Virus mass detection sensor and measuring device

Abstract

To provide an inspection device capable of detecting viruses suspended in air in an aerosol state, especially, a mass detection sensor and a measuring device.SOLUTION: A mass detection sensor forms a cavity on a substrate, forms a movable film in a bridge structure of graphene by opening an opening part of the cavity with the graphene, and detects the mass of a substance sticking on the movable film from variation in resonance frequency when the movable film is vibrated. The mass detection sensor also comprises an adsorbate which is carried on the surface of the graphene at least within a range where the movable film is formed and has adsorption ability for nucleic acid molecules, and aptamer which is adsorbed by the adsorbate and has bonding ability for specific viruses. A measuring device comprises excitation means which uses the mass detection sensor to vibrate the movable film, amplitude detection means which detects an amplitude of the movable film which is vibrating, and determination means which determines a resonance frequency from the detected amplitude.SELECTED DRAWING: Figure 2

Description

【Technical Field】 【0001】 The present invention relates to a sensor for detecting an increase in mass due to virus attachment and a measuring device using this sensor. 【Background Art】 【0002】 Since the beginning of 2020, the novel coronavirus disease (COVID-19) that has started to spread has caused a global pandemic, and as of 2022, it has repeated mutations and shows no sign of convergence, leading to situations such as the crisis of medical collapse and the stagnation of economic activities. In such a situation, in order to promote economic activities, the development of sensing devices that can continuously monitor SARS-CoV-2 (coronavirus) in the environment and high-throughput diagnostic and testing technologies is required. At present, the PCR method is exclusively used for the detection of SARS-CoV-2 (coronavirus). However, this PCR method has a problem in that it is difficult to detect SARS-CoV-2 simply and in a short time because the process of extracting and amplifying the RNA in the envelope of the virus is essential. On the other hand, as a simple test method, the antigen test method or the antibody test method is used. However, the antigen test method also targets antigens in the envelope, and the antibody test method measures antibodies after infection. Therefore, it does not directly measure SARS-CoV-2 present in the environment before infection. 【0003】 Therefore, as a small sensor for detecting the virus itself, a biosensor using an ISFET applying FET technology has been studied. For example, focusing on the conductivity that changes in response to the charge of proteins, viruses, etc. that are in contact with the surface of graphene, which exhibits very high electron mobility, a FET-type biosensor that electrically detects a target has been studied (see Patent Documents 1 to 3 and Non-Patent Document 1). In addition, it has been reported that a virus was detected by immobilizing an antibody that specifically adsorbs the spike protein of SARS-CoV-2 on a graphene channel (see Non-Patent Document 2). 【0004】 However, electrical biosensors have the problem of Debye shielding, which limits the measurement range, and it is difficult to detect biomolecules larger than 10 nm under physiological salt concentrations. In contrast, cross-linked graphene that is free-standing from the substrate (hereinafter sometimes simply referred to as "cross-linked graphene") does not suffer from electron scattering by optical phonons of the substrate, and therefore has a higher carrier mobility (200,000 cm²) than graphene fixed to the substrate. 2 It exhibits a (Vs) ratio, and it is anticipated that highly sensitive sensing can be achieved by using thin, lightweight cross-linked graphene. In fact, it has been reported that a single molecule of carbon dioxide has been detected on cross-linked graphene (see Non-Patent Document 3). 【0005】 However, according to the above-mentioned reports, the changes in physical properties when molecules are physically adsorbed onto crosslinked graphene were measured, and the molecules to be measured were not selectively detected. Therefore, the inventors of the present invention have developed a technique to provide crosslinked graphene on a cavity by forming a cavity in a substrate and sealing the opening with a graphene film (see Non-Patent Document 4), and further developed a technique to enable selective detection of molecules based on the deformation of the graphene film that occurs when molecules are adsorbed, by providing a device on the crosslinked graphene with a substance that has molecular adsorption ability (see Non-Patent Document 5). 【0006】 The above technology uses an antibody as a substance with molecular adsorption capacity, and enables the adsorption of an antigen onto cross-linked graphene that has been chemically functionalized by this antibody (utilizing an antigen-antibody reaction). The static deformation of the film due to the cross-linked graphene at this time was measured by surface stress measurement. [Prior art documents] [Patent Documents] 【0007】 [Patent Document 1] Japanese Patent Publication No. 2016-047777 [Patent Document 2] International Publication No. WO2020 / 170799 【Patent document 3】 Special Publication No. 2021-076529 【Non-licensed literature】 【0008】 【Non-licensed literature 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-licensed document 2] G.Seo, G.Lee, M.Jeong, S.-H.Beak, M.Choi, KBKu, C.-S.Lee, S.Jun, D.Park, HGKim, S.