WO2014196606A1 - Membrane-type surface stress sensor having antibody or antigen immobilized thereon, method for producing same, and immunoassay method using same - Google Patents

Membrane-type surface stress sensor having antibody or antigen immobilized thereon, method for producing same, and immunoassay method using same Download PDF

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Publication number
WO2014196606A1
WO2014196606A1 PCT/JP2014/064997 JP2014064997W WO2014196606A1 WO 2014196606 A1 WO2014196606 A1 WO 2014196606A1 JP 2014064997 W JP2014064997 W JP 2014064997W WO 2014196606 A1 WO2014196606 A1 WO 2014196606A1
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antibody
thin film
antigen
silicon thin
surface stress
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PCT/JP2014/064997
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French (fr)
Japanese (ja)
Inventor
元起 吉川
弘太 柴
奈生 細川
衛 中町
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独立行政法人物質・材料研究機構
富士レビオ株式会社
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Priority to JP2015521490A priority Critical patent/JPWO2014196606A1/en
Publication of WO2014196606A1 publication Critical patent/WO2014196606A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Definitions

  • the present invention relates to a membrane-type surface stress sensor (MSS) in which an antibody or antigen is immobilized on the surface of a sensor element, a manufacturing method thereof, and an immunoassay method using the same.
  • MSS membrane-type surface stress sensor
  • Non-Patent Document 1 Non-Patent Document 1
  • an antibody is immobilized in advance as a receptor layer on the surface of the piezoresistive cantilever, and a dedicated antibody binding and antigen binding chamber is designed.
  • An immunoassay based on an antibody reaction is performed.
  • PSA prostate-specific antigen
  • the output voltage accompanying the change in the electrical resistance value of the piezoresistive portion increases, and the antigen concentration can be quantified.
  • the detection sensitivity is not sufficient, and further improvement in detection sensitivity has been desired.
  • Patent Document 1 a biosensor manufacturing method that can determine the performance of a biosensor in a non-destructive manner during the manufacturing process.
  • the biosensor manufacturing method described in Patent Document 1 uses a working electrode and a counter electrode formed on an insulating substrate to detect a reaction between a liquid sample and a reagent, and determines a contact area between the liquid sample and the working electrode. By measuring, it is said that the performance of the biosensor can be determined non-destructively during the manufacturing process, and biosensors of the same quality can be stably produced.
  • immunosensors used for immunoassay can only be evaluated by performing antigen or antibody treatment and actually confirming the antigen-antibody reaction, and once immunosensor or antigen treatment has been performed, it cannot be used and discarded. There was nothing else to do. For this reason, 100% inspection of the manufactured immunosensors was not feasible, although it was used in fields such as medical treatment, diagnosis, and food analysis and required high reliability.
  • the present invention is capable of measurement with much higher sensitivity than the conventional piezoresistive cantilever type immunosensor, and moreover, the quality that has been a major problem for biosensors in general. It is an object to provide a new technical means that guarantees the guarantee in a highly stable and stable manner, and that is easy to manufacture the sensor.
  • the present inventor has intensively studied to solve the above problems, and in the process, has focused on utilizing the features of the piezoresistive surface stress sensor newly developed by the present inventors as a means of precision measurement.
  • Patent Document 2 Non-Patent Documents 2 and 3
  • the present inventor has completed the present invention as a measure that enables high detection sensitivity, high quality assurance, and simple production that could not be conventionally expected in immunoassays.
  • a first aspect of the present invention is a membrane-type surface stress sensor, wherein a silicon thin film on which an antibody or an antigen is immobilized is supported at a plurality of points by a support including a piezoresistive element.
  • the second aspect of the present invention is characterized in that an antibody or an antigen is immobilized on one or both sides of a silicon thin film.
  • the antibody or antigen is immobilized by either an ink jet spotting method or an immersion method.
  • a sensor chip is characterized in that one or more film-type surface stress sensors according to any of the first to third inventions are arranged on a semiconductor substrate.
  • a membrane type capable of detecting the binding between the antigen and the antibody or quantifying the amount of the binding by detecting the deformation of the silicon thin film caused by the binding of the antibody to the antigen with a piezoresistive element.
  • a method of manufacturing a surface stress sensor the step of cleaning a surface of a silicon thin film supported by a plurality of supports including at least ⁇ 1> a piezoresistive element; ⁇ 2> a step of bringing a compound that forms a self-assembled film into contact with the surface of the cleaned silicon thin film; ⁇ 3> a step of bringing an antibody or antigen solution into contact with a silicon thin film having a self-assembled film formed on the surface; ⁇ 4>
  • a voltage is applied to the film-type surface stress sensor and a change in the output voltage is monitored; It is characterized by including.
  • an immunoassay method using the membrane-type surface stress sensor according to any one of the first to third inventions wherein at least the following step ⁇ 1> a membrane on which an antibody or antigen is immobilized Bringing the sample solution into contact with the silicon thin film of the mold surface stress sensor; ⁇ 2> a step of applying a voltage to the film-type surface stress sensor and measuring an electrical signal; It is characterized by including.
  • the binding between the antibody or antigen immobilized on the silicon thin film of the membrane type surface stress sensor and the antigen or antibody in the sample solution is determined by membrane type. It is characterized by immersing the surface stress sensor in a sample solution.
  • the stress on the silicon thin film caused by the binding of the antibody or antigen on the silicon thin film and the antigen or antibody contained in the sample solution is concentrated on the portion of the piezoresistive element.
  • the sensitivity can be improved by 20 times or more. This makes it possible to detect an antigen or an antibody with high sensitivity only with a primary antibody, unlike a second antibody system used in immunoassays such as conventional ELISA (Enzyme-Linked Immunosorbent Assay) and CLEIA (Chemiluminescent Enzyme Immunoassay). It becomes like this. Further, when supplying the sample solution to the sensor, it is not necessary to provide a flow path or the like to flow the sample solution.
  • the second and third inventions even if the sensor surface is coated on both sides, stress is concentrated on the piezoresistive element portion, so that high sensitivity can be maintained. Moreover, since the covering efficiency of the sensor is improved, mass production of the sensor can be facilitated.
  • the fourth invention it is possible to mass-produce sensor chips having a plurality of membrane-type surface stress sensors using a semiconductor production line, and it is possible to reduce the manufacturing cost of the sensor chip and to dispose the sensor chip. It becomes. Also, it is easy to make a multi-channel with one sensor chip.
  • the step of cleaning the surface of the silicon thin film; the step of contacting the surface of the cleaned silicon thin film with the compound that forms the self-assembled film; and the self-assembled film formed on the surface In each step of bringing the antibody or antigen solution into contact with the silicon thin film, the change in the electrical resistance value according to the stress applied to the sensor surface is monitored in real time as the output voltage to evaluate whether each step is successful. It becomes possible to do. As a result, a sensor that does not satisfy the evaluation criteria can be constructed to be removed from the production line at that time.
  • immunoassay can be performed with high sensitivity and high accuracy as compared with a measurement method using a conventional piezoresistive cantilever array sensor.
  • FIG. 5 is a schematic diagram of deformation such as bending that occurs when an antigen or an antibody binds to the surfaces of an optical cantilever sensor, a piezoresistive cantilever sensor, and a membrane surface stress sensor that are coated on one or both sides with an antibody or antigen.
  • A It is the photograph of the sensor chip which has arrange
  • (B) It is an enlarged photograph of the sensor of the present invention.
  • (C) It is a figure which shows the example of the measurement system in the case of measuring by the flow method using the sensor of this invention. It is a figure showing the double-sided coating process of the silicon thin film by the hand dipping (manual immersion) method. The graph on the right shows three signals of the same type of membrane type surface stress sensor coated with both sides of polymer (Polyvinylpyrrolidone; PVP) by hand dipping (manual dipping) method. The signals of the three sensors are almost perfectly overlapped.
  • . 6 shows a sensor chip holder used when a sensor chip similar to the sensor chip of FIG. 5 is immersed in a sample solution or the like.
  • FIG. 6 shows that a sensor chip similar to the sensor chip of FIG. 5 is immersed in a sample solution or the like using a commercially available 96-well microplate.
  • 3 shows the results of an antigen-antibody binding experiment when a membrane surface stress sensor of the present invention having a silicon thin film on which an anti-AFP antibody is immobilized is immersed in an AFP solution.
  • the monitoring data during the phosphonic acid treatment of the silicon thin film in the manufacturing process of the film type surface stress sensor of the present invention are shown.
  • 7 shows monitoring data when the phosphonic acid treatment is performed within 5 minutes after the plasma cleaning of the silicon thin film in the manufacturing process of the film type surface stress sensor of the present invention.
  • 6 shows monitoring data when a phosphonic acid treatment is performed 30 minutes after the plasma cleaning of the silicon thin film in the manufacturing process of the film-type surface stress sensor of the present invention.
  • 7 shows monitoring data when a silicon thin film treated with an EDC / NHS solution (DMF) is immersed in an antibody solution in the manufacturing process of the membrane type surface stress sensor of the present invention.
  • the signal diagram on the left shows the results when the silicon thin film treated with the EDC / NHS solution (DMF) was immersed in the anti-AFP antibody solution and the MES containing no anti-AFP antibody (2 -Morpholinoethanesulfonic acid) Monitoring data when immersed in buffer.
  • the right histogram shows the anti-mouse IgG antibody-ALP label added to the silicon thin film immersed in the anti-AFP antibody solution and the silicon thin film immersed in the MES buffer not containing the anti-AFP antibody. It shows that the anti-AFP antibody is immobilized.
  • the present invention has the characteristics as described above, and an embodiment thereof will be described in detail with reference to FIG.
  • FIG. 1 is a schematic diagram of a film type surface stress sensor (MSS) in the present invention.
  • a silicon thin film in which a receptor layer is formed by immobilizing an antibody or an antigen is a support including a piezoresistive element. Is supported by multiple points. It is preferable to use n-type Si (100) for the silicon thin film of the film-type surface stress sensor. As the piezoresistive element, it is preferable to use p-type Si formed on a silicon thin film.
  • the support of the silicon thin film is exemplified by four-point support as shown in FIG. 1, but is not limited to four points.
  • the membrane type surface stress sensor By bringing the membrane type surface stress sensor into contact with the sample solution to be measured, the antibody or antigen immobilized on the silicon thin film of the membrane type surface stress sensor and the antigen or antibody in the sample solution are combined. At that time, the silicon thin film undergoes deformation such as bending due to the surface stress due to the antigen-antibody bond. In each piezoresistive element that supports the silicon thin film, a stress corresponding to the deformation amount of the silicon thin film is generated, and the resistance value of the piezoresistive element changes in proportion to the stress.
  • an output voltage proportional to stress can be obtained by the change in the resistance value described above, and detection of the presence or absence of an antigen or antibody And the amount of antigen or antibody.
  • an output voltage proportional to stress can be obtained by the change in the resistance value described above, and detection of the presence or absence of an antigen or antibody And the amount of antigen or antibody.
  • An antibody or an antigen may be immobilized on a silicon thin film.
  • the antibody is immobilized on the silicon thin film
  • the antigen is immobilized on the silicon thin film.
  • the antibody may be a polyclonal antibody or a monoclonal antibody.
  • the antibody may be any isotype of immunoglobulin such as IgG, IgM, IgA, IgD, IgE, IgY, for example.
  • the antibody may also be a full-length antibody.
  • “Full length antibody” refers to an antibody comprising heavy and light chains, each comprising a variable region and a constant region (eg, an antibody comprising two Fab portions and an Fc portion).
  • the antibody may also be an antibody fragment derived from the full-length antibody.
  • the antibody fragment is a part of a full-length antibody, and examples thereof include F (ab ′) 2, Fab ′, Fab, and Fv.
  • the antibody may also be a modified antibody such as a single chain antibody.
  • modified antibodies such as single chain antibodies include anti-IgG antibodies, anti-IgM antibodies, anti-IgA antibodies, anti-IgE antibodies, anti-prostate specific antigen (PSA) antibodies, anti- ⁇ -fetoprotein (AFP) antibodies, anti-GAD antibodies, Examples include anti-HBV antibody, anti-HCV antibody, anti-HTLV-I antibody, HIV antibody, tuberculosis antibody, mycoplasma antibody, anti-allergen antibody and the like.
  • PSA anti-prostate specific antigen
  • AFP antigen antibodies
  • anti-GAD antibodies examples include anti-HBV antibody, anti-HCV antibody, anti-HTLV-I antibody, HIV antibody, tuberculosis antibody, mycoplasma antibody, anti-allergen antibody and the like.
  • the antigen may be a high molecular substance or a low molecular substance.
  • Polymer substance refers to a compound having a molecular weight of 1,500 or more.
  • Low molecular weight substance refers to a compound having a molecular weight of less than 1,500.
  • polymer substance examples include proteins and nucleic acids.
  • proteins include proteins having affinity binding ability and proteins having aggregation ability.
  • Examples of the protein having affinity binding ability include, for example, the above-mentioned antibodies, receptors on cell membranes such as G protein-coupled receptors, immunoglobulins such as IgG, IgM, IgA, and IgE, extracellular domains cleaved from receptors on cell membranes, and Ligand-dependent proteins such as nuclear receptors, transcription factors, nucleic acid binding proteins such as nucleic acid protection or transport proteins, proteins that form protein complexes such as adapter proteins, extracellular matrix proteins such as intercellular adhesion proteins, tyrosine kinases And enzymes such as serine / threonine kinase, glycoproteins and the like.
  • Examples of the protein having an aggregating ability include pathogenic proteins such as denatured proteins and neurodegenerative proteins such as ⁇ -amyloid.
  • proteins include, for example, apoprotein AI, apoprotein AII, apoprotein B, apoprotein E, rheumatoid factor, D-dimer, glycoalbumin, T3, T4, C-reactive protein (CRP), prostate Specific antigen (PSA), ⁇ -fetoprotein (AFP), DUPAN-2, carcinoembryonic antigen (CEA), CA19-9, CA-125, PIVKA-II (Protein induced by vitamin K absence-II), parathyroid hormone (PTH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), insulin, C-peptide, pepsinogen, influenza A antigen, influenza B antigen, coronavirus antigen, HBV antigen, HCV antigen, HTLV-I Antigen, glycoalbumin, hemoglobin A1c, adiponectin , Cystatin C, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), tropon
  • nucleic acids examples include DNA, RNA, methoxylated RNA, and the like.
  • Low molecular weight substances include ligands, hormones, lipids, fatty acids, vitamins, opioids, neurotransmitters such as catecholamines, nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, amino acids, and oligopeptides, or drugs, toxicants, and Examples include cytokines and metabolites. These low molecular weight substances may be natural substances or synthetic substances.
  • low molecular weight substances examples include hormones such as estrogen and triiodothyronine, cholesterol such as oxidized LDL and saccharified LDL, vitamins such as vitamin A and vitamin D, mold toxins such as DON, NIV and T2, and bisphenol A.
  • endocrine disrupting substances such as nonylphenol, dibutyl phthalate, polychlorinated biphenyls (PCBs), dioxins, p, p′-dichlorodiphenyltrichloroethane, and tributyltin.
  • the antigen may be an allergen (allergen).
  • allergen include fungi such as Escherichia coli, food allergic substances such as eggs, milk, wheat, buckwheat, and peanuts, allergic substances derived from mites such as mite and cypress mites, pollen, mold spores, and the like. .
  • the antibodies or antigens immobilized on the silicon thin film of the membrane surface stress sensor may be the same type, or two or more different types of antibodies or antigens may be used.
