JP7050878B2 - Detection device and detection method - Google Patents

Description

The embodiment relates to a detection device and a detection method.

Among the influenza A viruses, for example, avian influenza represented by H5N1 subtype has a high mortality rate and is called highly virulent because the infection spreads to organs throughout the body. At present, the H5N1 subtype is not pandemic because it is difficult to infect humans, but it is expected that catastrophic damage will occur if a new influenza that infects humans occurs due to mutation. ..

On the other hand, although the immunochromatography method has been put into practical use for the rapid diagnostic test for influenza, the rapid test technique for distinguishing between strong toxicity and weak toxicity has not yet been established, and at present, PCR (Polymerase Chain Reaction: polymerase) is used. Genetic testing after gene amplification by the (chain reaction) method is required. When a new influenza outbreak occurs, it is expected that the infection will spread rapidly, so it is expected that it will be difficult to stop the spread of the infection without a rapid diagnostic test method.

Further, as a technique for specifically detecting a virus, a method of capturing using a probe molecule that binds to a marker on the virus surface is known, but in this method, impurities are other than the probe molecule and the probe molecule. There is also a problem that it is difficult to distinguish the case where the virus is non-specifically adsorbed to the portion and it is difficult to increase the sensitivity. Furthermore, if there are viruses with surface markers that bind to the same probe molecule, there is also the problem that they cannot be identified.

International Publication No. 2017/1266617

The embodiment provides a detection device and a detection method capable of rapid evaluation of a detection target.

The detection device of the embodiment comprises a sensor element and a probe molecule that associates with a receptor exposed on the surface of the detection target. The probe molecule is immobilized on the sensor element, the detection target comprises a first detection target, and the first detection target is exposed to the surface of the first detection target and cleaved by a specific protease. The sensor element includes the presence or absence of fluctuations in the electrical characteristics of the sensor element due to the supply of the detection target , and the electricity of the sensor element due to the further supply of the protease. Detects the presence or absence of fluctuations in characteristics.

The figure which shows the structure of influenza A virus. The figure which shows the classification of influenza A. The figure which shows the infection mechanism of influenza virus. (A) is a diagram showing the molecular structure of sugar chains expressed in the upper respiratory tract of humans, and (b) is a diagram showing the molecular structure of sugar chains expressed in the upper respiratory tract of birds. (A) and (b) are schematic views showing the deformation of the cleaved HA due to pH. The schematic diagram which shows the structure of a lipid bilayer membrane. The schematic diagram which shows the outline of the detection apparatus of embodiment. The schematic diagram which shows the state of capturing the human infectious influenza virus by using the detection apparatus of embodiment. The schematic diagram which shows the state of detecting the virulent influenza virus using the detection apparatus of embodiment. The schematic diagram which shows the appearance of detecting that the HA of a virus was cleaved by using the detection apparatus of embodiment. The schematic diagram which shows the appearance of detecting the membrane fusion after the opening and subordination of the virus HA by using the detection apparatus of embodiment. The schematic diagram which shows the structure which fixed the reagent with hydrogel on the sensor element in the detection apparatus of embodiment. The schematic diagram which shows the structure which dispersed the reagent in the ionic liquid on the sensor element in the detection apparatus of embodiment. (A) to (h) are diagrams showing the molecular structure of an example of an ionic liquid. The schematic diagram which shows the molecular structure of an organic electrolyte oligomer. The schematic diagram of another example of a sensor element in the detection apparatus of embodiment. The schematic diagram of the structure in which the graphene film floated from the base in the detection device of an embodiment. (A) and (b) are enlarged schematic views of the vicinity of the graphene film in the detection device shown in FIG. The schematic diagram which shows the protection structure of the phospholipid membrane in the detection apparatus of embodiment. The schematic diagram which shows the protection structure of the phospholipid membrane in the detection apparatus of embodiment. The schematic diagram which shows the protection structure of the phospholipid membrane in the detection apparatus of embodiment. The schematic diagram which shows the protection structure of the phospholipid membrane in the detection apparatus of embodiment. (A) is a schematic diagram of a phospholipid membrane formed on a hydrophobic substrate, and (b) is a schematic diagram of a phospholipid membrane formed on a hydrophilic substrate.

Hereinafter, embodiments will be described with reference to the drawings. In each drawing, the same elements are designated by the same reference numerals.

