JP3892292B2 - Biopolymer detection device and biopolymer detection method, carbon nanotube structure used therefor, and disease diagnosis apparatus - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention provides a biopolymer detection device and a biopolymer detection method capable of easily and reliably detecting a target biopolymer present in a sample and capable of efficiently diagnosing a disease, and the like. The present invention relates to a carbon nanotube structure and a disease diagnosis apparatus.
[0002]
[Prior art]
The Human Genome Project is a plan to share all human genes (DNA) in various countries around the world, and has been implemented since the 1990s. In the summer of 2000, a draft was written that included information about all human genes (DNA). A version has been published. If it becomes clear what parts of all human genes (DNA) are related to what biological functions, life science-related technologies will be further developed, including technologies for diagnosing and treating diseases. It is expected to be.
[0003]
For example, conventionally diagnosed diabetes is simply classified as type I or type II based on the level of insulin producing ability in the patient's body. In the case of diabetes, the balance of functions and amounts in a plurality of proteins that function in a complex manner such as a receptor for blood glucose and an enzyme that synthesizes or degrades insulin according to the amount of the blood glucose level is lost. It develops due to incomplete adjustment of the value, but the conventional diagnosis of diabetes has a problem that the direct cause of diabetes cannot be known. However, the decoding information of all human genes (DNA) revealed by the Human Genome Project is the total of gene (DNA) information encoding amino acid sequences of various proteins such as enzymes and receptors involved in the regulation of blood glucose level. By analyzing this gene (DNA) information, it is possible to know the protein that causes the abnormal regulation of blood glucose level. Therefore, it can be classified into more detailed subtypes rather than classified as type II, and more appropriate diagnosis and treatment of diabetes can be performed. In the near future, by analyzing the functions and abundances of a series of functionally closely related proteins, it is expected that the era of more appropriate diagnosis and treatment of diseases will arrive.
[0004]
By the way, at present, the only method that can easily measure the abundance of a plurality of proteins that are closely related to each other as described above is based on the combined use of two-dimensional electrophoresis and mass spectrometry. It has not been. However, this method has a problem that information effective for diagnosing or treating a disease cannot be obtained sufficiently and measurement cannot be easily performed.
On the other hand, with respect to DNA, a DNA in a sample bound with complementary DNA strands arranged in an array when a DNA to be measured is amplified (increase) in advance by PCR reaction (polymerase chain reaction). There is provided a DNA chip capable of easily quantifying the amount of DNA in the sample by the fluorescence intensity based on the above. However, for proteins, there is no amplification method corresponding to the PCR reaction, and since the reactivity of the protein and the fluorescent labeling dye is different, when multiple proteins are mixed, the fluorescent labeling dye is uniformly introduced into each protein. Currently, no chip capable of quantifying the amount of protein in a sample has yet been provided. For this reason, there is a demand for the development of an array chip that can easily quantify a specific protein and related technology without using a fluorescent labeling dye.
[0005]
[Problems to be solved by the invention]
The present invention has been made in response to such a demand, and solves the above-described problems and achieves the following objects. That is, the present invention can easily and reliably detect abundance of a plurality of target biopolymers having a series of functionally close relationships existing in a sample, and can efficiently diagnose diseases, etc. It is an object of the present invention to provide a biopolymer detection device and a biopolymer detection method applicable to array chip technology, a carbon nanotube structure used therefor, and a disease diagnosis apparatus.
[0006]
[Means for Solving the Problems]
Means for solving the above problems are as follows.
<1> vibration inducing means for inducing vibration, Formed of carbon nanotubes, A coupling means capable of resonating by vibration induced by the vibration inducing means and capable of binding or interacting with the target biopolymer; and whether or not the coupling means has bound or interacted with the target biopolymer. It is a biopolymer detection device characterized by having a detection means to detect.
In the biopolymer detection device according to <1>, the vibration inducing unit induces vibration. Formed with carbon nanotubes The coupling means resonates due to the vibration induced by the vibration inducing means. Since the binding means can bind to or interact with the target biopolymer, if the target biopolymer is present in the sample, it binds to or interacts with the target biopolymer. The detection means detects whether or not the binding means has bound or interacted with the target biopolymer, whereby the amount of the target biopolymer in the sample can be quantified easily.
