Method and device for detecting biomolecule

Abstract

One embodiment of the present disclosure provides a method for detecting a biomolecule. In some embodiments, this method comprises: (a) providing an aptamer that recognizes a target biomolecule; (b) providing a probe that hybridizes with the aptamer; (c) binding the aptamer to the target biomolecule; (d) allowing the aptamer that has not bound to the target biomolecule (unbound aptamer) to hybridize with the probe; and (e) measuring the amount of the unbound aptamer that has hybridized with the probe, thereby determining the amount of the target biomolecule.

Description

Method and device for detecting biomolecules

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

[0002] In biosensing, there is a need for more accurate and / or simpler techniques for determining the amount or presence of biomolecules in solutions such as body fluids. These techniques are also useful for point-of-care testing. They are also desirable for testing minute amounts of samples.

[0003] Here, for example, but not by way of limitation, there is recognized a need to more accurately identify and measure biomolecules to be measured from among various substances (such as proteins) present in body fluids.

[0004] According to some embodiments of the present disclosure, there is provided a method for detecting a biomolecule, in some embodiments, the method comprises providing an aptamer that recognizes a biomolecule of interest, providing a probe that hybridizes to the aptamer, allowing the aptamer to bind to the biomolecule of interest, allowing the probe to bind to the aptamer that is not bound to the biomolecule of interest, and measuring the amount of the probe that hybridizes to the unbound aptamer.

[0005] This allows for more accurate measurement of biomolecules for which no highly specific antibodies are known but for which good aptamers exist. For example, the aptamer and probe are composed of nucleic acids, providing a measurement method and sensing device that are relatively stable against thermal and chemical environmental conditions. For example, by performing two nucleic acid recognition steps, noise sources such as nonspecific adsorption can be suppressed.

[0006]

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

[0007] 1 shows a flowchart of a method for detecting biomolecules according to an embodiment; 2 shows a schematic diagram illustrating steps of a method for detecting biomolecules according to an embodiment; 3 shows a schematic diagram illustrating steps of a method for detecting biomolecules according to an embodiment; 4 shows a schematic diagram illustrating steps of a method for detecting biomolecules according to an embodiment; 5 shows a schematic diagram illustrating steps of a method for detecting biomolecules according to an embodiment; 6 shows a schematic diagram illustrating steps of a method for detecting biomolecules according to an embodiment; 7 shows a schematic diagram of a cross section of a device for detecting biomolecules according to an embodiment; 8 shows electrical characteristics of an ITO device when the channel thickness is 31 nm according to an example; 9 shows electrical characteristics of an ITO device when the channel thickness is 10 nm according to an example; 10 shows the relationship between etching time and drain current (Id) (a) and the V-Id characteristic (b) according to an example; 11 shows a schematic diagram illustrating a step of immobilizing a DNA probe on the surface of an ITO channel according to an example; 12 shows the relationship between the change in interfacial potential and a concentration series of glycated albumin (GA) in a measurement solution (a), and comparative examples (b and c). 1 shows a schematic diagram illustrating steps of a method for detecting a biomolecule according to an embodiment; 2 shows a schematic diagram illustrating steps of a method for detecting a biomolecule according to an embodiment; 3 shows a schematic diagram illustrating steps of a method for detecting a biomolecule according to an embodiment; 4 shows a schematic diagram illustrating steps of a method for detecting a biomolecule according to an embodiment; 5 shows a schematic diagram illustrating steps of a method for detecting a biomolecule according to an embodiment; 6 shows a schematic diagram illustrating steps of a method for detecting a biomolecule according to an embodiment; 7 shows a schematic diagram illustrating steps of a method for detecting a biomolecule according to an embodiment; 8 shows a schematic diagram illustrating steps of a method for detecting a biomolecule according to an embodiment; 9 shows a schematic diagram illustrating steps of a method for detecting a biomolecule according to an embodiment;1 shows a schematic diagram illustrating steps of a method for detecting biomolecules, according to an embodiment.

[0008] 1 shows a process flow (S100) of a method for detecting a biomolecule according to one embodiment of the present disclosure. First, an aptamer is bound to a target biomolecule (S101). Aptamers that are not bound to the target biomolecule (unbound aptamers) are hybridized to a DNA probe (S102). The amount of unbound aptamers hybridized to the DNA probe is quantified (S103).

[0009] The term "biomolecule" as used herein generally refers to a biological molecule, a molecule that exists or functions in a living organism. Biological molecules include, but are not limited to, proteins, oligonucleotides, nucleic acids (DNA, RNA), amino acids, peptides, lipids, cells, vesicles, sugars, carbohydrates, antibodies, and modified or altered versions thereof.

[0010] Proteins as target biomolecules include, but are not limited to, glycoalbumin, albumin, and hemoglobin. In some embodiments, the target biomolecule may be glycoalbumin (glycated albumin). In some aspects, the target biomolecule may be glucose. Other target biomolecules include, but are not limited to, neurotransmitters such as dopamine, metabolic substances, diabetes markers, cancer markers, allergy-related substances such as histamine, and Alzheimer's-related substances such as amyloid beta.

[0011] Biomolecules according to the present disclosure include, but are not limited to, biomolecules such as nucleic acids and proteins derived from bacteria or viruses. Viruses include, but are not limited to, influenza virus, coronavirus, norovirus, Ebola virus, etc. In some embodiments, biomolecules include extracellular vesicles (EVs). The extracellular vesicles (EVs) may be exosomes.

[0012] In some embodiments, the biomolecule of interest comprises a glycated biomolecule. In some aspects, the biomolecule of interest may be a glycated protein. In some aspects, the biomolecule of interest may be glycoalbumin.

[0013] Target biomolecules include, but are not limited to, glycated hemoglobin (HbA1c); sialic acid-containing glycoproteins and related glycopeptides that are highly expressed on the surface of cancer cells; and advanced glycation end products (AGEs) including Nε-carboxymethyllysine (CML), Nε-carboxyethyllysine (CEL), argpyrimidine, pentosidine, pyrraline, crosslin, GA-pyridine, Nω-carboxymethylarginine (CMA), furoylfuranylimidazole, and glucospan.

[0014] In some embodiments, the biomolecules of interest may comprise multiple types of biomolecules of interest.

[0015] Biomolecules may be naturally occurring, natural substances, or artificially produced.

[0016] 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.

[0017] 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.

[0018] The liquid containing the target biomolecule may be a solution (also referred to as a "target 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.

[0019] 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 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.

[0020] 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.

[0021] 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.

[0022] As used herein, the term "aptamer" is used interchangeably with the term "nucleic acid ligand" and refers to a DNA, RNA, oligonucleotide, or peptide molecule that binds to a specific target molecule. Aptamers may be single-stranded. Aptamers are generally relatively inexpensive and have a long lifespan. Unless otherwise specified, the term "aptamer" in the present disclosure refers to an aptamer that specifically recognizes a target biomolecule of the present disclosure.

[0023] In some embodiments, the aptamer may be an aptamer that specifically binds to a protein. The aptamer may be single-stranded.

[0024] In some embodiments, aptamers may be introduced to a target biomolecule in an amount significantly greater than the expected amount of the target biomolecule. Not all of the introduced aptamers will necessarily recognize the target biomolecule. Some of the introduced aptamers may recognize the target biomolecule, while others may end up not binding to the target biomolecule. Therefore, in some embodiments, it is preferable to introduce an amount of aptamer greater than the amount that will bind to the target molecule. For example, in a mixture containing a target biomolecule and an aptamer, it is preferable that the molar number of the aptamer is greater than that of the aptamer.

