State detection sensor of detection object substance, and measuring device and sensing device using the same

Abstract

To provide a state detection sensor of a detection object substance, etc. capable of distinguishing a state of specific adsorption of the detection object substance from that of non-specific adsorption of impurities in an environment where the impurities such as proteins, are present, even when the impurities are non-specifically adsorbed.SOLUTION: A state detection sensor of a detection object substance, forms, on a substrate 1, a movable film 20 by a crosslinked structure of graphene 2 and includes vibration state detection means for detecting a vibration state of the movable film. An adsorbent having adsorption capacity for specific molecules is supported on a surface of the graphene within a range where the movable film is formed, the adsorbent adsorbing specific molecules having binding capacity with the detection object substance, and the graphene is connected to a power source for energizing the range where the movable film is formed. The state detection sensor measures resonance frequency of the movable film and vibration amplitude during the resonance operation, while vibrating the movable film by alternately repeating heating and cooling the graphene through generation and stop of Joule heat associated with periodic energization of the graphene.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a sensor for detecting the state, such as the mass or quantity, of a target substance such as a virus, and to a measuring apparatus and a sensing device that utilize this sensor. [Background technology]

[0002] The novel coronavirus disease (COVID-19), which began spreading around early 2020, has caused a global pandemic, leading to a critical situation involving the collapse of medical systems and the stagnation of economic activity. Looking ahead to a post-COVID-19 society in 2023, measures to revive economic activity are being implemented. In this situation, the development of sensor systems capable of continuously monitoring viruses in the environment (not only for COVID-19 but also for other viruses) and high-throughput diagnostic and testing technologies is required to promote economic activity. Currently, PCR, the method used to detect viruses, requires the extraction and amplification of RNA from the viral envelope, making it difficult to detect viruses simply and quickly. Meanwhile, antigen and antibody tests are used as simple testing methods. However, antigen tests detect antigens in the envelope, while antibody tests measure antibodies produced in the blood after infection. Both methods are used to determine the presence or absence of infection. Therefore, they do not directly measure the state of the virus present in the environment before infection.

[0003] Therefore, ISFET-based biosensors that apply FET technology are being researched as small sensors that can detect viruses themselves. For example, FET-type biosensors that use graphene, which exhibits extremely high electron mobility, are being researched to electrically detect targets by focusing on the conductivity that changes in response to the charge of proteins or viruses that come into contact with the graphene surface (see Patent Documents 1 to 3 and Non-Patent Document 1). It has also been reported that viruses can be detected by immobilizing antibodies that specifically adsorb the spike protein of SARS-CoV-2 on a graphene channel (see Non-Patent Document 2).

[0004] However, electrical biosensing suffers from the problem of Debye shielding, which limits the measurement range, making it difficult to detect biomolecules larger than 10 nm at physiological salt concentrations. Furthermore, because FET biosensors are based on the principle of detecting targets in solution, their application to environmental measurements, such as detecting viruses present in the air, is problematic. In response to this problem, biosensor technologies that can operate in air have been developed, such as SPR sensors that utilize surface plasmons (see Non-Patent Document 3) and QCM sensors that measure the mass of adsorbed molecules (see Non-Patent Document 4). However, the sensitivity of these sensors remains insufficient, making it difficult to detect minute amounts of viruses dispersed in the air.

[0005] Therefore, researchers including some of the inventors of the present application have proposed a fabrication technique for a structure in which the cavity below suspended graphene is sealed in order to realize a MEMS sensor that chemically modifies graphene with receptors and selectively detects molecules with high sensitivity (see Non-Patent Document 5), and a report has been made on selective molecular detection using an antigen-antibody reaction (see Non-Patent Document 6). According to these techniques, suspended graphene that is free-standing from a substrate is not subject to electron scattering by the optical phonons of the substrate, and therefore has a higher carrier mobility (200,000 cm) than graphene fixed to a substrate. 2 / Vs), and it is expected that the use of thin, lightweight suspended graphene can be used to create an ultrasensitive mass sensor.

[0006] The structure described above is robust against the solution treatments required for chemical modification, allowing chemical treatments to be performed using the same protocols as for graphene immobilized on a substrate. Furthermore, surface stress measurements were realized to detect static deformation of the film that occurs upon antigen adsorption on cross-linked graphene chemically functionalized with an antibody. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-047777 [Patent Document 2] International Publication No. WO2020 / 170799 [Patent Document 3] Patent Publication No. 2021-076529 [Patent Document 4] Japanese Patent Publication No. 2023-107255 [Non-patent literature]

[0008] [Non-Patent Document 1] Y.Ohno, K.Maehashi, K.Matsumoto, “Label-free biosensors based on aptamer-modified graphene field-effect transistors”, J.AM.Chem.Soc. 132, pp.18012-18013 (2010) [Non-patent document 2] G.Seo, G.Lee, M.Jeong, S.-H.Beak, M.Choi, K.B.Ku, C.-S.Lee, S.Jun, D.Park, H.G.Kim, S.-J.Kim, J.Lee, B.T.Kim, E.C.Park, S.I.Kim, “Rapid detection of COVID-19 causative virus(SARS-CoV-2) in human nasopharyngeal swab specimens using field-effect transistor-based biosensor”, ACS Nano 14, pp.5135-5142 (2020) [Non-Patent Document 3] G. Alison, A.K. Sana, Y. Ogawa, H. Kato, K. Ueno, H. Misawa, K. Hayashi, and H. Suzuki, “A Fabry-Perot cavity coupled surface plasmon photodiode for electrical biomolecular sensing,” Nature communications 12, 6483(2021) [Non-Patent Document 4] A. Afzal, A. Mujahid, R. Schirhagl, S.Z. Bajwa, U. Latif, and S. Feroz, “Gravimetric viral diagnostics: QCM based biosensors for early detection of viruses,” Chemosensors 5(1), 7(2017) [Non-Patent Document 5] K. Takahashi, H. Ishida and K. Sawada, “Vacuum-sealed microcavity formed from suspended graphene by using a low-pressure dry-transfer technique,” Appl. Phys. Lett. 112, 041901(2018) [Non-Patent Document 6] S.Kidane, H.Ishida, K.Sawada, and K.Takahashi, “A suspended graphene-based optical interferometric surface stress sensor for selective biomolecular detection,” Nanoscale Adv. 2, pp.1431-1436(2020) [Non-Patent Document 7] Shin Kitane, Kazuaki Sawada, Kazuhiro Takahashi, "Ultrasensitive optical interference multimodal biosensor using suspended graphene," 12th Integrated MEMS Symposium, October 26-28, 2020, Online, 28A3-AP-1 [Non-patent document 8] K.Arano, AABKusaini, E.Furusawa, J.Uesaka, Y.-J.Choi, T.Noda, T.Goda, Y.Miyahara, K.Sawada, K.Takahashi, “A suspended graphene-based resonant mass sensor for label-free virus detection”, 2021 Int. Conf. on Solid State Devices and Materials, On-line, G-6-01 [Non-Patent Document 9] Ken Niino, Amirn Khusaini, Junpei Uesaka, Eriko Furusawa, Yongjun Choi, Tatsuro Goda, Yuji Miyahara, Toshihiko Noda, Kazuaki Sawada, Kazuhiro Takahashi, "Detection of influenza virus in droplets using graphene resonant sensors," 82nd Autumn Meeting of the Japan Society of Applied Physics, September 10-13, 2021, Online, 13p-N322-8 Summary of the Invention [Problem to be solved by the invention]

[0009] The technology disclosed in the aforementioned Non-Patent Document 6 relies on surface stress measurement. However, this surface stress measurement measures the deformation of the membrane, which changes depending on the charge of antigen molecules. Therefore, although it can measure changes in concentration, it cannot quantify the actual number of molecules adsorbed. Researchers, including some of the present inventors, further discovered that when cross-linked graphene is vibrated, the resonant frequency changes depending on the mass of the adsorbed substance on the cross-linked graphene. They then developed a resonant mass measurement method to detect this resonant frequency shift (see Patent Document 4). Using this resonant mass measurement, they demonstrated the possibility of measuring the mass of antigens adsorbed by antigen-antibody reactions (Non-Patent Document 7) and the possibility of measuring the mass of adsorbed human influenza viruses (Non-Patent Documents 8 and 9). These demonstration experiments used antibodies or sialoglycans as receptors, and simultaneously demonstrated the specific adsorption of proteins or human influenza viruses onto cross-linked graphene. Furthermore, by changing the receptor to a DNA aptamer that specifically adsorbs the COVID-19 causative virus, resonant mass measurement after inactivation treatment became possible.