According to the embodiment described below, a detection device and a detection method for detecting a virus as a detection target are provided. In particular, it is possible to quickly distinguish between highly virulent and attenuated influenza viruses and between avian-infected type and human-infected type, and detection that can specifically detect human-infected highly virulent influenza virus that has not yet appeared. Equipment and detection methods are provided.

FIG. 1 is a diagram showing the structure of influenza A virus.

Influenza virus is a vesicle with a diameter of about 100 nm wrapped in a lipid double membrane (surface membrane) called an envelope 31, and has a single-stranded RNA (ribonucleic acid) as a genome inside the envelope 31. Hemagglutinin (HA) and neuraminidase (NA), which are spike proteins (proteins pierced into the membrane), are exposed on the surface of the envelope 31. In the following description, hemagglutinin may be referred to as HA and neuraminidase may be referred to as NA.

Influenza virus has types A, B, and C. Type C infects children, but is less pathogenic and often has no symptoms. In addition, type C infects only humans. The epidemic of type B repeats every year, but the scale of the epidemic is small because the gene is stable and immunity to the virus may be maintained for a long time. Type B also infects only humans. Influenza A has many variants and is prone to a global pandemic. In addition, there are other types of type A that infect other than humans.

In addition, A type and B type have HA and NA, and there is no big difference in structure. Type C lacks HA and NA and instead expresses hemagglutinin-esterase (HE).

FIG. 2 is a diagram showing the classification of influenza A.
FIG. 3 is a diagram showing the infection mechanism of influenza virus.
4A and 4B are diagrams showing the molecular structure of sugar chains expressed in the upper respiratory tract of humans, and FIG. 4B is a diagram showing the molecular structure of sugar chains expressed in the upper respiratory tract of birds. be.

There are two types of influenza A virus, one that infects birds and the other that infects humans. This is due to the fact that the molecular structure of sugar chains expressed in the cells of the upper respiratory tract that are first adsorbed when infected with influenza differs between birds and humans. When the influenza virus is adsorbed on cells, HA exposed on the surface of the virus recognizes and binds to sugar chains exposed on the surface of the cell membrane.

As shown in FIG. 4 (b), the sugar chains exposed in the upper respiratory tract of birds have an α2,3 structure, and as shown in FIG. 4 (a), the sugar chains exposed in the upper respiratory tract of humans. Has an α2,6 structure. These differences are due to the difference in which carbon the sialic acid at the tip of the sugar chain and the second galactose are bound to each other.

Viruses expressing HA that specifically binds to α2,3 type sugar chains infect birds, and viruses expressing HA that specifically binds to α2,6 type sugar chains infect humans. HA is a receptor or sugar chain recognition site.

As shown in FIG. 2, influenza A includes attenuated influenza that spreads mainly to the respiratory tract and highly virulent influenza that spreads to organs throughout the body. The Spanish flu, which has caused many casualties in the past, and the swine flu that prevailed in North and Central America in 2009 are classified as attenuated, just like seasonal flu. On the other hand, avian influenza such as H5N1 type, which is also called poultry plague and is feared, is classified as highly virulent.

As shown in FIG. 3, the influenza virus is adsorbed on the host cell by recognizing and binding to the sugar chain. After this, the influenza virus is taken up into the cells by the phagocytosis of the cells called endocytosis.

Endocytosis traps the influenza virus in vesicles called endosomes. The protease in the endosome cleaves HA. Further, when the endosome becomes acidic, the three-dimensional structure of the cleaved HA molecule changes, and the virus envelope 31 fuses with the endosome membrane to release the virus genome into the cell.

5 (a) and 5 (b) are schematic views showing the deformation of the cleaved HA due to pH.

HA forms a trimer as shown in FIG. 5 (a). FIG. 5B is a diagram in which one HA is taken out.

The alteration of HA begins with cleavage (cleavage) of a specific site by a protease (proteolytic enzyme) present in endosomes. The cleaved HA is divided into HA1 bound to a sugar chain on the endosome membrane side and HA2 immobilized on the virus envelope 31.

On the other hand, inside the endosome, protons are sent by a proton pump formed through the endosome membrane and become acidic.

When the pH becomes weakly acidic, about 5.0 to 5.5, the cleaved HA is deformed, and the hydrophobic group 32 at the cleaved portion protrudes.

FIG. 6 is a schematic diagram showing the general structure of the lipid bilayer membrane.