  • a mixture of multiple types of antibodies that bind at different sites of the viral antigen protein may be immobilized on a silicon thin film. it can.
  • a mixture of a plurality of different antigens derived from the virus can be immobilized on a silicon thin film.
  • Antibody or antigen is immobilized on one or both sides of the silicon thin film.
  • 2A and 2B show coatings on one side and both sides for the case of (a) (b) optical cantilever, (c) (d) piezoresistive cantilever, and (e) (f) piezo film type surface stress sensor of the present invention. Is shown schematically. As shown in FIGS. 2 (e) and 2 (f), in the film type surface stress sensor of the present invention, stress concentration is recognized in the piezoresistive element portion in both the single-sided coating and the double-sided coating, and FIG. Compared with the cantilever type sensor of (d), it is possible to detect a molecule to be detected efficiently even with double-sided coating. Here, when comparing the sensitivity of the single-sided film-type surface stress sensor and the double-sided film-type surface stress sensor, the single-sided coating is more sensitive (FIG. 3).
  • the optical cantilever with double-sided coating shown in FIG. 2 (b) and the piezoresistive cantilever with single-sided and double-sided coating shown in FIGS. Detection output cannot be obtained.
  • the single-side coated optical cantilever shown in FIG. 2 (a) can produce a detection output, but cannot be used when the sample solution does not transmit light, and is measured when the sample solution is flowing. Is not stable and difficult to use.
  • FIG. 2A suggests that the measurement result may be affected by the change in the refractive index accompanying the change in the concentration of the sample solution.
  • a multi-channel sensor chip in which one or more film-type surface stress sensors are arranged on a semiconductor substrate can be configured.
  • the antibodies or antigens immobilized on the silicon thin film of each membrane type surface stress sensor may be the same type, or different types of antibodies or antigens may be used for each type of membrane type surface stress sensor. Good.
  • two or more different types of antibodies or antigens may be used in one membrane-type surface stress sensor.
  • FIG. 4A shows an embodiment of a sensor chip provided with a plurality of membrane type surface stress sensors according to the present invention, and shows a new configuration MSS chip in which the sensor portion is a two-dimensional array.
  • FIG. 4B shows an enlarged view of the sensor of the present invention.
  • FIG. 4C shows a system configuration of measurement detection by the flow method when the sensor of the present invention is used for measurement.
  • an immersion method may be used, and the apparatus configuration of FIG.
  • an antibody or an antigen can be immobilized only on one side of a silicon thin film using an inkjet spotting method.
  • the ink jet spotting method it is easy to immobilize different types of antibodies or antigens for each silicon thin film of a film-type surface stress sensor arranged on a multi-channel sensor chip. For this reason, it becomes possible to manufacture a multi-channel film-type surface stress sensor provided with antibodies or antigens that can bind to a plurality of types of antigens or antibodies on a single semiconductor substrate.
  • the sensor chip is inverted after immobilizing the antibody or antigen on one side of the silicon thin film as compared with the ink jet spotting method.
  • the manufacturing process is simplified as shown in FIG.
  • the immersion method since a plurality of chips can be dipped in the same solution at the same time, an advanced production facility is not required. For this reason, the immersion method makes it possible to manufacture a one-chip, one-channel film-type surface stress sensor in large quantities and at low cost.
  • the required membrane-type surface stress sensor can be obtained by simply immersing a silicon thin film in an antibody or antigen solution manually or with a simple device without using an advanced and large-scale production facility. Can be manufactured. Moreover, as described in the examples below, it is possible to manufacture by monitoring the presence or absence of a signal of the output voltage observed when the silicon thin film is immersed in the chemical solution and the behavior of the signal change, and feeding back. It is possible to confirm that the processing is successful in each processing step. Therefore, when a large-scale infectious disease occurs, the required mass production system for the sensor can be set up quickly, and a sensor with guaranteed operation can be produced with high efficiency. There is a remarkable effect of making it possible to make a high diagnosis inexpensively.
  • the sensor chip incorporates an electrode for reading a signal resulting from the binding of an antigen or antibody. Therefore, when the silicon thin film is coated on both sides by the dipping method, it is necessary to protect the electrodes and the like from coming into contact with the antibody or the antigen solution.
  • a sensor chip holder (see FIG. 6 and FIG. 7) that can immerse only the sensor element portion in an antibody solution, a sample solution, etc. while maintaining a state where a specific portion such as an electrode of the sensor chip does not contact the liquid sample. It is also preferred to use H).
  • Such a sensor chip holder (H) may be configured to simply fix the sensor chip, or includes a connector that can connect a conductive wire that electrically couples the sensor chip to the outside of the sensor chip holder (H). It may be.
  • the method of manufacturing a film-type surface stress sensor includes a step of cleaning the surface of a silicon thin film supported by a plurality of supports including at least ⁇ 1> piezoresistive elements; ⁇ 2> a step of bringing a compound that forms a self-assembled film into contact with the surface of the cleaned silicon thin film; ⁇ 3> a step of bringing an antibody or antigen solution into contact with a silicon thin film having a self-assembled film formed on the surface; ⁇ 4> In each of the above steps ⁇ 1> to ⁇ 3>, a voltage is applied to the film-type surface stress sensor and a change in the output voltage is monitored; It is characterized by including.
  • Each of the above steps ⁇ 1> to ⁇ 3> is performed by bringing a chemical solution described below in detail into contact with the silicon thin film.
  • a method of manufacturing a double-sided sensor by the dipping method will be described below, but a method of manufacturing a single-sided sensor by the inkjet spotting method is also possible.
  • the surface of the silicon thin film supported by a plurality of supports including the piezoresistive element is cleaned.
  • the method for cleaning the surface of the silicon thin film include a method using a solvent and a method such as plasma cleaning.
  • the solvent used for cleaning the surface of the silicon thin film include acetone, 2-propanol, and ultrapure water.
  • the cleaning of the surface of the silicon thin film is preferably performed a plurality of times using two or more kinds of solvents while changing the solvent.
  • a compound that forms a self-assembled film is brought into contact with the surface of the cleaned silicon thin film.
  • the compound that forms a self-assembled film on the surface of the silicon thin film include phosphonic acid compounds such as Glyphosine and 10-CDPA, but are compounds that can retain the antibody without denaturing or inactivating it.
  • phosphonic acid compounds such as Glyphosine and 10-CDPA
  • the surface of the silicon thin film for immobilizing the antibody or antigen.
  • the surface modification of the silicon thin film include conversion of a carboxyl group contained in the self-assembled film coated on the surface of the silicon thin film into an active ester.
  • N-hydroxysuccinimide (NHS) N-hydroxysulfo in the presence of carbodiimide such as N, N′-dimethylcarbodiimide (DIC), 1-ethyl-3- (3dimethylaminopropyl) carbodiimide hydrochloride (EDC), etc.
  • NHS N-hydroxysuccinimide
  • DIC N, N′-dimethylcarbodiimide
  • EDC 1-ethyl-3- (3dimethylaminopropyl) carbodiimide hydrochloride
  • succinimide such as succinimide.
  • the treatment of the succinimide in the presence of the carbodiimide is specifically performed by immersing the carbodiimide and the succinimide in a mixed solution in which each of them is dissolved in a solvent such as N, N-dimethylformamide (DMF) for about 1 to several hours. Can be done.
  • a solvent such as N, N-dimethylformamide (DMF)
  • an antibody or antigen is brought into contact with the surface of the silicon thin film.
  • the antibody or antigen is dissolved in a buffer solution such as MES buffer or carbonate buffer, and diluted to an appropriate concentration and added to the silicon thin film that has undergone the surface modification step. And allowed to stand at 36 ° C. for 1 hour or at 4 ° C. overnight to bring the silicon thin film into contact with the antibody or antigen.
  • excess components for example, unbound antibody or antigen
  • PBS phosphate buffer
  • TBS Tris buffer
  • the cleaning liquid may contain skim milk, bovine serum albumin (BSA), or the like in the blocking step.
  • a voltage is applied to the film type surface stress sensor to monitor the change in the output voltage.
  • Successful work in each manufacturing process by monitoring and feeding back the output voltage signal behavior and signal change behavior observed when the silicon thin film is in contact with the above chemical solution. Can be confirmed. For this reason, it is possible to construct a system in which sensors that do not satisfy the evaluation criteria are removed from the production line at that time.
  • the immunoassay method using the membrane-type surface stress sensor of the present invention comprises at least the following step ⁇ 1> a step of bringing a sample solution into contact with the silicon thin film of the membrane-type surface stress sensor on which an antibody or antigen is immobilized; ⁇ 2> a step of applying a voltage to the film-type surface stress sensor and measuring an electrical signal; It is characterized by including.
  • a sample solution (eg, for example, from a living body such as a human, monkey, mouse, rat, sheep, horse, etc.) is applied to a silicon thin film of a membrane-type surface stress sensor on which the antibody or antigen is immobilized. Blood) is collected, and the sample solution diluted by pretreatment as necessary is brought into contact with the antibody or antigen immobilized on the surface of the membrane surface stress sensor, and the antigen or antibody in the sample solution Are combined.
  • the origin of the sample solution is not limited to the natural sample derived from the living body.
  • the step of bringing the sample solution into contact with the silicon thin film of the membrane surface stress sensor to which the antibody or antigen is immobilized is preferably performed by immersing the membrane surface stress sensor in the sample solution.
  • a voltage is applied to the membrane type surface stress sensor before and after the first step, and an electric signal is measured. It is possible to detect an antigen-antibody reaction by measuring the presence or absence of a signal of the output voltage observed when the silicon thin film is brought into contact with the sample solution and the behavior of the signal change. Specifically, the silicon thin film undergoes deformation such as bending due to surface stress due to the antigen-antibody bond. In each piezoresistive element that supports the silicon thin film, a stress corresponding to the deformation amount of the silicon thin film is generated, and the resistance value of the piezoresistive element changes in proportion to the stress.
  • an output voltage proportional to the stress can be obtained by the change in the resistance value described above, and in the sample solution It is possible to quantify the presence or absence of the antigen or antibody and the amount (concentration) of the antigen or antibody.
  • the present invention is based on such a measurement principle, it is possible to provide a highly sensitive immunoassay in the medical and diagnostic fields as compared with a conventional immunoassay.
  • Example 1 As illustrated in FIG. 1, the silicon thin film is supported at four points by a support including a piezoresistive element, and the silicon thin film of the sensor structure before the antibody is bonded is acetone, 2-propanol, ultrapure water (MilliQ water). ; Water in the order of MQ water), and finally the silicon thin film was cleaned by performing plasma cleaning.
  • the cleaned silicon thin film was immersed in a 1 mM phosphonic acid solution (Glyphosine / MQ water) and allowed to stand at room temperature for 60 minutes.
  • the silicon thin film immersed in phosphonic acid was washed with MQ water and ethanol, removed from the socket after drying, and annealed at 140 ° C. for 1 hour to modify the surface of the silicon thin film with phosphonic acid.
  • phosphonic acid modification was performed on a mixed solution of 5 mM N-hydroxysuccinimide (NHS) and 5 mM 1-ethyl 3- (3 dimethylaminopropyl) carbodiimide hydrochloride (EDC) / N, N-dimethylformamide (DMF) solution.
  • NHS N-hydroxysuccinimide
  • EDC dimethylaminopropyl carbodiimide hydrochloride
  • DMF N, N-dimethylformamide
  • the silicon thin film treated with EDC / NHS solution (DMF) was connected to the sensor chip holder, and the sensor chip was added to the anti-AFP ( ⁇ -fetoprotein) antibody solution (MES buffer, pH 5.5) dispensed to each well of the 96-well plate.
  • the silicon thin film connected to the holder was immersed and allowed to stand overnight at 4 ° C. to bind to the anti-AFP antibody.
  • the silicon thin film to which the anti-AFP antibody was bound was washed with a washing buffer containing Triton X-100 as a surfactant, and allowed to stand at 37 ° C. for 60 minutes for masking. By washing this with a masking buffer, an anti-AFP antibody could be immobilized on the sensor chip.
  • a silicon thin film immersed in a MES buffer not containing an anti-AFP antibody was used instead of the anti-AFP antibody solution (MES buffer, pH 5.5).
  • the membrane type surface stress sensor (antibody binding sensor) and the control sensor thus obtained were connected to a sensor chip holder, and 2000 ng / mL (about 28.5 ⁇ M) dispensed into each well of a 96-well plate.
  • the sensor was immersed in the AFP solution.
  • the output voltage of the signal of the piezoresistive cantilever array sensor described in Non-Patent Document 1 is 7.5 ⁇ V per unit bias voltage
  • the membrane type surface stress sensor of the present invention is described in Non-Patent Document 1. It was observed that the sensitivity reached about 40 times compared to the piezoresistive cantilever array sensor. Further, when comparing the detectable low antigen concentration, the piezoresistive cantilever array sensor described in Non-Patent Document 1 has a value of 0.3 nM, whereas the membrane-type surface stress sensor of the present invention theoretically has 7 It was found that 5 pM of antigen could be detected.
  • Example 2 The signal of the silicon thin film during the phosphonic acid solution treatment was measured in the surface treatment process of the silicon thin film in the production process of the film type surface stress sensor. The results are shown in FIG. In FIG. 9, signal patterns A, B, and C are shown. Note that Ch1 to Ch4 (channel 4) in FIG. 9 indicate detection voltages of four sensors arranged in one sensor chip. A slight difference between detection voltages is considered to be due to a lot difference at the time of manufacturing a silicon thin film, a difference in the amount of immobilized antibody, and the like.
  • the signal pattern will be explained.
  • the silicon thin film was cleaned by immersing the silicon thin film in a solvent in the order of acetone, 2-propanol and ultrapure water (MilliQ water; MQ water), and finally performing plasma cleaning. .
  • the signal when the sensor after this plasma cleaning is immersed in MQ water is the pattern of A.
  • a signal when the silicon thin film is moved from MQ water to MQ water is a pattern B.
  • the signal when the silicon thin film is transferred from the MQ water to the phosphonic acid solution is a C pattern.
  • the voltage of the C signal started to change immediately after the silicon thin film was immersed in the phosphonic acid solution, and the change in voltage settled after about 1000 seconds. This change in voltage is considered to capture the state in which phosphonic acid is bonded to the surface of the silicon thin film.
  • FIG. 10 shows a signal when the silicon thin film is immersed in the phosphonic acid solution after 30 minutes from the plasma cleaning.
  • the signal when the silicon thin film was immersed in the phosphonic acid solution within 5 minutes after the plasma cleaning was an output voltage of about 700 ⁇ V, whereas the silicon thin film was immersed in the phosphonic acid solution 30 minutes after the plasma cleaning. In this case, the output voltage was greatly reduced to about 200 ⁇ V. From this, by performing the phosphonic acid solution treatment in a short time after the plasma cleaning, it is possible to perform a treatment using a silicon thin film having a high surface activity, but if the standing time after the cleaning is long, It was suggested that the surface activity of the silicon thin film decreases.
  • Example 4 In the step of binding the antibody to the silicon thin film surface of the membrane type surface stress sensor, the signal during the treatment with the anti-AFP antibody solution was measured (FIG. 12). First, a silicon thin film treated with an EDC / NHS solution (DMF) was immersed in a MES buffer, and then the silicon thin film was transferred from the MES buffer to the MES buffer. Then, the silicon thin film was moved from the MES buffer to the anti-AFP antibody solution, and the signal during this period was continuously measured.
