WO2006046509A1 - カンチレバーセンサ、センサシステム及び検体液中の検出対象物質の検出方法 - Google Patents
カンチレバーセンサ、センサシステム及び検体液中の検出対象物質の検出方法 Download PDFInfo
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- WO2006046509A1 WO2006046509A1 PCT/JP2005/019498 JP2005019498W WO2006046509A1 WO 2006046509 A1 WO2006046509 A1 WO 2006046509A1 JP 2005019498 W JP2005019498 W JP 2005019498W WO 2006046509 A1 WO2006046509 A1 WO 2006046509A1
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- cantilever
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- sugar chain
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- metal film
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
- G01N5/02—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by absorbing or adsorbing components of a material and determining change of weight of the adsorbent, e.g. determining moisture content
Definitions
- the present invention relates to a cantilever sensor, a sensor system using the cantilever sensor, and a method for detecting a detection target substance in a sample liquid.
- Viruses and bacteria may be detected in the fields of biological analysis and medicine.
- immunochromatography and PCR Polymerase chain reaction
- PCR Polymerase chain reaction
- the immunochromatography method is a simple diagnostic method using an antigen-antibody reaction (Patent Document 1).
- the PCR method is a highly sensitive diagnostic method in which detection is performed by amplifying an arbitrary part of a gene (Patent Document 2).
- Patent Document 1 International Publication No. WO2002Z088737 Pamphlet
- Patent Document 2 Japanese Unexamined Patent Application Publication No. 2004-201679
- the immunochromatography method is simple but has low detection sensitivity.
- a virus is detected by immunochromatography in a subject in the early infection stage or in the healing stage, false negatives appear, that is, when the virus is actually present. In some cases, no virus was detected.
- the PCR method has high detection sensitivity, the pretreatment time is long, and there is a possibility of erroneous diagnosis due to contamination, so that the operation is complicated and difficult. Furthermore, the PCR method in particular has a certain base sequence of genes such as viruses and bacteria to be detected. There is a restriction that it cannot be applied unless it is specified, and the scope of application may be limited.
- the present invention was devised in view of the above problems, and a cantilever sensor capable of detecting a detection target substance such as a virus or a bacterium with high sensitivity in a short time with a simple configuration, It is another object of the present invention to provide a sensor system using the same and a method for detecting a detection target substance in a sample liquid.
- a cantilever having a sugar chain immobilized thereon is used to allow the sugar chain to interact with a detection target substance such as a virus or a bacterium.
- a detection target substance such as a virus or a bacterium.
- the gist of the present invention is a cantilever sensor for detecting a detection target substance, comprising a cantilever and a sugar chain that can interact with the detection target substance fixed to the cantilever.
- the cantilever sensor is characterized in that it bends when the substance to be detected interacts with the sugar chain. (Claim 1).
- the detection target substance can be detected with high sensitivity and in a short time with a simple configuration.
- the sugar chain may be directly immobilized on the cantilever or may be indirectly immobilized.
- the detection target substance preferably has two or more sites capable of interacting with the sugar chain (claim 2).
- Such a detection target substance can interact with a sugar chain on the cantilever at two or more sites. Therefore, when one detection target substance interacts with a sugar chain, interactions occur at many reaction points, For this reason, the stress generated by the interaction also increases. As a result, the amount of deflection of the cantilever sensor increases, so that the detection sensitivity can be improved.
- examples of preferred U and detection target substances include viruses and bacteria (claim 3).
- Viruses and bacteria usually interact with sugar chains at two or more points, so the stress generated when interacting with sugar chains increases, and the amount of deflection of the cantilever sensor increases. Therefore, detection sensitivity can be improved.
- viruses include hepatitis B virus, hepatitis C virus, reo winoles, lunar myocarditis winoles, AIDS winoles, mouth tawinores, corona winores, norevovirus, Sendai virus, Newcastle disease
- viruses include viruses, herpes type 1 viruses, tenguils and influenza viruses (Claim 4).
- the sugar chain preferably contains sialic acid or substituted sialic acid.
- the sugar chain preferably contains fluorinated sialic acid.
- the sugar chain is formed only on one side of the cantilever (claim 5).
- the amount of deflection due to the interaction can be increased, and it is preferable to form a metal film only on the one surface of the cantilever (claim 6).
- the sugar chain can be easily fixed only on one side.
- the cantilever sensor has a metal film provided on the surface of the cantilever and an organic molecule fixed on the metal film, and the sugar chain is immobilized on the organic molecule. (Claim 7). As a result, the sugar chain can be easily and reliably fixed to the cantilever.
- the cantilever sensor has a metal film provided on the surface of the cantilever, an organic molecule fixed on the metal film, and a porous matrix bonded on the organic molecule,
- the sugar chain is preferably immobilized on the porous matrix (claim 8).
- hydrogel As such a porous matrix, it is preferable to use a hydrogel (claim 9). If a hydrogel is used, sugar chains can be fixed easily and with high density.
- the sugar chain is fixed by a covalent bond.
- the organic molecule is fixed to the metal film by a “sulfur metal bond”. Is preferred (Claim 10). Thereby, the organic molecule can be easily fixed on the metal film.
- the outermost layer of the metal film is preferably made of gold (claim 11). Also by this, the organic molecule can be easily fixed on the metal film.
- an uneven pattern is formed on the surface of the metal film (claim 12). It is also preferable that a concavo-convex pattern is formed on the surface of the cantilever (claim 13).
- the uneven pattern is preferably formed periodically. Thereby, formation of a concave-convex pattern can be facilitated.
- the width of the concave / convex pattern is preferably from lOnm to 100 m.
- the depth of the uneven pattern is preferably 10 nm or more and 100 m or less.
- the immobilization density of the sugar chain is 1. OX 10 _1 ° mol Zcm 2 or more and 1.0 X 10 " 2 mol / cm 2 or less (claim 14).
- another gist of the present invention is a sensor system for detecting a detection target substance, which is an interaction with the above-described cantilever sensor, that is, the cantilever and the detection target substance fixed to the cantilever.
- a cantilever sensor that has a sugar chain that can be bent, and causes a deflection when the detection target substance and the sugar chain interact with each other; a sample liquid contact portion that makes a sample liquid contact the sugar chain; and the cantilever sensor And a deflection amount measuring unit for measuring the deflection amount of the sensor system (claim 15).
- the detection target substance can be detected with high sensitivity and in a short time with a simple configuration.
- the sensor system includes a correction cantilever, a correction deflection measuring unit for measuring a deflection amount of the correction cantilever, a deflection amount of the cantilever sensor, and a deflection amount of the correction cantilever. It is preferable that a deflection amount difference output unit for outputting a difference between the two and the difference is output (claim 16). This eliminates the influence of deflection due to the external environment and enables accurate measurement of the amount of deflection due to the above interaction.
- the correction cantilever has a non-fixed portion where the sugar chain is not immobilized on the entire surface. It is preferable to have it in the body (Claim 17). As a result, the correction cantilever is prevented from being bent due to the above-described interaction, so that correction for eliminating the influence of deflection due to the external environment or the like can be reliably performed.
- the correction cantilever has a non-deposition portion where no metal film is provided on the entire surface (claim 18). This facilitates the production and handling of the correction cantilever.
- the correction cantilever preferably has a metal film having an outermost layer formed of a metal other than gold on one side (claim 19).
- a metal film having an outermost layer formed of a metal other than gold on one side claim 19
- the sugar chain can be easily fixed to only one surface of the cantilever sensor, and more precise correction can be performed.
- the metal other than gold it is preferable to use at least one metal selected from the group consisting of aluminum, copper and silver (claim 20).
- the correction cantilever is formed with a correction sugar chain fixing part in which a correction sugar chain having a different interaction magnitude with respect to the detection target substance is fixed. (Claim 21). This also makes it possible to reliably perform correction to eliminate the influence of deflection due to the external environment.
- the sensor system preferably includes a cantilever vibrating section that vibrates the cantilever (claim 22).
- a cantilever vibrating section that vibrates the cantilever (claim 22).