[0007]
<2> The vibration inducing means is connected to the base electrode provided at one end of the coupling means, the vibration inducing electrode disposed in the vicinity of the coupling means, the base electrode and the vibration inducing electrode in a conductive manner, and an alternating current The biopolymer detection device according to <1>, further comprising an AC power supply capable of applying a voltage.
In the biopolymer detection device according to <2>, the base electrode is provided at one end of the coupling unit, the vibration-inducing electrode is disposed in the vicinity of the coupling unit, and the two are electrically connected to each other. When the AC power supply applies an AC voltage, an AC electric field is generated between the coupling means and the vibration-inducing electrode, and the coupling means is connected to the AC voltage due to its conductivity. Vibrates according to frequency.
[0008]
<3> The biopolymer detection device according to <1> or <2>, wherein the vibration inducing means is a piezoelectric element. In the biopolymer detection device according to <3>, when the piezoelectric element is driven, the coupling unit resonates with vibration by the piezoelectric element.
[0009]
<4> Any one of <1> to <3>, wherein the coupling unit includes a sensitive unit capable of resonating by vibration applied by the vibration inducing unit and a coupling unit capable of coupling or interacting with the target biopolymer. The biopolymer detection device described. In the biopolymer detection device according to <4>, the sensitive part resonates due to vibration induced by the vibration inducing means. If the target biopolymer is present in the sample, the binding unit binds to or interacts with the target biopolymer.
[0010]
<5> The biopolymer detection device according to <4>, wherein the sensitive part is a carbon nanotube. In the biopolymer detection device according to <5>, the carbon nanotube resonates due to vibration induced by the vibration inducing means.
[0011]
<6> The bonding means is formed by bonding the bonding portion to the tip of the carbon nanotube, and reacting the material forming the bonding portion with the dangling bond at the tip of the carbon nanotube generated by processing the carbon nanotube. The biopolymer detection device according to any one of <3> to <5>, which is manufactured by bonding. In the biopolymer detection device according to <6>, the carbon nanotube resonates due to vibration induced by the vibration inducing unit. The bonding part bonded to the tip of the carbon nanotube by reacting the material forming the bonding part with a dangling bond generated by treatment in an oxygen plasma atmosphere is bonded to the target biopolymer in the sample. If present, it binds to or interacts with the target biopolymer.
[0012]
<7> Any of the above <4> to <6>, wherein the binding part is at least one selected from a substance, an antibody and an antibody fragment capable of binding to or interacting with a target biopolymer under physiological conditions A biopolymer detection device according to claim 1. In the biopolymer detection device according to <7>, since the binding portion can specifically bind to or interact with the target biopolymer, the specific target biopolymer is easily quantified.
[0013]
<8> Can bind to or interact with the target biopolymer And formed with carbon nanotubes A biopolymer detection method comprising: a vibration inducing step for inducing vibration in a coupling means; and a detection step for detecting a vibration change of the coupling means when the coupling means is coupled to the target biopolymer. It is.
In the biopolymer detection method according to <8>, in the vibration induction step, Formed with carbon nanotubes Vibration is induced in the coupling means. Since the binding means can bind to or interact with the target biopolymer, if the target biopolymer is present in the sample, it binds to or interacts with the target biopolymer. In the detection step, it is detected whether or not the binding means has bound or interacted with the target biopolymer. As a result, the amount of the target biopolymer in the sample is easily quantified.
[0014]
<9> A bond part that can bind to or interact with the target biopolymer is formed at the tip of the carbon nanotube, and the bond part is formed on the dangling bond at the tip of the carbon nanotube generated by treating the carbon nanotube. It is a carbon nanotube structure manufactured by reacting and bonding materials that form the structure. The carbon nanotube structure according to <9> is preferably used in the biopolymer detection device, the biopolymer detection method, and the like.