  • EDC / NHS solution DMF
  • Example 5 Connect the silicon thin film treated with EDC / NHS solution (DMF) to the sensor chip holder and immerse the silicon thin film in 100 ⁇ g / mL anti-AFP antibody solution (MES buffer, pH 5.5) dispensed to each well of 96-well plate did.
  • MES buffer 100 ⁇ g / mL anti-AFP antibody solution
  • a silicon thin film was immersed in a MES buffer not containing an anti-AFP antibody.
  • Example 6 The silicon thin film treated with EDC / NHS solution (DMF) was connected to the sensor chip holder, and the silicon thin film was immersed in an anti-AFP antibody solution (MES buffer, pH 5.5) dispensed to each well of the 96-well plate. The antibody was immobilized overnight at 0 ° C. The next day, the silicon thin film was washed with a washing buffer containing a surfactant (Triton X-100) and masked at 37 ° C. for 60 minutes. By washing this with a masking buffer, the anti-AFP antibody could be immobilized on the silicon thin film. As a control, a silicon thin film immersed in a MES buffer not containing an anti-AFP antibody was used instead of the anti-AFP antibody solution (MES buffer, pH 5.5).
  • MES buffer pH 5.5
  • 0.1 ⁇ g / mL of anti-mouse IgG antibody labeled with alkaline phosphatase (ALP) dispensed into each well of the 96-well plate is contained.
  • the aforementioned silicon thin film was immersed in the solution at 37 degrees for 30 minutes. Thereafter, the silicon thin film was washed with a washing solution containing a surfactant (Triton X-100), reacted with a luminescent substrate solution of ALP at 37 ° C. for 5 minutes, and then a chemiluminescence signal was measured.
  • a surfactant Triton X-100

Abstract

A membrane-type surface stress sensor characterized in that a thin silicone membrane having an antibody or antigen immobilized thereon is supported at multiple points by a supporting member comprising a piezo-resistance element, and the deformation of the thin silicone membrane, which is induced by the binding of the antibody or antigen to an antigen or antibody in a sample solution, is detected by the piezo-resistance element so that the antigen-antibody biding can be detected or the binding amount thereof can be determined.

Description

抗体または抗原を固定化した膜型表面応力センサとその製造方法並びにこれを用いた免疫測定方法Membrane surface stress sensor immobilizing antibody or antigen, method for producing the same, and immunoassay method using the same
　本発明は、センサ素子表面に抗体または抗原を固定化した膜型表面応力センサ（membrane-type surface stress sensor、ＭＳＳ）とその製造方法並びにこれを用いた免疫測定方法に関する。  The present invention relates to a membrane-type surface stress sensor (MSS) in which an antibody or antigen is immobilized on the surface of a sensor element, a manufacturing method thereof, and an immunoassay method using the same.
　従来、抗体と抗原の結合反応を利用して、生体試料中に含まれる抗原の濃度を測定する免疫測定方法では様々な工夫がなされてきている。例えば、光学的検知方法などが開発され、近年では、より簡便な測定を可能とするための方法として、ピエゾ抵抗型カンチレバー方式での測定が提案されている（非特許文献１）。 Conventionally, various contrivances have been made in an immunoassay method for measuring the concentration of an antigen contained in a biological sample using a binding reaction between an antibody and an antigen. For example, an optical detection method has been developed, and in recent years, measurement using a piezoresistive cantilever method has been proposed as a method for enabling simpler measurement (Non-Patent Document 1).
　非特許文献１記載のピエゾ抵抗型カンチレバー方式による免疫測定においては、ピエゾ抵抗型カンチレバー表面に予め受容体層として抗体を固定化し、専用の抗体結合用、抗原結合用のチャンバーを設計して、抗原抗体反応に基づく免疫測定を行うようにしている。この方法では、抗原として用いた前立腺特異抗原（ＰＳＡ）の濃度に依存して、ピエゾ抵抗部の電気抵抗値変化に伴う出力電圧が大きくなり、抗原濃度の定量も可能である。しかしながら、検出感度が十分とは言えず、さらなる検出感度の向上が望まれていた。 In immunoassay by the piezoresistive cantilever method described in Non-Patent Document 1, an antibody is immobilized in advance as a receptor layer on the surface of the piezoresistive cantilever, and a dedicated antibody binding and antigen binding chamber is designed. An immunoassay based on an antibody reaction is performed. In this method, depending on the concentration of prostate-specific antigen (PSA) used as an antigen, the output voltage accompanying the change in the electrical resistance value of the piezoresistive portion increases, and the antigen concentration can be quantified. However, the detection sensitivity is not sufficient, and further improvement in detection sensitivity has been desired.
　ところで、従来のバイオセンサの品質保証に関する研究では、統計手法を用いた抜き取り法を行っているため、バイオセンサ群の感度を全数管理あるいは保証することは不可能であった。さらに、ロット単位で抜き取り検査し、抜き取り検査結果が不良と判定されたロットは全数破棄していた。このような品質管理手法では、良品まで破棄してしまうという無駄が発生するだけはなく、完成した製品の良品率が統計的にある程度以上であることは保証されるが、個々の製品が良品であることは必ずしも保証されていない。これは、誤診が重大な問題を引き起こす可能性のある、本発明の適用分野では好ましいことではない。 By the way, in the research on quality assurance of the conventional biosensor, since the sampling method using the statistical method is performed, it is impossible to manage or guarantee the sensitivity of the biosensor group. Further, a sampling inspection was performed in units of lots, and all the lots for which the sampling inspection result was determined to be defective were discarded. Such a quality control method not only causes waste of discarding non-defective products, but also guarantees that the non-defective product rate of the finished product is statistically above a certain level. It is not always guaranteed that there is. This is not desirable in the field of application of the present invention, where misdiagnosis can cause serious problems.
　このような課題を解決するために、バイオセンサの性能を製造工程中に非破壊で判定することが可能なバイオセンサの製造方法が提案されている（特許文献１）。特許文献１記載のバイオセンサの製造方法は、絶縁性の基板上に形成した作用電極と対電極とを用いて、液体試料と試薬との反応を検出し、液体試料と作用電極の接触面積を測定することによって、バイオセンサの性能を製造工程中に非破壊で判定し、同一品質のバイオセンサを安定して生産することができるとされている。 In order to solve such a problem, a biosensor manufacturing method that can determine the performance of a biosensor in a non-destructive manner during the manufacturing process has been proposed (Patent Document 1). The biosensor manufacturing method described in Patent Document 1 uses a working electrode and a counter electrode formed on an insulating substrate to detect a reaction between a liquid sample and a reagent, and determines a contact area between the liquid sample and the working electrode. By measuring, it is said that the performance of the biosensor can be determined non-destructively during the manufacturing process, and biosensors of the same quality can be stably produced.
　しかしながら、免疫測定に用いるイムノセンサは、抗原または抗体処理を行い、実際に抗原抗体反応を確認することでしか性能を評価できず、一度抗原または抗体処理を行ったイムノセンサは、利用できなくなり破棄するより他はなかった。このため、医療や診断、食品分析などの分野で用いられ、高い信頼性を要求されているにもかかわらず、製造したイムノセンサの全数検査は実現不可能であった。 However, immunosensors used for immunoassay can only be evaluated by performing antigen or antibody treatment and actually confirming the antigen-antibody reaction, and once immunosensor or antigen treatment has been performed, it cannot be used and discarded. There was nothing else to do. For this reason, 100% inspection of the manufactured immunosensors was not feasible, although it was used in fields such as medical treatment, diagnosis, and food analysis and required high reliability.
　そこで、以上のとおりの背景から、本発明は、従来のピエゾ抵抗型カンチレバー方式によるイムノセンサよりもはるかに高感度の測定が可能であって、しかも従来よりバイオセンサ一般の大きな課題であった品質保証を高度に、かつ安定して確実なものとし、かつセンサ製造も簡便な新しい技術的手段を提供することを課題とする。 Therefore, based on the background as described above, the present invention is capable of measurement with much higher sensitivity than the conventional piezoresistive cantilever type immunosensor, and moreover, the quality that has been a major problem for biosensors in general. It is an object to provide a new technical means that guarantees the guarantee in a highly stable and stable manner, and that is easy to manufacture the sensor.
　本発明者は、上記課題を解決するために鋭意検討を進め、その過程において、本発明者らが精密測定の手段として新たに開発したピエゾ抵抗膜型表面応力センサの特徴を生かすことに着目した（特許文献２、非特許文献２、３）。その結果、本発明者は、免疫測定において従来では予期し得ない高い検出感度と高い品質保証、さらには簡便な製造を可能とする方策として本発明を完成した。 The present inventor has intensively studied to solve the above problems, and in the process, has focused on utilizing the features of the piezoresistive surface stress sensor newly developed by the present inventors as a means of precision measurement. (Patent Document 2, Non-Patent Documents 2 and 3). As a result, the present inventor has completed the present invention as a measure that enables high detection sensitivity, high quality assurance, and simple production that could not be conventionally expected in immunoassays.
　本発明は、第１には、膜型表面応力センサであって、抗体または抗原が固定化されたシリコン薄膜が、ピエゾ抵抗素子を含む支持体によって複数点支持されており、前記抗体または抗原が試料中の抗原または抗体と結合することによって引き起こされるシリコン薄膜の変形をピエゾ抵抗素子によって検出することにより、抗原と抗体との結合を検出またはその結合量を定量可能とすることを特徴とする。 A first aspect of the present invention is a membrane-type surface stress sensor, wherein a silicon thin film on which an antibody or an antigen is immobilized is supported at a plurality of points by a support including a piezoresistive element. By detecting the deformation of the silicon thin film caused by binding to the antigen or antibody in the sample with a piezoresistive element, the binding between the antigen and the antibody can be detected or the amount of the binding can be quantified.
　また、本発明は、第２には、抗体または抗原が、シリコン薄膜の片面または両面に固定化されていることを特徴とする。第３には、抗体または抗原が、インクジェットスポッティング法、浸漬法のいずれかで固定化されていることを特徴とする。第４には、センサチップであって、上記第１から第３の発明のいずれかの膜型表面応力センサを半導体基板上に１つ以上配置されていることを特徴とする。 The second aspect of the present invention is characterized in that an antibody or an antigen is immobilized on one or both sides of a silicon thin film. Third, the antibody or antigen is immobilized by either an ink jet spotting method or an immersion method. Fourth, a sensor chip is characterized in that one or more film-type surface stress sensors according to any of the first to third inventions are arranged on a semiconductor substrate.
　そして、第５には、抗体が抗原と結合することによって引き起こされるシリコン薄膜の変形をピエゾ抵抗素子によって検出することにより、抗原と抗体との結合を検出またはその結合量を定量可能とする膜型表面応力センサの製造方法であって、少なくとも
＜１＞ピエゾ抵抗素子を含む複数の支持体により支持されたシリコン薄膜の表面を洗浄する工程；
＜２＞洗浄したシリコン薄膜の表面に、自己組織化膜を形成する化合物を接触させる工程；
＜３＞表面に自己組織化膜が形成されたシリコン薄膜に、抗体または抗原溶液を接触させる工程；
＜４＞上記＜１＞～＜３＞の各工程において、膜型表面応力センサに電圧を印加して、出力電圧の変化をモニタリングする工程；
を含むことを特徴とする。 And fifth, a membrane type capable of detecting the binding between the antigen and the antibody or quantifying the amount of the binding by detecting the deformation of the silicon thin film caused by the binding of the antibody to the antigen with a piezoresistive element. A method of manufacturing a surface stress sensor, the step of cleaning a surface of a silicon thin film supported by a plurality of supports including at least <1> a piezoresistive element;
<2> a step of bringing a compound that forms a self-assembled film into contact with the surface of the cleaned silicon thin film;
<3> a step of bringing an antibody or antigen solution into contact with a silicon thin film having a self-assembled film formed on the surface;
<4> In each of the above steps <1> to <3>, a voltage is applied to the film-type surface stress sensor and a change in the output voltage is monitored;
It is characterized by including.
　さらに、第６には、上記第１から第３の発明のいずれかの膜型表面応力センサを用いた免疫測定方法であって、少なくとも以下の工程
＜１＞抗体または抗原が固定化された膜型表面応力センサのシリコン薄膜に、試料溶液を接触させる工程；
＜２＞前記膜型表面応力センサに電圧を印加し、電気シグナルを測定する工程；
を含むことを特徴とする。 Further, sixthly, an immunoassay method using the membrane-type surface stress sensor according to any one of the first to third inventions, wherein at least the following step <1> a membrane on which an antibody or antigen is immobilized Bringing the sample solution into contact with the silicon thin film of the mold surface stress sensor;
<2> a step of applying a voltage to the film-type surface stress sensor and measuring an electrical signal;
It is characterized by including.
　第７には、上記第６の発明の免疫測定方法であって、膜型表面応力センサのシリコン薄膜に固定化された抗体または抗原と、試料溶液中の抗原または抗体との結合を、膜型表面応力センサを試料溶液に浸漬して行うことを特徴とする。 Seventhly, in the immunoassay method of the sixth invention, the binding between the antibody or antigen immobilized on the silicon thin film of the membrane type surface stress sensor and the antigen or antibody in the sample solution is determined by membrane type. It is characterized by immersing the surface stress sensor in a sample solution.
　第１の発明によれば、シリコン薄膜上の抗体または抗原と試料溶液中に含まれる抗原または抗体が結合することによって生じたシリコン薄膜上の応力が、ピエゾ抵抗素子の部分に集中するため、従来のピエゾ抵抗カンチレバーアレイセンサに比べて、例えば２０倍以上の感度の向上が可能となる。これにより、従来のＥＬＩＳＡ（Ｅｎｚｙｍｅ－Ｌｉｎｋｅｄ　ＩｍｍｕｎｏＳｏｒｂｅｎｔ　Ａｓｓａｙ）やＣＬＥＩＡ（Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｔ　Ｅｎｚｙｍｅ　Ｉｍｍｕｎｏａｓｓａｙ）などの免疫測定法で使用される二抗体系と異なり、一次抗体のみで感度よく抗原または抗体を検出することができるようになる。また、試料溶液をセンサに供給する際、流路等を設けて試料溶液を流す必要がない。 According to the first invention, the stress on the silicon thin film caused by the binding of the antibody or antigen on the silicon thin film and the antigen or antibody contained in the sample solution is concentrated on the portion of the piezoresistive element. Compared to the piezoresistive cantilever array sensor, for example, the sensitivity can be improved by 20 times or more. This makes it possible to detect an antigen or an antibody with high sensitivity only with a primary antibody, unlike a second antibody system used in immunoassays such as conventional ELISA (Enzyme-Linked Immunosorbent Assay) and CLEIA (Chemiluminescent Enzyme Immunoassay). It becomes like this. Further, when supplying the sample solution to the sensor, it is not necessary to provide a flow path or the like to flow the sample solution.
　また、第２、第３の発明によれば、センサ表面を両面被覆してもピエゾ抵抗素子部に応力が集中するため、高い感度を維持することが可能となる。また、センサの被覆効率が向上するため、センサの大量生産を容易にすることができる。 Further, according to the second and third inventions, even if the sensor surface is coated on both sides, stress is concentrated on the piezoresistive element portion, so that high sensitivity can be maintained. Moreover, since the covering efficiency of the sensor is improved, mass production of the sensor can be facilitated.