[0010] However, when attempting to measure the state of a substance to be detected, impurities such as proteins are suspended in the indoor space used as the measurement target. Particularly in indoor spaces used by many people at the same time, such as conference rooms or stores, a large amount of proteins in saliva can become aerosolized and float, causing nonspecific adsorption to the sensing area of ​​the sensor. To avoid this type of nonspecific adsorption of impurities, a method is generally used in which a specific interface is formed in the sensing area by using a nonspecific adsorption inhibitor molecule (blocking agent) such as polyethylene glycol. However, because impurities such as large amounts of proteins originating from saliva are present in high concentrations in actual indoor environments, it is difficult to completely avoid this nonspecific adsorption.

[0011] Furthermore, when measuring the state (especially the mass) of the substance to be detected using the above-mentioned resonant mass measurement, impurities in addition to the substance to be detected are adsorbed to the movable membrane, which can make it difficult to distinguish between them based solely on changes in the resonant frequency.

[0012] The present invention has been made in consideration of the above points, and its purpose is to provide a state detection sensor for a substance to be detected that can distinguish between specific adsorption of the substance to be detected and non-specific adsorption of impurities in an environment where impurities such as proteins are present, even if the impurities are non-specifically adsorbed, and a measuring apparatus and sensing device that use the same. [Means for solving the problem]

[0013] Therefore, as a result of intensive research, the inventors discovered that when a movable membrane made of graphene is vibrated by utilizing Joule heat, electricity is periodically passed through the graphene, and the impedance at this time changes depending on the quantity of substances attached to the graphene. This led to the completion of an invention that can detect the state of a substance to be detected based on changes in both the vibration behavior of the movable membrane due to changes in mass and quantity when substances with different molecular weights are adsorbed together.

[0014] That is, the present invention relating to a sensor for detecting the state of a substance to be detected comprises forming a cavity on a substrate, closing the opening of the cavity with graphene to form a movable membrane with a cross-linked structure of the graphene, and detecting the state of a substance attached to the movable membrane from a change in state when the movable membrane is vibrated, and is characterized in that the sensor comprises vibration state detection means for detecting the vibration state of the movable membrane, and an adsorbent having an ability to adsorb specific molecules is carried on the surface of the graphene at least in the area where the movable membrane is formed, and specific molecules having an ability to bind to the substance to be detected are adsorbed to the adsorbent, the graphene is connected to a power source for applying electricity to the area where the movable membrane is formed, and the movable membrane is vibrated by alternately heating and cooling the graphene by generating and stopping Joule heat due to the cyclic application of electricity to the graphene, while the vibration state detection means measures the resonant frequency of the movable membrane and the vibration amplitude during resonant operation.

[0015] According to the invention having the above configuration, specific molecules capable of binding to a detection target substance are adsorbed to the adsorbent supported on the graphene surface of the movable membrane, and when these specific molecules bind to the detection target substance, the detection target substance is specifically adsorbed to the graphene surface. On the other hand, impurities are nonspecifically adsorbed to the graphene surface, but the adsorption of these substances (detection target substance and impurities) to the movable membrane surface (graphene surface) changes the vibration state of the movable membrane, and by utilizing this, the state of the adsorbed substances can be detected.

[0016] In other words, substances adsorbed to the surface of the movable membrane can be classified into the detection target substance and impurities, and although the two have different molecular weights and clearly different masses, and the amount adsorbed also varies with each measurement, if at least some substance is adsorbed, the vibration state of the movable membrane changes. By analyzing the vibration state of this movable membrane, it is possible to detect the adsorption state of the detection target substance.

[0017] Therefore, the driving method for this vibration is to utilize Joule heat generated by periodically passing electricity through the graphene. By manipulating the generation and stopping of Joule heat, it is possible to alternately heat and cool the graphene, which thermally contracts when heated, and the movable graphene membrane vibrates by repeatedly contracting and relaxing. By changing the period of the current flow, the vibration frequency of the movable membrane can be freely changed, making it possible to vibrate at a resonant frequency.

[0018] The movable film, which is made of a bridged graphene structure, is a single-layer film with a thickness of 0.34 nm, the thickness of one carbon atom layer. It is known that electron mobility is reduced due to phonon scattering from the substrate and scattering from surface deposits. Therefore, as the number of substances adsorbed on the movable film increases, the resistance of graphene increases. Therefore, when a current is applied from a constant-voltage power supply, the current flowing through the graphene decreases, and the amount of heat generated by Joule heating also decreases. This reduces the driving force of the vibration, and as a result, the vibration amplitude also decreases.

[0019] The vibration state of the movable membrane can be measured optically, or can be calculated from the magnitude of the impedance when current is applied. In other words, the motional impedance (the impedance in the resonant state) can be detected from the change in impedance when the vibration frequency is changed. To measure this impedance, an impedance measuring device or other impedance measuring means can be used.

[0020] When measuring the amplitude (impedance) while changing the vibration frequency, the weight (mass) of the substance adsorbed to the movable membrane affects the resonant frequency (motional impedance), and the amplitude (impedance) during resonant operation (motional impedance) also changes. Therefore, even when vibrating at the same resonant frequency (i.e., the total mass of the adsorbed substance is the same), the quantity and mass (total weight) of the adsorbed substance can be obtained by comparing the degree of decrease in vibration amplitude (degree of increase in impedance) during resonant operation. Therefore, if the target substance has a molecular weight significantly larger than that of the impurities, it is possible to distinguish between the response when a small amount of the target substance is adsorbed and the response when a large amount of the impurities is adsorbed. Because the decrease in vibration amplitude during resonant operation is due to the increase in graphene resistance, comparing the degree of increase in impedance does not necessarily have to be limited to resonant operation. As long as a comparable state is possible, it is also possible to understand the state by simply comparing the impedance values ​​(resistance values) when powered by a DC power source. However, if the impedance of the graphene is measured when detecting the motional impedance, the measured impedance value during resonant operation can be used. Note that when a voltage from a constant current power supply is applied to the graphene, the Joule heat increases due to a decrease in impedance, and the vibration state of the movable membrane behaves in the opposite way to the above.

[0021] In the above invention, a blocking agent that inhibits adsorption of substances other than the target substance to be detected can be adsorbed onto either or both of the surface of the graphene and the surface of the adsorbent.

[0022] According to the above configuration, non-specific adsorption of impurities is eliminated as much as possible, and it is possible to observe changes in the vibration state of the movable membrane when a small number of impurities are adsorbed, thereby improving the detection accuracy of the target object. In other words, when the total amount of substance adsorbed to the movable membrane (total number of target substance and impurities) is large, the amplitude becomes extremely small, making it difficult to determine the resonant frequency (motional impedance). However, by reducing the number of adsorbed impurities, the total number becomes small, which moderates the decrease in amplitude and makes it easy to derive the resonant frequency (motional impedance).

[0023] Furthermore, in the invention having the above configuration, the specific molecule may be a nucleic acid molecule that specifically adsorbs the spike protein of SARS-CoV-2, and the substance to be detected may be SARS-CoV-2.