The lipid bilayer is composed of a phospholipid molecule 51 in which a hydrophilic phosphate group (hydrophilic group) 52 and a hydrophobic long-chain fatty acid (hydrophobic group) 53 are bound. Further, in many cases, two fatty acids 53 are bound to one phosphoric acid group 52. In an aqueous solution such as a biological fluid, the hydrophilic groups 52 are exposed on the surface (aqueous solution side), and the fatty acids 53 (hydrophobic groups) tend to aggregate with each other. Here, when only one hydrophobic fatty acid 53 is attached, the hydrophilic phosphate group 52 forms a spherical micelle structure facing outward, but when two fatty acids 53 are attached, the hydrophilic phosphate group is formed. Since the thicknesses of 52 and the fatty acid 53 are close to each other, they cannot be surrounded in a micelle shape, and two molecules facing each other of the fatty acids 53 have a structure in which they are arranged in a layer. This is the lipid bilayer membrane.

The above-mentioned virus membrane (envelope) 31, the cell membrane infected with the virus, and the endosome membrane incorporating the virus all have the above-mentioned lipid double membrane structure.

As described above, the lipid bilayer membrane forming the endosome membrane is mostly formed of the hydrophobic fatty acid 53 except for the phosphate groups 52 on the front and back surfaces, which is shown in FIG. 5 (a). Thus, the hydrophobic group 32 protruding from HA by cleavage can pierce the endosome membrane by hydrophobic interaction. This triggers a membrane fusion between the endosomal membrane and the viral envelope 31.

Cleavage of HA is caused by proteases (proteolytic enzymes). Proteases recognize and cleave the amino acid sequence of a protein. This protease does not have the virus itself, but the protease in the infected cell and its surroundings is used.

There is a protease called trypsin in the human airway, and the human infectious influenza virus expresses HA that is cleaved by trypsin.

In addition, TMPRSS (transmembrane protease / serine) 2, which is a type II transmembrane serine protease, is also expressed on the cell surface of the human airway, and HA of the human infectious influenza virus is also cleaved by TMPRSS2.

However, since these proteases are rarely present in human organs other than the respiratory tract, the current human infectious influenza virus spreads only to the respiratory tract and rarely spreads to organs throughout the body.

For example, a protease called FURIN is present in organs throughout the body. FURIN is a protease that is abundantly present in the Golgi apparatus, which is a tissue of cells.

Some avian influenza viruses such as H5N1 express HA having an amino acid sequence cleaved by FURIN.

Since FURIN is present throughout the body, infection with the above-mentioned types of influenza spreads to systemic organs, resulting in serious symptoms. This is why it is called poultry plague or highly virulent influenza.

In addition, some highly virulent influenza expresses HA that is not cleaved by FURIN but is cleaved by a protease called TMPRSS13 / MSPL.

As mentioned above, the difference between the avian-infected type and the human-infected type is due to the difference in the structure of the sugar chain that HA recognizes and binds to, and the difference between attenuated and highly virulent is due to the difference in the protease that HA cleaves. are doing.

At present, it is believed that the highly virulent influenza virus that infects humans has not yet appeared. However, since pigs can be infected with both avian-infected virus and a human-infected virus, a hybrid type of avian-infected type and a human-infected type occurs in the body of a pig infected with both viruses. It has been pointed out that there is a risk that it will end up.

In view of the above, the embodiment of the present invention provides a detection device and a detection method for rapidly and specifically detecting a human infectious type highly virulent influenza that is a threat to cause a pandemic.

Further, in the embodiment of the present invention, the human infectious type utilizes the property that influenza virus recognizes and binds to a specific sugar chain structure by HA exposed on the surface and the property that HA is cleaved by a specific protease. Allows specific detection of highly virulent influenza virus.

Furthermore, according to the embodiment of the present invention, it is possible to specifically detect all four types classified by the two properties of infectious type and toxicity shown in FIG.

Furthermore, the detection target is not limited to influenza virus, but it is possible to detect if a receptor exposed on the surface recognizes and binds to a specific target and the receptor has the property of being cleaved by a prosthesis. be.