　第４の発明によれば、膜型表面応力センサを複数備えたセンサチップを、半導体の製造ラインを用いて大量生産することが可能となり、センサチップの製造コストの低下やセンサチップの使い捨てが可能となる。また、一つのセンサチップで多チャンネル化することが容易である。 According to the fourth invention, it is possible to mass-produce sensor chips having a plurality of membrane-type surface stress sensors using a semiconductor production line, and it is possible to reduce the manufacturing cost of the sensor chip and to dispose the sensor chip. It becomes. Also, it is easy to make a multi-channel with one sensor chip.
　また、第５の発明によれば、シリコン薄膜の表面を洗浄する工程；洗浄したシリコン薄膜の表面に、自己組織化膜を形成する化合物を接触させる工程；表面に自己組織化膜が形成されたシリコン薄膜に、抗体または抗原溶液を接触させる工程；の各工程において、センサ表面に印加された応力に応じた電気抵抗値の変化を出力電圧としてリアルタイムでモニタリングし、各工程が成功しているか評価することが可能となる。これにより、評価基準を満たさないセンサは、その時点で生産ラインから取り除くシステムを構築することができる。その結果、表面に自己組織化膜が形成されたシリコン薄膜に、抗体または抗原溶液を接触させる工程まで終了したセンサは、全製造工程が確実に成功していることが保証されており、製造したセンサの全数検査と同等の効果を得ることができる。したがって、医療や診断分野において高品質で、極めて信頼性の高い検査用キット等を提供することが可能となる。 Further, according to the fifth invention, the step of cleaning the surface of the silicon thin film; the step of contacting the surface of the cleaned silicon thin film with the compound that forms the self-assembled film; and the self-assembled film formed on the surface In each step of bringing the antibody or antigen solution into contact with the silicon thin film, the change in the electrical resistance value according to the stress applied to the sensor surface is monitored in real time as the output voltage to evaluate whether each step is successful. It becomes possible to do. As a result, a sensor that does not satisfy the evaluation criteria can be constructed to be removed from the production line at that time. As a result, a sensor that has been completed up to the step of bringing an antibody or antigen solution into contact with a silicon thin film having a self-assembled film formed on the surface is guaranteed to have been successfully manufactured. It is possible to obtain the same effect as 100% sensor inspection. Therefore, it is possible to provide a high-quality and highly reliable test kit in the medical and diagnostic fields.
　第６、７の発明によれば、従来のピエゾ抵抗カンチレバーアレイセンサを用いた測定方法と比較して、高感度かつ高精度に免疫測定を行うことができる。 According to the sixth and seventh inventions, immunoassay can be performed with high sensitivity and high accuracy as compared with a measurement method using a conventional piezoresistive cantilever array sensor.
本発明における膜型表面応力センサ（ＭＳＳ）の模式図である。It is a schematic diagram of a film type surface stress sensor (MSS) in the present invention. 抗体または抗原を片面被覆または両面被覆した光学式カンチレバーセンサ、ピエゾ抵抗型カンチレバーセンサ、および膜型表面応力センサの表面に、抗原または抗体が結合した際に生じる撓み等の変形の模式図である。FIG. 5 is a schematic diagram of deformation such as bending that occurs when an antigen or an antibody binds to the surfaces of an optical cantilever sensor, a piezoresistive cantilever sensor, and a membrane surface stress sensor that are coated on one or both sides with an antibody or antigen. ポリマー（ｐｏｌｙ（ｓｏｄｉｕｍ　４－ｓｔｙｒｅｎｅｓｕｌｆｏｎａｔｅ）、ＰＳＳ）を片面被覆または両面被覆した膜型表面応力センサを、水蒸気に曝露した際に得られるシグナルを示すグラフである。It is a graph which shows the signal obtained when the film | membrane type surface stress sensor which coat | covered the polymer (poly (sodium 4-styrenesulfonate), PSS) on one side or both sides was exposed to water vapor | steam. （ａ）本発明における膜型表面応力センサを複数配置させたセンサチップの写真である。（ｂ）本発明のセンサの拡大写真である。（ｃ）本発明のセンサを用いてフロー法で測定する場合の測定システムの例を示す図である。(A) It is the photograph of the sensor chip which has arrange | positioned multiple film type surface stress sensors in this invention. (B) It is an enlarged photograph of the sensor of the present invention. (C) It is a figure which shows the example of the measurement system in the case of measuring by the flow method using the sensor of this invention. ハンドディッピング（手動浸漬）法によるシリコン薄膜の両面被覆工程を表す図である。右のグラフは、ハンドディッピング（手動浸漬）法によってポリマー（Polyvinylpyrrolidone；ＰＶＰ）が両面被覆された同一型の膜型表面応力センサ３つのシグナルを示し、３つのセンサのシグナルはほぼ完璧に重なっている。It is a figure showing the double-sided coating process of the silicon thin film by the hand dipping (manual immersion) method. The graph on the right shows three signals of the same type of membrane type surface stress sensor coated with both sides of polymer (Polyvinylpyrrolidone; PVP) by hand dipping (manual dipping) method. The signals of the three sensors are almost perfectly overlapped. . 図５のセンサチップと同様のセンサチップを試料溶液などに浸漬する際に用いるセンサチップホルダーを示している。6 shows a sensor chip holder used when a sensor chip similar to the sensor chip of FIG. 5 is immersed in a sample solution or the like. 市販の９６穴マイクロプレートを用いて、図５のセンサチップと同様のセンサチップを試料溶液などに浸漬することを示している。6 shows that a sensor chip similar to the sensor chip of FIG. 5 is immersed in a sample solution or the like using a commercially available 96-well microplate. 抗ＡＦＰ抗体を固定化したシリコン薄膜を備えた本発明の膜型表面応力センサを、ＡＦＰ溶液に浸漬した際の抗原－抗体結合実験の結果を示している。3 shows the results of an antigen-antibody binding experiment when a membrane surface stress sensor of the present invention having a silicon thin film on which an anti-AFP antibody is immobilized is immersed in an AFP solution. 本発明の膜型表面応力センサの製造過程で、シリコン薄膜をホスホン酸処理中のモニタリングデータを示している。The monitoring data during the phosphonic acid treatment of the silicon thin film in the manufacturing process of the film type surface stress sensor of the present invention are shown. 本発明の膜型表面応力センサの製造過程で、シリコン薄膜のプラズマ洗浄後、５分以下でホスホン酸処理をした際のモニタリングデータを示している。7 shows monitoring data when the phosphonic acid treatment is performed within 5 minutes after the plasma cleaning of the silicon thin film in the manufacturing process of the film type surface stress sensor of the present invention. 本発明の膜型表面応力センサの製造過程で、シリコン薄膜のプラズマ洗浄後、３０分経過後ホスホン酸処理をした際のモニタリングデータを示している。FIG. 6 shows monitoring data when a phosphonic acid treatment is performed 30 minutes after the plasma cleaning of the silicon thin film in the manufacturing process of the film-type surface stress sensor of the present invention. 本発明の膜型表面応力センサの製造過程で、ＥＤＣ／ＮＨＳ溶液（ＤＭＦ）処理したシリコン薄膜を抗体溶液に浸漬した際のモニタリングデータを示している。7 shows monitoring data when a silicon thin film treated with an EDC / NHS solution (DMF) is immersed in an antibody solution in the manufacturing process of the membrane type surface stress sensor of the present invention. 左のシグナル図は、本発明の膜型表面応力センサの製造過程で、ＥＤＣ／ＮＨＳ溶液（ＤＭＦ）処理したシリコン薄膜を、抗ＡＦＰ抗体溶液に浸漬した際と抗ＡＦＰ抗体を含まないＭＥＳ（2-Morpholinoethanesulfonic acid）バッファーに浸漬した際のモニタリングデータを示している。右のヒストグラムは、抗ＡＦＰ抗体溶液に浸漬したシリコン薄膜と抗ＡＦＰ抗体を含まないＭＥＳバッファーに浸漬したシリコン薄膜に抗マウスＩｇＧ抗体－ＡＬＰ標識体を添加し、化学発光させることによって、シリコン薄膜上に抗ＡＦＰ抗体が固定化されていることを示している。The signal diagram on the left shows the results when the silicon thin film treated with the EDC / NHS solution (DMF) was immersed in the anti-AFP antibody solution and the MES containing no anti-AFP antibody (2 -Morpholinoethanesulfonic acid) Monitoring data when immersed in buffer. The right histogram shows the anti-mouse IgG antibody-ALP label added to the silicon thin film immersed in the anti-AFP antibody solution and the silicon thin film immersed in the MES buffer not containing the anti-AFP antibody. It shows that the anti-AFP antibody is immobilized.
　本発明は、上記のとおりの特徴をもつものであるが、図１に基づいて、その実施の形態について詳しく説明する。 The present invention has the characteristics as described above, and an embodiment thereof will be described in detail with reference to FIG.
　図１は、本発明における膜型表面応力センサ（ＭＳＳ）の模式図である。 FIG. 1 is a schematic diagram of a film type surface stress sensor (MSS) in the present invention.
　本発明の膜型表面応力センサにおいては、図１に示したように、抗体または抗原が固定化されて受容体層（Receptor Layer）が形成されているシリコン薄膜が、ピエゾ抵抗素子を含む支持体によって複数点支持されている。膜型表面応力センサのシリコン薄膜は、ｎ型のＳｉ（１００）を用いることが好ましい。また、ピエゾ抵抗素子としては、シリコン薄膜上に構成されたｐ型のＳｉを用いることが好ましい。 In the membrane type surface stress sensor of the present invention, as shown in FIG. 1, a silicon thin film in which a receptor layer is formed by immobilizing an antibody or an antigen is a support including a piezoresistive element. Is supported by multiple points. It is preferable to use n-type Si (100) for the silicon thin film of the film-type surface stress sensor. As the piezoresistive element, it is preferable to use p-type Si formed on a silicon thin film.
　シリコン薄膜の支持は、例えば、図１に示したように４点支持が例示されるが、４点に限らない。 The support of the silicon thin film is exemplified by four-point support as shown in FIG. 1, but is not limited to four points.
　この膜型表面応力センサと測定対象の試料溶液を接触させることで、膜型表面応力センサのシリコン薄膜上に固定化された抗体または抗原と試料溶液中の抗原または抗体が結合する。その際、シリコン薄膜は、抗原－抗体結合による表面応力を受けて撓み等の変形が生じる。シリコン薄膜を支持するそれぞれのピエゾ抵抗素子には、シリコン薄膜の変形量に応じた応力が発生し、この応力に比例してピエゾ抵抗素子の抵抗値が変化する。 By bringing the membrane type surface stress sensor into contact with the sample solution to be measured, the antibody or antigen immobilized on the silicon thin film of the membrane type surface stress sensor and the antigen or antibody in the sample solution are combined. At that time, the silicon thin film undergoes deformation such as bending due to the surface stress due to the antigen-antibody bond. In each piezoresistive element that supports the silicon thin film, a stress corresponding to the deformation amount of the silicon thin film is generated, and the resistance value of the piezoresistive element changes in proportion to the stress.
　本発明は、例えば、４つのピエゾ抵抗素子によって構成されるホイートストンブリッジに電圧を印加することで、上記の抵抗値の変化によって、応力に比例した出力電圧が得られ、抗原または抗体の有無の検出および抗原または抗体量を測定するものである。既知の抗原または抗体量とそれに応じて生じる出力電圧との間で検量線を作成することで、試料溶液中の抗原または抗体の有無および抗原または抗体量（濃度）を定量することが可能となる。 In the present invention, for example, by applying a voltage to a Wheatstone bridge composed of four piezoresistive elements, an output voltage proportional to stress can be obtained by the change in the resistance value described above, and detection of the presence or absence of an antigen or antibody And the amount of antigen or antibody. By creating a calibration curve between the known antigen or antibody amount and the output voltage generated accordingly, the presence or absence of the antigen or antibody in the sample solution and the antigen or antibody amount (concentration) can be quantified. .
　シリコン薄膜上には、抗体を固定化してもよいし、抗原を固定化してもよい。試料溶液中に含まれる測定対象物質が抗原の場合はシリコン薄膜上に抗体を固定化し、試料溶液中に含まれる測定対象物質が抗体の場合はシリコン薄膜上に抗原を固定化する。 An antibody or an antigen may be immobilized on a silicon thin film. When the measurement target substance contained in the sample solution is an antigen, the antibody is immobilized on the silicon thin film, and when the measurement target substance contained in the sample solution is an antibody, the antigen is immobilized on the silicon thin film.
　抗体は、ポリクローナル抗体でもよいし、モノクローナル抗体でもよい。 The antibody may be a polyclonal antibody or a monoclonal antibody.
　抗体は、例えば、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＹ等の免疫グロブリンのいずれのアイソタイプであってもよい。 The antibody may be any isotype of immunoglobulin such as IgG, IgM, IgA, IgD, IgE, IgY, for example.
　抗体はまた、全長抗体であってもよい。「全長抗体」とは、可変領域および定常領域を各々含む重鎖および軽鎖を含む抗体（例、２つのＦａｂ部分およびＦｃ部分を含む抗体）をいう。 The antibody may also be a full-length antibody. “Full length antibody” refers to an antibody comprising heavy and light chains, each comprising a variable region and a constant region (eg, an antibody comprising two Fab portions and an Fc portion).
　抗体はまた、上記全長抗体に由来する抗体断片であってもよい。抗体断片は、全長抗体の一部であり、例えば、Ｆ（ａｂ’）２、Ｆａｂ’、 Ｆａｂ、Ｆｖ等が例示される。 The antibody may also be an antibody fragment derived from the full-length antibody. The antibody fragment is a part of a full-length antibody, and examples thereof include F (ab ′) 2, Fab ′, Fab, and Fv.
　抗体はまた、単鎖抗体等の改変抗体であってもよい。 The antibody may also be a modified antibody such as a single chain antibody.
　単鎖抗体等の改変抗体としては、例えば、抗ＩｇＧ抗体、抗ＩｇＭ抗体、抗ＩｇＡ抗体、抗ＩｇＥ抗体、抗前立腺特異抗原（ＰＳＡ）抗体、抗α－フェトプロテイン（ＡＦＰ）抗体、抗ＧＡＤ抗体、抗ＨＢＶ抗体、抗ＨＣＶ抗体、抗ＨＴＬＶ－Ｉ抗体、ＨＩＶ抗体、結核抗体、マイコプラズマ抗体、抗アレルギー物質抗体等が例示される。 Examples of modified antibodies such as single chain antibodies include anti-IgG antibodies, anti-IgM antibodies, anti-IgA antibodies, anti-IgE antibodies, anti-prostate specific antigen (PSA) antibodies, anti-α-fetoprotein (AFP) antibodies, anti-GAD antibodies, Examples include anti-HBV antibody, anti-HCV antibody, anti-HTLV-I antibody, HIV antibody, tuberculosis antibody, mycoplasma antibody, anti-allergen antibody and the like.
　抗原は、高分子物質であってもよいし、低分子物質であってもよい。「高分子物質」とは、分子量１，５００以上の化合物をいう。「低分子物質」とは、分子量１，５００未満の化合物をいう。 The antigen may be a high molecular substance or a low molecular substance. “Polymer substance” refers to a compound having a molecular weight of 1,500 or more. “Low molecular weight substance” refers to a compound having a molecular weight of less than 1,500.
　高分子物質としては、例えば、タンパク質や核酸等が例示される。 Examples of the polymer substance include proteins and nucleic acids.
　タンパク質としては、例えば、親和的結合の能力を有するタンパク質、凝集能を有するタンパク質等が例示される。 Examples of proteins include proteins having affinity binding ability and proteins having aggregation ability.