[0024] With this configuration, SARS-CoV-2, the target substance, is specifically adsorbed by specific nucleic acid molecules. SARS-CoV-2 connects to the graphene via its spike protein and nucleic acid molecules, resulting in contact over a very limited area. Meanwhile, contaminants such as proteins are nonspecifically adsorbed to the graphene surface, resulting in direct contact with the graphene surface. Furthermore, because the volume and molecular weight of contaminants such as proteins are significantly smaller than SARS-CoV-2, their contact area with the graphene is also small, resulting in a small mass of nonspecifically adsorbed particles. As a result, when only one SARS-CoV-2 specifically adsorbs, for example, the vibration state of the movable membrane changes in resonant frequency (the vibration of the movable membrane changes depending on the mass of the substance), but the decrease in vibration amplitude (the decrease in Joule heat due to increased impedance) is minimal. In contrast, if only impurities (proteins, etc.) of approximately the same mass as SARS-CoV-2 are nonspecifically adsorbed to graphene, the change in mass will cause a corresponding change in the resonant frequency, and a significant decrease in vibration amplitude (a significant decrease in Joule heat due to a significant increase in impedance). Therefore, the total number of adsorbed substances (both due to specific adsorption and nonspecific adsorption) can be determined from the decrease in vibration amplitude (a decrease in Joule heat due to an increase in impedance) or the increase in the graphene impedance value, and the total mass of all substances can be determined from the change in resonant frequency. By processing this rationally, the mass and quantity of the substance to be detected can be determined.

[0025] The present invention also relates to a measuring device for the mass of a detection target substance, which uses any one of the state detection sensors configured as above to measure the mass of the detection target substance, and includes the state detection sensor for the detection target substance, a power supply device that periodically applies and stops current from a constant voltage power supply or voltage from a constant current power supply to the movable film, amplitude detection means that detects the vibration amplitude of the movable film, determination means that determines a resonance frequency based on the detected amplitude, impedance detection means that detects the impedance of the graphene, and a mass measurement device that calculates the mass of the detection target substance from the detected value of the impedance of the graphene and fluctuations in the resonance frequency of the movable film. and a processing means for calculating the total mass of the substance adhering to the movable film based on the rate of change in the resonance frequency of the movable film before and after the sensor is placed in the environment to be measured, and for calculating the total quantity of the substance adhering to the movable film based on the change in the impedance of the graphene before and after the sensor is placed in the environment to be measured or the change in the vibration amplitude during resonant operation of the movable film, and for calculating the total mass of the substance to be detected from the change in the resonance frequency due to only the substance to be detected based on the correlation between the total mass and the total quantity of the substance adhering to the movable film.

[0026] According to the invention having the above configuration, the power supply device periodically applies and stops current or voltage from a constant-voltage power supply to the movable membrane, thereby applying current to the graphene at a predetermined cycle. By adjusting the cycle, the movable membrane can be vibrated at a desired cycle. The vibration amplitude is detected by the amplitude detection means while appropriately changing the cycle of application of the current. The frequency at which the vibration amplitude reaches its maximum is determined by the determination means, thereby identifying the resonant frequency. Furthermore, since the impedance of the graphene during application of the current can be detected by the impedance detection means, the processing means can calculate the mass of only the target substance based on the change in the resonant frequency and the change in impedance (or the change in the vibration amplitude during resonant operation of the movable membrane). In this case, the processing means calculates the change in the mass of the entire adsorbed substance based on the rate of change in the resonant frequency of the movable membrane before and after installing the state detection sensor in the measurement environment, and calculates the total quantity of the substance attached to the movable membrane based on the change in the impedance of the graphene (or the change in the vibration amplitude during resonant operation of the movable membrane). The change in the impedance of the graphene at this time can be obtained by referring to and comparing the value when the movable membrane is resonating, but is not limited to this, and any comparable impedance can be referenced. After calculating these, based on the correlation between the total mass (fluctuation rate of the resonant frequency) of the substances attached to the movable membrane and the total quantity (impedance during resonant operation), that is, by utilizing the difference between the change in mass (resonant frequency) relative to the quantity of the detection target substance and the change in mass (resonant frequency) relative to the quantity of impurities, the change in resonant frequency due to only the detection target substance can be derived, and the total mass of the detection target substance can be calculated from the mass of the detection target substance corresponding to the change in resonant frequency.

[0027] On the other hand, the present invention relating to a measuring device for the quantity of a detection target substance is a quantity measuring device for a detection target substance that uses a state detection sensor of any of the above configurations to measure the quantity of the detection target substance, and includes the state detection sensor for the detection target substance, a power supply device that periodically applies and stops current from a constant voltage power supply or voltage from a constant current power supply to the movable film, amplitude detection means that detects the vibration amplitude of the movable film, determination means that determines a resonance frequency based on the detected amplitude, impedance detection means that detects the impedance of the graphene, and processing means that calculates the quantity of a substance adhered to the movable film from fluctuations in the detected value of the impedance of the graphene and the resonance frequency of the movable film, and the processing means ... the total mass of the substance adhering to the movable membrane is calculated based on a rate of change in the resonance frequency of the movable membrane before and after the sensor is installed, and the total quantity of the substance adhering to the movable membrane is calculated based on a change in the impedance of the graphene or a change in the vibration amplitude during resonant operation of the movable membrane before and after the sensor is installed in the environment to be measured, and the amount of change in the impedance of the graphene or the amount of change in the vibration amplitude during resonant operation of the movable membrane is derived based on the correlation between the total mass and the total quantity of the substance adhering to the movable membrane, thereby calculating the amount of the substance to be detected from the amount of change in the impedance of the graphene due to only the substance to be detected or the amount of change in the vibration amplitude during resonant operation of the movable membrane.

[0028] According to the above configuration, as in the case of the mass measuring device described above, the resonant frequency can be identified and the impedance of the graphene can be detected when current is applied. Therefore, the processing means can calculate the quantity of only the target substance based on the change in the resonant frequency and the change in the impedance. In this case, as in the case of the mass measuring device, the processing means calculates the change in the mass of the entire adsorbed substance based on the rate of change in the resonant frequency of the movable membrane before and after installing the state detection sensor in the measurement environment, and calculates the total quantity of substances adhering to the movable membrane based on the change in the impedance of the graphene or the change in the vibration amplitude during resonant operation of the movable membrane. After calculating these, by utilizing the difference between the change in the resonant frequency with respect to the quantity of the target substance and the change in the resonant frequency with respect to the quantity of impurities, the change in impedance due to only the target substance or the change in the vibration amplitude during resonant operation of the movable membrane can be calculated, thereby calculating the quantity of only the target substance corresponding to the change in impedance.

[0029] The present invention, which relates to a sensing device for a detection target substance, is a multimodal sensing device that uses any one of the state detection sensors configured as above to measure the quantity and mass of a detection target substance, and includes the state detection sensor for the detection target substance, a power supply device that periodically applies and stops current from a constant voltage power supply or voltage from a constant current power supply to the movable film, amplitude detection means that detects the vibration amplitude of the movable film, determination means that determines a resonance frequency based on the detected amplitude, impedance detection means that detects the impedance of the graphene, and processing means that calculates the quantity and mass of a substance adhered to the movable film from the detected value of the impedance of the graphene and fluctuations in the resonance frequency of the movable film, and the processing means calculates the quantity and mass of a substance adhered to the movable film from fluctuations in the resonance frequency of the movable film before and after placing the sensor in a measurement environment. The total mass of the substance adhered to the movable membrane is calculated based on the ratio, and the total quantity of the substance adhered to the movable membrane is calculated based on the change in impedance of the graphene or the change in vibration amplitude during resonant operation of the movable membrane before and after placing the sensor in the environment to be measured.The amount of change in resonance frequency due to only the substance to be detected and the amount of change in impedance of the graphene due to only the substance to be detected or the amount of change in vibration amplitude during resonant operation of the movable membrane are derived based on the correlation between the total mass and total quantity of the substance adhered to the movable membrane, thereby calculating the total mass of the substance to be detected from the amount of change in resonance frequency due to only the substance to be detected, and calculating the quantity of the substance to be detected from the amount of change in impedance of the graphene due to only the substance to be detected or the amount of change in vibration amplitude during resonant operation of the movable membrane.