-J.Kim, J.Lee, BTKim, ECPark, SIKim, “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-licensed document 3] J.Sun, M.Muruganathan, H.Mizutani, “Room temperature detection of individual molecular physisorption using suspended bilayer graphene”, Sci.Adv.2, e1501518 (2016) 【Non-licensed Document 4】 K.Takahashi, H.Ishida, 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 5] S.Kidane, H.Ishida, K.Sawada, 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 6] Shin Kishide, Kazuaki Sawada, and Kazuhiro Takahashi, "Ultra-high sensitivity optical interference type multimodal biosensor using cross-linked graphene," 12th Symposium on Integrated MEMS, October 26-28, 2020, online, 28A3-AP-1 [Non-Patent Document 7] 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 8] Ken Niino, Amilun Ksaini, Junpei Uesaka, Eriko Furusawa, Yongjun Choi, Tatsuro Goda, Yuji Miyahara, Toshihiko Noda, Kazuaki Sawada, Kazuhiro Takahashi, "Detection of Influenza Virus in Droplets using a Graphene Resonance Sensor," The 82nd Autumn Meeting of the Japan Society of Applied Physics, September 10-13, 2021, Online, 13p-N322-8 [Non-Patent Document 9] Y.Song, J.Song, X.Wei, M.Huang, M.Sun, L.Zhu, B.Lin, H.shen, Z.Zhu, C.Yang, “Discovery of aptamers targeting the receptor-binding domain of the SARS-COV-2 spike gilcoprotein”, Anal.Chem.92, pp.9895-9900 (2020) [Overview of the Initiative] [Problems that the invention aims to solve] 【0009】 The technique disclosed in Non-Patent Document 5, mentioned above, is based on surface stress measurement. However, this surface stress measurement measures the amount of film deformation, which changes depending on the charge of the antigen molecule. Therefore, while it can measure changes in concentration, it could not quantify the actual number of adsorbed molecules. Accordingly, the inventors of the present invention discovered that when cross-linked graphene is vibrated, the resonance frequency changes according to the mass of the adsorbed substance on the cross-linked graphene, and developed a resonance mass measurement method that detects this resonance frequency shift. Using this resonance mass measurement method, they demonstrated the possibility of measuring the mass of antigens adsorbed by antigen-antibody reactions (Non-Patent Document 6) and the possibility of measuring the mass of adsorbed human influenza virus (Non-Patent Documents 7 and 8). In these demonstration experiments, antibodies and sialycoglycans were used as receptors, and the specific adsorption of proteins or human influenza virus onto cross-linked graphene was also demonstrated. 【0010】 Incidentally, in the antigen mass measurement described above, a liquid reagent of an appropriate concentration was dropped onto cross-linked graphene to simulate antigen detection in bodily fluids. In the human influenza virus mass measurement, 1.6 HAU (Hemagglutination Units) of virus was forcibly modified by spraying to simulate droplet infection, and then the resonant frequency was measured. 【0011】 However, it has been reported that SARS-CoV-2 spreads by floating in the air as an aerosol, and therefore, detecting antigens contained in bodily fluids or viruses contained in droplets is insufficient for detecting SARS-CoV-2. For this reason, there has been a strong demand for a detection device that can prevent viral infection by detecting viruses in the environment, and in particular by detecting the quantitative concentration of the virus. Non-patent document 9 discloses an aptamer that has the property of specifically adsorbing the spike protein of SARS-CoV-2. 【0012】 The present invention has been made in view of the above points, and its object is to provide an inspection device, in particular a mass detection sensor and a measuring device, that can detect viruses floating in the air in the form of aerosols. [Means for solving the problem] 【0013】 Therefore, the present invention relating to a virus mass detection sensor is a mass detection sensor that forms a cavity on a substrate, closes the opening of the cavity with graphene to form a movable membrane with a crosslinked structure of graphene, and detects the mass of a substance attached to the movable membrane by a change in the resonant frequency when the movable membrane is vibrated, characterized in that it comprises an adsorbent substance supported on the surface of graphene in an area that forms the movable membrane and has the ability to adsorb to nucleic acid molecules, and an aptamer that is adsorbed to the adsorbent substance and has the ability to bind to a specific virus. 【0014】 According to the above-described configuration, an aptamer that can bind to a specific virus is adsorbed onto the surface of the movable film made of graphene via an adsorbent. When the aptamer specifically adsorbs (binds) to the specific virus, the resonance frequency of the movable film changes according to the mass of the adsorbed specific virus. By detecting this change in the resonance frequency, the presence of a specific virus in the environment can be detected, and it is also possible to detect the quantitative concentration of the specific virus according to the degree of change in the resonance frequency. Here, since the film thickness of the movable film made of graphene is extremely thin at 0.34 nm, the mass sensitivity during vibration is extremely high, and the analytical value is 10 to 100 yg / Hz. Therefore, even if a small amount of a specific virus floating in the air in the form of an aerosol is adsorbed, it can be detected. 【0015】 In the invention having the above configuration, it is preferable to adsorb a blocking agent that inhibits the adsorption of substances other than the measurement target on either one or both of the surface of the graphene and the surface of the adsorbent. By adsorbing the blocking agent on the surface of the movable film, the adsorption of substances other than the specific virus that can specifically adsorb to the aptamer is excluded, and the change in the resonance frequency of the movable film due to the adsorption of the specific virus as the measurement target can be measured. 