- Still another subject matter of the present invention is the above-described cantilever sensor, that is, a cantilever and a sugar chain that can interact with the detection target substance immobilized on the cantilever, and the detection target substance A substance to be detected in a sample liquid, characterized in that the sample liquid is brought into contact with the sugar chain of a cantilever sensor that generates a deflection when interacting with the sugar chain, and the amount of deflection of the cantilever sensor is measured. (Claim 23).
- the detection target substance can be detected with high sensitivity in a short time with a simple configuration.
- the used cantilever sensor is cleaned to remove the detection liquid. It is also preferable to perform detection using the washed cantilever sensor (claim 24). As a result, the detection cost can be reduced.
- the cantilever sensor in which the sugar chain is fixed again is used. It is also preferable to perform detection. This also makes it possible to reduce the detection cost.
- the detection target substance such as virus or bacteria can be detected with high sensitivity and in a short time with a simple configuration. can do.
- FIG. 1 is a schematic diagram for explaining a virus.
- FIG. 2 is a schematic perspective view showing an essential part of a cantilever sensor for explaining an embodiment of the present invention.
- FIG. 3 is a diagram for explaining an embodiment of the present invention.
- FIG. 3 is a schematic enlarged view of the vicinity of a sugar chain fixing part when the sugar chain fixing part is formed by the first immobilization method.
- FIG. 3 is a diagram for explaining an embodiment of the present invention.
- FIG. 3 is a schematic enlarged view of the vicinity of a sugar chain fixing part when the sugar chain fixing part is formed by the first immobilization method.
- FIG. 4 is for explaining an embodiment of the present invention, and is a schematic enlarged view of the vicinity of a sugar chain immobilization part when the sugar chain immobilization part is formed by the second immobilization method.
- FIG. 4 is for explaining an embodiment of the present invention, and is a schematic enlarged view of the vicinity of a sugar chain immobilization part when the sugar chain immobilization part is formed by the second immobilization method.
- FIG. 5 is a schematic diagram for explaining an embodiment of the present invention and showing a state in which a substance to be detected interacts with a sugar chain at a sugar chain immobilization part.
- FIG. 6 is a schematic diagram for explaining the main part of an example of a sensor system using a cantilever sensor, for explaining one embodiment of the present invention.
- FIG. 7 is a cross-sectional view schematically illustrating the vicinity of the sensor unit when the sensor system is used, for explaining one embodiment of the present invention.
- FIG. 8 is a schematic diagram for explaining an embodiment of the present invention and explaining how deflection occurs when a detection target substance interacts with a cantilever sensor.
- Fig. 9 is a schematic outline view for explaining a piezoresistive element which is a cantilever oscillating portion, explaining one embodiment of the present invention.
- Fig. 10 is a schematic outline diagram for explaining an embodiment of the present invention and explaining a main part of another example of the sensor system.
- FIG. 11 is a reaction formula for explaining the method for synthesizing the sugar compound (1) carried out in Example 1 of the present invention.
- FIG. 12 is a schematic diagram for explaining a main configuration of the sensor system used in Example 1 of the present invention.
- FIG. 13 is a graph showing the results of Example 1 of the present invention.
- FIG. 14 is a graph showing the results of Comparative Example 1.
- the cantilever sensor of the present invention is a sensor for detecting a detection target substance, and has a force cantilever and a sugar chain immobilized on the cantilever and capable of interacting with the detection target substance.
- the substance to be detected is a substance to be detected using a cantilever sensor. There are no particular restrictions on the type or state, but it is usually used for detection in a state dissolved or dispersed in the sample liquid. Further, the detection target substance may be detected alone or in any combination of two or more.
- the detection target substances those having two or more sites capable of interacting with a sugar chain can be detected with high detection sensitivity, and thus are detected with the cantilever sensor of the present invention.
- Suitable for That is, in the case of a detection target substance having two or more sites that can interact with a sugar chain, one detection target substance can interact with two or more sites.
- the change in the surface stress on the cantilever surface is derived from the change in the surface free energy due to the interaction. For this reason, in the detection of a detection target substance that interacts at two or more sites, the deflection caused by the change in surface stress increases.
- a detection target substance that can interact with a sugar chain at two or more sites is usually detected with higher detection sensitivity than a detection target substance that interacts with a sugar chain only at a single site. You can go out.
- the change in surface stress caused by the interaction is larger when the interaction can occur in a large number of sites, it preferably has 5 or more sites that can interact with the sugar chain, and more than 10 More preferred to have.
- binding protein a protein that binds to a sugar chain on the surface of an infected cell
- binding protein a protein that binds to a sugar chain on the surface of an infected cell
- FIG. 1 is a schematic diagram for explaining a virus.
- the binding protein 2 of virus 1 is usually distributed in large numbers on the surface of virus 1. did Therefore, when sugar chain 4 on cantilever sensor 3 and virus 1 bind, sugar chain 4 binds to virus 1 rather than the number of viruses 1 bound to sugar chain 4 of cantilever sensor 3. The number of becomes larger. That is, since the sugar chain 4 on the cantilever sensor 3 and the virus 1 can bind at two or more sites, the change in surface stress can be increased. The same is true for bacteria.
- bacteria that are examples of detection target substances include Escherichia coli, Vibrio cholerae, Staphylococcus, Bacillus anthracis, Neisseria gonorrhoeae, Plague, Legionella, Shigella, Salmonella typhi, Helicobacter pylori, Tuberculosis, Borinus Examples include fungi, tetanus, and diphtheria.
- viruses that are examples of detection target substances include hepatitis B virus, hepatitis C virus, reovirus, encephalomyocarditis virus, AIDS virus, rotavirus, coronawinores, panolevowinores, Examples include Sendai Winores, New Katsunoue Disease Winores, Henopes Type 1 Virus, Proboscis Virus, and Influenza Virus.
- influenza virus include human influenza virus and avian influenza virus.
- the "interaction" between the specific substance and the substance to be detected is not particularly limited, but usually a covalent bond, a hydrophobic bond, a hydrogen bond, a van der Waals bond, and an electrostatic force. It shows the effect of forces acting between molecules that also generate at least one of the bonds due to.
- the term “interaction” in this specification should be construed in the broadest sense and should not be construed as limiting in any way.
- FIG. 2 is a schematic perspective view showing the main part of the cantilever sensor of the present embodiment.
- the cantilever sensor 3 of the present embodiment has a cantilever 5 and a sugar chain 4 fixed to the cantilever 5.
- the part of the cantilever 5 where the sugar chain 4 is fixed is called a sugar chain fixing part 6. 2 that are the same as those in FIG. 1 are denoted by the same reference numerals. In FIG. 2, the sugar chain 4 is not shown.
- a known cantilever without limitation on the cantilever 5 used in the present invention can be arbitrarily used.
- cantilever 5 There is no limit to the material of cantilever 5, and the force that can be used with any material. A flexible one is used. Specific examples of the material of the cantilever 5 include silicon and silicon nitride.
- the shape of the cantilever 5 is not limited, the cantilever 5 is usually formed as a rectangular parallelepiped member having a free end and a fixed end. As another example, a shape having one side of a triangle as a fixed end, or a shape in which the inside of the triangle is punched out is also possible. In the present embodiment, the cantilever 5 will be described as a member that extends from the support member 7 into a rectangular parallelepiped shape as shown in FIG.
- the size of the cantilever 5 is not limited, the length L (that is, the distance from the free end to the fixed end) is usually a deflection caused by the interaction between the sugar chain 4 and the detection target substance. It is preferable that it is formed long enough to reliably measure.
- length L is 10 m to 1000 m
- width W is 5 ⁇ m to 500 ⁇ m
- thickness T is 0.1 ⁇ m to 5 ⁇ m. It is preferable to set each in the range of m.
- a known method can be arbitrarily used without any limitation on the method for producing the cantilever 5.
- it can be fabricated in the same manner as a cantilever used in an AFM (Atomic Force Microscope) by an existing semiconductor process.
- a concavo-convex pattern 8 on the surface of the cantilever 5 so that the concavo-convex portion is formed in the sugar chain fixing portion 6 where the sugar chain 4 is fixed. This is to increase the detection sensitivity of the cantilever sensor 3. Details will be described later together with the explanation of the sugar chain fixing part 6.