[0025] In some embodiments, the amount of aptamer introduced is predetermined. When the aptamer is introduced in solution, its concentration may be determined. When the aptamer is introduced in solid form, its weight or number of moles may be determined. The amount of aptamer introduced may be determined according to the volume of the solution estimated to contain the target biomolecule. For example, the amount of aptamer introduced may be set so that the amount of unbound aptamer bound to the probe to be finally measured falls within the dynamic range of the measurement system.

[0026] Generally, the amount of target biomolecules in the provided sample can be estimated in advance, and the amount of aptamer to be introduced can be determined according to the estimated amount. According to some embodiments of the present disclosure, a predetermined amount of GA aptamer is introduced into the provided sample, and the GA aptamer is allowed to recognize glycated albumin in the sample, and the amount of unbound GA aptamer is measured. Therefore, it is preferable to estimate the amount of albumin and the amount of glycated albumin in the sample in advance, and then determine the amount of GA aptamer to be introduced.

[0027] The concentration of a target biomolecule may vary between multiple measurements or between multiple samples. For example, when measuring the amount of a target biomolecule that may be present in a subject's body fluid, the concentration of the target biomolecule may vary between subjects or between multiple samples collected from the same subject at different times. In such cases, applying the same amount of aptamer makes it difficult to obtain accurate measurement results.

[0028] In some embodiments, when determining a ratio of a target biomolecule, both the amount of the denominator and the amount of the numerator related to the ratio may be measured. For example, when determining the glycation level of a protein, the amount of glycated protein may be measured using an aptamer for the glycated protein, and the amount of protein in the same sample may also be measured. For example, when determining the GA value (amount of glycated albumin / amount of albumin), the amount of glycated albumin may be measured using a GA aptamer, and the amount of albumin in the same sample may also be measured. For example, when determining the glycation level of a protein, the amount of protein may first be measured, and the amount of aptamer to be introduced may be determined based on that amount.

[0029] In some embodiments, multiple aptamer solutions with different amounts or concentrations of aptamer may be prepared to cover the entire range of the amount of target biomolecule that may vary between multiple measurements. For example, a provided sample may be aliquoted into multiple amounts, and multiple prepared aptamer solutions may be introduced into each aliquot. Multiple aptamer solutions may be prepared so that at least one aptamer solution reacts with the amount of target biomolecule in the provided sample within the dynamic range of the measurement system that can subsequently quantify unbound aptamer.

[0030] In some embodiments, allowing the aptamer to recognize the target biomolecule may include mixing a solution containing the aptamer with a solution containing the target biomolecule. In many practical cases, the target biomolecule is provided in solution. In some embodiments, a solution containing the target biomolecule (target solution) may be mixed with a solution containing the aptamer (aptamer solution). In some embodiments, the target solution may be mixed with a solid, e.g., powdered, aptamer.

[0031] The term "probe" as used herein generally refers to a nucleic acid that can hybridize with an aptamer, or a molecule that includes such a nucleic acid sequence as a portion thereof. In other words, a "probe" has a complementary strand to an aptamer, or a nucleic acid that can form a complementary complex with an aptamer. In the present disclosure, a "probe" may also be referred to as a "DNA probe." A "probe" may have a molecular structure other than a nucleic acid that has a complementary strand to an aptamer, and in that case, it may also be referred to as a "DNA probe." In other words, the term "DNA probe" as used herein includes probes consisting only of DNA, and also includes probes having molecular structures other than DNA.

[0032] In some embodiments, the probe may be provided in a liquid (solution). For example, the probe may be provided dissolved in a so-called Tris-EDTA (TE) buffer. In some embodiments, the probe may be provided in a solid or dry state. A solid probe may be introduced into a solution containing the aptamer or a complex of the aptamer and the target biomolecule.

[0033] Examples of combinations of target biomolecules, aptamers, and probes are shown below. These combinations are illustrative, and the present disclosure should not be construed as being limited to these combinations. Table 1 lists the following combinations:

[0034] In some embodiments, the target biomolecule may be glycoalbumin. In some embodiments, the base sequence of the glycoalbumin aptamer may be: 5'-TGCGGTTGTAGTACTCGTGGCCG-3' (SEQ ID NO: 1). The sequence of the probe that hybridizes to this aptamer may be: 5'-CGGCCACGAGTACTACAACCGCA-3' (SEQ ID NO: 12).

[0035] In some embodiments, the base sequence of the aptamer for glycoalbumin may be: 5'-GGTGCGGTTCGTGCGGTTGTAGTACTCGTGGCCGATAGAGGTAG-3' (SEQ ID NO: 2) The sequence of the probe that hybridizes to this aptamer may be: 5'-CTACCTCTATCGGCCACGAGTACTACAACCGCACGAACCGCACC-3' (SEQ ID NO: 13)

[0036] In some embodiments, the target biomolecule may be HSA. In some embodiments, the base sequence of an aptamer for HSA may be: 5'-ATACCAGCTTATTCAATTCCCCCGGCTTTGGTTTAGAGGTAGTTGCTCATTACTTGTACGCTCCGGATGAGATAGTAAGTGCAATCT-3' (SEQ ID NO: 3). The sequence of a probe that hybridizes to this aptamer may be: 5'-AGATTGCACTTACTATCTCATCCGGAGCGTACAAGTAATGAGCAACTACCTCTAAACCAAAGCCGGGGGAATTGAATAAGCTGGTAT-3' (SEQ ID NO: 14).

[0037] In some embodiments, the base sequence of the HSA aptamer may be: 5'-GTCTCAGCTACCTTACCGTATGTGGCCCAAAGCGTCTGGATGGCTATGAA-3' (SEQ ID NO: 4) The sequence of the probe that hybridizes to this aptamer may be: 5'-TTCATAGCCATCCAGACGCTTTTGGGCCACATACGGTAAGGTAGCTGAGAC-3' (SEQ ID NO: 15)

[0038] In some embodiments, the target biomolecule may be HSA. In some embodiments, the base sequence of the HbA1c aptamer may be: 5'-GGGGACACAGCAACACACCCACCCACCAGCCCCAGCATCATGCCCATCCGTCGTGTGTG-3' (SEQ ID NO: 5). The sequence of the probe that hybridizes to this aptamer may be: 5'-CACACACGACGGATGGGCATGATGCTGGGGCTGGTGGGTGGGTGTGTTGCTGTGGTCCCC-3' (SEQ ID NO: 16).

[0039] In some embodiments, the base sequence of the HbA1c aptamer may be: 5'-ACGCACACCAGAGACAAGTAGCCCCCCAAACGCGGCCACGGAACGCAGCACCTCCATGGC-3' (SEQ ID NO: 6) The sequence of the probe that hybridizes to this aptamer may be: 5'-GCCATGGAGGTGCTGCGTTCCGTGGCCGCGTTTGGGGGGCTACTTGTCTCTGGTGTGCGT-3' (SEQ ID NO: 17)

[0040] In some embodiments, the biomolecule to which the aptamer specifically binds may be a protein expressed on a cell surface or a membrane protein on a cell surface. In some embodiments, the aptamer may be an aptamer that specifically binds to a membrane protein on the surface of a leukemia cancer cell, and the base sequence of the aptamer may be as follows: 5'-AAAAAAAAAAAAATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3' (SEQ ID NO: 7) The sequence of a probe that hybridizes to this aptamer may be as follows: 5'-TCTAACCGTACAGTATTTTCCCGGCGGCGCAGCAGTTAGATTTTTTTTTTT-3' (SEQ ID NO: 18)

[0041] In some embodiments, the aptamer may be an aptamer that specifically binds to a virus. The virus may be, for example, but not limited to, a coronavirus. The coronavirus may be, for example, but not limited to, the SARS virus. The coronavirus may be the SARS-COVID-19 virus. In some embodiments, the base sequence of the aptamer may be: 5'-GCAATGGTACGGTACTTCCGGATGCGGAAAACTGGCTAATTGGTGAGGCTGGGGCGGT-3' (SEQ ID NO: 8). The sequence of the probe that hybridizes to this aptamer may be: 5'-ACCGCCCCAGCCTCACCAATTAGCCAGTTTCCGCATCCGGAAGTACCGTACCATTGC-3' (SEQ ID NO: 19).