【0016】 Also, in the invention of each of the above configurations, it is preferable that the inside of the cavity closed by the graphene is maintained in a reduced-pressure environment. In the case of such a configuration, since the inside of the cavity is in a reduced-pressure environment, even when static deformation (bulging deformation) of the film occurs due to intermolecular stress caused by the adsorption of the aptamer and the specific virus, the bulging deformation of the movable film can be suppressed, and the vibration of the movable film during resonance frequency measurement becomes smooth. Also, for the purpose of eliminating the air resistance to the vibration of the movable film, even when the resonance frequency measurement is performed in a reduced-pressure chamber, the deformation of the movable film due to the pressure difference can be suppressed. 【0017】 Furthermore, in the inventions of the above-described respective configurations, the aptamer may be a nucleic acid molecule that specifically adsorbs the spike protein of SARS-CoV-2, and the particular virus may be SARS-CoV-2. 【0018】 According to the above-mentioned configuration, the attachment of SARS-CoV-2 to the movable membrane can be achieved by specifically adsorbing the spike protein of the SARS-CoV-2, and it becomes possible to capture the SARS-CoV-2 in the form of aerosol floating in the air when it comes into contact with the aptamer. And, as described above, since the movable membrane made of graphene has extremely high mass sensitivity during vibration, even when a small amount of SARS-CoV-2 floating in the air in the form of aerosol is adsorbed, the change in the resonance frequency can be measured. 【0019】 On the other hand, the invention related to the virus measuring device is a measuring device that uses any of the mass detection sensors of the above-described respective configurations to detect aerosolized viruses, and includes the mass detection sensor, vibration means for vibrating the movable membrane, amplitude detection means for detecting the amplitude of the movable membrane when vibrating, and determination means for determining the resonance frequency from the detected amplitude. 【0020】 According to the above configuration, by vibrating the movable membrane constituting the mass detection sensor at different frequencies by the vibration means and determining the resonance frequency based on the amplitude detected by the amplitude detection means, it becomes possible to measure the change in the resonance frequency of the movable membrane. When this resonance frequency changes, it is possible to detect the presence of the virus, and it is also possible to measure (mass measurement) the quantitative concentration of the virus according to the degree of change in the resonance frequency. 【0021】 Here, as the vibration means, in addition to the photoexcitation method such as thermal drive by light irradiation, it can be by an electrical method such as Joule heat drive or electrostatic drive. As the amplitude detection means, in addition to the method of measuring by the change in interference color due to the change in the gap between the cavity and the movable membrane, there is an electrical detection method by dynamic impedance measurement. 【Effect of the Invention】 【0022】 According to the present invention relating to a mass detection sensor, when a virus floating in the air in the form of an aerosol comes into contact with a movable membrane, the virus can be captured, and the presence or absence of the virus can be determined by detecting the resonant frequency in that state. Depending on the degree of change in the resonant frequency at this time, it is possible to appropriately detect the mass (quantitative concentration) of the virus. 【0023】 On the other hand, according to the present invention relating to a virus measuring device, since a mass detection sensor as described above is used, it is possible to detect viruses and measure their mass, and by providing an excitation means, an amplitude detection means, and a determination means, it can be used as a measuring device capable of measuring the mass of viruses. [Brief explanation of the drawing] 【0024】 [Figure 1] This is an explanatory diagram showing an embodiment of a mass detection sensor. [Figure 2] This is an explanatory diagram showing the state of the movable membrane of a mass detection sensor. [Figure 3] This is an explanatory diagram illustrating a vibrator and a frequency analyzer. [Figure 4] This is an explanatory diagram showing other examples of vibrators and frequency analyzers. [Figure 5] This is an explanatory diagram showing the procedure for fabricating a mass detection sensor. [Figure 6] This is a graph showing the experimental results. [Modes for carrying out the invention] 【0025】 Hereinafter, embodiments of the present invention will be described based on the drawings. 【0026】 <Mass detection sensor> Figure 1 shows an embodiment of a virus mass detection sensor. As shown in Figure 1(a), the mass detection sensor A of this embodiment has a configuration in which a single layer of graphene is bonded to the surface of a silicon substrate 1. This silicon substrate 1 is formed by thermally oxidizing the surface of the substrate body 11 to form a silicon oxide film 12, and then partially forming a cavity 3 by reactive ion etching (RIE). The single layer of graphene 2 is laminated in the region of the silicon substrate 1 that includes the portion (opening) 30 where the cavity 3 opens, and the opening 30 is closed, sealing the cavity 3. In this state, a crosslinked structure is formed in the single layer of graphene 2, and the portion with this crosslinked structure functions as a vibrating movable membrane. The shape of the cavity 3 can be rectangular in plan view, circular, or other shapes, but considering vibration as described later, a circular or elliptical shape that does not form a vertex at the boundary of the crosslinked structure is preferred. 