[0030] According to the above configuration, as in the case of the mass measuring device described above, the resonant frequency can be identified and the impedance of the graphene can be detected when a current is applied. Therefore, the processing means can calculate the quantity of only the target substance based on the change in the resonant frequency and the change in the impedance (or the change in the vibration amplitude during resonant operation of the movable membrane). In this case, as in the case of the mass measuring device, the processing means calculates the change due to the mass of the entire adsorbed substance based on the rate of change in the resonant frequency of the movable membrane before and after installing the state detection sensor in the measurement environment, and calculates the total quantity of substances adhering to the movable membrane based on the change in the impedance of the graphene (or the change in the vibration amplitude during resonant operation of the movable membrane). After these calculations, the difference between the amount of change in the resonant frequency with respect to the quantity of the target substance and the amount of change in the resonant frequency with respect to the quantity of impurities can be used to derive the amount of change in the resonant frequency due only to the target substance, and the total mass of the target substance can be calculated from the mass of the target substance corresponding to the amount of change in the resonant frequency. Similarly, by utilizing the difference between the amount of change in resonant frequency with respect to the quantity of the substance to be detected and the amount of change in resonant frequency with respect to the quantity of impurities, the amount of change in the impedance of the graphene due to only the substance to be detected or the amount of change in the vibration amplitude during resonant operation of the movable membrane can be calculated, making it possible to calculate the quantity of only the substance to be detected corresponding to that amount of change. [Effects of the Invention]

[0031] According to the present invention, the sensor for detecting the state of a substance to be detected can distinguish whether a decrease in the resonant frequency of the movable membrane is due to the virus or the impurities, when the substance to be detected is a virus or the like in an environment where impurities such as proteins with a smaller mass than the virus or the impurities are present. Therefore, even in an environment where the substance to be detected and the impurities are mixed in different mass proportions, and the impurities are nonspecifically adsorbed, the state of specific adsorption of the substance to be detected and the state of nonspecific adsorption of the impurities can be distinguished and measured. The use of such a state detection sensor can realize a device for measuring the mass or quantity of the substance to be detected, and can also be used as a multimodal sensing device. [Brief explanation of the drawings]

[0032] [Figure 1] 1 is a schematic diagram showing the configuration of an embodiment of a state detection sensor for a detection target substance. [Figure 2] 1 is an explanatory diagram showing the configuration of an embodiment of a state detection sensor for a detection target substance. [Figure 3] FIG. 1 is an explanatory diagram illustrating the principle of mass measurement. [Figure 4] FIG. 1 is an explanatory diagram showing the principle of quantity measurement. [Figure 5] FIG. 10 is an explanatory diagram showing changes in the resonant frequency and vibration amplitude when a detection target substance and impurities are adsorbed to single-layer graphene. [Figure 6] 1 is an explanatory diagram showing the configuration of an embodiment of an apparatus for measuring the mass of a detection target substance. [Figure 7] FIG. 10 is an explanatory diagram showing a modified example of the embodiment of the device for measuring the mass of a detection target substance. [Figure 8] 1A to 1C are explanatory diagrams showing a method for producing a sensor for detecting the state of a detection target substance. [Figure 9] Graph (a) shows the results of the experiment, and (b) shows the vibration state of the movable membrane, showing the results of a comparative experiment. [Figure 10] This is an SEM image of the movable membrane used in the experiment. DETAILED DESCRIPTION OF THE INVENTION

[0033] Hereinafter, an embodiment of the present invention will be described with reference to the drawings. <Sensor for detecting the state of the substance to be detected> 1 and 2 show the configuration of an embodiment of a sensor for detecting the state of a substance to be detected. As shown in these figures, the basic configuration of this embodiment is a silicon substrate 1 having a cavity 3 formed therein, on which a single-layer graphene 2 is laminated with the opening of the cavity 3 closed. The silicon substrate 1 is formed by thermally oxidizing the surface of a substrate body 11 to form a silicon oxide film 12, and the cavity 3 is formed by partially eroding the silicon substrate 1 using reactive ion etching (RIE). The single-layer graphene 2 is laminated on the surface of the silicon substrate 1, including the opening of the cavity 3, and forms a bridge structure at the opening of the cavity 3. This bridge structure functions as a vibrating movable membrane 20. While the cavity 3 shown in the figure has a circular cross section, the cavity 3 may have a rectangular or any other cross-sectional shape. A pair of electrodes 4 and 5 are connected to both surfaces of the single-layer graphene 2, which are provided to pass electricity through the single-layer graphene 2, as described below.

[0034] The state detection sensor of this embodiment is intended to detect the state of any substance (substance to be detected) from the state of a substance adsorbed (specifically adsorbed or non-specifically adsorbed) to the single-layer graphene 2 (particularly the movable film 20). Specifically, by measuring the state (resonance frequency, vibration amplitude, etc.) when the movable film 20 is vibrated, the degree of difficulty of vibrating the movable film 20 is quantified, and state detection is possible based on this.

[0035] Therefore, as shown in Figure 2, Joule heat acting on the single-layer graphene 2 is used to excite (vibrate) the movable membrane 20, and an AC power supply 6 for this purpose is connected to the single-layer graphene 2. The AC power supply (constant voltage power supply or constant current power supply) 6 is connected to one electrode 4, and the other electrode 5 is connected to ground GND, thereby applying a current (or a voltage) from the constant voltage power supply to the single-layer graphene 2. The AC power supply 6 periodically applies a current or voltage, and by switching ON and OFF using a square wave at a predetermined cycle, the generation and cessation of Joule heat in the single-layer graphene 2 is achieved. Heating and cooling of the movable membrane 20 are alternately and cyclically performed, and vibrational motion is generated in response to the expansion and contraction of the movable membrane 20.

[0036] When the movable film 20 is excited (excited) by expansion and contraction, it is preferable that the bulging direction of the movable film 20 alternate between outward (rising from the substrate) and inward (depressing into the cavity). Therefore, the AC power supply 6 is also connected to the substrate body 11 of the silicon substrate 1, and this voltage application causes electrostatic forces (attractive and repulsive forces) to act, adjusting the bulging direction of the movable film 20 when it expands due to heating. In this case, the effect of the electrostatic force on the excitation (excitation) of the movable film 20 is extremely limited, and does not affect the vibration amplitude of the movable film 20.

[0037] Such a vibration state of the movable film 20 can be measured from a change in impedance occurring in the single-layer graphene 2. Therefore, by calculating the impedance from fluctuations in the current value (or voltage value) applied to the single-layer graphene 2, the resonance frequency can be identified from the motional impedance based on the change in impedance per period (frequency) when the movable film 20 is vibrated, and the degree (magnitude) of the impedance during the resonant operation of the movable film 20 can be obtained. Therefore, an impedance meter (impedance measuring means) is connected to a pair of electrodes 4, 5 provided on both sides of the single-layer graphene 2, and is configured to measure the impedance at the timing of application of the current (or voltage).

[0038] In this embodiment, the surface of the single-layer graphene 2 (at least the movable membrane 20) is supported with an adsorbent that adsorbs a specific molecule 7. The specific molecule 7 is a molecule that has the property of specifically adsorbing a detection target substance 8, thereby enabling the detection target substance 8 to be adsorbed by the specific molecule. For example, if the detection target substance 8 is SARS-CoV-2, the specific molecule 7 is a nucleic acid molecule (aptamer) that specifically adsorbs the spike protein of SARS-CoV-2, and the surface of the single-layer graphene 2 (at least the movable membrane 20) is supported with an adsorbent that can adsorb the nucleic acid molecule (aptamer). However, substances (impurities) other than the detection target substance may also be nonspecifically adsorbed to the surface of the single-layer graphene 2, and a blocking agent may be adsorbed to the adsorbent. In this case, the phenomenon of impurities being nonspecifically adsorbed to the surface of the single-layer graphene 2 may also occur.

[0039] <Detection principle> Here, we will explain the principle of state detection when a substance is adsorbed on the movable film 20. Figure 3 shows a detection method in mass measurement, and Figure 4 shows a detection method in quantity measurement.

[0040] First, as shown in Figure 3, when a small amount of substances (detection target substance 8 and impurities 9) are adsorbed to the movable membrane 20 and the total mass is relatively small (see Figure 3(a)), the movable membrane 20 vibrates relatively easily and resonates in a high frequency band. In contrast, when a large amount of substances 8, 9 are adsorbed to the movable membrane 20 and the total mass is large (see Figure 3(b)), the movable membrane 20 vibrates relatively difficultly and resonates in a low frequency band.