[0042] In some embodiments, the base sequence of the SARS-COVID-19 virus aptamer may be: 5'-CAGCACCGACCTTGTGCTTTTGGGAGTGCTGGTCCAAGGGCGTTAATGGACA-3' (SEQ ID NO: 9) The sequence of the probe that hybridizes to this aptamer may be: 5'-TGTCCATTAACGCCCTTGGACCAGCACTCCCAAAGCACAAGGTCGGTGCTG-3' (SEQ ID NO: 20)

[0043] In some embodiments, the base sequence of the SARS-COVID-19 virus aptamer may be: 5'-ATCCAGAGTGACGCAGCATTTCATCGGGGTCCAAAGGGGCTGCTCGGGATTGCGGATATGGACACGT-3' (SEQ ID NO: 10) The sequence of the probe that hybridizes to this aptamer may be: 5'-ACGTGTCCATATCCGCAATCCCGAGCAGCCCCTTTTGGACCCGATGAAATGCTGCGTCACTCTGGAT-3' (SEQ ID NO: 21)

[0044] In some embodiments, the aptamer may be an aptamer that specifically binds to cortisol. In some embodiments, the base sequence of the cortisol aptamer may be as follows: 5'-GCCCGCATGTTCCATGGATAGTCTTGACTAGTCGTCCC-3' (SEQ ID NO: 11). The sequence of the probe that hybridizes to this aptamer may be as follows: 5'-GGGACGACTAGTCAAGACTATCCATGGAACATGCGGGC-3' (SEQ ID NO: 22).

[0045] Table 1 lists the above combinations.

[0046] In the present disclosure, expressions such as "probe base sequence" and "aptamer base sequence" refer to a base sequence that constitutes at least a portion of a "probe" or "aptamer." The present disclosure includes cases where a "probe" or "aptamer" is composed only of a "probe base sequence" or an "aptamer base sequence." However, this should not be interpreted as limiting the "probe" or "aptamer" to having only the base sequence described. In other words, these refer to the base sequences of the portions of the "probe" or "aptamer" that correspond to complementary strands, and do not exclude the "probe" or "aptamer" from having nucleic acid or non-nucleic acid portions other than the base sequences of the portions that correspond to complementary strands (the portions of the base sequences defined by the "probe base sequence" and the "aptamer base sequence").

[0047] In some embodiments, the amount of unbound aptamer bound to the probe may be measured. As used herein, the term "unbound aptamer" refers to an aptamer that is present without binding to the target biomolecule after the aptamer is introduced to the target biomolecule. In the present disclosure, the amount of unbound aptamer can be determined by quantifying the amount of the original target biomolecule.

[0048] In some embodiments, the probe may be immobilized on a solid surface. For example, the probe may be immobilized on the surface of a plate-like substrate. For example, the probe may be immobilized on the surface of a solid particle. For example, the probe may be immobilized on the surface of a magnetic particle. In some embodiments, the probe may be provided without being immobilized on a solid surface. For example, the probe may be dissolved in solution and introduced into a mixed solution containing unbound aptamers.

[0049] In some embodiments, a probe immobilized on a solid surface may be provided, and unbound aptamers bound to the probe may be measured. In some embodiments, unbound aptamers bound to the probe immobilized on a solid surface may be measured. The probe may have one end or portion immobilized on a solid surface or sensing surface, and the other end or portion may be free.

[0050] In some embodiments, substances other than unbound aptamers, i.e., contaminants, may be removed from the vicinity of the sensing surface. For example, the solid surface on which the probe is immobilized may be washed while the unbound aptamers remain bound to the probe. This allows the target biomolecule or a complex of the target biomolecule and the aptamer to be removed from the vicinity of the solid surface. This reduces the influence of contaminants other than the unbound aptamer to be measured, allowing the unbound aptamer bound to the probe to be measured with higher accuracy. A buffer solution may be used for washing.

[0051] In some embodiments, the probes may be immobilized on a surface of a sensor (also referred to as a "sensing surface"). As used in this disclosure, the term "sensing surface" refers to the surface of a solid material that may be configured to detect hybridization between the immobilized probes and the aptamer. The "sensing surface" may have electrodes entirely or partially.

[0052] Molecules other than probes (also referred to as "non-probe molecules") may be immobilized on the sensing surface. The non-probe molecules or membrane may have the function of suppressing the reaction of molecules other than the unbound aptamer to be detected with the sensing surface. For example, the non-probe molecules may suppress the reaction of the bound aptamer or a complex of the aptamer and the target biomolecule with the sensing surface. The non-probe molecules may suppress the nonspecific adsorption of such contaminants to the sensing surface. Examples of non-probe molecules include, but are not limited to, polyethylene glycol (PEG), ethanolamine, surfactants (e.g., Pluronic F-127), 2-methacryloyloxyethyl phosphorylcholine (MPC), etc.

[0053] The electrode can detect the electrical properties of the vicinity resulting from hybridization between the probe and the aptamer. Nucleic acids have negative charges uniformly distributed along the backbone due to ionization of the OH groups of the phosphates in the sugar-phosphate backbone. The charge increases near the electrode according to the amount of aptamer that has hybridized with the probe. This increase in charge can be sensed by the electrode.

[0054] In some embodiments, the sensing surface may be a surface of a channel of a transistor, which may be a field effect transistor (FET).

[0055] In some embodiments, a biotransistor or biosensor having a FET is provided. For example, at least the channel of the FET may be made of ITO. For example, the drain, channel, and gate of the FET may be made of continuous ITO.

[0056] The sensing surface may also be configured to detect hybridization of the immobilized probe with the aptamer by other methods, including, but not limited to, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), cantilever methods, etc.

[0057] In some embodiments, the sensing device may have multiple sensing surfaces, and the same type of probes may be disposed on multiple sensing surfaces, or different probes may be disposed on each of the multiple sensing surfaces.

[0058] The multiple sensing surfaces may include a first sensing surface and a second sensing surface. A first probe may be disposed on the first sensing surface, and a second probe different from the first probe may be disposed on the second sensing surface. For example, a first probe hybridizing with a first aptamer that recognizes a modified target biomolecule (also referred to as a "modified biomolecule") may be disposed on the first sensing surface. For example, a second probe hybridizing with a second aptamer that recognizes an unmodified target biomolecule (also referred to as an "unmodified target biomolecule") or both modified and unmodified target biomolecules (also referred to as a "total target biomolecule") may be disposed on the second sensing surface. This allows, for example, the modification ratio of the target biomolecule to be determined.

[0059] In some embodiments, the target biomolecule is a protein, the modified target biomolecule is a glycated protein, the unmodified target biomolecule is an unglycated protein, and the total target biomolecule can be total protein. The protein can be albumin. The protein can be hemoglobin.