　親和的結合の能力を有するタンパク質としては、例えば、上記抗体、Ｇタンパク共役型レセプター等の細胞膜上レセプター、ＩｇＧ、ＩｇＭ、ＩｇＡ、ＩｇＥ等の免疫グロブリン、細胞膜上レセプターから切断された細胞外ドメインおよび核内レセプター等のリガンド依存性タンパク質、転写因子、核酸の保護または輸送タンパク質等の核酸結合タンパク質、アダプタータンパク質等のタンパク質複合体を形成するタンパク質、細胞間接着タンパク質等の細胞外マトリクスタンパク質、チロシンキナーゼ、セリン／スレオニンキナーゼ等の酵素、糖タンパク質等が例示される。 Examples of the protein having affinity binding ability include, for example, the above-mentioned antibodies, receptors on cell membranes such as G protein-coupled receptors, immunoglobulins such as IgG, IgM, IgA, and IgE, extracellular domains cleaved from receptors on cell membranes, and Ligand-dependent proteins such as nuclear receptors, transcription factors, nucleic acid binding proteins such as nucleic acid protection or transport proteins, proteins that form protein complexes such as adapter proteins, extracellular matrix proteins such as intercellular adhesion proteins, tyrosine kinases And enzymes such as serine / threonine kinase, glycoproteins and the like.
　凝集能を有するタンパク質としては、例えば、変性タンパク質、βアミロイド等の神経変性タンパク質等の病原性タンパク質等が例示される。 Examples of the protein having an aggregating ability include pathogenic proteins such as denatured proteins and neurodegenerative proteins such as β-amyloid.
　また、その他のタンパク質としては、例えば、アポ蛋白ＡＩ、アポ蛋白ＡＩＩ、アポ蛋白Ｂ、アポ蛋白Ｅ、リウマチファクター、Ｄ－ダイマー、グリコアルブミン、Ｔ３、Ｔ４、Ｃ－反応性蛋白（ＣＲＰ）、前立腺特異抗原（ＰＳＡ）、α－フェトプロテイン（ＡＦＰ）、ＤＵＰＡＮ－２、癌胎児性抗原（ＣＥＡ）、ＣＡ１９－９、ＣＡ－１２５、ＰＩＶＫＡ－ＩＩ（Protein induced by vitamin K absence-II）、副甲状腺ホルモン（ＰＴＨ）、ヒト絨毛性ゴナドトロピン（ｈＣＧ）、甲状腺刺激ホルモン（ＴＳＨ）、インスリン、Ｃ－ペプタイド、ペプシノーゲン、インフルエンザＡ型抗原、インフルエンザＢ型抗原、コロナウイルス抗原、ＨＢＶ抗原、ＨＣＶ抗原、ＨＴＬＶ－Ｉ抗原、グリコアルブミン、ヘモグロビンＡ１ｃ、アディポネクチン、シスタチンＣ、心房性ナトリウム利尿ペプチド（ＡＮＰ）、脳性ナトリウム利尿ペプチド（ＢＮＰ）、トロポニンＴ、トロポニンＩ、クレアチニンキナーゼ－ＭＢ（ＣＫ－ＭＢ）、ミオグロビン、Ｈ－ＦＡＢＰ（ヒト心臓由来脂肪酸結合蛋白）等が例示される。 Other proteins include, for example, apoprotein AI, apoprotein AII, apoprotein B, apoprotein E, rheumatoid factor, D-dimer, glycoalbumin, T3, T4, C-reactive protein (CRP), prostate Specific antigen (PSA), α-fetoprotein (AFP), DUPAN-2, carcinoembryonic antigen (CEA), CA19-9, CA-125, PIVKA-II (Protein induced by vitamin K absence-II), parathyroid hormone (PTH), human chorionic gonadotropin (hCG), thyroid stimulating hormone (TSH), insulin, C-peptide, pepsinogen, influenza A antigen, influenza B antigen, coronavirus antigen, HBV antigen, HCV antigen, HTLV-I Antigen, glycoalbumin, hemoglobin A1c, adiponectin , Cystatin C, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), troponin T, troponin I, creatinine kinase-MB (CK-MB), myoglobin, H-FABP (fatty acid binding protein derived from human heart) Etc. are exemplified.
　核酸としては、ＤＮＡ、ＲＮＡ、メトキシ化ＲＮＡ等が例示される。 Examples of nucleic acids include DNA, RNA, methoxylated RNA, and the like.
　低分子物質としては、リガンド、ホルモン、脂質、脂肪酸、ビタミン、オピオイド、カテコールアミン等の神経伝達物質、ヌクレオシド、ヌクレオチド、オリゴヌクレオチド、単糖、オリゴ糖、アミノ酸、およびオリゴペプチド、あるいは医薬、毒物、およびサイトカイン類、代謝産物等が例示される。これらの低分子物質は、天然物質であってもよいし、合成物質であってもよい。 Low molecular weight substances include ligands, hormones, lipids, fatty acids, vitamins, opioids, neurotransmitters such as catecholamines, nucleosides, nucleotides, oligonucleotides, monosaccharides, oligosaccharides, amino acids, and oligopeptides, or drugs, toxicants, and Examples include cytokines and metabolites. These low molecular weight substances may be natural substances or synthetic substances.
　低分子物質としては、例えば、エストロゲンやトリヨードサイロニン等のホルモン、酸化ＬＤＬ、糖化ＬＤＬ等のコレステロール、ビタミンＡやビタミンＤ等のビタミン類、ＤＯＮ、ＮＩＶ、Ｔ２等のカビ毒類、ビスフェノールＡ、ノニルフェノール、フタル酸ジブチル、ポリ塩素化ビフェニル（ＰＣＢ）類、ダイオキシン類、ｐ，ｐ’－ジクロロジフェニルトリクロロエタン、トリブチルスズ等の内分泌撹乱物質類等が例示される。 Examples of low molecular weight substances include hormones such as estrogen and triiodothyronine, cholesterol such as oxidized LDL and saccharified LDL, vitamins such as vitamin A and vitamin D, mold toxins such as DON, NIV and T2, and bisphenol A. And endocrine disrupting substances such as nonylphenol, dibutyl phthalate, polychlorinated biphenyls (PCBs), dioxins, p, p′-dichlorodiphenyltrichloroethane, and tributyltin.
　また、抗原は、アレルギー物質（アレルゲン）であってもよい。アレルギー物質としては、例えば、大腸菌等の菌類、卵、乳、小麦、そば、落花生等の食物アレルギー物質やコナヒョウダニやトヤヒョウダニ等のダニ類由来のアレルギー物質、花粉、カビ類の胞子等が例示される。 Further, the antigen may be an allergen (allergen). Examples of allergic substances include fungi such as Escherichia coli, food allergic substances such as eggs, milk, wheat, buckwheat, and peanuts, allergic substances derived from mites such as mite and cypress mites, pollen, mold spores, and the like. .
　膜型表面応力センサのシリコン薄膜に固定化する抗体または抗原は、同一種類であってもよいし、異なる種類の抗体または抗原を二種類以上用いてもよい。例えば、免疫測定によりウイルスの抗原を検出することで該ウイルスの感染を診断する場合、該ウイルスの抗原タンパク質の異なる部位で結合する複数種の抗体を混合したものをシリコン薄膜に固定化することができる。また、ウイルスに対する抗体を検出することで該ウイルスの感染を診断する場合、該ウイルス由来の異なる複数の抗原をそれぞれ混合したものをシリコン薄膜に固定化することができる。 The antibodies or antigens immobilized on the silicon thin film of the membrane surface stress sensor may be the same type, or two or more different types of antibodies or antigens may be used. For example, when diagnosing viral infection by detecting viral antigens by immunoassay, a mixture of multiple types of antibodies that bind at different sites of the viral antigen protein may be immobilized on a silicon thin film. it can. Moreover, when diagnosing the infection of the virus by detecting an antibody against the virus, a mixture of a plurality of different antigens derived from the virus can be immobilized on a silicon thin film.
　抗体または抗原は、シリコン薄膜の片面または両面に固定化されている。図２には、（ａ）（ｂ）光学式カンチレバー、（ｃ）（ｄ）ピエゾ抵抗型カンチレバー、（ｅ）（ｆ）本発明のピエゾ膜型表面応力センサの場合について、片面および両面の被膜について模式的に示している。図２（ｅ）（ｆ）に示すように、本発明の膜型表面応力センサにおいては、片面被覆と両面被覆のいずれにおいても、ピエゾ抵抗素子部に応力集中が認められ、図２（ｃ）（ｄ）のカンチレバー方式のセンサと比較して、両面被覆であっても効率良く検出対象の分子を検出することが可能である。ここで、片面被覆の膜型表面応力センサと両面被覆の膜型表面応力センサの感度を比較すると、片面被覆の方が高感度である（図３）。 Antibody or antigen is immobilized on one or both sides of the silicon thin film. 2A and 2B show coatings on one side and both sides for the case of (a) (b) optical cantilever, (c) (d) piezoresistive cantilever, and (e) (f) piezo film type surface stress sensor of the present invention. Is shown schematically. As shown in FIGS. 2 (e) and 2 (f), in the film type surface stress sensor of the present invention, stress concentration is recognized in the piezoresistive element portion in both the single-sided coating and the double-sided coating, and FIG. Compared with the cantilever type sensor of (d), it is possible to detect a molecule to be detected efficiently even with double-sided coating. Here, when comparing the sensitivity of the single-sided film-type surface stress sensor and the double-sided film-type surface stress sensor, the single-sided coating is more sensitive (FIG. 3).
　また、従来のカンチレバーを使用したセンサでは、図２（ｂ）の両面被覆の光学的カンチレバー、図２（ｃ）（ｄ）の片面および両面被覆のピエゾ抵抗型カンチレバーの構成では、実際にはほとんど検出出力が得られない。さらに、図２（ａ）の片面被覆の光学式カンチレバーにおいては、検出出力が得られるものの、試料溶液が光を透過しない場合には使用できず、試料溶液が流動している場合には、測定が安定しないため使用しづらい、という問題がある。また、図２（ａ）では、試料溶液の濃度変化に伴う屈折率変化により、測定結果が影響を受ける可能性が示唆される。 In addition, in the sensor using the conventional cantilever, the optical cantilever with double-sided coating shown in FIG. 2 (b) and the piezoresistive cantilever with single-sided and double-sided coating shown in FIGS. Detection output cannot be obtained. In addition, the single-side coated optical cantilever shown in FIG. 2 (a) can produce a detection output, but cannot be used when the sample solution does not transmit light, and is measured when the sample solution is flowing. Is not stable and difficult to use. Further, FIG. 2A suggests that the measurement result may be affected by the change in the refractive index accompanying the change in the concentration of the sample solution.
　本発明では、図４に示したように、膜型表面応力センサが半導体基板上に１つ以上配置されたマルチチャネルのセンサチップを構成することができる。この場合には、それぞれの膜型表面応力センサのシリコン薄膜に固定化する抗体または抗原が、同一種類であってもよいし、膜型表面応力センサごとに異なる種類の抗体または抗原を用いてもよい。あるいはひとつの膜型表面応力センサに異なる種類の抗体または抗原を二種類以上用いてもよい。図４（ａ）は、本発明の膜型表面応力センサを複数備えたセンサチップの一実施形態であり、センサ部分が二次元配列となっている新しい構成のＭＳＳチップを示している。図４（ｂ）は本発明のセンサの拡大図を示している。そして、図４（ｃ）は、本発明のセンサを測定に用いた場合のフロー法による測定検出のシステム構成を示している。なお、本発明のセンサを用いた測定方法としては、浸漬法を用いてもよく、必ずしも図４（ｃ）の装置構成をとる必要はない。 In the present invention, as shown in FIG. 4, a multi-channel sensor chip in which one or more film-type surface stress sensors are arranged on a semiconductor substrate can be configured. In this case, the antibodies or antigens immobilized on the silicon thin film of each membrane type surface stress sensor may be the same type, or different types of antibodies or antigens may be used for each type of membrane type surface stress sensor. Good. Alternatively, two or more different types of antibodies or antigens may be used in one membrane-type surface stress sensor. FIG. 4A shows an embodiment of a sensor chip provided with a plurality of membrane type surface stress sensors according to the present invention, and shows a new configuration MSS chip in which the sensor portion is a two-dimensional array. FIG. 4B shows an enlarged view of the sensor of the present invention. FIG. 4C shows a system configuration of measurement detection by the flow method when the sensor of the present invention is used for measurement. In addition, as a measuring method using the sensor of the present invention, an immersion method may be used, and the apparatus configuration of FIG.
　シリコン薄膜の片面または両面に抗体または抗原を固定化する方法としては、インクジェットスポッティング法、浸漬法のいずれかであることが好ましい。 As a method for immobilizing an antibody or an antigen on one side or both sides of a silicon thin film, either an ink jet spotting method or an immersion method is preferable.
　例えば、インクジェットスポッティング法を用いて、シリコン薄膜の片面のみに抗体または抗原を固定化することができる。インクジェットスポッティング法では、マルチチャネルのセンサチップ上に配置された膜型表面応力センサのシリコン薄膜ごとに異なる種類の抗体または抗原を固定化することが容易である。このため、一枚の半導体基板上に複数種の抗原または抗体と結合可能な抗体または抗原を備えたマルチチャンネルの膜型表面応力センサを製造することが可能となる。 For example, an antibody or an antigen can be immobilized only on one side of a silicon thin film using an inkjet spotting method. In the ink jet spotting method, it is easy to immobilize different types of antibodies or antigens for each silicon thin film of a film-type surface stress sensor arranged on a multi-channel sensor chip. For this reason, it becomes possible to manufacture a multi-channel film-type surface stress sensor provided with antibodies or antigens that can bind to a plurality of types of antigens or antibodies on a single semiconductor substrate.
　一方、浸漬法によってシリコン薄膜の両面に一種類の抗体または抗原を固定化する場合は、インクジェットスポッティング法と比較して、シリコン薄膜の片面に抗体または抗原を固定化した後、センサチップを反転させる等の操作を必要としないので、図５に示したように製造工程が簡略化される。特に、浸漬法では、複数のチップを同一の溶液に同時に浸漬することが可能であるため、高度な生産設備を必要としない。このため、浸漬法では、１チップ１チャンネルの膜型表面応力センサを大量かつ安価に製造することが可能となる。具体的には、両面被覆の場合には、高度かつ大規模な生産設備を使用せず、単にシリコン薄膜を手動あるいは簡単な装置で抗体または抗原溶液に浸漬するだけで所要の膜型表面応力センサを製造することができる。しかも、後述の実施例においても説明しているが、シリコン薄膜を化学物質の溶液に浸漬した際に観測される出力電圧のシグナルの有無やシグナル変化の挙動をモニタリングし、フィードバックすることによって、製造の各処理工程において処理が成功していることを確かめることが可能である。このため、大規模な感染症が発生した際など、必要とされるセンサの量産体制を迅速に立ち上げることができ、また、動作が保証されたセンサを高い効率で生産できるので、信頼性の高い診断を安価に行うことを可能にするという顕著な効果を奏する。 On the other hand, when one kind of antibody or antigen is immobilized on both sides of the silicon thin film by the immersion method, the sensor chip is inverted after immobilizing the antibody or antigen on one side of the silicon thin film as compared with the ink jet spotting method. Thus, the manufacturing process is simplified as shown in FIG. In particular, in the dipping method, since a plurality of chips can be dipped in the same solution at the same time, an advanced production facility is not required. For this reason, the immersion method makes it possible to manufacture a one-chip, one-channel film-type surface stress sensor in large quantities and at low cost. Specifically, in the case of double-sided coating, the required membrane-type surface stress sensor can be obtained by simply immersing a silicon thin film in an antibody or antigen solution manually or with a simple device without using an advanced and large-scale production facility. Can be manufactured. Moreover, as described in the examples below, it is possible to manufacture by monitoring the presence or absence of a signal of the output voltage observed when the silicon thin film is immersed in the chemical solution and the behavior of the signal change, and feeding back. It is possible to confirm that the processing is successful in each processing step. Therefore, when a large-scale infectious disease occurs, the required mass production system for the sensor can be set up quickly, and a sensor with guaranteed operation can be produced with high efficiency. There is a remarkable effect of making it possible to make a high diagnosis inexpensively.