【0027】 On the surface of the cross-linked graphene structure (cross-linked graphene) configured in this way, an adsorbent material capable of adsorbing specific nucleic acid molecules (aptamers) is supported. This adsorbent material can be applied before bonding the single-layer graphene or after bonding. The adsorbent material is selected depending on the aptamer to be adsorbed. The adsorbent material may be supported over the entire surface of the single-layer graphene, but it should be supported at least in the region containing the cross-linked graphene (movable membrane). 【0028】 For example, as shown in Figure 1(b), when an adsorbent is specifically supported on a cross-linked graphene (movable membrane) 20 of a single-layer graphene, the aptamer 4 can be adsorbed onto the surface of the movable membrane. After adsorbing the aptamer 4, further adsorption of a blocking agent 5 can be made to eliminate the adsorption of substances other than the specific virus that can be specifically adsorbed between the aptamer 4 and the blocking agent 5. The adsorption state of the blocking agent 5 can be either adsorbed onto the cross-linking agent or adsorbed onto the surface of the graphene 2, and it may be either one or both. The key point is to stabilize the detection accuracy by preventing the attachment of substances other than the target virus 6 to the movable membrane, and the specific adsorption situation of the virus 6 does not change depending on the adsorption state of the blocking agent. As a result, only the virus 6 can be adsorbed by contact with the virus 6 floating in the environment in aerosol form. 【0029】 Furthermore, by keeping the inside of cavity 3 under reduced pressure (approximately -1 atmosphere gauge pressure) while it is sealed with single-layer graphene, deformation (bulging) of the movable membrane 20 due to intermolecular stress during the adsorption of aptamer 4, blocking agent 5, and virus 6 can be suppressed. 【0030】 <Principle of Mass Detection> As shown in Figure 2, the mass detection sensor A has the above configuration, so the movable film 20 of the single-layer graphene 2, which has a cross-linked structure due to the formation of the cavity 3, can be vibrated within the deformable range inherent in the graphene itself. At this time, the natural frequency of the movable film 20 changes according to the total mass of the movable film 20, so by detecting the resonant frequency when vibrating, the change in the total mass of the movable film 20 can be detected. 【0031】 In other words, as shown in Figure 2(a), by pre-measuring the resonance frequency of the movable membrane 20 when it is vibrated in a state where it can specifically adsorb to the specific virus 6 (pre-adsorption state), and then measuring the resonance frequency of the movable membrane 20 again after observation, it becomes possible to detect the presence and mass of the specific virus based on the difference. 【0032】 For example, when a mass detection sensor A is placed in a predetermined environment for a certain period of time, and the resonance frequency is measured after the single-layer graphene surface is exposed to the air in that environment, as shown in Figure 2(b), if a specific virus 6 is adsorbed onto the aptamer 4, it will resonate at a different frequency. Therefore, the virus 6 can be detected based on the difference in the resonance frequency. Furthermore, since the resonance frequency naturally differs depending on the amount (mass) of adsorbed virus 6, the mass can be calculated based on that resonance frequency. 【0033】 Here, the mass of virus 6 adsorbed onto the movable membrane can be calculated using the following formula. 【number】 【0034】 Therefore, for example, if the resonant frequency (f0) before adsorption is 6.88 MHz and the resonant frequency during measurement is 5.54 MHz, the frequency shift (Δf) of the resonant frequency will be -1.34 MHz. Here, if the mass of the movable membrane 20 is 45.5 fg, the mass of the adsorbed substance (specific virus) can be calculated to be 17.7 fg. 【0035】 Furthermore, when a substance is adsorbed onto the movable membrane, the mass sensitivity S m This can be expressed by the following equation. Therefore, by constructing the movable membrane from single-layer graphene, the weight of the movable membrane can be reduced, and the mass sensitivity can be improved. 【0036】 【number】 【0037】 <Excitation means, amplitude detection means, and determination means> As described above, if the movable membrane 20 can be vibrated and the resonant frequency can be measured, the mass of the adsorbed substance (specific virus) can be detected using the above formula. Therefore, an exciter (excitation means) and a resonant frequency analysis device (amplitude detection means and determination means) will be explained as examples. The exciter can be made to vibrate by heating the movable membrane 20 by periodically applying Joule heat or periodically irradiating it with an excitation laser, and utilizing the thermal contraction of graphene. That is, by periodically heating the graphene, it can be excited by repeatedly causing contraction and relaxation according to the frequency. 【0038】 Figure 3 illustrates an electrical vibrator and frequency analyzer. The vibrator shown in Figure 3(a) applies an AC power supply to the single-layer graphene 2 while adjusting the frequency of the output voltage using a function generator 7. By applying voltage to the graphene while changing the frequency, the amount of current flowing through the single-layer graphene 2 also changes periodically, causing the Joule heat generated at this time to fluctuate periodically (ON / OFF). In response to this periodic change in Joule heat, the movable membrane 20 repeatedly contracts and relaxes, thereby inducing vibration. 