[0015]
<10> A disease diagnosis apparatus comprising the biopolymer detection device according to any one of <1> to <7>. Since the disease diagnosis apparatus according to <10> includes the biopolymer detection device, the abundance in the sample of the protein causing the disease as the target biopolymer can be reduced in a short time. Since it can be quantified, the disease can be diagnosed easily and in detail.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
(Biopolymer detection device)
The biopolymer detection device of the present invention includes vibration inducing means, coupling means, and detection means, and further includes other means appropriately selected as necessary.
[0017]
-Vibration induction means-
The vibration inducing means is not particularly limited as long as it has a function of inducing vibration, and can be appropriately selected from known ones according to the purpose. However, it has an electric field, current, sound wave, magnetism, light having a frequency. And those capable of inducing vibration by at least one selected from mechanical stimuli.
[0018]
The vibration inducing means capable of inducing vibration by the current includes, for example, a base electrode provided at one end of the coupling means, a vibration inducing electrode disposed in the vicinity of the coupling means, the base electrode, and the vibration inducing means. Preferred examples include an electric circuit having an AC power source that is electrically connected to the electrode and capable of applying an AC voltage, and a piezoelectric element. Among the electric circuits, it is preferable that the base electrode is fixed, the coupling means is disposed in an upright state, and the vibration-inducing electrode is disposed in the vicinity of the peripheral side surface of the coupling means. In this case, when an AC voltage is applied from the AC power source to the base electrode and the vibration inducing electrode, the coupling means can be vibrated in a substantially horizontal direction corresponding to the frequency of the AC voltage.
[0019]
As the vibration inducing means capable of inducing vibration by the sound wave, for example, an ultrasonic oscillating device or the like is preferably exemplified. In the case of this ultrasonic oscillating device, the coupling means can be vibrated in accordance with the frequency of the ultrasonic wave oscillated by the ultrasonic oscillating device.
[0020]
As the vibration inducing means capable of inducing vibration by the magnetism, for example, a rotatable permanent magnet having both poles, an electromagnet, and the like disposed in the vicinity of the coupling means to which a magnetic polarity is imparted are preferably exemplified. In the case of the rotatable permanent magnet, the coupling means can be vibrated in accordance with the polarity change period applied to the coupling means by rotating the permanent magnet. In the case of the electromagnet, the coupling means can be vibrated corresponding to the cycle by switching the electromagnet to ON-OFF at a constant cycle.
[0021]
As the vibration inducing means capable of inducing vibration by the light, for example, a material whose conformation structure can be changed in accordance with the presence or absence of exposure or a change in the wavelength of exposure light can be preferably cited. If the coupling means is coupled to such a material, the coupling means can be vibrated in accordance with the ON / OFF switching cycle of exposure or the switching cycle of the type of exposure light to be exposed.
[0022]
Suitable examples of the vibration inducing means capable of inducing vibration by mechanical stimulation include a piezoelectric element, a shaking device, and a shaker. In these cases, the coupling means can be vibrated corresponding to the vibration frequency.
[0023]
There is no restriction | limiting in particular as a frequency of the vibration induced by the said vibration induction means, Although it can select suitably according to the objective, For example, 1-10 MHz is preferable and 1-10 kHz is more preferable.
[0024]
There is no restriction | limiting in particular as an amplitude of the vibration which the said vibration induction means induces, Although it can select suitably according to the objective, For example, 0.1 nm-10 micrometers are preferable, and 1 nm-100 nm are more preferable.
[0025]
-Coupling means-
The coupling means is not particularly limited as long as it can resonate by vibrations induced by the vibration inducing means and can be coupled to or interacts with the target biopolymer. Preferred examples include those having a sensitive part capable of resonating by vibration applied by the vibration inducing means and a binding part capable of binding to or interacting with the target biopolymer.
Moreover, there is no restriction | limiting in particular about the shape of the said coupling | bonding means, a structure, a magnitude | size, a material, etc., It can select suitably according to the objective.
[0026]
The sensitive part is preferably a flexible part. In this case, it is advantageous in that the sensitive part can be vibrated efficiently. Further, the sensitive part is preferably one having conductivity.
[0027]
Examples of the sensitive portion having the flexibility and the conductivity include carbon nanotubes, silicon thin films, crystal oscillators, etc. Among these, durability, vibration controllability, ease of manufacture, etc. Carbon nanotubes are particularly preferred in this respect.