FIG. 7 is a schematic diagram showing an outline of the detection device (biosensor) of the embodiment.
FIG. 8 is a schematic diagram showing how the detection device of the embodiment is used to capture a human infectious influenza virus.
FIG. 9 is a schematic diagram showing how a highly virulent influenza virus is detected using the detection device of the embodiment.
FIG. 10 is a schematic diagram showing how the detection device of the embodiment is used to detect that the HA of the virus has been cleaved.
FIG. 11 is a schematic diagram showing how the detection device of the embodiment is used to detect the membrane fusion of the virus HA after subordination.

As shown in FIG. 7, the detection device of the embodiment includes a sensor element and a probe molecule 21 fixed to the sensor element. The sensor element is, for example, a charge detection element including a graphene film 13. The probe molecule 21 is an α2,6 type sugar chain and associates with HA exposed on the surface of influenza virus.

The base film 12 is provided on the substrate 11, and the graphene film 13 is provided on the base film 12. Alternatively, the graphene film 13 may be provided on the surface of the substrate 11 without providing the base film 12. Further, a circuit or a transistor (not shown) may be formed on the substrate 11.

As the material of the substrate 11, for example, silicon, silicon oxide, glass, or a polymer material can be used. The base film 12 is an insulating film such as a silicon oxide film or a fluororesin. Further, the undercoat film 12 can be provided with the function of a chemical catalyst for forming the graphene film 13.

Further, the phospholipid membrane 14 (phospholipid monomolecular membrane) covers the graphene membrane 13, and the probe molecule 21 is either the graphene membrane 13 or the phospholipid membrane 14 (phospholipid monomolecular membrane) via the linker 22. It is fixed to the crab. The linker 22 adjusts the distance between the probe molecule 21 and the surface of the sensor element.

In some drawings including FIGS. 7 to 11, a part of the phospholipid membrane 14 (phospholipid monomolecular membrane) is intentionally not shown so that the internal structure can be easily understood, and the underlying graphene membrane is not shown. 13 is visible. Further, the probe molecule 21 is shown in a state where three monosaccharides are bound, but it is not always necessary to have this number.

The sensor element has at least two electrodes (first electrode 16 and second electrode 17) having, for example, a FET (field effect transistor) structure. The graphene film 13 is electrically connected to the first electrode 16 and the second electrode 17. One of the first electrode 16 and the second electrode 17 functions as a drain electrode, and the other functions as a source electrode. A current can flow between the first electrode 16 and the second electrode 17 through the graphene film 13.

The graphene membrane 13, the phospholipid membrane 14 (phospholipid monomolecular membrane), and the probe molecule 21 are arranged in a well surrounded by the side wall 18. An injection port 15 for supplying a sample solution or the like is formed above the well.

When a sample solution obtained from a subject (for example, pharyngeal swab or mouthwash) is dropped into the well, if the subject is infected with human infectious influenza, the HA of influenza virus is a probe as shown in FIG. It recognizes the molecule (α2,6 type sugar chain) 21 and binds to the probe molecule (α2,6 type sugar chain) 21.

The same applies when the droplets scattered in the air due to coughing are captured. Even when the captured droplets contain droplets from the cough of a human-infectious influenza-infected person, the HA of influenza virus binds to a probe molecule (α2,6 type sugar chain) as shown in FIG.

The virus captured by the sugar chain is close to the graphene membrane 13. In general, influenza virus has a surface charge, and when the charge of the virus is close to the graphene film 13, the graphene potential fluctuates, and the source-drain current flowing through the graphene film 13 fluctuates. Therefore, the presence or absence of human-infectious influenza virus can be determined by reading the fluctuation of the source-drain current.

In addition, when the HA of the highly virulent influenza virus is cleaved and the protease present in systemic organs is supplied to the well, the influenza virus captured by the probe molecule (α2,6 type sugar chain) 21 is highly virulent. If so, as shown in FIG. 9, HA bound to the α2,6 type sugar chain is cleaved.

When the HA is cleaved, as shown in FIG. 10, the fixation of the virus to the sensor element becomes unstable, and this can be detected as a fluctuation in the electrical characteristics.

Alternatively, when the sample liquid 26 is acidified, as shown in FIG. 11, the cleaved HA is deformed into a phospholipid membrane 14 (phospholipid monomolecular membrane) in which the hydrophobic group of HA covers the surface of the graphene membrane 13. Stick. By detecting the fluctuation of the electrical characteristics of the sensor element due to this event, it is possible to detect that the HA has been cleaved with high sensitivity. Further, by detecting the change in the electrical characteristics of the sensor element due to the subsequent fusion of the virus envelope 31 with the phospholipid membrane 14 (phospholipid monolayer), it is possible to detect the cleavage of HA with high sensitivity. can.

