode that is related to a reaction between the molecular recognition pH indicator and the target biomolecule upon introduction of the target biomolecule.

12. A device for measuring albumin, comprising: a recognition unit including PEDOT and BCP or BCG; and a transducer for detecting a reaction between the BCP or BCG and the albumin when the albumin is introduced.

13. A GA value measuring device comprising: a unit for measuring albumin, the unit comprising: a recognition unit including PEDOT and BCP or BCG; and a transducer for detecting a reaction between the BCP or BCG and the albumin that occurs when albumin is introduced; and a unit for measuring glycoalbumin.

14. A fluidic device for measuring GA values, comprising: an albumin measuring chamber having disposed therein a recognition unit including PEDOT and BCP or BCG; and a transducer for detecting a reaction between the BCP or BCG and the albumin that occurs when albumin is introduced; and a glycoalbumin measuring chamber having disposed therein a unit for measuring glycoalbumin.

15. A fluidic device according to claim 14, wherein the albumin measurement chamber and the glycoalbumin measurement chamber are fluidly connected, and the glycoalbumin measurement chamber is disposed downstream of the albumin measurement chamber.

16. A fluidic device according to claim 14, wherein a sample introduced into the fluidic device is divided and introduced into each of the albumin measurement chamber and the glycated albumin measurement chamber.

17. A fluidic device according to claim 14, further comprising a measuring fluid chamber, the measuring fluid chamber accommodating therein both a unit for measuring albumin and a unit for measuring glycated albumin.

Images