【0039】 On the other hand, the resonant frequency analysis device consists of an impedance measuring instrument 8 that measures the impedance of the current flowing through the aforementioned single-layer graphene 2. For the alternating current whose frequency has been adjusted by the function generator 7, the impedance measuring instrument 8 detects the momentary impedance (impedance in the resonant state) from the state in which the impedance measured by the impedance measuring instrument 8 changes significantly. The resonant frequency is analyzed by outputting the phase and amplitude when the momentary impedance is detected. Furthermore, depending on the magnitude of the impedance when the momentary impedance is detected, it can be converted into the amplitude when the movable diaphragm 20 vibrates. 【0040】 Furthermore, in addition to utilizing the thermal contraction of graphene due to Joule heating as an electrical excitation method, electrostatic drive is also possible. For example, as shown in Figure 3(b), the cavity 3 formed in the silicon substrate 1 is used as a gap, and an electrostatic attraction is applied between the silicon and the single-layer graphene 2, driving the movable film 20 like an electrostatic actuator to induce vibration. In this case, an AC voltage with a frequency adjusted by the impedance meter 8 can be applied, and at the same time, the change in impedance is measured by the impedance meter 8, and the resonant frequency is measured by identifying the motional impedance. 【0041】 On the other hand, optical methods can also be used. For example, Figure 4(a) shows a resonant frequency analysis device that is an optical device. That is, the vibrator is an alternating current that is supplied to the movable film 20 by Joule heating, and an example of a configuration in which the amplitude is detected optically is shown. Optical amplitude detection is performed by irradiating the movable film 20 with a CW laser as a probe laser and detecting the reflected light. The probe laser is a laser beam of a specific wavelength (e.g., 638 nm) that is continuously oscillated from a probe laser oscillator 81 and irradiated onto the movable film 20, and the reflected light is input to a spectrum analyzer 83 via a photodiode 82, and the frequency and amplitude are detected by the change and degree of the wavelength of the reflected light. 【0042】 Furthermore, when employing an optical method for the excitation of the movable membrane 20, a photo-excitation measurement method using an excitation laser can be used, as shown in Figure 4(b). The excitation laser is an excitation laser of a specific wavelength (e.g., 405 nm) continuously oscillated by a laser oscillator 71 onto the movable membrane 20. Vibration detection is performed using a probe laser, where a laser beam of a specific wavelength (e.g., 638 nm) continuously oscillated from a probe laser oscillator 81 is used to detect the frequency and amplitude of the reflected light using a spectrum analyzer 83. Therefore, in order to distinguish the excitation laser from the probe laser, a laser with a wavelength sufficiently outside the range expected to change due to the vibration of the movable membrane 20 irradiated by the probe laser is selected. In addition, a bandpass filter 84 is provided immediately before the photodiode 82 in order to input only the reflected light of the probe laser to the spectrum analyzer 83. 【0043】 In any of the above embodiments, the resonant frequency of the movable membrane 20 can be detected by vibrating the movable membrane 20 while changing its frequency and by detecting the state (amplitude) of the vibration. Therefore, by using a virus mass detection sensor and combining the above-described excitation device and frequency measuring device, an embodiment of the virus measuring device of the present invention is constructed. 【0044】 When detecting the resonant frequency of the movable membrane 20, simple measurements can be performed under atmospheric pressure, but for precise measurements, it is preferable to perform them under reduced pressure (for example, in a vacuum chamber) to eliminate subtle air resistance. In this case, a simple measurement device can be provided with the vibrator and resonant frequency analyzer integrated with the mass detection sensor, while a precise measurement device can be configured to have the vibrator and resonant frequency analyzer installed in a vacuum chamber, with the mass detection sensor installed at a predetermined position within the vacuum chamber. [Examples] 【0045】 The virus mass detection sensor was fabricated as follows: As shown in Figure 5, a silicon substrate 1 and a single-layer graphene 2 were fabricated separately and then bonded together. The graphene was grown by CVD on the surface of a catalyst copper foil Cu, as shown in Figures 5(a) to (d). To remove the copper foil Cu from the single-layer graphene 2, polymethyl methacrylate resin (PMMA) was spin-coated onto the surface of the graphene 2 as a support film, and polydimethylsiloxane (PDMS) was pressed onto the PMMA and the outer periphery of the graphene. While supporting the PMMA and graphene with this PDMS, the copper foil Cu was immersed in an etching solution (Etch) with FeCl3 to remove the copper foil Cu. 【0046】 On the other hand, as shown in Figures 5(e) to (h), a silicon oxide film 12 was formed on the upper surface of the silicon body 11 by thermal oxidation, a resist Res was transferred to areas other than the region where the cavity was to be formed, and then a circular cavity with a diameter of 8 μm and a depth of 1 to 2 μm was formed by reactive ion etching (RIE). 【0047】 As described above, the individually fabricated single-layer graphene and silicon substrate 1 were bonded to the surface of the silicon substrate 1 on the side where the cavity was formed, as shown in Figures 5(i) and (j). The substrate was then heated under reduced pressure to a temperature above the glass transition point of PMMA (approximately 125°C) to soften the PMMA and cause the graphene to adhere closely to the silicon substrate 1. Subsequently, the excess PMMA was removed and the PDMS was peeled off to obtain a laminate of silicon substrate 1 and single-layer graphene. The single-layer graphene obtained a structure with a cross-linked structure having a gap due to the cavity of the substrate 1. Furthermore, by performing the heat treatment under reduced pressure (gauge pressure of -1 atmosphere), the inside of the cavity was maintained in a reduced pressure state, improving the adhesion with the graphene. 