[0028]
The carbon nanotube is not particularly limited and may be appropriately selected depending on the purpose. For example, the carbon nanotube may be linear (having a substantially linear axis) or curved It may be a shape (having a curved axis), but is preferably linear from the viewpoint of vibration controllability, and the structure may be a single wall structure, A multi-wall structure may be used, but a single-wall structure is preferable from the viewpoint of good flexibility. The size is preferably about 0.4 to 10 nm in diameter.
[0029]
There is no restriction | limiting in particular as a manufacturing method of the said carbon nanotube, Although it can manufacture by a well-known method, from a viewpoint of manufacturing the said linear carbon nanotube, it manufactures by a chemical vapor deposition method (CVD) under a direct current electric field. And a method of manufacturing by chemical vapor deposition (CVD) using a structure having a substantially linear cavity under a direct current electric field is more preferable.
[0030]
The structure having the substantially linear cavity is not particularly limited and may be appropriately selected depending on the intended purpose. For example, anodized alumina is preferable.
Moreover, when manufacturing a carbon nanotube by the said chemical vapor deposition method (CVD), a catalyst layer is used and a carbon nanotube is grown on this catalyst layer. In this case, preferred examples of the material for the catalyst layer include transition metals such as iron, nickel, and cobalt.
When carbon nanotubes are produced by the chemical vapor deposition method (CVD), the raw material gas is not particularly limited and can be appropriately selected according to the purpose. For example, methane and the like are preferably used. Is not particularly limited and can be appropriately selected according to the purpose. For example, thermal CVD, plasma CVD, and the like are preferable, and an apparatus for performing the chemical vapor deposition method is not particularly limited and is known. The conditions for the chemical vapor deposition are not particularly limited and may be appropriately selected according to the purpose. Examples thereof include conditions for setting the substrate temperature to 500 ° C. or higher.
[0031]
The silicon thin film or the like can be manufactured by, for example, a micromachine manufacturing process.
[0032]
The binding part is not particularly limited and can be appropriately selected according to the purpose. At least selected from substances, antibodies, and antibody fragments that can bind to or interact with the target biopolymer under physiological conditions. One type is preferred.
[0033]
The target biopolymer is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include nucleic acids, lipids, sugars, and proteins.
[0034]
The substance is not particularly limited and may be appropriately selected depending on the intended purpose.For example, nucleic acid, protein, lipid, sugar, substance serving as an enzyme substrate, allosteric regulator, agonist for receptor protein, antagonist or These derivatives, proteins that interact with the target biopolymer in vivo, and proteins that form a complex with the target biopolymer in vivo. The substance may be obtained by appropriately synthesizing or isolated and purified.
[0035]
The antibody is not particularly limited and may be appropriately selected depending on the intended purpose. For example, anti-target biopolymer IgG antibody, anti-target biopolymer IgA antibody, anti-target biopolymer IgM antibody, anti-target biomolecule High molecular IgD antibodies, anti-target biopolymer IgE antibodies, and the like can be mentioned, and among these, anti-target biopolymer IgG antibodies are preferable. The antibody may be a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody, and can be appropriately obtained by a known method.
[0036]
The antibody fragment is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a Fab fragment of the antibody and a fragment containing a part or all of the variable region of the antibody. The antibody fragment can be obtained by appropriately known methods, for example, a method of treating the antibody with an enzyme, a method of gene recombination of lymphocyte cells producing the antibody, and the like.
[0037]
These binding parts may be used singly or in combination of two or more. However, when quantifying a plurality of target biopolymers at a time, different binding parts may be used. Two or more types are preferably used in combination, and a plurality of the binding portions are present in a fractionated manner, and a mode in which the target biopolymers capable of binding or interacting with each of the fractionated binding portions is more preferable.
[0038]
The bond between the sensitive part and the bond part is not particularly limited and may be appropriately selected according to the purpose. For example, any of chemical bond, physical adsorption, etc. may be used. From the viewpoint, a chemical bond is preferable, and among them, a covalent bond, a coordinate bond, and the like are more preferable.