[0060] In some embodiments, the sensing device may have a flow channel or fluid chamber (sometimes simply referred to as a "flow channel" in this disclosure), and the first sensing surface and the second sensing surface may be arranged in the same flow channel. This allows the modification ratio of the target biomolecule to be determined using the same solution without dispensing the target solution. Therefore, the modification ratio of the target biomolecule can be determined more accurately.

[0061] In some embodiments, the sensing device may have multiple flow paths, where multiple flow paths may have the same type of sensing surface therein, or multiple flow paths may have different types of sensing surfaces therein.

[0062] Embodiment 1 With reference to FIGS. 2A to 2E, steps of a method for detecting biomolecules according to one embodiment of the present disclosure will be described.

[0063] A target biomolecule 201 and an aptamer 202 that specifically recognizes the target biomolecule 201 are mixed to prepare a mixed solution 205 (FIG. 2A). The amount of the aptamer 202 is preferably greater than the amount of the predicted target biomolecule 201.

[0064] 2B , some of the aptamers 202 specifically recognize and bind to the target biomolecule 201. As a result, the mixture 205 contains a complex 203 in which the target biomolecule 201 and the aptamer 202 (binding aptamer 202b) bind to each other, and aptamers 202 that did not bind to the target biomolecule 201 (non-binding aptamers 202n).

[0065] 2B , all target biomolecules 201 are schematically shown to be recognized by aptamers 202. In reality, there may be target biomolecules 201 that are not recognized by aptamers 202. Furthermore, the binding between the target biomolecules 201 and aptamers 202 is a reversible chemical reaction determined by an equilibrium constant depending on the environmental conditions in the solution, and statistically, a certain amount of target biomolecules 201 may exist in a state that is not recognized by aptamers 202. Within the range permitted by the environmental conditions in the solution, substantially the maximum amount of target biomolecules 201 is recognized by aptamers 202. The absolute amount or concentration of the aptamers 202 to be mixed may be set so that the target biomolecules 201 are sufficiently recognized by the aptamers 202.

[0066] A sensor 210 is provided that has a DNA probe 215 having a complementary strand of an aptamer 202. The sensor 210 shown in Figures 2C and 2D has an electrode 211, which has a sensing surface 212 and is configured to be able to sense electrical properties or changes therein in the vicinity of the sensing surface 212. In Figures 2C and 2D, electrical circuits other than the electrode 211 are omitted. A detailed example will be described in the Examples. A DNA probe 215 is immobilized at one end on the sensing surface 212. A mixed solution 205 containing a target biomolecule-aptamer complex 203 and unbound aptamer 202n is introduced onto this sensing surface 212 (Figure 2C).

[0067] Unbound aptamers 202n in the mixture 205 hybridize with DNA probe 215 (FIG. 2D).

[0068] The DNA has charges at its phosphate moieties and at evenly spaced intervals along its length. Electrode 211 senses the opposite charge (plus sign in the figure) corresponding to the charge (minus sign in the figure) of unbound aptamer 202n hybridized to DNA probe 215 (FIG. 2E).

[0069] The amount of unbound aptamer 202n hybridized to DNA probe 215 depends on the amount or concentration of unbound aptamer 202n present in mixed solution 205. Therefore, sensor 210 can estimate the amount or concentration of unbound aptamer 202n present in mixed solution 205. Furthermore, from this, the amount or concentration of the original target biomolecule 201 can be estimated.

[0070] 3 is a schematic diagram of a sensing system 300 that can be used in the above embodiment. The sensing system 300 has a sensor 310 and a transducer 320. The sensing system 300 may have a signal processing unit including a CPU or the like that processes the output signal of the transducer 320. The sensor 310 may have a flow path that accommodates a solution. The sensing system 300 may also have a liquid delivery system that controls the solution, a controller for the system, a temperature control system, and the like.

[0071] In FIG. 3 , the sensor 310 has a conductor 311 formed on an insulating substrate 316. The conductor 311 may be made of ITO, for example. A portion of the conductor 311 is thinned to form the gate or channel of a transistor. The surface functions as a sensing surface 312, and a probe is disposed on the surface (not shown). The surface of the conductor 311 is covered with an insulator or resist 317 and patterned to expose a portion. The sensing surface 312, which corresponds to the drain D and source S of the transistor, is configured to be accessible from the outside. A space 318 above the sensing surface 312 is configured to accommodate a target solution 330.

[0072] The transducer 320 is configured to be connected to a reference electrode R configured to be in contact with the target solution 330 contained in the space 318, and to a drain D and a source S. The transducer 320 applies a gate voltage V Ｇ is applied and controlled, and the drain voltage V Ｄ and the drain current I Ｄ For example, the relationship between the drain voltage V Ｄ is applied to the drain current I Ｄ For example, a constant drain current I Ｄ Applying a drain voltage V Ｄ Alternatively, the drain voltage V Ｄ and the drain current I Ｄ You can also look for a relationship with

[0073] In general, Graham's equation (Equation 1) holds between the amount of charge (called "surface charge") per unit area present near the surface of an electrode (called "surface charge") (called "surface charge density") and the potential of that electrode (called "surface potential"). where σ is the surface charge density, ε ｗ is the relative dielectric constant of water, ε ０is the dielectric constant of a vacuum, k is Avogadro's constant, T is temperature, C is the ion density of the solution, e is the elementary charge, and φ is the surface potential. This surface potential φ is the potential difference due to a change in surface charge when the electric double layer generated at the interface between the solid surface and the aqueous solution is considered as a capacitor. In some embodiments, based on Graham's equation, the surface charge density σ on the electrode surface generated by hybridization of unbound aptamer (increased from the case where no hybridization occurs) is detected as an electrical signal called the surface potential φ, and the amount of hybridized unbound aptamer can be estimated from this.

[0074] Example: Fabrication of Sensor Fabrication of ITO-TFT A glass substrate was ultrasonically cleaned, blown with nitrogen, and dried by heating. The dried glass substrate was spin-coated with photoresist (OFPR-800, Tokyo Ohka Kogyo Co., Ltd.) at 500 rpm for 5 seconds and at 3000 rpm for 30 seconds, and then pre-baked at 110°C for 5 minutes. For exposure, a film mask was placed on the substrate, and ultraviolet light was irradiated for 40 seconds using a photolithography system (PhL Q-2001QT, QUINTEL). The exposed substrate was developed with a developer (NMD-3, Tokyo Ohka Kogyo Co., Ltd.) and washed in ultrapure water for approximately 1 minute. The substrate was then post-baked at 110°C for 5 minutes.

[0075] A 100 nm thick thin film of indium tin oxide (hereinafter referred to as "ITO") was formed on the patterned substrate by radio frequency (RF) sputtering. Sputter deposition was carried out for 25 minutes at a rate of approximately 4 nm / min in an atmosphere of 20% argon and 0% oxygen. Because the film was deposited without heating, the ITO thin film was largely amorphous.

[0076] In this example, the thickness of the ITO thin film was 100 nm, but this is just an example, and other thicknesses may be used. ITO is required to be conductive. For example, the thickness of the ITO thin film may be equal to or smaller than the thickness of the depletion layer. Therefore, the thickness of the ITO thin film may be 20 to 30 nm or more, which does not make it a semiconductor.