- An example of a method for measuring the deflection amount of the cantilever sensor 3 is an electric method.
- the surface of the cantilever 5 (usually one side) may be patterned with a piezoresistive element.
- the length is long or near the length L of the cantilever 5 itself.
- the piezoelectric resistance element part is formed, and the deflection amount of the large ridge part cannot be measured. Therefore, the piezoelectric resistance is expanded over a wider range in the length direction of the cantilever 5 where the deflection occurs. This is because it is preferable to pattern the element portion.
- cantilever 5 The ratio force of the length of the piezoresistive element portion to the length L is usually 50% or more, preferably 70% or more.
- a piezoelectric resistance element portion (not shown) is provided on the surface of the cantilever 5 below the metal film 9, and the piezoelectric resistance is insulated from the metal film 9 on the surface of the support member 7.
- a metal film pattern (not shown) that functions as the wiring of the element portion is formed.
- a sugar chain 4 is fixed on the surface of the cantilever 5 so that the cantilever sensor 3 bends when the above-described interaction occurs. At this time, the sugar chain 4 may be directly immobilized on the cantilever 5 or may be indirectly immobilized.
- sugar chain 4 There are no restrictions on the type of sugar chain 4, and a suitable sugar chain can be adopted and used from known sugar chains according to the type of substance to be detected. However, in particular, when detecting a detection target substance such as a virus or a bacterium, it is preferable to use a sugar chain 4 that allows one detection target substance to interact with each other at two or more sites. This is because, as described above, it is possible to cause a larger change in surface stress and increase the detection sensitivity of the cantilever sensor.
- the number of carbon atoms excluding the substituent portion of the monosaccharide constituting the sugar chain 4 is usually 4 or more, preferably 6 or more, and usually 12 or less, preferably 10 or less. Outside this range, there is a possibility that the binding to a site (binding protein, etc.) that can interact with the sugar chain on the surface of the detection target substance is not performed effectively.
- the monosaccharides constituting the sugar chain 4 may be used alone or in combination of two or more in any combination and ratio.
- the number of monosaccharides constituting the sugar chain 4 is usually 1 or more, preferably 3 or more, and usually 20 or less, preferably 10 or less. If the upper limit of this range is exceeded, the cost of synthesizing sugar chains increases, which may make it impractical.
- the sugar chain 4 is composed of monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylyldarcosamine, N-acetylylgalatatosamine, sialic acid, and derivatives thereof.
- monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylyldarcosamine, N-acetylylgalatatosamine, sialic acid, and derivatives thereof.
- formic acid or substituted sialic acid! / An example of a substituted sialic acid is fluorinated sialic acid, and a specific example is 3-fluorocyanuric acid. This has the advantage that sugar chains are not easily degraded by enzymes contained in the specimen.
- the darcoside bond part of sugar is a nitrogen atom, a carbon atom, or a sulfur atom instead of an oxygen atom.
- sugar chain 4 examples include sialylacto type I and II sugar chains having the [NeuAca2-6 (3) Galj81-4 (3) GlcNA C j81-] structure, [ ⁇ . ⁇ ; 2— 6 (3) 0 & 1
- Examples include sialylatato sugar chains having a structure 1S, but are not limited thereto.
- the density of the sugar chain 4 immobilized on the cantilever 5 is not limited, but the density (immobilization density) of the sugar chain 4 per area in the sugar chain immobilization part 6 is usually 1.0X 10 _) m O lZcm 2 than on, preferably 1. 0X 10 _9 mol / cm 2 or more, more preferably 1. OX 10 _8 mol / cm 2 or more on, and usually 1. OX 10 _2 mol / cm 2 or less Preferably, it is 1. OX 10 _3 mol / cm 2 or less, more preferably 1.0 X 10 _4 molZcm 2 or less.
- the detection sensitivity may be lowered.
- the surface density of sugar chain 4 is too high, the interaction between sugar chain 4 and the detection target substance is inhibited. This is because there is a risk of being lost.
- sugar chain 4 one type may be used alone or two or more types may be used in any combination and ratio depending on the application.
- FIG. 3 shows an enlarged view of the vicinity of the sugar chain immobilization part 6 when sugar chain 4 is fixed by the first immobilization method.
- FIG. 3 for the sake of explanation, the flat portion of the uneven pattern 8 on the surface of the cantilever 5 is shown.
- the same reference numerals as those in FIGS. 1 and 2 denote the same elements as those in FIGS.
- the sugar chain immobilization part 6 has a metal film 9 formed on the surface of the cantilever 5, an organic molecule 10 immobilized on the metal film 9, and a sugar chain 4 immobilized on the organic molecule 10. And formed as a part having.
- FIG. 3 for the sake of explanation, the planar portion of the uneven pattern 8 on the surface of the cantilever 5 is shown. Further, for the purpose of explanation, the organic molecules 10 are not drawn individually but as a layer in which the organic molecules 10 are assembled.
- the metal film 9 can be formed of any material without any limitation as long as the organic molecules 10 can be fixed on the surface thereof.
- the metal film 9 may be a single-layer film formed by only one layer, or may be a film having a structure in which two or more layers are laminated in any combination and thickness.
- the outermost layer of the metal film 9 is formed of gold. That is, when the metal film 9 has a single layer structure, the metal film 9 itself is formed of gold. When the metal film 9 has a laminated structure, the outermost layer to which the organic molecules 10 are fixed is gold. Is preferably formed. As a result, the organic molecules 10 can be easily fixed to the metal film 9.
- the metal film 9 has a laminated structure, it is preferable that the metal film 9 has a layer having a chromium force between the surface of the cantilever 5 and the outermost layer of the metal film 9. ,. This provides the advantage that the adhesion between the metal film 9 and the cantilever 5 surface is improved.
- the thickness of the metal film 9 is not limited and is arbitrary, but is usually 1 nm or more, preferably 10 ⁇ m or more, and usually 10 ⁇ m or less, preferably 5 ⁇ m or less. If the lower limit of this range is not reached, the organic molecules 10 may not be fixed sufficiently, and if the upper limit is exceeded, a good metal film may not be formed.
- the metal film 9 is formed by sputtering, vapor deposition, or the like.
- the cantilever 5 itself is made of metal, the surface of the cantilever 5 can be used as the metal film 9.
- An organic molecule 10 is fixed on the metal film 9.
- the organic molecule 10 can be fixed to the metal film 9, and there is no limitation on the type as long as the sugar chain 4 can be fixed on the organic molecule 10.
- Organic molecular force Any suitable one can be used depending on the type of sugar chain 4 to be fixed.
- the organic molecule 10 preferably has a mercapto group (one SH group) at its end.
- the organic molecule 10 since the organic molecule 10 is fixed to the metal film 9 by a stable “sulfur-metal bond”, the organic molecule 10 can be firmly fixed to the metal film 9.
- Specific examples of the organic molecule 10 include 16 mercaptohexadecanoic acid.
- organic molecule 10 may be used alone, or two or more organic molecules 10 may be used in any combination and in any ratio.
- the amount of the organic molecule 10 to be fixed is arbitrary, but it is usually preferable to fix it at a high density. This has the advantage that the thickness of the immobilized organic molecular layer can be made uniform.
- the organic molecules 10 may be fixed two-dimensionally on the surface of the metal film 9, but may be configured to be three-dimensionally stacked. Furthermore, when the organic molecule 10 is formed as a layer, it may be a single layer structure or a laminated structure. However, it is usually desirable to have a single layer structure. This is because it is easier to control the film thickness of the organic molecular layer in the single-layer structure than in the multi-layer structure.
- FIG. 3 shows an example in which organic molecules are formed in a single layer.
- the sugar chain 4 is immobilized on the organic molecule 10, and thereby, the sugar chain 4 is immobilized on the surface of the cantilever 5 via the metal film 9 and the organic molecule 10, so that the sugar chain 4 is immobilized.
- a fixed part 6 is formed.