Here, as the pH adjusting solution for acidifying the sample solution 26 in order to deform HA, an acidic buffer solution such as Acetate-NaOH can be used.

Further, for example, FURIN can be used as a protease that cleaves HA of the highly virulent influenza virus and is present in systemic organs.

Alternatively, TMPRSS13 / MSPL, which is known to cleave the HA of highly virulent avian-infectious influenza, is used when a new influenza virus in which this type of avian-infectious highly virulent influenza is mutated to a human-infectious type appears. Can also detect the new influenza virus. Since TMPRSS13 / MSPL is a kind of transmembrane serine protease, it can be fixed to the phospholipid membrane 14 (phospholipid monomolecular membrane) covering the surface of the graphene membrane 13.

Since the specimens such as the pharyngeal swab and the droplets from the cough are all from the upper respiratory tract of humans, they may contain trypsin.

If the sample solution contains a sufficient amount of trypsin, HA will be cleaved even if the influenza virus is weakly virulent, resulting in false positives for the virulent influenza virus.

As a method of avoiding this concern, for example, a trypsin inhibitor can be supplied to the well.

When the trypsin inhibitor specifically inhibits the enzymatic activity of trypsin and does not affect the enzymatic activity of FURIN or TMPRSS13 / MSPL, the trypsin inhibitor is supplied as shown in FIG. Further, for example, if FURIN is supplied and HA is cleaved, the highly virulent influenza virus can be detected.
When it is detected that the virus is immobilized on the probe molecule (α2,6 type sugar chain) 21 by using the above procedure, and cleavage is observed in either FURIN or TMPRSS13 / MSPL. , Human infectious type highly virulent influenza, that is, it can be judged to be an extremely dangerous new influenza.

When the trypsin inhibitor also inhibits the enzymatic activity of FURIN and TMPRSS13 / MSPL, the trypsin inhibitor is supplied as shown in FIG. 8, and the probe molecule (α2,6 sugar chain) 21 is infected with human influenza. After fixing the virus, it can be washed to eliminate trypsin inhibitors and trypsin from the wells, and then FURIN or TMPRSS13 / MSPL can be supplied to the wells to detect HA cleavage behavior.

If the virus was detected to be immobilized on the probe molecule (α2,6 type sugar chain) 21 using the above procedure, and no cleavage was observed in either FURIN or TMPRSS13 / MSPL, human infection was observed. It can be determined that it is a type of attenuated influenza, that is, seasonal influenza.

In order to judge this more reliably, trypsin is further supplied. If HA cleavage is observed by the supply of trypsin, it can be confirmed that the influenza is a human infectious type attenuated influenza.

Alternatively, two sensor elements, a first sensor element that supplies FURIN and TMPRSS13 / MSPL to the well and a second sensor element that supplies trypsin to the well, are prepared, and the sample liquid is supplied to both of them. It is also possible to determine toxicity with a sensor element. The well of the first sensor element and the well of the second sensor element are separated by, for example, a side wall 18.

If the result on the first sensor element is positive, the detected virus is human infectious virulent influenza, if the result on the first sensor element is negative and the result on the second sensor element is positive. , The detected virus is human infectious attenuated influenza.

However, even if the results of the first sensor element and the second sensor element are both negative, the existence of bird-infected influenza cannot be denied.

Therefore, if the above procedure is performed using α2,3 type sugar chains as the probe molecule 21, bird-infected highly virulent influenza and attenuated influenza can be specifically detected in the same manner as described above.

The α2,6 type sugar chain is fixed on the sensor element as the probe molecule 21, and the element (region) capable of detecting human infectious influenza virus and the α2,3 type sugar chain are fixed on the sensor element as the probe molecule 21. , An element (region) capable of detecting a bird-infecting influenza virus can coexist in the same well.

The above-mentioned reagents such as a protease for cleaving HA, a protease inhibitor for inhibiting non-specific cleavage of HA, and a pH adjusting solution for promoting membrane fusion after cleavage of HA are referred to as a sample solution. Similarly, it can be supplied to the well through the inlet 15. Alternatively, the reagents may be placed in a liquid reservoir formed on the substrate 11 separately from the wells and supplied to the wells from there.