　センサチップには、抗原または抗体の結合に起因する信号を読み取るための電極等が組み込まれている。そのため、浸漬法によってシリコン薄膜の両面被覆を行う際には、電極等が抗体または抗原溶液等に接触しないよう保護する必要がある。図６および図７に示すような、センサチップの電極等の特定部分が液体状試料に接触しない状態を保ちながら、センサ素子部分のみを抗体溶液および試料溶液等に浸すことができるセンサチップホルダー（Ｈ）を用いることも好ましい。 The sensor chip incorporates an electrode for reading a signal resulting from the binding of an antigen or antibody. Therefore, when the silicon thin film is coated on both sides by the dipping method, it is necessary to protect the electrodes and the like from coming into contact with the antibody or the antigen solution. A sensor chip holder (see FIG. 6 and FIG. 7) that can immerse only the sensor element portion in an antibody solution, a sample solution, etc. while maintaining a state where a specific portion such as an electrode of the sensor chip does not contact the liquid sample. It is also preferred to use H).
　このようなセンサチップホルダー（Ｈ）は、単にセンサチップを固定するための構成であってもよいし、センサチップをセンサチップホルダー（Ｈ）外部に電気的に結合する導線を接続できるコネクターを備えていてもよい。
膜型表面応力センサの製造方法は、少なくとも
＜１＞ピエゾ抵抗素子を含む複数の支持体により支持されたシリコン薄膜の表面を洗浄する工程；
＜２＞洗浄したシリコン薄膜の表面に、自己組織化膜を形成する化合物を接触させる工程；
＜３＞表面に自己組織化膜が形成されたシリコン薄膜に、抗体または抗原溶液を接触させる工程；
＜４＞上記＜１＞～＜３＞の各工程において、膜型表面応力センサに電圧を印加して、出力電圧の変化をモニタリングする工程；
を含むことを特徴としている。上記＜１＞～＜３＞の各工程は、いずれもシリコン薄膜に、以下に詳述する化学物質の溶液を接触させることで行われる。例えば、以下に浸漬法による両面被覆のセンサの製造方法を説明するが、インクジェットスポッティング法による片面被覆のセンサの製造方法も可能である。 Such a sensor chip holder (H) may be configured to simply fix the sensor chip, or includes a connector that can connect a conductive wire that electrically couples the sensor chip to the outside of the sensor chip holder (H). It may be.
The method of manufacturing a film-type surface stress sensor includes a step of cleaning the surface of a silicon thin film supported by a plurality of supports including at least <1> piezoresistive elements;
<2> a step of bringing a compound that forms a self-assembled film into contact with the surface of the cleaned silicon thin film;
<3> a step of bringing an antibody or antigen solution into contact with a silicon thin film having a self-assembled film formed on the surface;
<4> In each of the above steps <1> to <3>, a voltage is applied to the film-type surface stress sensor and a change in the output voltage is monitored;
It is characterized by including. Each of the above steps <1> to <3> is performed by bringing a chemical solution described below in detail into contact with the silicon thin film. For example, a method of manufacturing a double-sided sensor by the dipping method will be described below, but a method of manufacturing a single-sided sensor by the inkjet spotting method is also possible.
　第１工程として、前記ピエゾ抵抗素子を含む複数の支持体により支持されたシリコン薄膜の表面を洗浄する。シリコン薄膜の表面の洗浄方法としては、溶媒を用いる方法やプラズマ洗浄などの方法が例示される。シリコン薄膜の表面の洗浄に用いられる溶媒としては、例えば、アセトン、２－プロパノール、超純水などが例示される。シリコン薄膜の表面の洗浄は、２種類以上の溶媒を用いて、溶媒を変更しつつ複数回洗浄することが好ましい。 As a first step, the surface of the silicon thin film supported by a plurality of supports including the piezoresistive element is cleaned. Examples of the method for cleaning the surface of the silicon thin film include a method using a solvent and a method such as plasma cleaning. Examples of the solvent used for cleaning the surface of the silicon thin film include acetone, 2-propanol, and ultrapure water. The cleaning of the surface of the silicon thin film is preferably performed a plurality of times using two or more kinds of solvents while changing the solvent.
　つづいて、第２工程として、洗浄したシリコン薄膜の表面に、自己組織化膜を形成する化合物を接触させる。シリコン薄膜の表面に自己組織化膜を形成する化合物としては、例えば、Glyphosineや10-CDPAなどのホスホン酸化合物が例示されるが、抗体を変性、失活させることなく保持できるような化合物であって、自己組織化膜を形成することができるものであれば、特に限定されない。 Subsequently, as a second step, a compound that forms a self-assembled film is brought into contact with the surface of the cleaned silicon thin film. Examples of the compound that forms a self-assembled film on the surface of the silicon thin film include phosphonic acid compounds such as Glyphosine and 10-CDPA, but are compounds that can retain the antibody without denaturing or inactivating it. As long as a self-assembled film can be formed, there is no particular limitation.
　また、第２工程において、抗体または抗原の固定化のためのシリコン薄膜の表面修飾を行うことが好ましい。シリコン薄膜の表面修飾としては、例えば、シリコン薄膜の表面に被覆した自己組織化膜に含まれるカルボキシル基の活性エステルへの変換等が例示される。例えばＮ，Ｎ’－ジメチルカルボジイミド（ＤＩＣ）、１－エチル－３－（３ジメチルアミノプロピル）カルボジイミド・ヒドロクロライド（ＥＤＣ）等のカルボジイミドの存在下でＮ－ヒドロキシスクシンイミド（ＮＨＳ）、Ｎ－ヒドロキシスルホスクシンイミド等のスクシンイミドで処理することによって行うことができる。前記カルボジイミドの存在下での前記スクシンイミドの処理は、具体的には、前記カルボジイミド及び前記スクシンイミドをそれぞれＮ，Ｎ－ジメチルホルムアミド（ＤＭＦ）などの溶媒に溶解させた混合溶液に１～数時間程度浸漬することによって行うことができる。 In the second step, it is preferable to modify the surface of the silicon thin film for immobilizing the antibody or antigen. Examples of the surface modification of the silicon thin film include conversion of a carboxyl group contained in the self-assembled film coated on the surface of the silicon thin film into an active ester. For example, N-hydroxysuccinimide (NHS), N-hydroxysulfo in the presence of carbodiimide such as N, N′-dimethylcarbodiimide (DIC), 1-ethyl-3- (3dimethylaminopropyl) carbodiimide hydrochloride (EDC), etc. It can be performed by treating with succinimide such as succinimide. The treatment of the succinimide in the presence of the carbodiimide is specifically performed by immersing the carbodiimide and the succinimide in a mixed solution in which each of them is dissolved in a solvent such as N, N-dimethylformamide (DMF) for about 1 to several hours. Can be done.
　第３工程として、シリコン薄膜の表面に抗体または抗原を接触させる。シリコン薄膜の表面に抗体または抗原を接触させる工程は、抗体または抗原をＭＥＳバッファーや炭酸バッファー等の緩衝液に溶解し、適当な濃度に希釈したものを、上記表面修飾工程を経たシリコン薄膜に添加し、３６℃で１時間もしくは４℃で１晩静置し、シリコン薄膜と抗体または抗原を接触させる。つづいて、リン酸緩衝液（ＰＢＳ）やトリス緩衝液（ＴＢＳ）などの洗浄液で、第３工程における余分な成分（例えば、未結合の抗体または抗原等）を洗浄する。なお、洗浄液には、ブロッキング工程では、スキムミルクや牛血清アルブミン（ＢＳＡ）などを含有させてもよい。 In the third step, an antibody or antigen is brought into contact with the surface of the silicon thin film. In the step of contacting the antibody or antigen with the surface of the silicon thin film, the antibody or antigen is dissolved in a buffer solution such as MES buffer or carbonate buffer, and diluted to an appropriate concentration and added to the silicon thin film that has undergone the surface modification step. And allowed to stand at 36 ° C. for 1 hour or at 4 ° C. overnight to bring the silicon thin film into contact with the antibody or antigen. Subsequently, excess components (for example, unbound antibody or antigen) in the third step are washed with a washing solution such as phosphate buffer (PBS) or Tris buffer (TBS). Note that the cleaning liquid may contain skim milk, bovine serum albumin (BSA), or the like in the blocking step.
　第４工程として、上記第１～第３工程において、膜型表面応力センサに電圧を印加して、出力電圧の変化をモニタリングする。シリコン薄膜を上記の化学物質の溶液に接触させた際に観測される出力電圧のシグナルの有無やシグナル変化の挙動をモニタリングし、フィードバックすることによって、製造の各工程において作業が成功していることを確かめることが可能である。このため、評価基準を満たさないセンサは、その時点で生産ラインから取り除くシステムを構築することができる。 As the fourth step, in the first to third steps, a voltage is applied to the film type surface stress sensor to monitor the change in the output voltage. Successful work in each manufacturing process by monitoring and feeding back the output voltage signal behavior and signal change behavior observed when the silicon thin film is in contact with the above chemical solution. Can be confirmed. For this reason, it is possible to construct a system in which sensors that do not satisfy the evaluation criteria are removed from the production line at that time.
　これまで用いられてきたイムノセンサの性能評価は、抗原または抗体処理を行うしかなく、性能評価に用いたイムノセンサは利用できなくなっていた。このため、製造したイムノセンサの全数検査は不可能であった。しかしながら、本発明において、各工程で処理が成功していることを確認しながらブロッキング工程まで終了したセンサは、全製造工程が確実に成功していることが保証されており、製造したセンサの全数検査と同等の効果を得ることができる。 The performance evaluation of immunosensors that have been used so far involves only antigen or antibody treatment, and the immunosensor used for performance evaluation cannot be used. For this reason, 100% inspection of the manufactured immunosensors was impossible. However, in the present invention, it is guaranteed that all the manufacturing processes have been successfully completed for the sensors that have been processed up to the blocking process while confirming that the processing has been successful in each process. The same effect as the inspection can be obtained.
　したがって、医療や診断分野において高品質で、極めて信頼性の高い検査用キット等を提供することが可能となる。 Therefore, it is possible to provide high-quality and highly reliable test kits in the medical and diagnostic fields.
　また、本発明によれば、イムノセンサの製造工程において特定の不良品を見つけだすことが可能となるため、抜き取り検査を行い、不良品を見つけると同一ロットのイムノセンサをすべて廃棄していた従来の品質管理手法に比べ、製造コストを低減させることにもつながる。 In addition, according to the present invention, since it becomes possible to find a specific defective product in the manufacturing process of the immunosensor, when the sampling inspection is performed and the defective product is found, all of the immunosensors in the same lot are discarded. Compared to quality control methods, this also leads to a reduction in manufacturing costs.
　本発明の膜型表面応力センサを用いた免疫測定方法は、少なくとも以下の工程
＜１＞抗体または抗原が固定化された膜型表面応力センサのシリコン薄膜に、試料溶液を接触させる工程；
＜２＞前記膜型表面応力センサに電圧を印加し、電気シグナルを測定する工程；
を含むことを特徴としている。 The immunoassay method using the membrane-type surface stress sensor of the present invention comprises at least the following step <1> a step of bringing a sample solution into contact with the silicon thin film of the membrane-type surface stress sensor on which an antibody or antigen is immobilized;
<2> a step of applying a voltage to the film-type surface stress sensor and measuring an electrical signal;
It is characterized by including.
　第１工程として、前記抗体または抗原が固定化された膜型表面応力センサのシリコン薄膜に、例えば、ヒトやサル、マウス、ラット、ヒツジ、ウマ等の生体から公知の方法で試料溶液（例えば、血液）を採取し、必要に応じて前処理を行って希釈調製した試料溶液を接触させることで、膜型表面応力センサの表面に固定された抗体または抗原と、試料溶液中の抗原または抗体とを結合させる。もちろん、試料溶液の由来は、上記生体由来である天然試料に限定されるものではない。
前記抗体または抗原が固定化された膜型表面応力センサのシリコン薄膜に、試料溶液を接触させる工程は、当該膜型表面応力センサを試料溶液に浸漬して行うことが好ましい。 As a first step, a sample solution (eg, for example, from a living body such as a human, monkey, mouse, rat, sheep, horse, etc.) is applied to a silicon thin film of a membrane-type surface stress sensor on which the antibody or antigen is immobilized. Blood) is collected, and the sample solution diluted by pretreatment as necessary is brought into contact with the antibody or antigen immobilized on the surface of the membrane surface stress sensor, and the antigen or antibody in the sample solution Are combined. Of course, the origin of the sample solution is not limited to the natural sample derived from the living body.
The step of bringing the sample solution into contact with the silicon thin film of the membrane surface stress sensor to which the antibody or antigen is immobilized is preferably performed by immersing the membrane surface stress sensor in the sample solution.
　第２工程として、上記第１工程の前後において膜型表面応力センサに電圧を印加し、電気シグナルを測定する。シリコン薄膜を上記の試料溶液に接触させた際に観測される出力電圧のシグナルの有無やシグナル変化の挙動を測定することによって、抗原抗体反応を検出することが可能となる。具体的には、シリコン薄膜は、抗原－抗体結合による表面応力を受けて撓み等の変形が生じる。シリコン薄膜を支持するそれぞれのピエゾ抵抗素子には、シリコン薄膜の変形量に応じた応力が発生し、この応力に比例してピエゾ抵抗素子の抵抗値が変化する。本発明の免疫測定法では、例えば、４つのピエゾ抵抗素子によって構成されるホイートストンブリッジに電圧を印加することで、上記の抵抗値の変化によって、応力に比例した出力電圧が得られ、試料溶液中の抗原または抗体の有無および抗原または抗体量（濃度）を定量することが可能となる。 As a second step, a voltage is applied to the membrane type surface stress sensor before and after the first step, and an electric signal is measured. It is possible to detect an antigen-antibody reaction by measuring the presence or absence of a signal of the output voltage observed when the silicon thin film is brought into contact with the sample solution and the behavior of the signal change. Specifically, the silicon thin film undergoes deformation such as bending due to surface stress due to the antigen-antibody bond. In each piezoresistive element that supports the silicon thin film, a stress corresponding to the deformation amount of the silicon thin film is generated, and the resistance value of the piezoresistive element changes in proportion to the stress. In the immunoassay method of the present invention, for example, by applying a voltage to a Wheatstone bridge composed of four piezoresistive elements, an output voltage proportional to the stress can be obtained by the change in the resistance value described above, and in the sample solution It is possible to quantify the presence or absence of the antigen or antibody and the amount (concentration) of the antigen or antibody.
　したがって、本発明は、このような測定原理に基づくため、医療や診断分野において、従来の免疫測定法と比較して高感度な免疫測定法を提供することが可能となる。 Therefore, since the present invention is based on such a measurement principle, it is possible to provide a highly sensitive immunoassay in the medical and diagnostic fields as compared with a conventional immunoassay.