[0077] The substrate was then left overnight in acetone and then ultrasonically cleaned to remove the photoresist (OFPR-800, Tokyo Ohka Kogyo Co., Ltd.) and the ITO thin film formed thereon. The water was then removed by nitrogen blowing, and the substrate was heated on a hot plate at 110°C for approximately 5 minutes to evaporate the water on the ITO thin film.

[0078] Next, photolithography was performed again to protect areas other than the etching area and the contact area with the conductor wire. Photoresist (SU-8 3005, Nippon Kayaku Co., Ltd.) was spin-coated at 500 rpm for 5 seconds and 6000 rpm for 30 seconds, and post-baked at 95°C. Next, a film mask was placed on the substrate, and it was irradiated with ultraviolet light for 7 seconds, followed by post-baking at 65°C for 2 minutes and 95°C for 5 minutes. The substrate was then developed in SU-8 developer for 3 minutes and in 2-propanol for 1 minute with stirring. Finally, the remaining 2-propanol was removed by spraying nitrogen gas.

[0079] In this example, a channel having a channel length (L) of 10 μm and a channel width (W) of 340 μm was formed in the ITO thin film.

[0080] The thickness was determined based on the IV characteristics. In this example, the conductivity of the channel was measured using a semiconductor parameter analyzer (B1500A, Agilent Technologies). Figures 4A and 4B show the IV characteristics when the ITO channel thickness was 31 nm and 10 nm, respectively. When the thickness was 31 nm, no semiconducting properties were observed. However, when the thickness was 10 nm, semiconducting properties were observed.

[0081] Thus, the IV characteristics of an ITO thin film change from conductive to semiconductive as the film becomes thinner. Therefore, a correlation between the film thickness, determined by etching conditions under predetermined conditions, and the IV characteristics is obtained in advance. Based on this correlation, during the etching process, the film thickness can be estimated by measuring the drain current (Id) at a constant drain-source voltage (Vds). Immediately after reaching a predetermined current value, i.e., film thickness, etching is quickly stopped by washing the channel portion with, for example, ultrapure water. This allows etching to be controlled so that the channel ITO thin film is within a film thickness range that indicates n-type semiconductor.

[0082] In this example, 1 M to 0.01 M hydrochloric acid was dropped to etch the ITO thin film in the exposed channel portion while applying 1 V between the source and drain, and the time change in Id due to etching was measured. Figure 5(a) shows the drain-source current (Id) during etching with 0.1 M hydrochloric acid (HCl). Id remained almost constant from 0 to 700 seconds of etching time, and then decreased rapidly.

[0083] The etching end time can be set by Id relative to the initial Id (Id(initial)). FIG. 5(b) shows the IV characteristics (Vg-Id characteristics) of the channel when Id(stop) / Id(initial), the ratio of Id at the end of etching (Id(stop)) to Id(initial), is 93%, 66%, 43%, and 3.4%. When Id(stop) / Id(initial) is 93% and 63%, the channel exhibits conductor characteristics. When Id(stop) / Id(initial) is 44%, the channel begins to exhibit transistor characteristics. When Id(stop) / Id(initial) is 3.4%, the channel exhibits transistor characteristics sufficient to indicate ON / OFF. In this example, Id(stop) / Id(initial) = 44% was used. The channel thickness at this time was found to be about 23 nm by atomic force microscopy (AFM).

[0084] Immediately after the etching of the channel reached a predetermined Id(stop) (in this example, Id(stop) / Id(initial)=44%), the channel portion was washed with ultrapure water to quickly stop the etching.

[0085] Immobilization of Probe DNA The ITO channel surface was modified with a thin film of polyserotonin (pST), which was used as an anchor layer. Chemical modification utilized the autoxidative polymerization of serotonin (ST), which proceeds at room temperature under basic conditions. A 1 mg / mL (5 mM) serotonin hydrochloride solution was prepared using a pH 9.0 phosphate buffer solution. A silicon ring was attached to the ITO channel to create a well, and the prepared serotonin hydrochloride solution was dropped into the well and sealed with a glass slide (Figure 6(a)).

[0086] The serotonin was left in the dark for 4, 8, or 24 hours. The reason for leaving it in the dark was to eliminate the effect of light on the auto-oxidation polymerization. After the auto-oxidation polymerization, the channel surface was washed with pure water, and residual water was removed by nitrogen blowing. The film was then stored in a vacuum desiccator for at least 1 hour to dry the pST thin film (Figure 6(b)).

[0087] The 5' end of a probe DNA (5'-CGGCCACGAGTACTACAACCGCA-3') was immobilized on the surface of an ITO channel modified with a pST thin film. The sequence of this probe DNA is complementary to the sequence of the GA aptamer described below.

[0088] First, a dimethyl sulfoxide (DMSO) / phosphate buffer solution (PBS) (1:9) containing 1 mM 3-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) was prepared. This solution was dropped onto the ITO channel surface and allowed to stand for 1 hour, allowing the amino groups of the ether-linked succinimidyl groups in the MBS to be substituted with the amino groups on the pST membrane surface (Figure 6(c)).

[0089] Then, 10 μL of the frozen 100 μM probe DNA was thawed and mixed with 90 μL of pure water and 100 μL of TBS (10 mM Tris-HCl + 10 mM KCl + 10 mM MgCl ２ The solution was diluted with PBS (pH 7.4) and then heated at 95°C for 30 minutes. After the heat treatment, a solution containing the probe DNA was dropped onto the MBS-modified surface, sealed with a slide glass, and left to stand overnight. This allowed the maleimide to bond with the thiol of the probe DNA. As a result, the probe DNA was immobilized on the pST thin film via the MBS (Figure 6(d)). Measurements were started immediately after the probe formation.

[0090] In this example, devices modified with thiolated PEG simultaneously with the probe DNA were also fabricated as needed. When modifying with PEG, the same concentration of thiolated PEG (molecular weight 500) was mixed with the probe DNA and added dropwise to the channel surface. PEG reacts with unreacted MBS, which is expected to suppress nonspecific adsorption of MGS and other substances to the electrode surface.

[0091] Example: Measurement of GA First, a GA sample was prepared. Determiner GA-L (Minaris Medical), a standard specimen for biochemical testing, was used. Albumin & IgG Depletion SpinTrap (Cytiva) was equilibrated with equilibration buffer (10 mM HEPES, 150 mM NaCl), and then 2 mL of Determiner GA-L diluted 1 / 1000 with the equilibration buffer was added to the column and thoroughly stirred. The unbound fraction was removed by centrifugation according to the instruction manual. The column was then washed with 10 mM MES buffer (pH 5.5), and 100 μL of pH 2.7 glycine buffer was added to elute the albumin fraction containing GA. The eluate was then neutralized by adding 10 μL of 1 M Tris buffer (pH 8.5). This was used as the GA sample. The GA concentration was determined by identifying the albumin concentration based on absorbance at 280 nm and then measuring the GA % by HPLC.

[0092] The GA sample was mixed with 5 μM GA aptamer (5'-TGCGGTTGTAGTACTCGTGGCCG-3') (Gene Design, Inc.) and phosphate pH standard buffer (pH 7.4, 40 mM) to prepare a mixture of GA and GA aptamer. The amount of phosphate pH standard buffer was adjusted to prepare each mixture so that the GA concentrations were 0 ng / mL, 33 ng / mL, 3.6 ng / mL, 7.2 ng / mL, 36 ng / mL, 72 ng / mL, and 360 ng / mL. After mixing, the mixture was left to stand at room temperature for 30 minutes. This allowed the GA aptamer to react with GA.