- the sugar chain 4 may be fixed to the organic molecule 10 by any bond. Usually, it is desirable to be immobilized by a covalent bond. As a result, the sugar chain 4 can be firmly fixed to the organic molecule 10. In such a case, it is desirable that the sugar chain 4 is fixed via, for example, an ester bond, an amide bond, a C ⁇ N bond, an ether bond, a thioether bond, or a bond by a carbon atom.
- the sugar chain 4 may be fixed by one kind of bond, or may be fixed by any two or more kinds of bonds.
- the sugar chain 4 may have a functional group for immobilization. Furthermore, the above functional group may be bonded to a monosaccharide constituting the sugar chain 4 via another functional group.
- sugar chain 4 is immobilized on the organic molecule 10
- the specific operation is arbitrary.
- sugar chain 4 is immobilized on organic molecule 10 by bringing solution of sugar chain 4 into contact with organic molecule 10.
- FIG. 4 is an enlarged cross-sectional view schematically showing the vicinity of the sugar chain fixing part 6 when the sugar chain 4 is fixed by the second fixing method.
- FIG. 4 for the sake of explanation, the flat portion of the uneven pattern 8 on the surface of the cantilever 5 is shown.
- the same reference numerals as those in FIGS. 1 to 3 denote the same elements as in FIGS.
- the organic molecules 10 are not drawn individually but are drawn as a layer in which the organic molecules 10 are assembled.
- the sugar chain immobilization part 6 has a metal film 9 formed on the surface of the cantilever 5, an organic molecule 10 fixed on the metal film 9, and a porous matrix 11 bonded on the organic molecule 10. And is formed as a part having sugar chains 4 immobilized on the porous matrix 11.
- the metal film 9 is formed on the surface of the cantilever 5, and the organic molecules 10 are fixed on the metal film 9.
- the metal film 9 in the second fixed key method is the same as the metal film 9 described in the first fixed key method. It is like.
- the organic molecule 10 in the second immobilization method is explained in the first immobilization method except that the porous matrix 11 is bound instead of the sugar chain 4 being immobilized thereon. Same as organic molecule 10.
- porous matrix 11 is a gel-like structure in which a conjugate of sugar chain 4 and other molecule 12 is immobilized on organic molecule 10, and sugar chain 4 and other molecule 12 are composed. It is configured as a porous matrix by the skeleton. If this porous matrix 11 is used, the sugar chain 4 can be fixed with high density, so that the sensitivity of the cantilever sensor 3 can be increased.
- the method for producing the porous matrix 11 is not limited, but usually a solution or dispersion containing the sugar chain 4 and the other molecules 12 constituting the porous matrix 11 is prepared, and this solution or dispersion is prepared. Is brought into contact with the organic molecule 10 on the cantilever 5. At this time, water is usually used as a solvent or a dispersion medium. Thereby, the porous matrix 11 can be produced as a hydrogel.
- the bond between the sugar chain 4 and the other molecule 12 is usually the bond exemplified in the first fixing method as the bond between the sugar chain 4 and the organic molecule 10. It is the same. Accordingly, the sugar chain 4 is covalently fixed to the porous matrix 11, and a specific example of the bond used for the fixation is also the first fixation method. This is the same as that exemplified in.
- the other molecules 12 that constitute the porous matrix 11 together with the sugar chains 4 are not limited, but organic molecules are usually used. Specific examples include polysaccharides such as agarose, dextran, force ragenan, alginic acid, starch and cellulose, and derivatives such as carboxymethyl derivatives thereof, and polyvinyl alcohol, polyacrylic acid, polyacrylamide, polyethylene glycol and the like. A water-swellable organic polymer is exemplified.
- the sugar chain 4 may be fixed by other immobilization methods without depending on the first and second immobilization methods. Therefore, the method for immobilizing sugar chain 4 is not limited to sugar. This can be done by arbitrarily using a method already known as a method for fixing an organic compound on the sensor surface.
- a method for immobilizing pyotinylated sugar is shown in International Publication No. WO01Z40796 pamphlet.
- a method for immobilizing by Diels-Alder reaction is described in “Chemistry & Biology, vol. 9, 443-454, 2002 ".
- an immobilization method by hydrophobic interaction is shown in rBiomacromolecules, 2002, 3, 41 1 414 ”.
- the method power “Nature Biotechnol., 20, 1011-1017, 2002” for fixing to a trocellulose membrane after reductive amination of sugar is shown in column f.
- the method of immobilizing glycan 4 is optimal depending on the type of glycan 4, the functional group bound to glycan 4, the chemical factors resulting from the surface properties of the cantilever 5 to be immobilized, and economic factors. You can choose anything.
- the sugar chain 4 is described as being fixed on the entire upper surface of the cantilever 5 by the first fixing method, but the second fixing method is used.
- the effects of the present invention can also be obtained when the sugar chain 4 is immobilized by other immobilization methods.
- FIG. 5 is a schematic diagram showing the state where the detection target substance 13 interacts with the sugar chain 4 in the sugar chain immobilization part 6.
- the metal film drawn in FIGS. 3 and 4 is used. 9 and organic molecule 10 are not shown.
- the shape of the concavo-convex pattern 8 is arbitrary. You may make it form smooth unevenness, Irregularities having discontinuous steps may be formed. Further, unevenness that is continuous in a groove shape may be formed, or intermittent unevenness such as a depression or a peak may be formed. In the present embodiment, it is assumed that an uneven pattern 8 in which a large number of grooves are formed in parallel in the width direction is provided.
- the size of the uneven pattern 8 is not limited and is arbitrary, but it is preferable to arbitrarily set the detection sensitivity so as to increase the detection sensitivity according to the type and amount of the detection target substance and sugar chain 4. .
- the width of the concavo-convex pattern 8 is usually lOnm or more, preferably 50 nm or more, more preferably lOOnm or more, and usually 100 ⁇ m or less, preferably 50 ⁇ m or less, more preferably 10 m or less. desirable. This is because if the lower limit of this range is not reached, the detection target substance may not enter the concave / convex pattern 8, and if the upper limit is exceeded, the effect may not be recognized as compared with the case where the concave / convex pattern 8 is not present.
- the depth of the concavo-convex pattern 8 is usually lOnm or more, preferably 50 nm or more, more preferably lOOnm or more, and usually 100 ⁇ m or less, preferably 50 ⁇ m or less, more preferably 10 ⁇ m or less. desirable. This is because if the lower limit of this range is not reached, the detection target substance may not enter the concave / convex pattern 8, and if the upper limit is exceeded, the effect may not be recognized particularly compared to the case where the concave / convex pattern 8 is not present.
- the uneven pattern 8 is formed periodically. This is because the formation of the concave / convex pattern 8 can be simplified, and advantages such as more precise control of the interaction between the sugar chain 4 and the substance to be detected can be obtained.
- the formation method for forming the concave / convex pattern 8 is not limited.
- the concave / convex pattern 8 can be formed by directly patterning the portion where the sugar chain 4 on the surface of the cantilever 5 is fixed.
- etching is performed with the surface partially protected with a resist. Etc.
- the uneven film pattern in the metal film 9 is changed by changing the film thickness of the metal film 9. 8 can also be formed.
- a known method is used.
- a method in which metal colloidal particles are adsorbed on the surface of a previously formed metal film 9 or a method in which the surface of the metal film 9 is partially protected with a resist or the like may be used.
- the uneven pattern 8 will be described as being formed by patterning the uneven pattern 8 directly on the surface of the cantilever 5.
- the deflection of the cantilever sensor 3 is caused by the difference in surface stress between both sides of the cantilever 5. Therefore, the cantilever 5 is configured so that a difference in surface stress occurs between both surfaces of the cantilever 5 (upper surface and lower surface in FIG. 2) when an interaction between the sugar chain 4 and the detection target substance occurs.
- the force of the specific configuration is arbitrary Normally, the sugar chain 4 is fixed only on one side of the cantilever 5, and the sugar chain 4 is not fixed on the opposite side. At this time, when the sugar chain 4 is immobilized using the metal film 9, the metal film 9 is formed only on one surface on which the sugar chain 4 is immobilized, and is not formed on the opposite surface. Then, the sugar chain 4 can be easily fixed on only one side (the one side).