[0041] By utilizing the above-mentioned properties and measuring the resonant frequency, it is possible to detect the change in the total mass of the substances 8 and 9 adsorbed to the movable film 20. Since the resonant frequency can be determined by the motional impedance, by applying vibrations at different frequencies, the frequency at the time of resonant operation can be determined and the total mass can be measured.

[0042] On the other hand, when a current is applied to the single-layer graphene 2, the electrons flowing through the single-layer graphene 2 are scattered by surface deposits (adsorbed substances) in addition to phonon scattering from the substrate 1, resulting in a decrease in electron mobility. Therefore, if there are few deposits (adsorbed substances) on the surface, the decrease in electron mobility is small and the resistance is low, but if there are many deposits (adsorbed substances) on the surface, the electron mobility decreases significantly and the resistance increases.

[0043] That is, as shown in FIG. 4(a), specific molecules 7 are adsorbed on the surface of the single-layer graphene 2 in advance, and the specific molecules 7 cause electrons e -This is the state in which scattering of electrons occurs. In other words, the vibration amplitude due to the decrease in electron mobility (increase in resistance) is at its maximum within the range corresponding to the number of specific molecules 7, and this can be the initial state. The impedance value at this point can be considered the initial value of the impedance during resonant operation. In this state, the target substance 8 is specifically adsorbed to the single-layer graphene 2 via the specific molecules 7, so the change in electron mobility due to the adsorption of the target substance 8 is limited. Note that the figure models a virus (SARS-CoV-2) as the target substance 8 and an aptamer (nucleic acid molecule) as the specific molecule 7. Although the target substance 8 is shown as floating due to the specific molecules 7, a target substance 8 with a large molecular weight can be attached to the surface of the single-layer graphene 2. However, even in this case, the area in contact with the single-layer graphene 2 is limited, so the change in electron mobility due to the adsorption of the target substance 8 can be extremely limited.

[0044] Conversely, as shown in FIG. 4(b), when a large number of impurities 9 are nonspecifically adsorbed on the surface of the single-layer graphene 2, the large number of impurities 9 adhere to the surface of the single-layer graphene 2 and the electrons e - In this case, the magnitude of the vibration amplitude during resonant operation becomes smaller than the initial state, and the increase can be clearly seen when compared with the impedance value considered to be the initial value.

[0045] Therefore, as shown in Figure 5, when a target substance (a substance with a large mass) is adsorbed, only the resonant frequency changes (see Figure 5(a)). When contaminants (substances with a small mass) are adsorbed, the vibration amplitude (during resonant operation) also changes along with the change in the resonant frequency (see Figure 5(b)). These changes in frequency and vibration amplitude can be analyzed from the change in impedance of the single-layer graphene 2, and the total number of adsorbed substances can be measured from the change in impedance value. The change in vibration amplitude can be compared with the impedance when the movable membrane is in a resonant state. However, because the change in vibration amplitude is due to a decrease in Joule heat caused by an increase in the resistance of graphene, it is not necessary for the membrane to be in a resonant state as long as it is in a comparable state. It is also possible to obtain the state of change by simply measuring and comparing the impedance.

[0046] From the measurement results described above, it is possible to measure the quantity and mass of the detection target substance. Essentially, the mass (molecular weight) of the detection target substance 8 must be at least 10 times (approximately 1,000 times) the mass (molecular weight) of the impurity (protein, etc.) 9. This is because, when the masses (molecular weights) are similar, the quantity and mass change over time, making it impossible to determine which substance is which by comparison. In particular, in this embodiment, the detection target substance is assumed to be SARS-CoV-2, and the impurity is assumed to be proteins floating in the environment, with a mass difference of approximately 1:1,000. Thus, when the masses (molecular weights) of the two substances are significantly different, the change in mass due to the attached substances 8 and 9 is the same when there is one detection target substance 8 and when there are 1,000 impurities 9. In this case, the resonant frequency of the vibrating movable membrane 20 shifts to a similarly low frequency. On the other hand, electrons moving through the single-layer graphene 2 are scattered by an amount equivalent to 1,000 electrons, and the resistance increases significantly, reducing the vibration amplitude (the impedance value of the single-layer graphene 2) during resonant operation. From the correlation between the two, it is possible to identify the number of detection target substances 8 specifically adsorbed to the movable film 20, and also to identify the total mass of the detection target substances 8 specifically adsorbed to the movable film 20.

[0047] Specifically, let us assume that the mass ratio (molecular weight ratio) of the target substance 8 to the impurities 9 per particle is 1:10. For example, let us assume that the degree of resonant frequency shift corresponds to "30 particles" when calculated in terms of the mass of the target substance 8, and that the rate of decrease in vibration amplitude during resonant operation corresponds to "210 particles" when converted to the number of adsorbed particles. In this case, based on the correlation between the two, the mass equivalent to "20 particles" of the target substance in the total mass of the adsorbed substance can be determined to be due to the number of impurities "200 particles." Therefore, the number of target substances is calculated as "10 particles," and the total mass of the target substances can be calculated as 10 / 210 of the total mass. In this way, similar calculations are possible even when the mass ratio (molecular weight ratio) per particle is 1:100 or 1:1000. If the mass (molecular weight) per particle of the target substance is calculated in advance, it can also be calculated using a formula.

[0048] Therefore, if precise mass measurement based on changes in resonant frequency and precise quantity measurement based on changes in vibration amplitude during resonant operation (or changes in the impedance of the monolayer graphene 2) were possible, it would be possible to measure changes in the number of individual contaminants. However, here we will limit ourselves to measurements based on the number of large molecular weight target substances. If the target substance is SARS-CoV-2 and the contaminants are proteins floating in the environment, the mass ratio of the two is 1:1000, resulting in 1000 times greater accuracy in detecting the state of SARS-CoV-2. Furthermore, if a blocking agent is adsorbed onto the surface of the monolayer graphene 2, the blocking effect can prevent adsorption by 1000 times, resulting in an overall improvement of 1,000,000 times greater detection accuracy in a typical environment.

[0049] <How to use> In the embodiment of the sensor for detecting the state of a substance to be detected described above, the entire silicon substrate 1 including at least the single-layer graphene 2 is placed in an environment to be detected, and the movable membrane 20 is excited (oscillated) after adsorbing (specifically adsorbing and nonspecifically adsorbing) substances floating in the environment. Measurement of the vibration state (resonance frequency, vibration amplitude, etc.) of the movable membrane 20 can basically be performed under atmospheric pressure (a general environment), but may also be performed under reduced pressure (vacuum, etc.) to increase the detected signal and make measurement easier. A power supply and an impedance measuring device (impedance measuring means) may be separately installed and connected during excitation (oscillation) and impedance measurement.

[0050] In this way, when actually measuring the vibration state of the movable film 20, a power supply is connected and the movable film 20 is excited (vibrated) by Joule heat while measuring the impedance of the single-layer graphene 2, and by measuring the resonant frequency and the vibration amplitude during resonant operation, the quantity of the substance to be detected can be calculated using the above-mentioned calculation method, and the total mass of the substance to be detected can be calculated.

[0051] <Embodiments of an Apparatus for Measuring the Mass of a Detection Target Substance> Next, an embodiment of an apparatus for measuring the mass of a detection target substance will be described. This embodiment uses the state detection sensor for the detection target substance described above, and is outlined in Fig. 6. As shown in Fig. 6, this embodiment is configured to include a state detection sensor A, a power supply device (not shown) connected to the single-layer graphene 2 of this sensor A, a function generator 60 that adjusts the frequency and waveform of the voltage output from the power supply device, an impedance measuring device (amplitude detecting means and impedance detecting means) 61 connected to the single-layer graphene 2, and a processing device (determining means and processing means) 62 that receives and processes detection data input from this impedance measuring device 61.

[0052] The voltage output from the power supply is converted into a square wave by a function generator 60, and a voltage can be applied to the single-layer graphene 2 at a predetermined frequency range while changing the ON / OFF cycle. Either a current from a constant voltage power supply or a voltage from a constant current power supply can be applied to the single-layer graphene 2, and here, a current from a constant voltage power supply is applied to the single-layer graphene 2 at a predetermined frequency range. The power supply (function generator 60) is connected to one electrode 4 of sensor A, and the other electrode is connected to an impedance measuring instrument 61, with the single-layer graphene 2 connected between the two electrodes 60 and 61.