[0093] 300 μL of this solution was dropped onto the surface of the ITO channel and allowed to stand at room temperature for 30 minutes, allowing the unbound GA aptamer to hybridize with the probe DNA on the surface of the ITO channel.

[0094] The ITO channel surface was washed several times with phosphate buffer solution (PBS, pH 6.8-7.4, 1-100 mM), which removed other substances, i.e., contaminants, from the vicinity of the ITO channel surface while leaving the unbound GA aptamer immobilized on the probe DNA on the ITO channel surface.

[0095] In the presence of this PBS, the electrical properties of the ITO channel caused by the charge of the GA aptamer hybridized to the probe DNA on the surface of the ITO channel were measured using a semiconductor parameter analyzer (Keysight).

[0096] In this example, while the GA aptamer and GA were reacting, PBS was introduced onto the sensing surface on which the probe DNA was immobilized, and the electrical characteristics of the ITO channel were measured. The potential at this time was defined as the initial potential. After the GA aptamer and GA were allowed to react sufficiently (after leaving it for 30 minutes), the mixed solution was introduced onto the sensing surface, and the potential was measured. In this disclosure, the difference between the potential in the mixed solution and the initial potential is defined as the "change in interfacial potential."

[0097] Figure 7 shows the change in interfacial potential (a in the figure). The change in interfacial potential was approximately constant at about 120 mV from when no GA was present (0 ng / mL) to when the GA concentration was increased to 72 ng / mL. The change in interfacial potential decreased when the GA concentration was increased from 36 ng / mL to 360 ng / mL.

[0098] As a comparative example, 5 μM thrombin (TB) aptamer was mixed with the GA sample instead of the GA aptamer. The change in interfacial potential was 28 mV, which was almost the same as that of the 360 ​​ng / mL GA sample (b in the figure).

[0099] As a further comparative example, no GA probe was used and only PEG was immobilized on the electrode surface. The change in the interfacial potential was 10 mV or less (c in the figure).

[0100] From these, it can be said that in this example, the upper limit of the GA concentration that could be measured was approximately 360 ng / mL, and the lower limit was approximately 36 ng / mL.

[0101] The measurable concentration range of the target biomolecule and the correlation between the GA concentration and the change in interfacial potential within that concentration range can be adjusted by adjusting the concentration of the target biomolecule present in the measurement solution, the concentration of the aptamer, the amount of probe, and other measurement parameters or conditions. These parameters may be set so that the amount of unbound aptamer bound to the probe that is ultimately measured falls within the dynamic range of the measurement system.

[0102] In some embodiments, the double strand of the probe and the unbound aptamer may be denatured, and the unbound aptamer may be separated (dissociated) from the probe. The unbound aptamer separated from the probe may be measured. This denaturation may be thermal denaturation. The probe and the unbound aptamer may be heated. Alternatively, the substrate may be heated.

[0103] Embodiment 2 Referring to Figures 8A-8F, a method for detecting biomolecules according to one embodiment will be described.

[0104] A target biomolecule 801 and an aptamer 802 that specifically recognizes the target biomolecule 801 are mixed to prepare a mixed solution 805 (FIG. 8A).

[0105] 8B , a portion of the aptamer 802 specifically recognizes and binds to the target biomolecule 801. As a result, the mixture 805 contains a complex 803 in which the target biomolecule 801 and the aptamer 802 (binding aptamer 802b) bind to each other, and aptamers 802 that did not bind to the target biomolecule 801 (unbinding aptamers 802n).

[0106] As shown in Figure 8C, a substrate 811 is provided having a DNA probe 815 having a complementary strand of aptamer 802n. The substrate 811 shown in Figures 8C and 8D does not need to be a conductor or be configured to be able to sense electrical properties near the surface of the substrate 811 (substrate surface 812). For example, the substrate 811 may be an insulator. One end of the DNA probe 815 is immobilized on the substrate surface 812. A mixture 805 containing a target biomolecule-aptamer complex 803 and unbound aptamer 802n is introduced onto this substrate surface 812 (Figure 8C).

[0107] Unbound aptamers 802n in mixture 805 hybridize with DNA probe 815 (FIG. 8D).

[0108] In this embodiment, the substrate surface 811 is washed ( FIG. 8E ). This makes it possible to remove the complex 803, unbound aptamer 802n (not shown) that remains without hybridizing with the probe 815, and the like from the vicinity of the substrate surface 812. This makes it possible to suppress the influence of contaminants on the measurement of the separated unbound aptamer, which is performed in the next step.

[0109] Unbound aptamers 802n that have hybridized to probes 812 are separated from the probes 812 (FIG. 8F). Unbound aptamers 802n released near the washed substrate surface 811 are measured. Unbound aptamers separated from the probes can be measured by various techniques.

[0110] 8C to 8F, the substrate 811 is depicted as a schematic flat member, but the substrate or probe support of the present disclosure should not be construed as being limited thereto. For example, the probe substrate or support (hereinafter simply referred to as "support") may be solid particles. In some embodiments, the probe support may be magnetic particles.

[0111] 9A to 9F, a method for detecting a biomolecule using a probe immobilized on a magnetic particle (which may be a magnetic bead) will be described.

[0112] A target biomolecule 901 and an aptamer 902 that specifically recognizes the target biomolecule 901 are mixed to prepare a mixed solution 905 (FIG. 9A).

[0113] 9B , a portion of the aptamer 902 specifically recognizes and binds to the target biomolecule 901. As a result, the mixture 905 contains a complex 903 in which the target biomolecule 901 and the aptamer 902 (binding aptamer 902b) bind to each other, and aptamers 902 that did not bind to the target biomolecule 901 (non-binding aptamers 902n).

[0114] As shown in Fig. 9C, magnetic beads 911 are provided, on the surface of which DNA probes 915 having a complementary strand to aptamer 902 are immobilized. The magnetic beads 911 are mixed with a mixture 905 containing a target biomolecule-aptamer complex 903 and unbound aptamer 902n (Fig. 9C).

[0115] The unbound aptamer 902n hybridizes to a DNA probe 915 immobilized on a magnetic bead 911 (FIG. 9D).

[0116] A magnetic field (not shown) is applied to the space containing the magnetic beads 911 and the mixing vessel 905, capturing the magnetic beads 911 in the magnetic field. In this state, washing is performed ( FIG. 9E ). This removes the magnetic beads 911, the DNA probe 915 immobilized on the magnetic beads 911, and the remaining unbound aptamers 902n that have not hybridized with the DNA probe 915 (none of which are shown). This reduces the influence of contaminants on the measurement of the separated unbound aptamers in the next step, enabling the unbound aptamers bound to the probes to be measured with higher accuracy.

[0117] Next, unbound aptamers 902n that have hybridized to the probe 915 are separated from the probe 915 ( FIG. 9F ). The unbound aptamers 902n that have been released near the washed magnetic beads 911 and DNA probe 915 are measured. The unbound aptamers separated from the probe can be measured by various techniques.

[0118] Measurement of Unbound Aptamers After Separation from the Probe In the above-described second or third embodiment or other embodiments, the unbound aptamers separated from the probe can be measured by various techniques. Examples of such measurements include, but are not limited to, absorbance, fluorescence, HPLC, polymerase chain reaction (PCR), mass spectrometry, etc. The PCR method may be quantitative PCR (qPCR), such as digital PCR or real-time PCR.