- the outermost layer of the metal film 9 (first metal film) on one side where the sugar chain 4 is fixed is fixed on the outer side of the metal film 9 (first metal film).
- Etc. the outermost layer of the metal film on the opposite side (second metal film) is a metal (for example, aluminum, copper, silver, etc.) other than gold If it is made of (metal), the sugar chain fixing part 6 can be easily formed only on one side (the one side).
- the amount of sugar chain 4 fixed to one surface of the both surfaces of the cantilever 5 and the other surface May be different.
- the type of sugar chain 4 fixed on one side of the cantilever 5 and the other side may be different.
- the sugar chain 4 is fixed only to the upper surface 5A in FIG. 2 of the cantilever 5, and the sugar chain 4 is not fixed to the lower surface 5B and the side surface 5C of the cantilever 5 in FIG.
- the difference in surface stress between the both surfaces of the cantilever 5 will be explained.
- the cantilever sensor 3 When measuring the deflection amount of the cantilever sensor 3 by an optical method, the cantilever sensor 3 is usually cantilevered. A reflective film is formed on the surface of the bar 5. At this time, the metal film 9 provided for fixing the sugar chain 4 on the cantilever 5 can be used as a reflection film.
- the metal film 9 can also be used as a piezoelectric resistance element portion.
- the metal film 9 is formed in the length direction of the cantilever 5 at least over a range where the piezoelectric resistance element portion is provided. This is because the deflection of the piezoelectric resistance element portion is detected as a signal, while the deflection other than the piezoelectric resistance element portion is not detected.
- the metal film 9 on the cantilever 5 has a function of fixing the sugar chain 4, it is preferable that the metal film 9 is not provided on the support member 7 of the cantilever 5. ,. This avoids immobilizing the sugar chain 4 at a position unrelated to detection.
- the metal film 9 is continuously formed on the support member 7 on the upper surface 5A of the cantilever 5 (see FIG. 8). ).
- any sensor system (measurement system) may be used as long as the deflection amount of the force cantilever sensor 3 can be measured.
- a sensor system including the above-described cantilever sensor 3, a sample liquid contact portion that makes the sample solution contact the sugar chain fixing portion 6, and a deflection amount measurement portion that measures the deflection amount of the cantilever 5 is used.
- FIG. 6 schematically illustrates an outline of a main part of an example of the sensor system.
- the sensor system of the present invention is not limited to the following example.
- the same reference numerals as those used in FIGS. 1 to 5 denote the same parts as in FIGS.
- this sensor system includes a cantilever sensor 15 to which the sugar chain 4 is fixed and a cantilever holder 15 to which a correction cantilever 14 is attached, and a detection that is a specimen liquid contact portion.
- the unit 16 includes a measuring device 17 that is a deflection amount measuring unit and a correction deflection amount measuring unit, a calculation device 18 that is a deflection amount difference output unit and a use limit detecting unit, and an arm 19 that is a cantilever vibrating unit.
- the cantilever holder 15 is a housing having a hollow opened downward, and the cantilever sensor 3 and the correction cantilever 14 are attached to the hollow inside.
- an inlet 15A for introducing the sample liquid into the cantilever holder 15 is provided on the side wall of the cantilever holder 15 so that the cantilever sensor 3 and the correction cantilever 14 can come into contact with the sample liquid during use.
- the cantilever holder 15 is in use, as shown by the arrow in FIG. 6, the force of the sample liquid flowing through the flow path 20 in the detection unit 16 is immersed in the sample liquid, and the sample liquid also flows through the cantilever holder 15.
- the cantilever sensor 3 and the correction cantilever 14 come into contact with each other through the flow passage (see FIG. 7).
- an electrode (not shown) is formed on a portion where the cantilever sensor 3 and the correction cantilever 14 on the upper portion of the cantilever holder 15 can be attached, and the cantilever sensor 3 and the correction cantilever are passed through this electrode. The amount of deflection of cantilever 14 is sent to measuring instrument 17 as a signal.
- the cantilever sensor 3 attached to the cantilever holder 15 is the cantilever sensor 3 described above with the sugar chain fixing part 6 formed on one side. Further, in the cantilever sensor 3, as described above, a piezoresistive element (not shown) is provided under the metal film 9 used for fixing the sugar chain 4, and the piezoresistive element is The wiring connected to the measuring instrument 17 is connected via the metal film pattern patterned so as to be insulated from the metal film 9 on the support member 7 and the electrode provided on the cantilever holder 15. RU
- the correction cantilever 14 is a cantilever used for the purpose of measuring a correction value for correcting the deflection amount of the cantilever sensor 3 so as to eliminate the influence of the deflection amount due to environmental change or the like.
- the amount of deflection of the cantilever sensor 3 changes not only due to the change in surface stress caused by the interaction between the detection target substance and the sugar chain 4, but also due to changes in the environment such as temperature and pressure. Therefore, when measuring the amount of deflection, it was caused by the interaction between the target substance to be detected and sugar chain 4 while eliminating the effect of deflection due to environmental changes. It is desirable to measure only the deflection.
- the correction cantilever 14 is used to eliminate the amount of deflection due to the environmental change described above.
- the correction cantilever 14 preferably has no sugar chain 4 immobilized on the entire surface.
- the entire surface 14A of the correction cantilever 14 is fixed with the sugar chain 4 !, and functions as a non-fixed portion.
- the correction cantilever 14 is formed in the same manner as possible with the cantilever sensor 3 to be corrected except that the sugar chain 4 is not fixed. Specifically, it is preferable that dimensions and materials are as equal as possible.
- the deflection amount of the cantilever sensor 3 is the sum of the deflection amount due to the interaction and the deflection amount due to the environmental change, while the deflection amount of the correction cantilever 14 is only the deflection amount due to the environmental change. By calculating, it becomes possible to accurately measure the amount of deflection caused by the interaction.
- the entire surface 14A of the correction cantilever 14 may be a non-film-deposited part where no metal film is provided.
- the sugar chain 4 cannot be fixed to the correction cantilever 14 via the metal film. Therefore, even if the same processing as that of the cantilever sensor 3 is performed, the surface of the correction cantilever 14 has no sugar. This is because the chain 4 is not fixed and, therefore, the correction cantilever can be easily manufactured and handled.
- the correction cantilever 14 may be provided with a metal film such that a metal other than gold, such as aluminum, copper, or silver, becomes the outermost layer.
- a metal other than gold such as aluminum, copper, or silver
- the binding force between the organic molecule 10 and the metal is smaller than when gold is used, so the ability to fix the sugar chain 4 of the correction force cantilever 14 is not This is smaller than the ability of the cantilever sensor 3 to immobilize the sugar chain 4. Therefore, even if the same processing as that of the cantilever sensor 3 is performed, the sugar chain 4 is not immobilized on the surface of the correction cantilever 14, and therefore the correction cantilever can be easily manufactured and handled.
- the sugar chain 4 fixed to the cantilever sensor 3 is detected on the correction cantilever 14.
- Another correction sugar chain (hereinafter referred to as “correction sugar chain” as appropriate) having a different interaction magnitude with respect to the target substance may be fixed.
- the portion where the correcting sugar chain is immobilized functions as a correcting sugar chain fixing portion. For example, when an appropriate correction sugar chain that does not interact with the detection target substance is fixed to the correction cantilever 14, a deflection very close to the deflection caused by the environmental change occurring in the cantilever sensor 3 is corrected. It is possible to more accurately measure the amount of deflection caused by the interaction generated in the cantilever sensor 3.
- the method for producing the correction cantilever 14 is not limited and is arbitrary. However, for example, in the description of the cantilever sensor 3, when the cantilever is manufactured using the semiconductor formation process exemplified as one manufacturing method, even if the cantilever is manufactured under the same conditions, There may be differences in film thickness and material between wafers, and even between locations within the same wafer. This can also occur in technologies other than semiconductor formation processes.