[0039]
When the sensitive part is a carbon nanotube, for example, after treating the carbon nanotube in an oxygen plasma atmosphere, an aqueous solution of a material forming the binding part (for example, the antibody) is sprayed toward the carbon nanotube. The aqueous solution is frozen by adiabatic expansion to produce a dried body of the material forming the binding part (for example, the antibody), the dried body is supplied to the carbon nanotube, and the dried body is attached to the tip of the carbon nanotube. Can be combined. In addition, there is no restriction | limiting in particular as said aqueous solution, Although it can select suitably according to the objective, For example, an ammonium carbonate pH buffer solution (pH7) etc. are mentioned suitably.
The carbon nanotube has a tube structure in which aromatic rings with six carbon atoms are continuous, and includes a carbon five-membered ring at the tip. When the carbon nanotube is treated in the oxygen plasma atmosphere, or when the carbon nanotube is treated by a milling process using argon plasma or the like, the five-membered ring portion is opened and a dangling bond is generated. When the material forming the bonding portion is sprayed and reacted therewith, the material forming the bonding portion efficiently chemically bonds to the dangling bond. According to this method, a desired material can be chemically bonded to the tip of the carbon nanotube easily, simply and efficiently.
[0040]
-Detection means-
The detection means is not particularly limited as long as it can detect whether or not the binding means has bound or interacted with the target biopolymer, and may be appropriately selected from known ones according to the purpose. For example, one that detects the presence / absence of energization, one that detects a vibration change as video information, and the like. These may be used individually by 1 type and may use 2 or more types together.
[0041]
The detection means for detecting the presence / absence of energization includes, for example, a measurement unit for detecting the presence / absence of energization, the measurement unit detects that there is energization, and the binding means detects the target biopolymer. Suitable examples include those that detect that they have bound or interacted with each other.
[0042]
The detection means for detecting the vibration change as video information includes, for example, a measurement unit that captures the vibration state of the coupling unit and detects the amplitude change of the coupling unit to detect the vibration change. However, it is preferable to detect a change in amplitude and detect that the binding means has bound or interacted with the target biopolymer.
[0043]
The vibration change to be detected includes natural vibration change, amplitude change, position change of standing wave node, etc., among these, from the viewpoint of detecting the change in the resonance state of the coupling means, Natural vibration changes are preferred.
[0044]
As the detection means, in addition to the measurement unit, the presence of the target biopolymer in the sample based on the detection result detected by the measurement unit and the dissociation constant between the target biopolymer and the binding unit It is preferable to have a data processing unit that calculates the amount, and it is preferable to have a data display unit that can display the detection result. A known computer or the like can be used as the data processing unit, the data display unit, or the like.
[0045]
The biopolymer detection device of the present invention can be used, for example, in a state where the binding means is arranged in a sample solution, and can be used for quantification of various proteins, diagnosis of diseases, etc. The biopolymer detection method of the invention and the disease diagnosis apparatus of the invention can be used particularly preferably.
[0046]
The sample solution is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include biological fluids such as blood, lymph and saliva, excreta such as digested products and urine, reaction solutions, and purified solutions. , Waste liquid, or diluted solutions thereof.
The disease is not particularly limited and can be appropriately selected according to the purpose. However, there is a disease that develops when at least one protein is reduced or increased from the required amount in a biological reaction involving a plurality of proteins. Suitable examples include, for example, diabetes, hypertension, hyperlipidemia, cancer, other multifactorial diseases, and the like.
[0047]
The detection sensitivity of the biopolymer detection device of the present invention varies depending on the molecular weight of the target biopolymer to which the binding means binds, and also changes depending on the binding constant between the target biopolymer and the binding means. However, for example, by arranging the coupling means having different coupling constants on a plurality of arrays, the detection sensitivity can be increased and the area can be widened.
[0048]
(Biopolymer detection method)
The biopolymer detection method of the present invention includes a vibration inducing step of inducing vibration in the coupling means capable of binding or interacting with the target biopolymer, and the binding means when the binding means is coupled to the target biopolymer. A detecting step for detecting a vibration change of the coupling means, and further including other steps appropriately selected as necessary.
The detection step can be suitably performed by the detection means. The biopolymer detection method can be preferably implemented using the biopolymer detection apparatus of the present invention.