[0119] Examples of real-time PCR methods include the intercalation method, the hybridization method, and the LUX method. Generally, the intercalation method uses a dye (SYBR green I) that specifically intercalates into double-stranded DNA and emits fluorescence. Generally, the hybridization method uses a probe (TaqMan probe) in which a fluorescent dye is modified onto an oligonucleotide specific to a DNA sequence. In some embodiments of the present disclosure, a kit containing reagents for use in the PCR method is provided.

[0120] As used herein, the term "processor" generally refers to a processing element capable of performing arithmetic operations such as analyzing measurement results, determining concentrations, calculating correction coefficients, and generating control signals, and may be implemented as a single or multiple integrated circuits, a microprocessor, or a combination thereof. The processor may be physically built into the device, or may be externally installed and capable of communicating with the device's control system.

[0121] In some embodiments, the device or system may contain multiple aptamer solutions with different concentrations. The aptamer concentrations may be prepared in stages, for example, 0.1 μM, 1 μM, and 10 μM. The sample may be divided into multiple portions, each of which may be reacted with a different aptamer solution. The measurement results obtained at each aptamer concentration may be evaluated to determine whether they are within a linear response range, and if so, the measurement values ​​may be used for analysis.

[0122] In some embodiments, the device or system may be configured to sequentially apply multiple sensors. Subsequent aptamer solutions may be selected by analysis based on the output of a previous measurement. This selection may be based on whether the output exceeds a predetermined threshold, such as a saturation level or a lower detection limit. The selection of an aptamer solution may be performed manually by a user or automatically by the device.

[0123] In some embodiments, a device or system may include multiple sensors with different sensitivity characteristics. For example, the sensors may include a high-sensitivity sensor and a low-sensitivity sensor. The sensors may be located on a single device, distributed across multiple devices, and used in parallel or simultaneously.

[0124] In some embodiments, a device or system may include multiple fluid channels containing one or more sensors, each of which may contain a sensor with sensitivity characteristics unique to that channel, or multiple sensors may be located within a single fluid channel.

[0125] In some embodiments, multiple sensors may be provided, either individually in different fluidic channels or with multiple sensors located within a single fluidic channel.

[0126] A configuration with multiple sensors with different sensitivities may be configured to avoid output saturation or insufficient sensitivity even when there is a large concentration variability between measurements or between individuals, which may be caused by changes over time within the same individual or by variations between different individuals.

[0127] The difference in sensitivity may be adjusted by varying the probe density at the sensing surface, the sensor material properties (e.g., indium tin oxide (ITO) channel thickness, patterning, etc.), or the transistor properties (e.g., gate threshold).

[0128] In some embodiments, only those outputs determined to be appropriate from the multiple measurement outputs may be selected and used for analysis. This selection may be based on whether the output falls within a predetermined range or on the linearity of the response curve (e.g., correlation coefficient). In some embodiments, multiple outputs may be weighted and integrated to be used as a representative value. For example, a fitting function may be applied to measurement results obtained at different aptamer concentrations to interpolate the relationship between concentration and response.

[0129] In some embodiments, the device or system may automatically select from among multiple sensors or measurement configurations. The selection may be performed by a controller based on the initial measurement result and matching it with stored predetermined switching criteria. Depending on the determination result, the sensor, fluid channel, or display output may be switched. This configuration allows for consistent results to be obtained without relying on the skill of the operator.

[0130] In some embodiments, the device or system may include a configuration for performing calibration using a standard sample having a known concentration. The standard sample may include multiple concentration levels, such as 10 ng / mL, 100 ng / mL, and 1000 ng / mL. The standard sample may be introduced through a different flow path or measurement system than the sample to be measured. Calibration may be performed before, during, or after measuring the sample to be measured.

[0131] In some embodiments, the device or system may include multiple fluid paths or measurement channels, each having a flow path for a standard sample and a flow path for a target sample, and a correction formula or factor may be calculated based on the standard sample and applied to the target sample measurement results to correct the estimated concentration.

[0132] In some embodiments, one sensor may be used to sequentially measure multiple standard samples with different concentrations. A calibration curve may be created based on the output obtained from the standard samples. Between application of the standard samples, the aptamer may be removed from the sensor surface, the surface may be washed, and the aptamer may be re-immobilized. Aptamer removal may be performed by changing the temperature, pH, or salt concentration.

[0133] In some embodiments, the device or system may be configured to automatically perform a calibration process using a standard sample. The controller may acquire an output corresponding to the standard sample and calculate a correction formula or correction factor based on the correlation with the stored known concentrations. The device or system may apply the correction to the measurement result of the sample being measured and display or record the corrected concentration. The correction may be performed using linear interpolation, nonlinear regression, or machine learning.

[0134] In some embodiments, the device may include a processor for analyzing the measurement results and controlling the determination or correction of concentration or the selection of an aptamer solution. The processor may be configured to perform calculations, determinations, or output of control signals, and may be implemented as a single or multiple integrated circuits, a microprocessor, or a combination thereof.

[0135] In some embodiments, the device or system may be configured to perform measurements by introducing an internal standard along with the sample to be measured. The internal standard can correct the measured value for the analyte based on the measurement results. The internal standard may be identified by an aptamer having a sequence different from that of the aptamer to be measured, and may be detected independently by another sensor or channel within the device.

[0136] In some embodiments, the device or system may be configured to remove the aptamer immobilized on the sensor surface after measurement, wash the sensor, and then re-immobilize the aptamer. Removal of the aptamer may be achieved by increasing the temperature, changing the pH, or changing the salt concentration, for example, to reduce non-specific binding and realize a reusable sensing configuration. The re-immobilized aptamer may be reacted again with a new sample or standard sample and used for multiple measurements.