【0048】 The composite of substrate 1 and graphene 2, fabricated in this manner, is subjected to chemical treatment to generate virus adsorption capacity. The surface of the single-layer graphene is modified by π-stacking with succinimidyl 1-pyrenebutanoate (PBSE), which has a pyrene group, as an adsorbent for nucleic acid molecules (aptamers). The pyrene group of PBSE is π-bonded to graphene, and the succinimidyl ester group on the opposite side reacts with the amino group at the end of the nucleic acid molecule to form an amide bond. Therefore, the PBSE modified on the surface of the single-layer graphene functions as a crosslinking agent, allowing nucleic acid molecules (aptamers) to be immobilized on the surface of the single-layer graphene. 【0049】 The nucleic acid molecules (aptamers) used here have the following structure: 5'-CAGCACCGACCTTGTGCTTTGGGAGTGCTGGTCCAAGGGCGTTAATGGACA-3' (51 base pairs) This aptamer is disclosed in the aforementioned Non-Patent Document 9 and has the property of specifically adsorbing the spike protein of SARS-CoV-2. 【0050】 As described above, a mass detection sensor was constructed by immobilizing an aptamer capable of specifically adsorbing the spike protein of SARS-CoV-2 onto the surface of cross-linked graphene. 【0051】 Next, as the vibrator, a photo-excitation measurement method using a photo-excitation laser was employed. As the frequency analysis device, a probe laser oscillator, a photodiode, and a spectrum analyzer were used. The probe laser oscillator continuously emitted laser light with a wavelength of 638 nm, and the photodiode and spectrum analyzer detected the vibration frequency and amplitude of the movable membrane from changes in reflected light. 【0052】 <Example of experiment> For the mass detection sensor with the above configuration, resonance frequency analysis was performed using the vibrator and frequency analysis device described above. Specifically, experiments were conducted to measure the resonance frequency of a movable membrane made only of crosslinked graphene, the resonance frequency in a state where the aptamer was immobilized via a crosslinking agent, and the resonance frequency in a state where spike proteins from inactivated SARS-CoV-2 were specifically adsorbed. 【0053】 First, as a preliminary experiment, a mass detection sensor was immersed in a solution of inactivated SARS-CoV-2, and the resonance frequency after drying was measured. PBSE was used as the crosslinking agent, and the sensor was modified using a 1 ng / mL solution containing 1% nonionic surfactant (Tween® 20) for a processing time of 5 minutes. Furthermore, 0.1 mL of an aptamer (5 μM) with the aforementioned specific adsorption capacity and consisting of 51 bases was used. The aptamer was adjusted to pH 8.5 using 100 mM trishydroxymethylaminomethane (tris) as a buffer, and modified for a processing time of 60 minutes. The target SARS-CoV-2 was 10 4 A 1.5 mL solution containing the viral load of a copy was used. 【0054】 Figure 6(a) shows the change in resonance frequency for each state at this time. As is clear from these detection results, the resonance frequency when only crosslinked graphene was vibrated was around 10.9 MHz, but in the state modified with the crosslinking agent and aptamer, the resonance frequency decreased to around 8.5 MHz. This is thought to be due to the increase in the mass of the movable membrane due to the adsorption of the crosslinking agent and aptamer. Furthermore, after immersion in a SARS-CoV-2 solution (after drying), the resonance frequency shifted to around 7.9 MHz. This is because SARS-CoV-2 was specifically adsorbed onto the aptamer, increasing the mass of the movable membrane, or in other words, the sensor response due to the adsorption of the virus onto the aptamer was obtained. 【0055】 If the initial mass of the mobile membrane made only of graphene is 45.5 fg, the mass of the adsorbed virus can be calculated based on the above formula (Equation 1). First, the mass increased by the adsorption of the crosslinking agent and aptamer is calculated to be 20.0 fg (-2 × 45.5 × (-2.4 / 10.9)). Then, using the mobile membrane with the added mass of the crosslinking agent and aptamer, the mass of the virus is further calculated from the change in resonance frequency before and after virus adsorption to be 9.2 fg (-2 × 65.5 × (-0.6 / 8.5)). 【0056】 In this way, the amount of virus specifically adsorbed onto the surface of the movable membrane made of graphene can be obtained as mass. Furthermore, in the calculation formula shown in equation (Equation 1) above, if the values ​​of the resonance frequency and mass in the state in which graphene is modified with a crosslinking agent and an aptamer are known in advance, then equation (Equation 1) becomes a mass conversion formula in which only the resonance frequency after virus adsorption is the variable. Thus, it was found that if the resonance frequency is detected, the amount of virus (mass) can be easily obtained. 【0057】 Therefore, as the main test, we experimented to see whether it was possible to detect the virus in aerosol form using the mass detection sensor described above. The mass detection sensor used in the experiment was the same as in the preliminary experiment, and the crosslinking agent and aptamer were modified under the same conditions. In this state, a compressor-type sprayer was used to produce sprayed particles of the virus solution with a particle size of approximately 3 μm, and the aerosol solution was supplied to the mass detection sensor, and the change in resonance frequency was observed. 