　以下、本発明について実施例及び比較例を用いて詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail using examples and comparative examples, but the present invention is not limited to these examples.
　（実施例１）
　図１に例示したように、シリコン薄膜がピエゾ抵抗素子を含む支持体によって４点支持され、抗体が結合される前のセンサ構成体のシリコン薄膜をアセトン、２-プロパノール、超純水（ＭｉｌｌｉＱ水；ＭＱ水）の順に溶媒中に浸漬し，最後にプラズマ洗浄を行うことで，シリコン薄膜を洗浄した。 (Example 1)
As illustrated in FIG. 1, the silicon thin film is supported at four points by a support including a piezoresistive element, and the silicon thin film of the sensor structure before the antibody is bonded is acetone, 2-propanol, ultrapure water (MilliQ water). ; Water in the order of MQ water), and finally the silicon thin film was cleaned by performing plasma cleaning.
　次に，洗浄したシリコン薄膜を１ｍＭのホスホン酸溶液（Glyphosine／ＭＱ水）に浸漬し、室温で６０分間静置した。ホスホン酸に浸漬したシリコン薄膜をＭＱ水、エタノールを用いて洗浄し、乾燥後にソケットから外し、１４０℃、１時間アニーリングすることによって、シリコン薄膜の表面をホスホン酸で修飾した。 Next, the cleaned silicon thin film was immersed in a 1 mM phosphonic acid solution (Glyphosine / MQ water) and allowed to stand at room temperature for 60 minutes. The silicon thin film immersed in phosphonic acid was washed with MQ water and ethanol, removed from the socket after drying, and annealed at 140 ° C. for 1 hour to modify the surface of the silicon thin film with phosphonic acid.
　その後、５ｍＭのＮ-ヒドロキシスクシンイミド（ＮＨＳ）と５ｍＭの１-エチル３-（３ジメチルアミノプロピル）カルボジイミドヒドロクロライド（ＥＤＣ）／Ｎ，Ｎ-ジメチルホルムアミド（ＤＭＦ）溶液の混合溶液にホスホン酸修飾したシリコン薄膜を浸漬し、室温で６０分間静置した。これをＤＭＥ、ＭＥＳバッファーの順に溶媒を用いて洗浄した。 Subsequently, phosphonic acid modification was performed on a mixed solution of 5 mM N-hydroxysuccinimide (NHS) and 5 mM 1-ethyl 3- (3 dimethylaminopropyl) carbodiimide hydrochloride (EDC) / N, N-dimethylformamide (DMF) solution. The silicon thin film was immersed and allowed to stand for 60 minutes at room temperature. This was washed with a solvent in the order of DME and MES buffer.
　ＥＤＣ／ＮＨＳ溶液（ＤＭＦ）処理したシリコン薄膜をセンサチップホルダーに接続し、９６穴プレートの各ウェルに分注した抗ＡＦＰ（α－フェトプロテイン）抗体溶液（ＭＥＳバッファー、ｐＨ５．５）に、センサチップホルダーに接続したシリコン薄膜を浸漬し、４℃で１晩静置して抗ＡＦＰ抗体と結合させた。抗ＡＦＰ抗体を結合させたシリコン薄膜を、界面活性剤としてＴｒｉｔｏｎＸ－１００を含む洗浄バッファーで洗浄し、３７℃で６０分間静置してマスキングした。これをマスキングバッファーで洗浄することによって、センサチップ上に抗ＡＦＰ抗体を固定化することができた。また、コントロールとして、抗ＡＦＰ抗体溶液（ＭＥＳバッファー、ｐＨ５．５）の代わりに、抗ＡＦＰ抗体を含まないＭＥＳバッファーに浸漬したシリコン薄膜を用いた。 The silicon thin film treated with EDC / NHS solution (DMF) was connected to the sensor chip holder, and the sensor chip was added to the anti-AFP (α-fetoprotein) antibody solution (MES buffer, pH 5.5) dispensed to each well of the 96-well plate. The silicon thin film connected to the holder was immersed and allowed to stand overnight at 4 ° C. to bind to the anti-AFP antibody. The silicon thin film to which the anti-AFP antibody was bound was washed with a washing buffer containing Triton X-100 as a surfactant, and allowed to stand at 37 ° C. for 60 minutes for masking. By washing this with a masking buffer, an anti-AFP antibody could be immobilized on the sensor chip. As a control, a silicon thin film immersed in a MES buffer not containing an anti-AFP antibody was used instead of the anti-AFP antibody solution (MES buffer, pH 5.5).
　このようにして得られた膜型表面応力センサ（抗体結合センサ）とコントロールのセンサを、センサチップホルダーに接続し、９６穴プレートの各ウェルに分注した２０００ｎｇ／ｍＬ（約２８．５μＭ）のＡＦＰ溶液中にセンサを浸漬した。 The membrane type surface stress sensor (antibody binding sensor) and the control sensor thus obtained were connected to a sensor chip holder, and 2000 ng / mL (about 28.5 μM) dispensed into each well of a 96-well plate. The sensor was immersed in the AFP solution.
　結果を図８に示す。図８では、抗体結合センサをＡＦＰ溶液に浸漬することで、出力電圧が降下した。一方、コントロールのセンサでは、出力電圧に変化が認められなかった。計測されたＡＦＰ検出のシグナルの出力電圧は、単位バイアス電圧あたりの出力差が３ｍＶであった。この結果を、非特許文献１に記載のピエゾ抵抗カンチレバーアレイセンサでＰＳＡ（前立腺特定的抗原）を検出した際に計測されたシグナルの出力電圧と、ホイートストンブリッジに印加されたバイアス電圧に注意して、単位バイアス電圧あたりで比較した。その結果、非特許文献１に記載のピエゾ抵抗カンチレバーアレイセンサのシグナルの出力電圧は、単位バイアス電圧あたり７．５μＶであって、本発明の膜型表面応力センサは、非特許文献１に記載のピエゾ抵抗カンチレバーアレイセンサと比較して、感度が約４０倍に達することが認められた。また、検出可能な低濃度の抗原濃度を比較すると、非特許文献１に記載のピエゾ抵抗カンチレバーアレイセンサでは、０．３ｎＭであるのに対し、本発明の膜型表面応力センサでは、理論上７．５ｐＭの抗原を検出可能であることが明らかになった。 The results are shown in FIG. In FIG. 8, the output voltage dropped by immersing the antibody binding sensor in the AFP solution. On the other hand, in the control sensor, no change was observed in the output voltage. The measured output voltage of the AFP detection signal had an output difference per unit bias voltage of 3 mV. This result is obtained by paying attention to the output voltage of the signal measured when the PSA (prostate specific antigen) is detected by the piezoresistive cantilever array sensor described in Non-Patent Document 1, and the bias voltage applied to the Wheatstone bridge. Comparison was made per unit bias voltage. As a result, the output voltage of the signal of the piezoresistive cantilever array sensor described in Non-Patent Document 1 is 7.5 μV per unit bias voltage, and the membrane type surface stress sensor of the present invention is described in Non-Patent Document 1. It was observed that the sensitivity reached about 40 times compared to the piezoresistive cantilever array sensor. Further, when comparing the detectable low antigen concentration, the piezoresistive cantilever array sensor described in Non-Patent Document 1 has a value of 0.3 nM, whereas the membrane-type surface stress sensor of the present invention theoretically has 7 It was found that 5 pM of antigen could be detected.
　また、アルカリフォスファターゼ（ＡＬＰ）で標識した抗マウスＩｇＧ抗体を用いて、シリコン薄膜に固定化したＡＦＰを発光検出したところ、１００μｇ／ｍＬの抗ＡＦＰ抗体を固定化したセンサでは、顕著な発光が確認された。一方、コントロールのセンサでは発光が認められなかった。したがって、本発明の膜型表面応力センサが、ＡＦＰを特異的に結合していることを確認できた。 In addition, when anti-mouse IgG antibody labeled with alkaline phosphatase (ALP) was used to detect luminescence of AFP immobilized on a silicon thin film, significant luminescence was confirmed with a sensor immobilized with 100 μg / mL anti-AFP antibody. It was done. On the other hand, no luminescence was observed in the control sensor. Therefore, it was confirmed that the membrane type surface stress sensor of the present invention specifically binds AFP.
　（実施例２）
　膜型表面応力センサの製造工程のシリコン薄膜の表面処理工程で、ホスホン酸溶液処理中のシリコン薄膜のシグナルを測定した。その結果を図９に示した。この図９では、シグナルのパターンＡ、Ｂ、Ｃが示されている。なお、図９におけるＣｈ１～Ｃｈ４（チャネル４）は、一つのセンサチップに配置した４つのセンサ各々の検出電圧を示している。検出電圧相互の若干の差異は、シリコン薄膜製造時のロット差や固定化した抗体量の差等によるものと考えられる。 (Example 2)
The signal of the silicon thin film during the phosphonic acid solution treatment was measured in the surface treatment process of the silicon thin film in the production process of the film type surface stress sensor. The results are shown in FIG. In FIG. 9, signal patterns A, B, and C are shown. Note that Ch1 to Ch4 (channel 4) in FIG. 9 indicate detection voltages of four sensors arranged in one sensor chip. A slight difference between detection voltages is considered to be due to a lot difference at the time of manufacturing a silicon thin film, a difference in the amount of immobilized antibody, and the like.
　シグナルのパターンについて説明すると、まず、シリコン薄膜をアセトン、２-プロパノール、超純水（ＭｉｌｌｉＱ水；ＭＱ水）の順に溶媒中に浸漬し，最後にプラズマ洗浄を行うことで，シリコン薄膜を洗浄した。このプラズマ洗浄後のセンサをＭＱ水に浸漬した際のシグナルが、Ａのパターンである。続いて、このシリコン薄膜をＭＱ水からＭＱ水に移動した際のシグナルが、Ｂのパターンである。そして、シリコン薄膜をＭＱ水からホスホン酸溶液に移動した際のシグナルが、Ｃのパターンである。 The signal pattern will be explained. First, the silicon thin film was cleaned by immersing the silicon thin film in a solvent in the order of acetone, 2-propanol and ultrapure water (MilliQ water; MQ water), and finally performing plasma cleaning. . The signal when the sensor after this plasma cleaning is immersed in MQ water is the pattern of A. Subsequently, a signal when the silicon thin film is moved from MQ water to MQ water is a pattern B. The signal when the silicon thin film is transferred from the MQ water to the phosphonic acid solution is a C pattern.
　図９に示すとおり、Ｃのシグナルは、ホスホン酸溶液にシリコン薄膜を浸漬した直後に電圧が変化し始め、約１０００秒経過後に電圧の変化が落ち着いた。この電圧の変化は、シリコン薄膜表面にホスホン酸が結合している様子を捉えていると考えられる。 As shown in FIG. 9, the voltage of the C signal started to change immediately after the silicon thin film was immersed in the phosphonic acid solution, and the change in voltage settled after about 1000 seconds. This change in voltage is considered to capture the state in which phosphonic acid is bonded to the surface of the silicon thin film.
　膜型表面応力センサの製造工程のシリコン薄膜の表面処理工程で、シリコン薄膜のプラズマ洗浄後、ホスホン酸溶液に浸漬するまでの時間を変化させて、ホスホン酸溶液処理中のシリコン薄膜のシグナルを測定した（図１０、図１１）。まず、シリコン薄膜をアセトン、２-プロパノール、超純水（ＭｉｌｌｉＱ水；ＭＱ水）の順に溶媒中に浸漬し、最後にプラズマ洗浄を行った。プラズマ洗浄後５分以内に、シリコン薄膜をホスホン酸溶液に浸漬したときのシグナルを図１０に示す。また、プラズマ洗浄後３０分経過後に、シリコン薄膜をホスホン酸溶液に浸漬したときのシグナルを図１１に示す。 Measure the silicon thin film signal during phosphonic acid solution treatment by changing the time from plasma cleaning of the silicon thin film to dipping in the phosphonic acid solution in the surface treatment process of the silicon thin film in the manufacturing process of the membrane type surface stress sensor (FIGS. 10 and 11). First, the silicon thin film was immersed in a solvent in the order of acetone, 2-propanol, and ultrapure water (MilliQ water; MQ water), and finally plasma cleaning was performed. The signal when the silicon thin film is immersed in the phosphonic acid solution within 5 minutes after the plasma cleaning is shown in FIG. FIG. 11 shows a signal when the silicon thin film is immersed in the phosphonic acid solution after 30 minutes from the plasma cleaning.
　プラズマ洗浄後５分以内に、シリコン薄膜をホスホン酸溶液に浸漬したときのシグナルは、出力電圧が約７００μＶであったのに対し、プラズマ洗浄後３０分経過後に、シリコン薄膜をホスホン酸溶液に浸漬したときのシグナルは、出力電圧が約２００μＶと大幅に低下した。このことから、プラズマ洗浄後、短時間のうちにホスホン酸溶液処理を行うことによって、表面活性が高い状態のシリコン薄膜を使用した処理を行うことができるが、洗浄後の放置時間が長いと、シリコン薄膜の表面活性が低下することが示唆された。 The signal when the silicon thin film was immersed in the phosphonic acid solution within 5 minutes after the plasma cleaning was an output voltage of about 700 μV, whereas the silicon thin film was immersed in the phosphonic acid solution 30 minutes after the plasma cleaning. In this case, the output voltage was greatly reduced to about 200 μV. From this, by performing the phosphonic acid solution treatment in a short time after the plasma cleaning, it is possible to perform a treatment using a silicon thin film having a high surface activity, but if the standing time after the cleaning is long, It was suggested that the surface activity of the silicon thin film decreases.
　したがって、シリコン薄膜表面のホスホン酸修飾レベルと電圧変化の間に相関性を見出すことができると考えられる。 Therefore, it is considered that a correlation can be found between the phosphonic acid modification level on the silicon thin film surface and the voltage change.
　（実施例４）
　膜型表面応力センサのシリコン薄膜表面に抗体を結合する工程で、抗ＡＦＰ抗体溶液処理中のシグナルを測定した（図１２）。まず、ＥＤＣ／ＮＨＳ溶液（ＤＭＦ）処理したシリコン薄膜をＭＥＳバッファーに浸漬し、続いて、このシリコン薄膜をＭＥＳバッファーからＭＥＳバッファーに移動した。そして、シリコン薄膜をＭＥＳバッファーから抗ＡＦＰ抗体溶液に移動し、この間のシグナルを計測し続けた。 Example 4
In the step of binding the antibody to the silicon thin film surface of the membrane type surface stress sensor, the signal during the treatment with the anti-AFP antibody solution was measured (FIG. 12). First, a silicon thin film treated with an EDC / NHS solution (DMF) was immersed in a MES buffer, and then the silicon thin film was transferred from the MES buffer to the MES buffer. Then, the silicon thin film was moved from the MES buffer to the anti-AFP antibody solution, and the signal during this period was continuously measured.
　図１２に示すとおり、ＭＥＳバッファーからＭＥＳバッファーへの移動に際しては、シグナル変動は顕著ではなく、シリコン薄膜をＭＥＳバッファーから抗ＡＦＰ抗体溶液に移動したときのシグナル変化は顕著であった。このため、膜型表面応力センサの製造の各工程でシグナル変化を確認することによって、各工程が成功していることを確認しながらセンサを製造することができると考えられる。 As shown in FIG. 12, when the MES buffer was moved from the MES buffer, the signal fluctuation was not significant, and the signal change was significant when the silicon thin film was moved from the MES buffer to the anti-AFP antibody solution. For this reason, it is thought that a sensor can be manufactured, confirming that each process is successful by confirming a signal change at each process of manufacture of a membrane type surface stress sensor.