- the difference in film thickness and material described above is small if the same production lots are used, and is further reduced if they are taken from the same wafer. Furthermore, even within the same wafer, the closer it is removed, the smaller the difference. By utilizing this, it is more preferable that the cantilever 5 for the cantilever sensor 3 and the correction cantilever 14 are manufactured at positions adjacent to each other even on a wafer that is preferably manufactured from the same wafer.
- the cantilever sensor 3 and the correction cantilever 14 are without being separated from each other. By not separating, it is clearly shown that measurement is performed by combining these two, and the mounting work of both cantilever sensors 3 and the correction cantilever 14 is simplified. Further, when the cantilever sensor 3 and the correction cantilever 14 are used without being separated from each other, the metal film 9 may not be formed on the surface of the correction cantilever 14 from the viewpoint of simplifying the production of the correction cantilever 14. preferable. As a result, sugar chain 4 is fixed only to one of the two cantilevers 5, 14 that are not separated (ie, cantilever 5), and sugar chain 4 is fixed to the other (ie, correction cantilever 14). It will be easier to keep away. This is because the sugar chain 4 is This is because the sugar chain 4 is not fixed to the correction cantilever 14 on which the metal film 9 is not formed when the fixing is performed.
- a reflective film for reflecting light from the light source may be formed on the correction cantilever 14 with a metal.
- the reflective film should be formed so that the metal that is difficult to fix the sugar chain 4 as the reflective film of the correction cantilever 14, specifically, the metal such as aluminum, copper, or silver is the outermost layer. That's fine.
- the binding force between the organic molecule 10 and the metal is weaker than when gold is used, so the ability to fix the sugar chain 4 of the correction cantilever 14 is not This is smaller than the ability of the forcech sensor 3 to fix the sugar chain 4.
- a cantilever formed in the same manner as the cantilever sensor 3 except that the sugar chain 4 is not immobilized, that is, the cantilever 5, the metal film 9, and the organic A cantilever constructed with molecules 10 shall be used.
- a piezoresistive element (not shown) is provided under the metal film 9, and the piezoresistive element is provided on the support member 7 of the cantilever 14 for correction.
- the wiring connected to the measuring instrument 17 is connected through the metal film pattern (not shown) patterned so as to be insulated from the metal film 9 and the electrode provided on the cantilever holder 15. ing.
- the cantilever holder 15 is configured to be detachable at the tip of an arm 19 that is movably provided, and can be attached to the arm 19 in use.
- the arm 19 can be moved up and down, and when the cantilever holder 15, the cantilever sensor 3, the correction cantilever 14, etc. are attached, removed, or exchanged, the arm 19 is raised to raise the arm 19, and when the detection target substance is detected, the arm 19 is lowered. Operate to lower arm 19 and insert cantilever holder 15 into channel 20! /
- the arm 19 is provided with a piezoresistive element portion for generating vibration (hereinafter referred to as “vibrating piezoelectric element” t; not shown) as necessary! As the element vibrates, the arm 19 can also vibrate.
- the vibration piezoelectric element is a member that expands and contracts by an applied voltage, and generates vibration using the expansion and contraction.
- the cantilever sensor 3 and the correction cantilever 14 The piezoresistive element provided in is an element whose resistance value changes according to the expansion and contraction of the element, and the configuration itself of the piezoresistive element is the same as that of the oscillating piezoelectric element, but its use is different.
- the cantilever holder 15 attached to the arm 19 also vibrates, and accordingly, the internal cantilever sensor 3 and the correction cantilever 14 also vibrate.
- the cantilever sensor 3 By vibrating the cantilever sensor 3 during detection of the detection target substance, it is possible to improve the selectivity when the detection target substance and the sugar chain 4 interact.
- the cantilever sensor 3 is first vibrated to dissociate substances other than the substance to be detected, then the vibration is stopped, and the deflection amount of the cantilever sensor 3 is measured. .
- the detection unit 16 is a member that serves as a place where the cantilever sensor 3 and the correction cantilever 14 are brought into contact with the sample liquid.
- the detection tube 16 is formed as a container having a flow path 20, and the specimen is introduced into the flow path 20 by a pump (not shown), and the cantilever sensor 3 and the correction cantilever 14. After touching, the detection unit 16 is discharged outside.
- an opening 21 for inserting the cantilever holder 15 into the flow path 20 is formed in the upper part of the detection unit 16, and the cantilever holder 15 is inserted into the flow path 20 through the opening 21.
- the cantilever sensor 3 and the correction cantilever 14 can come into contact with the sample liquid in the flow path 20.
- the opening 21 is formed larger than the cantilever holder 15 so as to have a predetermined play portion with the detection unit 16 when the cantilever holder 15 is inserted. The This is to prevent the cantilever holder 15 from contacting the detection nut 16 when the cantilever holder 15 is vibrated by the arm 19.
- the measuring device 17 is a deflection amount measuring device that detects the deflection amounts of the cantilever sensor 3 and the correction cantilever 14.
- any known measuring instrument for measuring the amount of deflection can be arbitrarily used.
- optical measuring instruments and electric measuring instruments as measuring instruments for measuring the deflection amount of the cantilever.
- An optical measuring instrument deflects light by illuminating a cantilever with light from a light source, reflecting the light on a cantilever, detecting the reflected light, and measuring the reflection angle of the reflected light. The amount is measured (see J. Vac. Sci. Technol. B, vol. 14, pp. 1383— 1385, 199 6). Therefore, when an optical measuring instrument is used, a reflective film is provided on the reflective surface side of the cantilever.
- an electrical measuring instrument uses a cantilever with a piezoresistive element patterned on one side and measures the change in the resistance value of the piezoresistive element when deflection occurs. (Refer to Ultramicroscopy, vol. 97, pp. 371-376, 2003) o In the case of this electrical measuring instrument, the deflection of the piezoresistive element is detected as a signal.
- an electric instrument is used as the measuring instrument 17, and the deflection amount is measured by the piezoresistive elements provided in the cantilever sensor 3 and the correction cantilever 14, respectively. Will be described. Further, the deflection amount of each of the cantilever sensor 3 and the correction cantilever 14 measured by the measuring instrument 17 is sent to the calculation device 18.
- the measuring device 17 is described as measuring the amount of deflection of both the cantilever sensor 3 and the correction cantilever 14, but the amount of deflection of both may be measured using separate measuring devices. Good.
- the calculation device 18 calculates the difference between the deflection amount of the cantilever sensor 3 and the deflection amount of the correction cantilever 14 from the sent measurement result, and outputs the difference to an output device (not shown) such as a printer or a display. It becomes like that and speaks.
- the calculation device 18 calculates the use limit of the cantilever sensor 3 from the measured deflection amount. Is supposed to be detected.
- the use limit here refers to the limit of the sugar chain 4 fixed to the cantilever sensor 3 that no longer interacts with the detection target substance, and no further detection target substance can interact.
- the amount of deflection corresponding to the usage limit is experimentally obtained in advance, and the limit value is recorded in a storage unit such as a memory of the calculation device 18, and the measured amount of deflection and the recording unit are recorded. It can be detected by comparing with the limit value.
- the sample liquid targeted by this sensor unit refers to any liquid that is a target for detection of the detection target substance, and any liquid can be used without any particular limitation.
- any liquid can be used without any particular limitation.
- nasal cavity suction liquid, nasal cavity wiping liquid, pharyngeal wiping liquid, or the like can be used as the sample liquid.
- the presence or absence of the subject's infection can be diagnosed by detecting these sample liquids.
- the nasal cavity aspirate is collected, for example, by suction with a tube force pump or the like inserted into the nasal cavity.
- the nasal wiping liquid is collected by, for example, inserting a cotton swab into the nasal cavity and scraping the mucosal epidermis.
- the scalp and pharyngeal wipes are collected, for example, by inserting a cotton swab into the pharynx and scraping the mucosal epidermis.
- sample solutions may be measured after being diluted with a solvent such as a buffer solution, physiological saline, ethanol, water or the like.
- a solvent such as a buffer solution, physiological saline, ethanol, water or the like.
- the sample collected in the solvent may be dissolved into a sample liquid by immersing the cotton swab in a solvent and squeezing out the wiped liquid. .