【0058】 Furthermore, since an aerosol is a mist-like droplet with a particle size of 5 μm or less that floats in the air for a while, droplets sprayed by a spraying device capable of supplying a spray particle size of approximately 3 μm are considered aerosols. In addition, the compressor spraying device used in this test had a spraying capacity of 0.35 mL / min or more and a maximum air supply flow rate of approximately 4 L / min. The volume of the sprayed virus solution was 3 mL, and the amount of virus (SARS-CoV-2) contained was 10 4I used the copy function. 【0059】 The experimental results at this time are shown in Figure 6(b). As is clear from these experimental results, a similar resonance frequency shift occurred even in the case of viruses in aerosol form. This revealed that it is possible to detect the mass of viruses in aerosol form floating in the air. Specifically, the resonance frequency when the movable membrane was graphene alone was around 10.85 MHz, but when the crosslinking agent and aptamer were modified, the resonance frequency decreased to 7.3 MHz, and it was clearly discernible that the resonance frequency further decreased to around 5.75 MHz after the sprayed virus solution adhered to it. Similarly, calculating the amount (mass) of virus, the mass of the crosslinking agent and aptamer was 29.8 fg (-2 × 45.5 × (-3.55 / 10.85)), so the mass of the virus was 31.98 fg (-2 × 75.3 × (-1.55 / 7.3)). 【0060】 In the above experiment, no modification of the crosslinking agent with a blocking agent was performed. However, by modifying the crosslinking agent with a blocking agent such as polyethylene glycol, it is possible to prevent the adsorption of substances other than the specific virus to be detected. In this case, the amount (mass) of the virus is detected by comparing it with the resonance frequency when the blocking agent, in addition to the crosslinking agent and aptamer, is adsorbed onto the movable membrane. 【0061】 Methods for modifying the blocking agent include adsorbing the blocking agent onto a crosslinking agent, and directly adsorbing the blocking agent onto the surface of graphene. As mentioned above, it is preferable to use PBSE, which has pyrene groups that can act as adsorbents for aptamers, as the crosslinking agent. 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 conceivable that there may be regions where the pyrene groups do not bond to graphene. Therefore, by directly adsorbing the blocking agent onto graphene, it is possible to prevent substances other than the target virus from adhering to the graphene. Thus, depending on the extent to which the graphene surface can be modified by PBSE, it is even more preferable to adsorb the blocking agent onto both PBSE and graphene. 【0062】 In either case, polyethylene glycol (PEG) can be used as the blocking agent. When adsorbing PEG onto the graphene surface, pyrene PEG, which has a pyrene group, is used to form a π bond with graphene. When adsorbing onto PBSE, amino PEG, which has an amino group, is used to form an amide bond with the succinimidyl ester group. The blocking agent is adsorbed after the aptamer has been bonded to the crosslinking agent. The two types of PEG may be used to modify the graphene simultaneously, or they may be used sequentially and individually. That is, if most of the PBSE used as a crosslinking agent has a succinimidyl ester group bonded to an amino group of a nucleic acid molecule, pyrene PEG will be bonded to the graphene surface. On the other hand, if there is PBSE remaining that does not have a nucleic acid molecule bonded to it, amino PEG will be adsorbed onto that PBSE. Therefore, by adsorbing PEG onto a region on the movable membrane where other substances can be adsorbed, the entire material can be modified by the two types of PEG. Thus, depending on the degree (range) to which the crosslinking agent is adsorbed onto the graphene surface, the adsorption state of the blocking agent may be adsorbed onto either the graphene surface or the crosslinking agent, but this is not uniformly selected; both may be formed in superposition, and in either of these states, the objective of eliminating the adsorption of other substances can be achieved. 【0063】 <How to use> Based on the experimental results above, the mass detection sensor fabricated in this embodiment can capture viruses in aerosol form. For example, when detecting viruses in the environment, the resonant frequency of the movable membrane after modification with the crosslinking agent and aptamer can be detected in advance, and the mass of the movable membrane in that state can be calculated in advance. Then, by exposing at least the movable membrane portion to the air in the environment and identifying the resonant frequency of the movable membrane, the amount (mass) of the virus can be detected. It is preferable to allow time for drying before measuring the resonant frequency in order to remove the moisture used when forming the aerosol. 【0064】 When exposing the sensor to air in such an environment, it may be used by leaving it installed for a long period of time and periodically observing the change in the resonant frequency. Alternatively, for short-term testing, air may be blown onto the movable membrane of the mass detection sensor for a certain period of time (e.g., 1 minute) using a blower or the like in that environment, and after drying it as appropriate, the resonant frequency of the movable membrane may be measured. 【0065】 In this case, if a simple test is performed to determine the presence or absence of a specific virus (e.g., SARS-CoV-2), it is possible to determine that the specific virus is present if a shift in the resonant frequency is observed, and that the virus is not present if no frequency shift is observed. 【0066】 Furthermore, to accurately measure the amount (mass) of the virus, multiple movable membranes may be formed in an array on a single substrate, and the resonant frequency of each of these movable membranes may be detected, and the average value of these frequencies may be calculated to correct for variations in the inspection. Moreover, considering that the formation of the movable membranes may be unsuitable, the amount (mass) of the virus may be calculated using only those movable membranes from the array that show a frequency shift. 