Further, it is also possible to provide a unit that automatically supplies the reagent to the well in response to the detection signal after the sensor element detects the association between the virus and the probe molecule.

FIG. 12 is a schematic diagram showing a structure in which the reagent is fixed on a sensor element with hydrogel.

As described above, the reagent can be coated and fixed on the sensor element as a water-soluble hydrogel 41.

FIG. 13 is a schematic diagram showing a structure in which the reagent is dispersed in an ionic liquid on a sensor element.

In this way, it is also possible to disperse the reagent in the hydrophilic ionic liquid 42 on the sensor element.

14 (a) to 14 (h) are views showing the molecular structure of an example of the ionic liquid 42.

As the ionic liquid 42, for example, a liquid composed of an imidazolium-based cation and a phosphonate-based anion as shown in FIG. 14A can be used. This type of ionic liquid is mild to biomaterials and can avoid significant damage to sugar chains, phospholipid membranes, and the like.

Further, since various molecular structures can be selected for the ionic liquid, for example, if an ionic liquid having a large molecular size and a high viscosity is used, it becomes easy to fix the ionic liquid together with the reagent on the sensor element. 14 (b) to 14 (h) show examples of ionic liquids having different molecular structures.

FIG. 15 is a schematic diagram showing the molecular structure of the organic electrolyte oligomer.

The viscosity of the ionic liquid 42 can also be increased by adding, for example, an organic electrolyte oligomer as shown in FIG. 15 as a gelling agent to the ionic liquid 42.

Both the hydrogel and the hydrophilic ionic liquid shown above are diluted when the sample liquid is supplied to reduce the viscosity.

Further, the hydrogel and the hydrophilic ionic liquid also have an effect that the sugar chain probe molecule 21 and the phospholipid film 14, which are biomaterials formed on the sensor element, can be retained in a wet environment.

FIG. 16 is a schematic diagram of another example of the sensor element according to the embodiment.

The sensor element shown in FIG. 16 has a target detection element 10 to which the probe molecule 21 is fixed and a reference element 100 to which the probe molecule is not fixed.

The target detection element 10 and the reference element 100 are formed on the same substrate 11. A reference element 100 to which the probe molecule is not fixed is formed next to the target detection element 10 to which the probe molecule 21 is fixed.

In the reference element 100 to which the probe molecule is not fixed, the signal due to the virus is not detected, but since the fluctuation due to the disturbance, for example, the pH change, is detected, it can be used for the correction of the disturbance noise for the target detection element 10. Further, although FIG. 16 shows the target detection element 10 and the reference element 100 formed in separate wells, they may coexist in the same well.

Further, according to the embodiment of the present invention, not only the influenza virus but also a receptor exposed on the surface recognizes and binds to a specific target, and the receptor has a property of being cleaved by a protease. It is possible to detect.

In fact, many enveloped viruses are known to invade infected cells by cleaving surface-exposed spike proteins (proteins or sugar chain-modified proteins) with proteases. For example, in HIV, the enveloped glycoprotein GP160 is cleaved by FURIN. In addition, SERS coronavirus is cleaved by trypsin for S protein.

When the spike protein is cleaved in this way, the viral envelope causes membrane fusion with the phospholipid membrane 14 (phospholipid monomolecular membrane) covering the surface of the graphene membrane 13. Alternatively, the hydrophobic group exposed by cleavage pierces the phospholipid membrane 14.

Since these changes occur in the vicinity of the graphene film 13 or in contact with the graphene film 13, the graphene potential fluctuates greatly. That is, a large signal can be obtained from one virus, and it can be detected even if the number of viruses is small.

The graphene film 13 is not limited to being formed on the base film (insulating film) 12, and may have a structure in which the graphene film 13 is floated from the base.

FIG. 17 is a schematic view of a detection device having a structure in which the graphene film 13 floats from the base.
FIG. 18A is an enlarged schematic view of the vicinity of the graphene film 13.

The graphene membrane 13 floats in the sample liquid 26 (weakly acidic buffer) in a state like a double-sided beam fixed by a source / drain electrode, and the phospholipid membrane 14 (phospholipid monomolecular membrane) is a graphene. It is formed not only on the front surface of the film 13 but also on the back surface. Further, the graphene film 13 is pore-processed in a mesh shape.

In the pore 13a of the graphene membrane 13, the phospholipid membrane 14 (phospholipid monomolecular membrane) on the front and back extends inside the pore 13a, and a phospholipid bilayer membrane is formed inside the pore 13a. ..