- FIG. 7 is a cross-sectional view schematically showing the vicinity of the detection unit 16 when the sensor system is used.
- the same components as those in FIGS. 1 to 6 are denoted by the same reference numerals. Since this sensor system is configured as described above, when detecting the detection target substance with this sensor system, as shown in FIG. 7, the arm 19 is moved down to bring the cantilever holder 15 into the flow path 20. In the inserted state, the sample liquid is circulated through the flow path 20. At the same time, the arm 19 starts to vibrate.
- FIG. 8 is a schematic diagram for explaining how the deflection occurs when the detection target substance 13 interacts with the cantilever sensor 3.
- the concave / convex pattern 8 is not shown.
- the deflection occurs by the same amount as the amount of deflection caused by the environmental change out of the total deflection amount generated in the cantilever sensor 3.
- the amount of deflection of the cantilever sensor 3 and the correction cantilever 14 is measured by the measurement unit 17, and the measurement result is sent to the calculation unit 18.
- the calculation unit 18 calculates the difference between the deflection amount of the cantilever sensor 3 and the deflection amount of the correction cantilever 14 from the measurement result sent.
- the calculated value is the amount of deflection due to the interaction between the detection target substance and sugar chain 4 without the influence of the amount of deflection due to environmental changes. Can be done.
- the calculated difference in deflection amount is output to an output device (not shown).
- the calculation unit 18 also detects the use limit of the cantilever sensor 3.
- a use limit is detected, a message to that effect is output to an output device (not shown). If the use limit is detected in this way, when the cantilever sensor 3 is used a plurality of times, the timing for replacing the cantilever sensor 3 can be properly grasped, so that the detection can be performed efficiently. Can be performed automatically.
- sample liquid that has finished flowing through the flow path 20 is discharged to the outside of the detection unit 16.
- the detection target substance is detected using the sensor system, the detection is performed.
- the target substance can be detected with high sensitivity in a short time with a simple configuration.
- sensors using cantilevers have been developed.
- DNA nobduction detection sensors Science, Vol. 288 (2000), pp. 316-318
- antigen-antibody reaction detection sensors Sensors
- Actuators B Vol. 79 (2001), pp. 115-126
- microcantilever 'biosensor pattern: WO9850773
- the conventional sensor as exemplified here does not have sufficient detection sensitivity, and has been unable to detect, for example, viruses and bacteria with practical sensitivity.
- the cantilever sensor of the present invention has sufficient sensitivity, it is possible to detect a detection target substance that has been difficult to detect with high sensitivity.
- the detection sensitivity can be further improved.
- the correction is performed using the correction cantilever 14, the influence of the deflection due to the environmental change is eliminated, and the deflection amount due to the interaction can be accurately measured.
- the detection sensitivity of the cantilever sensor 3 can be further increased.
- the selectivity of the detection target substance that interacts with the sugar chain 4 can be improved, and a more accurate analysis can be performed.
- cantilever sensor and sensor system of the present invention are not limited to those described above, and can be arbitrarily modified and implemented without departing from the gist of the present invention.
- the force with which the sample liquid flowing through the flow path 20 is discharged to the outside of the detection unit 16 may be returned to the detection unit 16 and circulated again.
- the sugar chain 4 on the cantilever sensor 3 interacts with the detection target substance more efficiently, and the detection sensitivity can be further improved.
- the sample liquid may be directly introduced into and discharged from the detection unit 16 with a pipette or the like without providing the flow path 20.
- the amount of deflection may be measured using a force-optical measuring device in which the amount of deflection is measured using an electric measuring device. good.
- the deflection amount measuring unit includes a light source that emits light to the cantilever sensor 3 and light emitted from the light source.
- a light detector that detects the reflected light reflected by the cantilever sensor 3, and the surface of the cantilever sensor 3 so that the light from the light source can be reflected (for example, a reflective film is formed).
- the cantilever sensor 3 is irradiated with light, and the reflected light reflected by the cantilever sensor 3 is detected by the photodetector and the reflection angle of the reflected light is measured. Can be measured.
- the correction deflection amount measuring unit it is also possible to configure the correction deflection amount measuring unit as an optical type.
- the configuration of the cantilever vibrating section for vibrating the cantilever sensor 3 is not limited to the above example, but is arbitrary.
- a vibration piezoelectric element 19A may be provided between the cantilever holder 15 and the arm 19 as shown in FIG. That is, the vibration piezoelectric element 19A may be provided in a portion where the cantilever holder 15 at the tip of the arm 19 is attached. Also by this, the cantilever holder 15 is vibrated by the vibration generated by the vibrating piezoelectric element 19A, and the cantilever sensor 3 can be vibrated.
- the vibration piezoelectric element 19A is supplied with electric power from a power source (not shown) and the vibration is controlled by a control device (not shown).
- the vibrating piezoelectric element 19A itself functions as a cantilever vibrating portion.
- FIG. 9 the same parts as those in FIGS. 1 to 8 are denoted by the same reference numerals.
- a vibrating piezoelectric element may be provided in the cantilever sensor 3 itself. Also in this case, the cantilever sensor 3 can be vibrated.
- a cantilever vibration part such as a piezoresistive element for vibration is actually provided outside the cantilever sensor 3.
- the force by which the base part of the cantilever sensor 3 is vibrated is inexpensive, and since the structure is simple, there is an advantage that it is difficult to break down.
- the vibration may be generated using a member other than the piezoresistive element for vibration.
- a vibration part such as the cantilever vibration part described in the above example is used to detect a detection target substance using a specific substance (for example, sugar chain 4 in the present invention) that specifically interacts with the detection target substance. It is extremely useful to use it as a sensor to detect. That is, even in sensors other than the cantilever sensor and sensor system of the present invention, when detecting a specific interaction between a detection target substance and a specific substance, the substance other than the detection target substance and the specific substance are non-specifically. There is a possibility that the signal due to the interaction may interfere with the reading of the signal due to the interaction between the detection target substance and the specific substance.
- a specific substance for example, sugar chain 4 in the present invention
- the members that generates vibration is arbitrary. At this time, it is usually preferable to use the vibration piezoelectric element as described above in order to effectively exclude non-specific interaction.
- the used cantilever sensor 3 is discarded, and each time detection is performed, the detection operation is performed by replacing the cantilever sensor 3 with the sugar chain 4 fixed. It may be. Thereby, accurate detection can be performed.
- the detection cost can be reduced.
- the washed cantilever is again used. It can be used for detection.
- the cantilever sensor 3 is repeatedly used for detection in this way, it is preferable to replace the cantilever sensor 3 when the use limit is reached.
- the number of sugar chains 4 immobilized on the cantilever sensor 3 that can interact with the detection target substance in the sample gradually decreases. This is because the number of sugar chains 4 that continue to interact with the detection target substance remaining on the cantilever sensor 3 after the cleaning treatment gradually increases.
- the use limit of the cantilever sensor 3 at which sufficient detection cannot be performed at a certain point in time is reached. Therefore, it is preferable to replace the cantilever sensor 3 when such a use limit is reached.
- the above-mentioned use limit is obtained by experimentally obtaining in advance a deflection amount corresponding to the use limit, and the limit value is calculated based on the deflection of the cantilever sensor 3 in use. It can be detected by comparing with an amount.
- the regeneration process for fixing the sugar chain 4 again is performed, and then the cantilever sensor 3 is used for detection. You may make it go out. That is, the sugar chain 4 fixed on the cantilever sensor 3 is once separated from the cantilever, and the sugar chain 4 is fixed again to regenerate the cantilever sensor 3, and the cantilever sensor 3 is used again for detection. Even so, it is good.
- the cantilever sensor 3 that has been repeatedly detected only by the cleaning process and has reached the use limit can be regenerated by such a regeneration process.
- a cartridge may be manufactured using the cantilever sensor 3, and detection may be performed using the cartridge.
- An example of the configuration at that time is shown in FIG.
- a container (sensor holder) 22 having a cantilever sensor 3 provided therein is prepared, and an electric signal indicating the amount of deflection of the cantilever sensor 3 is applied to the portion of the container 22 where the cantilever sensor 3 is attached.