[0053] The impedance measuring instrument 61 measures the impedance of the single-layer graphene 2, and the measured impedance data is processed by a processing device 62. The impedance measuring instrument 61 is connected to a power supply device (function generator 60) via the single-layer graphene 2, and can measure the impedance when current is applied in accordance with the voltage applied for each frequency of the output voltage.

[0054] A general-purpose PC is used as the processing device 62, and analysis software is installed on it. The analysis software stores the impedance of the single-layer graphene 2 measured by the impedance measuring device 61 when a voltage is applied for each frequency adjusted by the function generator 60, and performs calculations to determine various states and to determine states that can be derived from the impedance.

[0055] Specifically, the frequency at which the motional impedance (dynamic impedance) reaches its maximum value is detected, and this frequency is determined as the resonant frequency. To determine the resonant frequency, the resonant frequency of sensor A before the substance in the environment is adsorbed is determined in advance, and the resonant frequency after the substance in the environment is adsorbed is determined again. The mass of the adsorbed substance is calculated based on the amount of change (rate of change) between the two resonant frequencies. The calculated mass is the total mass of the adsorbed substance. The vibration amplitude is also compared before and after the substance is adsorbed in the environment, and the quantity of the adsorbed substance is calculated from the state of change (rate of change) of the vibration amplitude. The vibration amplitude can be calculated from the impedance during resonant operation, or it can be simply calculated by measuring the impedance of the single-layer graphene 2 and calculating the rate of change, which can then be converted into the change in vibration amplitude. In either case, the quantity of the adsorbed substance can be obtained. This quantity also becomes the total quantity of the adsorbed substance. The total mass of the target substance is calculated from the total mass and quantity of the adsorbed substance, the molecular weight of the target substance, and the molecular weight of the impurities. Therefore, in the above calculation, the mass (molecular weight) per particle of the detection target substance and the mass (molecular weight) per particle of the impurities, or the mass ratio between the two, etc., are input to the processing device 62. From these conditions, based on the correlation between the total mass and total quantity of the adsorbed substance, the amount of variation in the resonant frequency due only to the detection target substance can be derived, and since the amount of variation in the resonant frequency due only to the detection target substance is due to the mass of only the detection target substance, the total mass of the detection target substance is calculated based on this amount of variation (variation rate).

[0056] <Embodiment of the device for measuring the quantity of detection target substances> The configuration of the embodiment of the device for measuring the quantity of a detection target substance is basically the same as the embodiment of the mass measuring device shown in Fig. 6. The difference lies in the content of the processing by the processing device 62, which calculates the mass of the entire adsorbed substance, calculates the quantity of the entire adsorbed substance, and calculates the total quantity of the detection target substance based on the input mass (molecular weight) or mass ratio of the two. At this processing stage, based on the correlation between the mass and quantity of the entire adsorbed substance, the change in impedance of the single-layer graphene 2 due only to the detection target substance (or the change in vibration amplitude during resonant operation of the movable membrane) is derived. This change in impedance (or the change in vibration amplitude) can be determined to correspond to the quantity of the detection target substance, and the quantity of the detection target substance is calculated from this change.

[0057] <Embodiments Related to Multimodal Sensing Device> The configuration of the embodiment of the multimodal sensing device is also basically the same as the embodiment of the mass measuring device shown in FIG. 6 . The difference is again in the processing performed by the processing device 62. Because this is an apparatus that simultaneously senses multiple states of a substance, the processing device 62 is capable of simultaneously detecting (measuring) the mass and quantity of the target substance. Therefore, the total mass of the adsorbed substance is calculated, and the total quantity of the adsorbed substance is calculated. Based on the input mass (molecular weight) or mass ratio of the two, the total quantity and total mass of the target substance are calculated. During processing by the processing device 62, the amount of change in resonant frequency due to the target substance alone and the amount of change in impedance (or the amount of change in vibration amplitude during resonant operation of the movable membrane) due to the target substance alone can be derived based on the correlation between the total mass and quantity of the adsorbed substance alone. The total mass of the target substance can be calculated from the amount of change in resonant frequency due to the target substance alone, and the amount of the target substance alone can be calculated from the amount of change in impedance (or the amount of change in vibration amplitude during resonant operation of the movable membrane).

[0058] <Modification> The above-described mass measuring device, quantity measuring device, and multimodal sensing device have all been described as embodiments with the configuration shown in FIG. 6. However, if the movable membrane 20 in the sensor A can be excited (excited), the vibration amplitude of the movable membrane 20 can also be measured by another device (e.g., an optical detection device). In such a case, as shown in FIG. 7, a method is available in which a CW laser is used as a probe laser to irradiate the movable membrane 20 and detect the reflected light. Specifically, a laser beam of a specific wavelength (e.g., 638 nm) continuously oscillated from a probe laser oscillator 63 is irradiated onto the movable membrane 20, and the reflected light is input to a spectrum analyzer 65 via a photodiode 64, which outputs the change and degree of wavelength of the reflected light as frequency and vibration amplitude. The frequency and vibration amplitude values ​​output from the spectrum analyzer 65 are analyzed by a processing device 62, and can be calculated as the mass and number of the total adsorbed material, as described above. [Example]

[0059] The sensor for detecting the state of a substance to be detected was fabricated as follows. Specifically, as shown in FIG. 8, a silicon substrate 1 and a single-layer graphene 2 were fabricated separately and then bonded together. As shown in FIGS. 8(a) to 8(d), the single-layer graphene 2 was grown by CVD on the surface of a copper foil (Cu) catalyst. To remove the copper foil (Cu) from the single-layer graphene 2, a polymethyl methacrylate resin (PMMA) was spin-coated as a support film on the surface of the graphene 2, and polydimethylsiloxane (PDMS) was pressed onto the outer periphery of the PMMA and graphene. While the PMMA and graphene were supported by this PDMS, the copper foil (Cu) was immersed in an etching solution (Etch) containing FeCl3 solution to remove the copper foil (Cu).

[0060] On the other hand, as shown in Figures 8(e) to (h), a silicon oxide film 12 was formed on the upper surface of the silicon body 11 by thermal oxidation, and a resist Res was transferred to areas other than the area where the cavity was to be formed.Then, a circular cavity with a diameter of 6 μm and a depth of 1 to 2 μm was formed by reactive ion etching (RIE).

[0061] As shown in Figures 8(i) and 8(j), the single-layer graphene 2 and silicon substrate 1, which were individually prepared as described above, were bonded to the cavity-side surface of the silicon substrate 1. The substrate was then heated under reduced pressure above the glass transition point of PMMA (approximately 125 °C) to soften the PMMA and adhere the graphene to the silicon substrate 1. The unnecessary PMMA was then removed, and the PDMS was peeled off, yielding a laminate of the silicon substrate 1 and the single-layer graphene. The single-layer graphene 2 formed a bridged structure with a gap formed by the cavity in the substrate 1. The heat treatment was performed under reduced pressure (-1 atm gauge pressure), maintaining a reduced pressure inside the cavity and improving adhesion to the graphene. The single-layer graphene 2 was then patterned into the desired shape by photolithography, and the graphene was etched using O2 plasma. Finally, to form a pair of electrodes 4, 5 on both sides of the single-layer graphene 2, an Au / Ti double-layer film was deposited by sputtering, and then the electrodes 4, 5 and wire wiring were patterned by lift-off.

[0062] The resulting assembly of substrate 1 and single-layer graphene 2 is chemically treated to enhance its ability to adsorb target molecules. The surface of single-layer graphene 2 is modified with 1-pyrenebutanoic acid succinimidyl ester (PBSE) via π-stacking, serving as an adsorbent for specific molecules (e.g., nucleic acid molecules such as aptamers). The pyrene group of PBSE is π-bonded to graphene, and the succinimidyl ester group on the opposite side reacts with an amino group at the end of the nucleic acid molecule to form an amide bond. Therefore, the PBSE modified on the surface of single-layer graphene 2 functions as a crosslinker, allowing specific molecules (e.g., nucleic acid molecules such as aptamers) to be immobilized on the surface of single-layer graphene 2.