Here, if HA is cleaved by a protease and deformation of HA is caused by an acidic environment as described above, as shown in FIG. 18 (b), the virus envelope 31 and the pore 13a of the graphene membrane 13 are formed. The formed phospholipid membrane 14 (phospholipid bilayer membrane) is membrane-fused.

By this membrane fusion, inclusions containing RNA and the like inside the virus are released to the lower part of the graphene membrane 13. If a sensor element for detecting the inclusions is formed below the graphene film 13, virus membrane fusion can be detected with high sensitivity. Alternatively, the graphene membrane 13 may detect the membrane fusion.

The phospholipid film 14 coated on the sensor element described above had the phosphoric acid group exposed on the surface, but by covering the phosphoric acid group with the hydrophilic group, it became a contaminant. It is also possible to suppress non-specific adsorption.

19 to 22 are schematic views showing an example in which a hydrophilic group is coated on a phosphoric acid group.

For example, since the sugar chain used as the probe molecule 21 is also hydrophilic, as shown in FIG. 19, by forming the sugar chain probe molecule 21 at a high density, it is possible to suppress non-specific adsorption of contaminants. can.

Further, as shown in FIG. 20, at least sugar chains 44 that do not bind to impurities may be formed at a high density.

Further, as shown in FIG. 21, a hydrophilic chain polymer 45 such as polyethylene glycol (PEG) may be bound to the phosphoric acid portion of the phospholipid film 14.

Further, as shown in FIG. 22, a hydrophilic blocking agent 46 such as albumin may be coated on the phospholipid membrane 14.

The charge detection element used as the sensor element is not limited to graphene, and carbon nanotubes may be used. Further, the sensor element that optically or mechanically detects the cleavage of HA (HA cleavage behavior) is not limited to electrical detection, and a sensor element may be used. In addition to carbon-based charge detection elements, sensor elements include, for example, surface plasmon resonance elements, SAW (Surface Acoustic Wave) elements, FBAR (Film Bulk Acoustic Resonator) elements, QCM (Quartz Crystal Microbalance) elements, and IS-FETs (Ion sensitive). A FET) element, a MEMS (Micro Electro Mechanical System) cantilever element, or the like can be used.

FIG. 23 (a) is a schematic diagram of a phospholipid membrane formed on the hydrophobic substrate 113, and FIG. 23 (b) is a schematic diagram of the phospholipid membrane formed on the hydrophilic substrate 213.

As shown in FIG. 23A, when the surface of the sensor element is a hydrophobic substrate (for example, graphene) 113, the phospholipid film is formed as a monomolecular film. As shown in FIG. 23 (b), when the surface of the sensor element is a hydrophilic substrate 213, the phospholipid film is formed as a lipid bilayer film.

Further, when mechanical vibration such as QCM is used as the sensor element, the cleavage of HA can be detected mechanically, so that it is not always necessary to form a phospholipid film on the surface of the sensor element.

According to the embodiment described above, a probe molecule that specifically binds to the spike protein exposed on the surface of the virus and a sensor element that detects that the spike protein of the virus bound to the probe molecule is cleaved are used. Therefore, the virus can be detected with high sensitivity. Furthermore, by selecting a probe molecule and a protease that cleaves a spike protein, it becomes possible to specifically detect a specific virus.

The detection target is not limited to a virus, and any virus that has a receptor that associates with a probe molecule and has a function of expressing cleavage of the receptor can be detected. For example, it is possible to detect virus-like endoplasmic reticulum such as exosomes.

Although some embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other embodiments, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention, and are also included in the scope of the invention described in the claims and the equivalent scope thereof.