　（実施例５）
　ＥＤＣ／ＮＨＳ溶液（ＤＭＦ）処理したシリコン薄膜をセンサチップホルダーに接続し、９６穴プレートの各ウェルに分注した１００μｇ／ｍＬの抗ＡＦＰ抗体溶液（ＭＥＳバッファー、ｐＨ５．５）にシリコン薄膜を浸漬した。また、コントロールとして、抗ＡＦＰ抗体を含まないＭＥＳバッファーにシリコン薄膜を浸漬した。 (Example 5)
Connect the silicon thin film treated with EDC / NHS solution (DMF) to the sensor chip holder and immerse the silicon thin film in 100 μg / mL anti-AFP antibody solution (MES buffer, pH 5.5) dispensed to each well of 96-well plate did. As a control, a silicon thin film was immersed in a MES buffer not containing an anti-AFP antibody.
　図１３の左側のグラフに示すように、コントロールのＭＥＳバッファーにセンサチップを浸漬したところ、ＭＥＳバッファーに浸漬したシリコン薄膜のシグナル（Ａｂ（－）シグナル）は、溶液への浸漬の影響で、ベースラインが落ちこみ、その状態を維持した。一方、抗ＡＦＰ抗体溶液にセンサチップを浸漬したシリコン薄膜のシグナル（Ａｂ（＋）シグナル）はコントロールに比べて、緩やかかつ大幅に降下した後、規定レベルに落ち着いた。 この変動が規定レベルに落ち着くまでの挙動をモニタリングすることで、センサチップの表面に抗体が結合したことを保証することができると考えられる。 As shown in the graph on the left side of FIG. 13, when the sensor chip was immersed in the control MES buffer, the signal of the silicon thin film immersed in the MES buffer (Ab (−) signal) was affected by the immersion in the solution. The line fell and maintained that state. On the other hand, the signal (Ab (+) signal) of the silicon thin film in which the sensor chip was immersed in the anti-AFP antibody solution gradually and significantly dropped compared with the control, and then settled to the specified level.モ ニ タ リ ン グ It is considered that it is possible to guarantee that the antibody is bound to the surface of the sensor chip by monitoring the behavior until this fluctuation settles to the specified level.
　（実施例６）
　ＥＤＣ／ＮＨＳ溶液（ＤＭＦ）処理したシリコン薄膜をセンサチップホルダーに接続し、９６穴プレートの各ウェルに分注した抗ＡＦＰ抗体溶液（ＭＥＳバッファー、ｐＨ５．５）に、シリコン薄膜を浸漬し、４℃に１晩おいて抗体を固定化した。翌日、このシリコン薄膜を、界面活性剤（ＴｒｉｔｏｎＸ－１００）を含む洗浄バッファーを用いて洗浄し、３７℃に６０分間おいてマスキングした。これをマスキングバッファーで洗浄することによって、シリコン薄膜上に抗ＡＦＰ抗体を固定化することができた。また、コントロールとして、抗ＡＦＰ抗体溶液（ＭＥＳバッファー、ｐＨ５．５）の代わりに、抗ＡＦＰ抗体を含まないＭＥＳバッファーに浸漬したシリコン薄膜を用いた。 (Example 6)
The silicon thin film treated with EDC / NHS solution (DMF) was connected to the sensor chip holder, and the silicon thin film was immersed in an anti-AFP antibody solution (MES buffer, pH 5.5) dispensed to each well of the 96-well plate. The antibody was immobilized overnight at 0 ° C. The next day, the silicon thin film was washed with a washing buffer containing a surfactant (Triton X-100) and masked at 37 ° C. for 60 minutes. By washing this with a masking buffer, the anti-AFP antibody could be immobilized on the silicon thin film. As a control, a silicon thin film immersed in a MES buffer not containing an anti-AFP antibody was used instead of the anti-AFP antibody solution (MES buffer, pH 5.5).
　また、抗ＡＦＰ抗体溶液に浸漬したチップとコントロールのチップをブロッキング処理した後、９６穴プレートの各ウェルに分注したアルカリフォスファターゼ（ＡＬＰ）で標識した抗マウスＩｇＧ抗体を０．１μｇ／ｍＬ含有する溶液に、前述のシリコン薄膜を３７度で３０分浸漬した。その後、前記シリコン薄膜を、界面活性剤（ＴｒｉｔｏｎＸ－１００）を含む洗浄液で洗浄し、ＡＬＰの発光基質溶液で、３７℃で５分間反応させた後、化学発光シグナルを計測した。 Further, after blocking the chip immersed in the anti-AFP antibody solution and the control chip, 0.1 μg / mL of anti-mouse IgG antibody labeled with alkaline phosphatase (ALP) dispensed into each well of the 96-well plate is contained. The aforementioned silicon thin film was immersed in the solution at 37 degrees for 30 minutes. Thereafter, the silicon thin film was washed with a washing solution containing a surfactant (Triton X-100), reacted with a luminescent substrate solution of ALP at 37 ° C. for 5 minutes, and then a chemiluminescence signal was measured.
　図１３の右側のヒストグラムに示すように、コントロールのシリコン薄膜（Ａｂ（－））においては、ＡＬＰ標識抗マウスＩｇＧ抗体による発光が検出されなかったが、抗ＡＦＰ抗体溶液に浸漬したシリコン薄膜（Ａｂ（＋））においては、ＡＬＰ標識体による発光が強く検出された。したがって、抗ＡＦＰ抗体溶液に浸漬したシリコン薄膜（Ａｂ（＋））は、その表面に抗ＡＦＰ抗体が固定化しており、ＡＦＰを検出可能であることが確認された。 As shown in the histogram on the right side of FIG. 13, in the control silicon thin film (Ab (−)), no luminescence was detected by the ALP-labeled anti-mouse IgG antibody, but the silicon thin film immersed in the anti-AFP antibody solution (Ab In (+)), luminescence by the ALP label was strongly detected. Therefore, it was confirmed that the silicon thin film (Ab (+)) immersed in the anti-AFP antibody solution has the anti-AFP antibody immobilized on its surface and can detect AFP.
　従来のイムノセンサよりもはるかに高感度の測定が可能であって、しかも従来よりバイオセンサ一般の大きな課題であった品質保証を高度に、かつ安定して確実なものとし、かつセンサ製造も簡便な新しい技術的手段を実現できる。 Much higher sensitivity than conventional immunosensors can be measured, and quality assurance, which has been a major issue for biosensors in the past, is highly stable, reliable, and sensor manufacturing is simple. New technical means.
日本国公開特許公報第２００３－１８５６１８号Japanese Published Patent Publication No. 2003-185618 国際公開２０１１／１４８７７４１号のパンフレットPamphlet of International Publication No. 2011/1487741

Claims (7)

  1. 　抗体または抗原が固定化されたシリコン薄膜が、ピエゾ抵抗素子を含む支持体によって複数点支持されており、前記抗体または抗原が試料溶液中の抗原または抗体と結合することによって引き起こされるシリコン薄膜の変形をピエゾ抵抗素子によって検出することにより、抗原と抗体との結合を検出またはその結合量を定量可能とすることを特徴とする膜型表面応力センサ。 Silicon thin film to which an antibody or antigen is immobilized is supported at multiple points by a support including a piezoresistive element, and deformation of the silicon thin film caused by binding of the antibody or antigen to the antigen or antibody in the sample solution A membrane type surface stress sensor characterized by detecting the binding between an antigen and an antibody or quantifying the amount of the binding by detecting a piezoresistive element.
  2. 　前記抗体または抗原が、前記シリコン薄膜の片面または両面に固定化されていることを特徴とする請求項１に記載の膜型表面応力センサ。 The membrane type surface stress sensor according to claim 1, wherein the antibody or antigen is immobilized on one side or both sides of the silicon thin film.
  3. 　前記抗体または抗原が、インクジェットスポッティング法または浸漬法のいずれかで固定されていることを特徴とする請求項１または２に記載の膜型表面応力センサ。 The membrane type surface stress sensor according to claim 1 or 2, wherein the antibody or antigen is fixed by either an inkjet spotting method or an immersion method.
  4. 　請求項１から３のいずれか一項に記載の膜型表面応力センサを半導体基板上に１つ以上配置されていることを特徴とするセンサチップ。 A sensor chip comprising one or more film-type surface stress sensors according to any one of claims 1 to 3 disposed on a semiconductor substrate.
  5. 　抗体が抗原と結合することによって引き起こされるシリコン薄膜の変形をピエゾ抵抗素子によって検出することにより、抗原と抗体との結合を検出またはその結合量を定量可能とする膜型表面応力センサの製造方法であって、少なくとも
    ＜１＞ピエゾ抵抗素子を含む複数の支持体により支持されたシリコン薄膜の表面を洗浄する工程；
    ＜２＞洗浄したシリコン薄膜に、自己組織化膜を形成する化合物を接触させる工程；
    ＜３＞表面に自己組織化膜が形成されたシリコン薄膜の表面に、抗体または抗原溶液を接触させる工程；
    ＜４＞上記＜１＞～＜３＞の各工程において、膜型表面応力センサに電圧を印加して、出力電圧の変化をモニタリングする工程；
    を含むことを特徴とする膜型表面応力センサの製造方法。     A method of manufacturing a membrane-type surface stress sensor that can detect the deformation of a silicon thin film caused by the binding of an antibody to an antigen with a piezoresistive element, thereby detecting the binding between the antigen and the antibody or quantifying the amount of the binding. Cleaning the surface of the silicon thin film supported by a plurality of supports including at least <1> a piezoresistive element;
    <2> a step of bringing a compound that forms a self-assembled film into contact with the cleaned silicon thin film;
    <3> a step of bringing an antibody or antigen solution into contact with the surface of a silicon thin film having a self-assembled film formed on the surface;
    <4> In each of the above steps <1> to <3>, a voltage is applied to the film-type surface stress sensor and a change in the output voltage is monitored;
    A method for manufacturing a film-type surface stress sensor, comprising:
  6. 　請求項１から３のいずれか一項に記載された膜型表面応力センサを用いた免疫測定方法であって、少なくとも以下の工程
    ＜１＞抗体または抗原が固定化された膜型表面応力センサのシリコン薄膜に、試料溶液を接触させる工程；
    ＜２＞前記膜型表面応力センサに電圧を印加し、電気シグナルを測定する工程；
    を含むことを特徴とする免疫測定方法。     An immunoassay method using the membrane-type surface stress sensor according to any one of claims 1 to 3, wherein at least the following step <1> of a membrane-type surface stress sensor on which an antibody or an antigen is immobilized Bringing the sample solution into contact with the silicon thin film;
    <2> a step of applying a voltage to the film-type surface stress sensor and measuring an electrical signal;
    An immunoassay method comprising:
  7. 　請求項６に記載の膜型表面応力センサを用いた免疫測定方法であって、前記抗体または抗原が固定化された膜型表面応力センサのシリコン薄膜に、試料溶液を接触させる工程を、当該膜型表面応力センサを試料溶液に浸漬して行うことを特徴とする免疫測定方法。 7. An immunoassay method using the membrane-type surface stress sensor according to claim 6, wherein the step of bringing the sample solution into contact with the silicon thin film of the membrane-type surface stress sensor on which the antibody or antigen is immobilized is performed in the membrane. An immunoassay method comprising immersing a mold surface stress sensor in a sample solution.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017181440A (en) * 2016-03-31 2017-10-05 京セラ株式会社 Stress sensor and manufacturing method therefor
JP2019060763A (en) * 2017-09-27 2019-04-18 京セラ株式会社 Sensor element
WO2020110720A1 (en) * 2018-11-26 2020-06-04 国立研究開発法人物質・材料研究機構 Liquid sample analysis method and apparatus
CN112437873A (en) * 2018-09-03 2021-03-02 国立研究开发法人物质材料研究机构 Cleaning method for receptor layer of surface stress sensor
WO2021192641A1 (en) * 2020-03-26 2021-09-30 日本電気株式会社 Target analysis kit and analysis method using same
GB2599858B (en) * 2019-07-10 2024-01-03 Nec Corp Membrane-type surface stress sensor and analysis method using same

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004510130A (en) * 2000-06-02 2004-04-02 ツェプトゼンス　アクチエンゲゼルシャフト Kit and method for multi-analyte determination
JP2009538126A (en) * 2006-05-24 2009-11-05 コーニンクレッカ フィリップス エレクトロニクス エヌ ヴィ Biosensor solid substrate with integrated temperature control and method for making same
JP2011247859A (en) * 2010-05-31 2011-12-08 Universal Bio Research Co Ltd Biochip

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0562957A (en) * 1991-09-04 1993-03-12 Seiko Epson Corp Plasma cleaning method

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004510130A (en) * 2000-06-02 2004-04-02 ツェプトゼンス　アクチエンゲゼルシャフト Kit and method for multi-analyte determination
JP2009538126A (en) * 2006-05-24 2009-11-05 コーニンクレッカ フィリップス エレクトロニクス エヌ ヴィ Biosensor solid substrate with integrated temperature control and method for making same
JP2011247859A (en) * 2010-05-31 2011-12-08 Universal Bio Research Co Ltd Biochip

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Handotai Device no Kosho Kaiseki", 12 June 2006 (2006-06-12), Retrieved from the Internet <URL:http://japan.renesas.com/media/products/common_info/reliability/reliability_handbook/pdf/rjj2710001_05.pdf> [retrieved on 20140729] *
GENKI YOSHIKAWA: "Cho Kogata Cho Kokando Makugata Hyomen Oryoku Sensor (MSS) - Iryo Kankyo Security eno Oyo ni Kitai", EXPECTED MATERIALS FOR THE FUTURE, vol. 12, no. 4, 10 April 2012 (2012-04-10), pages 53 - 55 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017181440A (en) * 2016-03-31 2017-10-05 京セラ株式会社 Stress sensor and manufacturing method therefor
JP2019060763A (en) * 2017-09-27 2019-04-18 京セラ株式会社 Sensor element
CN112437873A (en) * 2018-09-03 2021-03-02 国立研究开发法人物质材料研究机构 Cleaning method for receptor layer of surface stress sensor
US11796408B2 (en) 2018-09-03 2023-10-24 National Institute For Materials Science Method for cleaning receptor layer of surface stress sensor
WO2020110720A1 (en) * 2018-11-26 2020-06-04 国立研究開発法人物質・材料研究機構 Liquid sample analysis method and apparatus
CN113167717A (en) * 2018-11-26 2021-07-23 国立研究开发法人物质材料研究机构 Liquid sample analysis method and device
JPWO2020110720A1 (en) * 2018-11-26 2021-09-30 国立研究開発法人物質・材料研究機構 Liquid sample analysis method and equipment
JP7080455B2 (en) 2018-11-26 2022-06-06 国立研究開発法人物質・材料研究機構 Liquid sample analysis method and equipment
GB2599858B (en) * 2019-07-10 2024-01-03 Nec Corp Membrane-type surface stress sensor and analysis method using same
WO2021192641A1 (en) * 2020-03-26 2021-09-30 日本電気株式会社 Target analysis kit and analysis method using same
JPWO2021192641A1 (en) * 2020-03-26 2021-09-30
JP7260058B2 (en) 2020-03-26 2023-04-18 日本電気株式会社 Target analysis kit and analysis method using it

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