[0063] Examples of specific molecules include nucleic acid molecules (aptamers) having the following structure: 5'-CAGCACCGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGCGTTAATGGACA-3' (51-mer) This type of aptamer is also described in the aforementioned Patent Document 4, and has the property of specifically adsorbing the spike protein of SARS-CoV-2. Therefore, when the substance to be detected is SARS-CoV-2, it can be specifically adsorbed. When detecting other substances, it is sufficient to select and use a specific molecule that can specifically adsorb depending on the substance to be detected.

[0064] In this way, the basic structure of a status detection sensor can be constructed by immobilizing specific molecules capable of specifically adsorbing a detection target substance on the surface of the single-layer graphene 2. By connecting a power supply and an impedance measuring instrument to the electrodes 4 and 5 of this sensor, it can function as a status detection sensor.

[0065] <Experimental Example> A resonance frequency analysis was performed on the state detection sensor with the above configuration. Specifically, an electric current was periodically passed through the single-layer graphene, causing it to vibrate due to Joule heat, and an experiment was conducted to measure the resonance frequency of the movable membrane and the amplitude during resonant operation. For comparison, measurements were performed on the single-layer graphene alone, on the aptamer immobilized via a crosslinker, and on the SARS-CoV-2 (inactivated) adsorbed state.

[0066] In the experiment, the above-mentioned state detection sensor was used to modify the graphene surface with a cross-linking agent (PBSE as mentioned above), and then specific molecules (nucleic acid molecules as mentioned above) were immobilized on the graphene surface. 5The state detection sensor was designed to specifically adsorb viruses (copies). Specific adsorption of viruses was achieved by immersing the state detection sensor in a solution containing the virus and then drying it. The resonance frequency and vibration amplitude were measured in each of the above states, and experiments were conducted to determine whether mass and quantity could be measured in a detectable state. For the state detection sensor in each of the above states, a current was applied to the single-layer graphene from a constant-voltage power supply, and the change in resonance frequency and the vibration amplitude during resonant operation were observed when the movable membrane was vibrated by Joule heat. The resonance frequency and vibration amplitude were measured using an optical method (a method shown as a modified example, see Figure 7).

[0067] The experimental results are shown in Figure 9(a). As can be seen from these results, the resonant frequency shifted. The frequency difference was 1.74 MHz. This indicates that the frequency decreased due to the (specific) adsorption of the virus and its overall mass. At the same time, the vibration amplitude during resonant operation also changed. The vibration amplitude decreased from -68 dBm to -71 dBm, and this decrease was determined to be due to the total amount of adsorbed material. To clarify that the decrease in vibration amplitude was due to an increase in the resistance of the graphene, the impedance (resistance) was measured in each state while a DC power supply was applied. The resistance value for graphene alone was 4.06 kΩ. The resistance value for graphene with a crosslinker (PBSE) and specific molecules (nucleic acid molecules) immobilized on the graphene was 12.5 kΩ, whereas the resistance value for the virus specifically adsorbed state was 21.8 kΩ.

[0068] The results of the above experiments revealed that the resonant frequency of the movable membrane shifts downward depending on the mass of the adsorbed substance. Furthermore, the adsorption of a large number of adsorbed substances to the movable membrane increases resistance, reducing the intensity of the vibration due to Joule heating and decreasing the vibration amplitude. The graphene resistance increases as the vibration amplitude decreases, clearly demonstrating a change in vibration amplitude corresponding to the increase in graphene resistance. The above experiments were conducted without the addition of contaminants (such as proteins contained in saliva). However, the results clearly demonstrate that if contaminants such as proteins, estimated to be 1 / 1000 the mass of SARS-CoV-2, are included, the increase in the quantity of adsorbed substances (decreased vibration amplitude) will be minimal compared to the increase in mass (shift in resonant frequency). Therefore, it is possible to distinguish between the adsorption state of the target substance and the adsorption state of contaminants. Note that the frequency and vibration amplitude in the above experiments were observed optically to ensure accuracy, but they can be determined from their relationship with the impedance of single-layer graphene.

[0069] In the above experiments, the crosslinker was not modified with a blocking agent, as this was based on the premise that nonspecific adsorption of contaminants would not occur. In environments containing contaminants, modifying the crosslinker with a blocking agent such as polyethylene glycol can significantly prevent nonspecific adsorption of contaminants. In this case, the resonant frequency and amplitude change when the crosslinker, aptamer, and blocking agent are adsorbed to the movable membrane. Comparisons can be made based on the resonant frequency and amplitude at this point. Furthermore, modification with a blocking agent significantly reduces the proportion of nonspecifically adsorbed contaminants, resulting in a reduction in the concentration of nonspecifically adsorbed contaminants relative to the concentration of contaminants present in the measurement environment (specific adsorption at a rate of 1 / 100 or 1 / 1000). This significantly improves identification accuracy (measurement accuracy).

[0070] <Comparative experiment> For comparison, an experiment was also conducted in which the movable membrane was vibrated by electrostatic actuation, which is an electrical actuation method. Electrostatic actuation uses a cavity in the silicon substrate as a gap, applies electrostatic attraction between the silicon and the single-layer graphene, and drives the movable membrane like an electrostatic actuator to induce vibration. This configuration is explained in the aforementioned Patent Document 4. Other conditions were the same as those in the experimental example.

[0071] The results of this comparative experiment are shown in Figure 9(b). According to the results of this comparative experiment, the resonant frequency shift was almost the same as in the experimental example (-1.72 MHz), but it can be seen that there was almost no change in the vibration amplitude during resonant operation. Since electrostatic actuation generates an electrostatic attraction between the silicon and the single-layer graphene, it is expected that the impedance of the single-layer graphene will have an effect, but in the comparative experiment, no significant change (a change that can be quantified) occurred.

[0072] <Summary of the experiment> According to the above experimental results, the change in impedance when a current is passed through single-layer graphene becomes significant when the movable membrane is vibrated by excitation (excitation) using Joule heat. As shown in the above experimental results, a configuration that allows the change in resonant frequency and the fluctuation in vibration amplitude during resonant operation to be observed simultaneously can eliminate measurement errors caused by impurities.

[0073] To confirm the adsorption of SARS-CoV-2 to the movable membrane used in the experiment (i.e., the movable membrane with a reduced resonant frequency), we performed SEM observations of the area around the movable membrane. Figure 10 shows the SEM image. As can be seen from this image, numerous particles measuring several tens of nanometers can be seen on the surface of the movable membrane. These particles were smaller than the molecular size of SARS-CoV-2 (100-200 nm in diameter). Therefore, although they were not whole viruses, they were determined to be SARS-CoV-2 spike proteins. The reason they were not whole viruses is that the solution used in the experiment contained a mixture of whole viruses and isolated spike proteins surrounding them. The extremely large number of spike proteins likely led to the observed attachment of spike proteins. In any case, it is clear that these adsorptions contribute to the shift in resonant frequency, and therefore the presence and extent of the virus can be confirmed.

[0074] <Summary> Although the embodiments and examples of the present invention have been described above, the present invention is not intended to be limited to these embodiments, etc. Therefore, each component in the above-described embodiments, etc. may be modified, or other components may be added to the configuration.

[0075] For example, in the above embodiments and examples, the case where a virus and a protein (impurity) are adsorbed has been described as a standard (however, the experiments were conducted only on viruses), but the object to be detected is not limited to the contents of the embodiments, etc., and it is also possible to distinguish between antigens that are specifically adsorbed by antigen-antibody reactions and impurities that are non-specifically adsorbed. In this case, by modifying the surface of single-layer graphene with a receptor (antibody) as a specific molecule, it is possible to specifically adsorb the antigen.

[0076] While the blocking agent modification method described above involves adsorbing the blocking agent onto a crosslinker, another method involves directly adsorbing the blocking agent onto the graphene surface. As mentioned above, the crosslinker preferably uses PBSE, which contains pyrene groups that can act as adsorbents for aptamers (nucleic acid molecules). However, since PBSE π-bonds the pyrene groups to graphene and amide-bonds the succinimidyl ester groups to the amino groups at the ends of nucleic acid molecules, it is possible that regions where the pyrene groups do not bond with graphene may be created. Therefore, direct adsorption of the blocking agent onto graphene can prevent substances other than the target virus from adhering to the graphene. Therefore, depending on the degree to which the graphene surface can be modified by PBSE, the blocking agent may be adsorbed onto both PBSE and graphene.