10 ... target detection element, 11 ... substrate, 12 ... base film, 13 ... graphene film, 14 ... phospholipid membrane, 15 ... inlet, 16,17 ... electrode, 21 ... probe molecule, 25 ... trypsin inhibitor, 31 ... Membrane, 41 ... Hydrogel, 42 ... Ionic liquid, 100 ... Reference element

Claims (24)

With the sensor element
Probe molecules that associate with receptors exposed on the surface of the detection target,
Equipped with
The probe molecule is fixed to the sensor element and
The detection target includes a first detection target.
The first detection target comprises a first receptor exposed on the surface of the first detection target and cleaved by a particular protease.
The sensor element detects whether or not the electrical characteristics of the sensor element change due to the supply of the detection target , and whether or not the electrical characteristics of the sensor element change due to the further supply of the protease. Detection device. The detection device according to claim 1, wherein the protease cleaves a specific amino acid sequence contained in the first receptor. The detection device according to claim 1 or 2, wherein the sensor element detects the detection target by a change in the electrical characteristics of the sensor element due to the supply of the detection target. The detection device according to claim 3, wherein the sensor element detects that the detection target is the first detection target depending on the presence or absence of fluctuations in the electrical characteristics of the sensor element due to the supply of the protease. The detection target further comprises a second detection target identified relative to the first detection target.
The detection device according to any one of claims 1 to 4, wherein the protease does not cleave the second receptor exposed on the surface of the second detection target. The sensor element is a charge detection element, a surface plasmon resonance element, a SAW (Surface Acoustic Wave) element, an FBAR (Film Bulk Acoustic Resonator) element, a QCM (Quartz Crystal Microbalance) element, an IS-FET (Ion sensitive FET) element, or , MEMS (Micro Electro Mechanical System) cantilever element,
The sensor element detects either a change in the position of the first detection target due to cleavage of the first receptor, a change in mobility, or a change in the shape of the first receptor. The detection device according to any one of claims 1 to 5. The detection device according to claim 6, wherein the charge detection element includes graphene or carbon nanotubes. The surface of the sensor element is coated with a phospholipid film, and the surface is coated with a phospholipid film.
The sensor element claims to detect either the invasion of the cleaved first receptor into the phospholipid membrane or the fusion of the surface membrane of the first detection target and the phospholipid membrane. The detection device according to any one of 1 to 7. The detection device according to any one of claims 1 to 8, wherein the probe molecule is a sugar chain. The detection device according to any one of claims 1 to 9, wherein the first detection target is a virus and the first receptor is a spike protein. The detection device according to claim 10, wherein the virus is an influenza virus and the spike protein is hemagglutinin. The first protease for cleaving the spiked protein and
A protease inhibitor for inhibiting cleavage of the spike protein by a second protease contaminated with the first detection target.
A pH regulator for promoting membrane fusion after cleavage of the spike protein, and
10. The detection device according to claim 10, wherein at least one of the above is supplied on the sensor element. The first protease is a protease that exists in addition to the human respiratory tract.
The detection device according to claim 12, wherein the protease inhibitor is a trypsin inhibitor. 12. The detection device according to claim 12 , wherein the first protease comprises at least one of FURIN and TMPRSS13 / MSPL. The detector according to claim 14, wherein the first protease comprises at least one of trypsin and TMPRSS2. The detection device according to any one of claims 1 to 15, wherein the probe molecule is either an α2,6 type sugar chain or an α2,3 type sugar chain. The detection device according to any one of claims 1 to 16, wherein the sensor element includes a target detection element to which the probe molecule is fixed and a reference element to which the probe molecule is not fixed. The first protease for cleaving the spiked protein and
A protease inhibitor for inhibiting cleavage of the spike protein by a second protease contaminated with the first detection target.
A pH adjuster for promoting membrane fusion after opening and inferiority of the spike protein, and
10. The detection device according to claim 10, wherein a gel containing at least one of the above is fixed on the sensor element, and the gel is water-soluble. The detection device according to claim 18, wherein the gel contains a hydrogel. The detection device according to claim 18, wherein the gel contains a hydrophilic ionic liquid. The detection device according to claim 20, wherein the ionic liquid contains a phosphonate compound as an anion. A receptor that associates with a probe molecule immobilized on a sensor element and is cleaved by a specific protease is a detection target whose surface is exposed, and the protease is supplied to the detection target captured by the probe molecule. death,
Detection in which the sensor element detects whether or not the electrical characteristics of the sensor element are changed due to the supply of the detection target and whether or not the electrical characteristics of the sensor element are changed due to the further supply of the protease. Method. 22. The detection method according to claim 22, wherein the detection target is a virus and the receptor is a spike protein. The first protease for cleaving the spiked protein and
A protease inhibitor for inhibiting cleavage of the spike protein by a second protease contaminated with the detection target.
A pH regulator for promoting membrane fusion after cleavage of the spike protein, and
23. The detection method according to claim 23, wherein at least one of the above is supplied onto the sensor element.

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