【0067】 Furthermore, if multiple mass detection sensors are manufactured simultaneously under the same conditions, or if sensors with more uniform performance can be manufactured under the same conditions, a calibration curve can be prepared in advance. This allows for immediate determination of the amount of virus (mass measurement) by measuring only the resonance frequency for each individual mass detection sensor during virus measurement (assuming the resonance frequencies are the same in the initial state). For simple tests, a threshold frequency can be set at the frequency at which the resonance frequency shifts to determine the presence or absence of the virus. 【0068】 The measuring device that uses the above-mentioned mass detection sensor consists of a vibrator and a frequency analyzer. However, if these devices are designed so that the mass detection sensor can be installed in its designated position, measurement can be performed simply by installing the mass detection sensor. In other words, there is no need to provide a separate vibrator or similar device for each individual mass detection sensor. 【0069】 Therefore, by bringing only the mass detection sensor into the environment to be measured and exposing it to the air in that environment, measurement becomes possible. For example, if one set of vibrator and frequency analyzer is possessed, tests can be conducted as many times as needed in the same environment, and the amount (mass) of the virus can be measured on the spot. For example, it can be used as a pre-opening test in a restaurant, and also for regular tests after opening. By aggregating these test results and information on the infection situation, it is expected that in the future, data such as the threshold for the amount (mass) of the virus indicating the possibility of infection can be obtained. In environments with low concentrations (low mass) where there is no possibility of infection, it is also expected that economic activities can continue without infection prevention measures. 【0070】 <Variation> While embodiments and examples of the present invention are as described above, these embodiments are merely examples of the present invention and do not mean that the present invention is limited thereto. Therefore, the components of the present invention may be modified or other components may be added. 【0071】 For example, in the above embodiments and examples, SARS-CoV-2 was used as an example of a specific virus, but if the specific virus is an influenza virus, the graphene surface may be modified with a sugar chain probe using a siloa sugar chain. In this case, aminopyrene can be used as the crosslinking agent. 【0072】 Furthermore, while the aforementioned 51-mer nucleic acid molecule was used as an aptamer capable of adsorbing to the SARS-CoV-2 spike protein, other nucleic acid molecules that can specifically adsorb to SARS-CoV-2 may be used. In addition, while Joule heating, electrostatic drive, and excitation laser were exemplified as means of exciting the movable membrane, configurations using other excitation means are also acceptable. [Explanation of symbols] 【0073】 1 circuit board 2 Graphene 3 Cavities 4 Aptamers 5 Blocking agent 6 Viruses 7 Function Generators 8 Impedance Meter 11 Silicon oxide film 12 Silicon substrate body 20. Movable membrane, cross-linked graphene (region with a cross-linked structure) 30 Cavity openings 60 Virus Spike Proteins 71 Laser Oscillator 81 Probe laser oscillator 82 Photodiodes 83. Spectrum Analyzer 84 Bandpass Filter

Claims

[Claim 1] A mass detection sensor for detecting the mass of a virus floating in the air in aerosol form, In a mass detection sensor in which a cavity is formed on a substrate, the opening of the cavity is closed with graphene to form a movable film with a cross-linked graphene structure, and the mass of a substance adhering to the movable film is detected by the change in the resonant frequency when the movable film is vibrated, A virus mass detection sensor comprising: an adsorbent substance supported on the surface of graphene in an area that forms a movable membrane and has the ability to adsorb nucleic acid molecules; an aptamer adsorbed on the adsorbent substance and having the ability to bind to a specific virus; a first blocking agent adsorbed on the adsorbent substance; and a second blocking agent adsorbed on the surface of the graphene. [Claim 2] The adsorbent has a pyrene group and a succinimidyl ester group, wherein the pyrene group is π-bonded to the graphene on the surface of the graphene, The aptamer and the first blocking agent have an amino group and are bonded to the succinimidyl ester group of the adsorbent by an amide bond. The virus mass detection sensor according to claim 1, wherein the second blocking agent has a pyrene group and is π-bonded to the graphene on the surface of the graphene. [Claim 3] The virus mass detection sensor according to claim 1 or 2, wherein the inside of the cavity sealed by the graphene is maintained in a reduced-pressure environment. [Claim 4] The virus mass detection sensor according to any one of claims 1 to 3, wherein the aptamer is a nucleic acid molecule that specifically adsorbs the spike protein of SARS-CoV-2, and the specific virus is SARS-CoV-2. [Claim 5] A measuring device for measuring the amount of aerosolized virus using a mass detection sensor according to any one of claims 1 to 4, A virus measuring device comprising the mass detection sensor, an excitation means for vibrating the movable membrane, an amplitude detection means for detecting the amplitude of the movable membrane when it vibrates, and a determination means for determining the resonant frequency from the detected amplitude.

Images