[0077] In either case, polyethylene glycol (PEG) can be used as a blocking agent. When adsorbing PEG to the graphene surface, pyrene PEG (Pyrene PEG) is used, which has a pyrene group and forms a π bond with the graphene. When adsorbing PEG to PBSE, amino PEG (Amino PEG) is used, which has an amino group and forms an amide bond with the succinimidyl ester group. The timing of adsorption of the blocking agent is after the aptamer is conjugated to the crosslinker. The two types of PEG can be modified simultaneously or sequentially. That is, for most PBSE used as a crosslinker, when the succinimidyl ester group is conjugated to the amino group of the nucleic acid molecule, pyrene PEG will bind to the graphene surface. On the other hand, when there is PBSE without nucleic acid molecules bound to it, amino PEG will adsorb to that PBSE. Therefore, by adsorbing PEG to areas of the movable membrane that can adsorb other substances, the entire membrane can be modified with the two types of PEG. Thus, depending on the degree (range) of adsorption of the cross-linking agent onto the graphene surface, the adsorption state of the blocking agent may be a state in which the blocking agent is adsorbed onto either the graphene surface or the cross-linking agent, but this is not a uniformly selected state, and both may be formed in a superimposed state, and in either of such states, the purpose of excluding adsorption of other substances can be achieved. [Industrial Applicability]

[0078] The status detection sensor of the present invention can distinguish between detection targets and impurities and can be used, for example, as a border control measure to prevent the entry of specific viruses. It can be installed in seats on aircraft, ships, etc., and measure the mass and quantity of detection targets in the environment to determine whether or not a person should enter the country. Furthermore, by testing individual breath samples, it is possible to measure the mass and quantity of specific viruses contained in the breath. When measuring the mass of specific viruses in such breath samples, the sensor can be used as a testing device at the entrances of buildings or rooms where many people gather. When implemented in a personal smartphone, the measurement results can also be used to create hazard maps. [Explanation of symbols]

[0079] 1. Silicon substrate 2. Single-layer graphene 3 Cavity 4,5 electrodes 6 AC power supply 7 Specific molecules 8. Substances to be detected 9. Contaminants 11 Board body 12 Silicon oxide film 20 Movable membrane 60 Function Generator 61 Impedance Measuring Instrument 62 Processing equipment 63 Probe laser oscillator 64 photodiodes 65 Spectrum Analyzer A Status detection sensor

Claims

1. A sensor includes a substrate having a cavity formed thereon, an opening of the cavity being closed with graphene to form a movable membrane having a bridge structure of the graphene, and the sensor detects the state of a substance attached to the movable membrane by a change in state when the movable membrane is vibrated, a vibration state detection means for detecting a vibration state of the movable film, an adsorbent having an adsorption ability for specific molecules is supported on at least a surface of the graphene in an area where the movable film is formed, and specific molecules having a binding ability with a detection target substance are adsorbed to the adsorbent; the graphene is connected to a power source for applying current to an area where the movable film is formed, A sensor for detecting the state of a substance to be detected, characterized in that the movable membrane is vibrated by alternately heating and cooling the graphene through the generation and cessation of Joule heat associated with the periodic application of electricity to the graphene, while the vibration state detection means measures the resonant frequency of the movable membrane and the vibration amplitude during resonant operation.

2. the vibration state detection means is an impedance measurement means that measures the impedance of the graphene when a current is applied to the graphene, 2. The sensor for detecting the state of a detection target substance according to claim 1, wherein the impedance measured by the impedance measuring means is converted into a resonance frequency and a vibration amplitude during resonance operation when the movable membrane vibrates.

3. The state detection sensor for a substance to be detected as described in claim 2, wherein a blocking agent that inhibits adsorption of substances other than the substance to be detected is adsorbed on either the surface of the graphene or the surface of the adsorbent, or both.

4. 4. The sensor for detecting the state of a substance to be detected according to claim 3, wherein the specific molecule is a nucleic acid molecule that specifically adsorbs a spike protein of SARS-CoV-2, and the substance to be detected is SARS-CoV-2.

5. A mass measurement device for a detection target substance that uses the state detection sensor for a detection target substance according to claim 2 to measure the mass of the detection target substance, a power supply device that periodically applies and stops current from a constant voltage power supply or voltage from a constant current power supply to the movable film; amplitude detection means that detects the vibration amplitude of the movable film; determination means that determines a resonance frequency based on the detected amplitude; impedance detection means that detects the impedance of the graphene; and processing means that calculates the mass of the detection target substance from the detected value of the impedance of the graphene and fluctuations in the resonance frequency of the movable film, The processing means calculates the total mass of the substance attached to the movable membrane based on the rate of change in the resonant frequency of the movable membrane before and after placing the sensor in the environment to be measured, calculates the total quantity of the substance attached to the movable membrane based on the change in impedance of the graphene before and after placing the sensor in the environment to be measured or the change in vibration amplitude during resonant operation of the movable membrane, and calculates the total mass of the substance to be detected from the change in resonant frequency due only to the substance to be detected based on the correlation between the total mass and total quantity of the substance attached to the movable membrane.

6. 3. A quantity measuring device for a detection target substance that uses the state detection sensor for a detection target substance according to claim 2 to measure the quantity of the detection target substance, a power supply device that periodically applies and stops current from a constant voltage power supply or voltage from a constant current power supply to the movable film; amplitude detection means that detects the vibration amplitude of the movable film; determination means that determines a resonance frequency based on the detected amplitude; impedance detection means that detects the impedance of the graphene; and processing means that calculates the quantity of the substance adhered to the movable film from the detected value of the impedance of the graphene and fluctuations in the resonance frequency of the movable film, The processing means calculates the total mass of the substance attached to the movable membrane based on the rate of change in the resonant frequency of the movable membrane before and after placing the sensor in the environment to be measured, and calculates the total quantity of the substance attached to the movable membrane based on the change in impedance of the graphene or the change in vibration amplitude during resonant operation of the movable membrane before and after placing the sensor in the environment to be measured, and derives the amount of change in impedance of the graphene or the amount of change in vibration amplitude during resonant operation of the movable membrane based on the correlation between the total mass and total quantity of the substance attached to the movable membrane, thereby calculating the quantity of the substance to be detected from the amount of change in impedance of the graphene due to only the substance to be detected or the amount of change in vibration amplitude during resonant operation of the movable membrane.

7. A multimodal sensing device that uses the state detection sensor for a detection target substance according to claim 2 to measure the quantity and mass of the detection target substance, a power supply device that periodically applies and stops current from a constant voltage power supply or voltage from a constant current power supply to the movable film; amplitude detection means that detects the vibration amplitude of the movable film; determination means that determines a resonance frequency based on the detected amplitude; impedance detection means that detects the impedance of the graphene; and processing means that calculates the quantity and mass of the substance attached to the movable film from the detected value of the impedance of the graphene and fluctuations in the resonance frequency of the movable film, a processing means for calculating the total mass of a substance adhered to the movable membrane based on a rate of change in the resonant frequency of the movable membrane before and after placing the sensor in the environment to be measured, and for calculating the total quantity of a substance adhered to the movable membrane based on a change in the impedance of the graphene before and after placing the sensor in the environment to be measured, and for deriving the amount of change in the resonant frequency due to only the substance to be detected and the amount of change in the impedance of the graphene due to only the substance to be detected or the amount of change in the vibration amplitude during resonant operation of the movable membrane based on the correlation between the total mass and the total quantity of the substance adhered to the movable membrane, thereby calculating the total mass of the substance to be detected from the amount of change in the resonant frequency due to only the substance to be detected, and for calculating the quantity of the substance to be detected from the amount of change in the impedance of the graphene due to only the substance to be detected or the amount of change in the vibration amplitude during resonant operation of the movable membrane.

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