[0137] The present disclosure also provides the following embodiments: A001 A method for detecting a biomolecule, comprising: (a) providing an aptamer that recognizes a target biomolecule; (b) providing a probe that hybridizes to the aptamer; (c) binding the aptamer to the target biomolecule; (d) hybridizing the aptamer that has not bound to the target biomolecule (unbound aptamer) to the probe; and (e) measuring the amount of unbound aptamer hybridized to the probe to determine the amount of the target biomolecule. A002 A method according to A001 or any of the embodiments, wherein binding the aptamer to the target biomolecule comprises mixing the target biomolecule and the aptamer to form a mixture. A005 A method according to A001 or any of the embodiments, further comprising washing the space near the probe after step (d). A011 The method according to A001, or any of the embodiments, wherein the target biomolecule is a glycated protein. A012 The method according to A001 or A011, or any of the embodiments, wherein the target biomolecule is selected from the group consisting of glycated albumin (GA) and glycated hemoglobin (HbA1c). A013 The method according to A001 or A011, or any of the embodiments, wherein the target biomolecule is glycated albumin. A031 The method according to A001, or any of the embodiments, wherein (b) providing the probe comprises providing a sensor having a sensing surface, the probe being immobilized at one end to the sensing surface, and (e) measuring the amount of the probe hybridized to the unbound aptamer comprises measuring an electrical characteristic of the probe corresponding to the amount of hybridization. A032 The method of A031 or any embodiment, wherein the electrical property is a charge or surface potential of the sensing surface corresponding to a negative charge of the aptamer hybridized to the probe.A101 The method according to A001, or any embodiment, wherein (b) providing the probe comprises providing a probe immobilized on a support, and (e) measuring the amount of unbound aptamers hybridized to the probe comprises separating the unbound aptamers hybridized to the probe from the probe, and measuring the amount of unbound aptamers separated from the probe. A111 The method according to A101, or any embodiment, wherein separating the unbound aptamers hybridized to the probe from the probe comprises heating. A121 The method according to A101 or A111, or any embodiment, further comprising washing the unbound aptamers while they are hybridized to the probe, before separating the unbound aptamers hybridized to the probe from the probe. A131 The method according to any one of A101 to A121 or any embodiment, wherein the support is magnetic particles. A132 The method according to A121 and A131, or any embodiment, wherein performing the washing comprises capturing the magnetic particles in a magnetic field and performing the washing while the magnetic particles are captured in the magnetic field. A141 The method according to any one of A101 to A132 or any embodiment, wherein measuring the amount of unbound aptamer separated from the probe comprises using a PCR method. A201 The method according to A001 or any embodiment, wherein the aptamer is provided as multiple aptamer solutions prepared at different concentrations. A202 The method according to A201 or any embodiment, further comprising dividing a single sample into multiple portions and reacting each divided sample with a different aptamer solution. A203 The method according to A202 or any of the embodiments, wherein, among the measurement results obtained at each aptamer concentration, a measurement value determined to be within a linear response range is used to calculate the amount of the target biomolecule.A205 The method according to A001 or any of the embodiments, further comprising introducing an internal standard together with the sample to be measured, and correcting the output value of the sample to be measured based on the output value for the internal standard. A206 The method according to A001 or any of the embodiments, further comprising removing the aptamer from the sensor surface after measurement and washing; and re-immobilizing the aptamer on the sensor surface and then performing another measurement. B001 A device for detecting a target biomolecule, comprising: a sensing surface capable of measuring electrical properties; and a probe that hybridizes with an aptamer of the target biomolecule, the probe having one end immobilized on the sensing surface. B002 The device according to B001 or any of the embodiments, configured to allow a mixed solution containing the aptamer and the target biomolecule to be introduced onto the sensing surface. B003 The device according to B001 or any of the embodiments, configured to measure the charge or potential of the sensing surface corresponding to the negative charge of the aptamer hybridized to the probe. B011 The device according to B001 or any of the embodiments, wherein the probe and a substance that suppresses non-specific adsorption of contaminants to the sensing surface are immobilized on the sensing surface. B012 The device according to B001 or any of the embodiments, wherein the probe and polyethylene glycol (PEG) are immobilized on the sensing surface. B021 The device according to B001 or any of the embodiments, wherein the sensing surface is electrically conductive. B022 The device according to B022 or any of the embodiments, wherein the sensing surface is formed of indium tin oxide (ITO). B031 The device of B021 or B022 or any embodiment, wherein the device comprises a transistor, and the sensing surface is formed as a channel of the transistor.B101 A reagent, kit, or device used for measuring a target biomolecule, comprising: a probe that hybridizes with an aptamer that recognizes the target biomolecule; and a support to which the probe is immobilized. B102 The reagent, kit, or device according to B101 or any of the embodiments, wherein the support to which the probe is immobilized is a magnetic particle. B111 The device according to B102 or any of the embodiments, comprising a magnetic field generator for capturing the magnetic particles. B121e The device according to B101 or any of the embodiments, wherein the support to which the probe is immobilized is a substrate having a flat surface, is flat, or is in the form of a plate. B131 A reagent used for measuring a target biomolecule, comprising: a probe that hybridizes with an aptamer that recognizes the target biomolecule; and magnetic beads to which the probe is immobilized. B201 The device according to B001 or any of the embodiments, comprising: a plurality of containers containing aptamer solutions prepared to different concentrations; a reaction system configured to react a respective portion of a common analyte with each of the aptamer solutions; and a processor configured to analyze measurement results determined to be within the linear response range. B202 The device according to B201 or any of the embodiments, wherein the aptamer solutions are prepared to have predetermined stepwise concentration differences. B203 The device according to B201 or any of the embodiments, wherein the processor is configured to determine, based on the measurement results, which concentration conditions of the aptamer solutions are within the linear response range, and select the corresponding measurement values ​​for analysis. B204 The device according to B201 or any of the embodiments, wherein the processor is configured to automatically determine the next aptamer solution to be applied based on the measurement results, and output a control signal instructing the use of the corresponding solution.The device of B205 B001 or any embodiment, further comprising: a configuration capable of introducing an internal standard together with the sample to be measured; and a processor configured to acquire an output value for the internal standard and correct the output value of the sample to be measured. The device of B206 B001 or any embodiment, further comprising a processor for controlling the process of removing, washing, and re-immobilizing the aptamer from the sensor surface.

[0138] 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 detecting a biomolecule, comprising: (a) providing an aptamer that recognizes a target biomolecule; (b) providing a probe that hybridizes with the aptamer; (c) binding the aptamer to the target biomolecule; (d) hybridizing the aptamer that has not bound to the target biomolecule (unbound aptamer) with the probe; and (e) measuring the amount of unbound aptamer hybridized with the probe to determine the amount of the target biomolecule.

2. The method of claim 1, further comprising, after step (d), cleaning the space adjacent to the probe.

3. The method according to claim 1, wherein the target biomolecule is a glycated protein.

4. The method according to claim 1, wherein the target biomolecule is glycated albumin or glycated hemoglobin.

5. The method of claim 1, wherein: (b) providing the probe comprises providing a sensor having a sensing surface, the probe being immobilized at one end to the sensing surface; and (e) measuring the amount of the probe hybridized to the unbound aptamer comprises measuring an electrical characteristic of the probe that corresponds to the amount of hybridization.

6. The method of claim 1, wherein the electrical property is a charge or surface potential of the sensing surface that corresponds to a negative charge of the aptamer hybridized to the probe.

7. The method of claim 1, wherein: (b) providing the probe comprises providing a probe immobilized on a support; and (e) measuring the amount of unbound aptamer hybridized to the probe comprises separating the unbound aptamer hybridized to the probe from the probe, and measuring the amount of unbound aptamer separated from the probe.

8. The method of claim 1, wherein separating unbound aptamers hybridized to the probe from the probe comprises heating.

9. The method of claim 7, wherein the support is a magnetic particle.

10. The method according to claim 9, comprising hybridizing the unbound aptamer to the probe, capturing the magnetic particles in a magnetic field, and washing the magnetic particles while they are captured in the magnetic field, before separating the unbound aptamer hybridized to the probe from the probe.

11. The method of claim 7, wherein measuring the amount of unbound aptamer separated from the probe comprises using PCR.

12. A device for detecting a target biomolecule, comprising: a sensing surface capable of measuring electrical properties; and a probe that hybridizes with an aptamer of the target biomolecule, one end of which is fixed to the sensing surface.

13. The device of claim 12, configured to measure a charge or potential on the sensing surface corresponding to a negative charge on the aptamer hybridized to the probe.

14. A device according to claim 12, wherein the probe and a substance that suppresses non-specific adsorption of contaminants to the sensing surface are immobilized on the sensing surface.

15. A device according to claim 13, wherein the device comprises a transistor, and the sensing surface is formed as a channel of the transistor.

16. The device of claim 15, wherein the sensing surface is formed of indium tin oxide (ITO).

17. A device used to measure a target biomolecule, comprising: a probe that hybridizes with an aptamer that recognizes the target biomolecule; and a support on which the probe is immobilized.

18. The device according to claim 17, wherein the support to which the probes are immobilized is a magnetic particle.

19. The device of claim 18, comprising a magnetic field generator for capturing the magnetic particles.

20. A reagent used for measuring a target biomolecule, comprising: a probe that hybridizes with an aptamer that recognizes the target biomolecule; and magnetic beads to which the probe is immobilized.

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