- the electrode 23 is formed for sending to the measuring device 17.
- an analyzer 26 equipped with a mounting part 25 for mounting the sensor cartridge 24 formed of the cantilever sensor 3 and the container 22 is prepared, and the sensor cartridge 24 is mounted on the mounting part 25.
- the measuring instrument 17 in the analyzer 26 can measure the deflection of the cantilever sensor 3. Using these, the sensor cartridge 24 is mounted on the mounting portion 25 of the analyzer 26, and even if the sample liquid is put into the container with a pipette or the like to measure the amount of deflection, the detection target substance in the sample liquid is detected. can do.
- the sensor system shown in FIG. 10 may be further modified and implemented, for example, in combination with the above-described configuration.
- Specific examples include a correction cantilever 14 attached to the sensor cartridge 24, a flow path through which the sample liquid is circulated, a calculation device 18 provided to the analyzer 26, and a cantilever sensor like the arm 19.
- a vibration part for vibrating 3 may be provided.
- FIG. 10 the same parts as those in FIGS. 1 to 9 are denoted by the same reference numerals.
- each component of the above-described cantilever sensor and sensor system can be used in any combination.
- the above-described cantilever sensor and sensor system may be used in combination with any other analyzer.
- the virus was cultured using Infnorenza type A (H3N2) ZFukuokaZC29Z85 according to the following procedure.
- the kidney kidney-derived cell line (MDCK cells) was cultured in a flask at 37 ° C for 3 days, and then the cell growth medium was removed.
- the virus was inoculated into MDCK cells and allowed to adsorb at room temperature for 1 hour. Then, the virus was removed, and an influenza medium containing 0.3% urine serum albumin (BSA) was added at 37 ° C. For 3 days.
- BSA urine serum albumin
- Infected cells exhibiting cytopathic effect (CPE) 3-4 + and culture supernatant were collected and centrifuged at 4 ° C and 3000 rpm for 10 minutes. The supernatant was dispensed in 2.5 ml aliquots as virus solution.
- the virus titer was measured and found to be 4 ⁇ 10 7 [TCID / ml].
- the sugar compound (1) was synthesized by the procedure shown in the reaction formula of FIG. Ts represents a tosyl group, DMF represents N, N-dimethylformamide, Bn represents a benzyl group, Me represents a methyl group, Ac represents a acetyl group, TMS represents a trimethylsilyl group, rt represents room temperature.
- the sialic acid derivative (5) and the compound (4) are reacted in DMF in the presence of CsF at ⁇ 10 ° C. to 0 ° C. for 15 hours for coupling, and after workup and purification, the compound ( 6) was obtained. Thereafter, this compound (6) was hydrolyzed in MeOH using water and LiOH at 0 ° C. to room temperature for 3 days to obtain compound (7) after workup and purification.
- This compound (7) was first treated with 5% -PdZcarbon in a mixed solvent of MeOH and 1N hydrochloric acid under a hydrogen atmosphere, subjected to a reduction reaction, filtered, and further NaOAc at room temperature for 30 minutes.
- a neutralization reaction was carried out, and a sugar compound (1) was obtained after purification. Except for the reduction reaction by PdZC, the reaction was carried out in a nitrogen atmosphere.
- the compounds (3) to (7) were purified by silica gel chromatography, and the sugar compound (1) was purified by gel filtration. The total yield of the sugar compound (1) from the compound (2) was 8.3%.
- One of the two cantilevers was used as a detection cantilever (cantilever sensor), and the surface was modified by the following process.
- N-hydro xysuccinimide and 1- Ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride were added to an aqueous solution adjusted to have concentrations of 5. OmM and 20. OmM, respectively.
- Immerse the detection cantilever for 20 minutes at room temperature After washing, it was thoroughly dried. Then, it was immersed in a 6.5 mM methanol solution of the above sugar compound (1) at room temperature for 19 hours, washed with methanol, and sufficiently dried.
- the sugar compound (1) was immobilized on the detection cantilever as a sugar chain.
- a 0.3% PSA (porcine serum albumin) buffer solution for 25 hours, washed with water, and then sufficiently dried to prepare a cantilever sensor.
- the remaining one cantilever was used as a correction cantilever, soaked in a 0.3% PSA (porcine serum albumin) buffer solution for 25 hours, washed with water, and sufficiently dried.
- PSA protein serum albumin
- the measuring cell has a structure in which a base made of Teflon (registered trademark) is covered with a glass substrate, the laser is irradiated to the cantilever sensor and the correction cantilever through the glass substrate, and the above cantilever sensor is used. And the reflected light from the correction cantilever can be taken out respectively.
- the measurement cell is provided with a liquid inlet, and the force is also used to replace the liquid in the measurement cell.
- a sensor system as shown in Fig. 12 was prepared.
- the sensor system shown in Fig. 12 is equipped with a He-Ne laser as a light source, and the output light from this He-Ne laser is condensed into a linear shape with a width of m and a length of lcm by the lens 2).
- the tip of the cantilever sensor and correction cantilever set in the measurement cell (not shown) can be irradiated simultaneously.
- the line segment XI in FIG. 12 represents a straight line portion where the light emitted from the He—Ne laser is collected, and the light hits the cantilever sensor and the correction cantilever. It ’s like that. In FIG. 12, the measurement cell is not shown.
- the sensor system of FIG. 12 includes a CCD camera as a light detection unit for detecting reflected light from the cantilever sensor and the correction cantilever.
- the CD camera can observe the reflected light of the cantilever sensor and the correcting cantilever force at the same time.
- the sensor system in FIG. 12 includes a personal computer (not shown; hereinafter referred to as “PC” for the sake of convenience) to calculate the amount of deflection of the reflected light force detected by the CCD camera. Is taken into the personal computer, and image processing is performed on the personal computer, so that the center position of the two reflected lights can be obtained, and the time change of the deflection amount of the cantilever sensor and the correction cantilever can be calculated.
- PC personal computer
- the amount of deflection of each of the cantilever sensor and the correction cantilever was measured using the sensor system described above, the virus solution as a sample solution, and the virus solution filled in the measurement cell. Specifically, with the virus solution filled in the measurement cell and the virus solution in contact with the cantilever sensor and the correction cantilever, the He-Ne laser force also irradiates the cantilever sensor and the correction cantilever with light. The reflected light from the cantilever sensor and the correction cantilever was detected by a CCD camera, and the output image was calculated by a personal computer to calculate the amount of deflection.
- the angle change at the tip of the cantilever was measured as the amount of deflection of the force cantilever.
- the difference obtained by subtracting the deflection amount of the correction cantilever from the deflection amount of the cantilever sensor was calculated by the above personal computer, and the change with time was obtained.
- Figure 13 shows the measurement results.
- Figure 14 shows the time variation of the difference obtained by subtracting the deflection amount of the cantilever for correcting the deflection force of the cantilever sensor.
- the cantilever sensor, the sensor system of the present invention, and the detection of the detection target substance in the sample liquid The dispensing method can be used in any industrial field, but is suitable for use in fields such as medical treatment, food analysis, and biological analysis.
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WO2008106469A1 (en) * | 2007-02-26 | 2008-09-04 | University Of Florida Research Foundation, Inc. | Living cell force sensors and methods of using same |
WO2009142045A1 (ja) * | 2008-05-20 | 2009-11-26 | 日本電波工業株式会社 | 圧電センサ及び感知装置 |
JP2010185890A (ja) * | 2010-06-02 | 2010-08-26 | Nippon Dempa Kogyo Co Ltd | 圧電センサ及び感知装置 |
CN105675430A (zh) * | 2016-03-31 | 2016-06-15 | 常州轻工职业技术学院 | 一种新型传感器 |
WO2016136905A1 (ja) * | 2015-02-27 | 2016-09-01 | 国立研究開発法人物質・材料研究機構 | 母材と粒状材料を混合した受容体層を被覆したセンサ |
JPWO2016121155A1 (ja) * | 2015-01-27 | 2017-08-31 | 国立研究開発法人物質・材料研究機構 | 多孔質材料または粒状材料を受容体層として有するセンサ |
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