[0049]
The biopolymer detection method of the present invention can be used, for example, in a state where the binding means is arranged in the sample solution, and can be suitably used for quantification of various proteins, diagnosis of diseases, and the like.
[0050]
(Carbon nanotube structure)
The carbon nanotube structure of the present invention has a binding part that can bind to or interact with the target biopolymer at the tip of the carbon nanotube, and further has other parts as necessary. The details of the carbon nanotube are as described above.
The carbon nanotube structure is manufactured by reacting and bonding a material forming the coupling portion to a dangling bond at the tip of the carbon nanotube generated by processing the carbon nanotube.
[0051]
There is no restriction | limiting in particular as a coupling | bonding of the said carbon nanotube and the said coupling | bond part, According to the objective, it can select suitably, For example, any, such as a chemical bond and physical adsorption, stability etc. From the viewpoint, a chemical bond is preferable, and among them, a covalent bond, a coordinate bond, and the like are more preferable.
[0052]
The binding method is not particularly limited and may be appropriately selected depending on the purpose. For example, after the carbon nanotube is treated, an aqueous solution of a material that forms the binding portion, for example, the antibody, is added to the carbon. By spraying toward the nanotubes, the aqueous solution is frozen by adiabatic expansion to produce a dried body of the material forming the binding portion (for example, the antibody), supplying the dried body to the carbon nanotubes, and drying A body can be bonded to the tip of the carbon nanotube. The carbon nanotube has a tube structure in which aromatic rings of carbon six atoms are continuous, and a carbon five-membered ring is included at the tip portion thereof. When the carbon nanotube is treated in the oxygen plasma atmosphere, or when the carbon nanotube is treated by a milling process using argon plasma or the like, the five-membered ring portion is opened and a dangling bond is generated. If the material which forms the said connection part is supplied and made to react there, the material which forms this connection part will chemically bond with the said dangling bond efficiently.
[0053]
The carbon nanotube structure of the present invention can be used for various applications, but as the binding means in the biopolymer detection device, the biopolymer detection method of the present invention, or in the following disease diagnosis device, etc. It can be preferably used.
[0054]
(Disease diagnosis device)
The disease diagnosis apparatus of the present invention includes the biopolymer detection device, and further includes other equipment and the like appropriately selected as necessary.
[0055]
In the disease diagnosis apparatus of the present invention, the target biopolymer that becomes the cause of the disease, the marker, or the like may be designed so as to be detectable alone, or may be designed so that two or more types can be detected.
[0056]
In the former case, the binding part of the binding means in the biopolymer detection device only needs to be able to bind to a specific one type of the target biopolymer. It is preferable to design so that it may become the monoclonal antibody or polyclonal antibody with respect to said target biopolymer.
[0057]
In the latter case, the binding part of the binding means in the biopolymer detection device may be capable of binding to two or more specific target biopolymers. For example, the binding part should be quantified. The type (number) of target biopolymers may be divided, and the respective target biopolymers that can be bound or interacted with may be designed to be different for each coupling portion. As the quantification unit, the biopolymer detection device has only the types (numbers) of the two or more specific target biopolymers to be quantified, and can be bound or interacted with for each binding part The target biopolymer may be designed to be different. Each binding portion is preferably designed to be a monoclonal antibody or a polyclonal antibody against the specific target biopolymer.
[0058]
The disease diagnosis apparatus of the present invention can be particularly suitably used for diagnosis of the presence or absence of a disease caused by a decrease or increase in at least one protein in a biological reaction involving a plurality of proteins. For example, it can be particularly suitably used for diagnosis of diabetes, hypertension, hyperlipidemia, cancer, and other multifactorial diseases.
[0059]
The sample liquid that is a measurement target of the disease diagnosis apparatus of the present invention is not particularly limited and can be appropriately selected according to the purpose. For example, biological fluid such as blood, lymph, saliva, digested material, urine And the like, and excretion liquids, reaction liquids, purified liquids, waste liquids, or diluted liquids thereof.
[0060]
According to the above-described biopolymer detection device or disease diagnosis apparatus of the present invention, the binding parts that bind to or interact with the target biopolymer, such as antibodies, are arranged in an array, and the target biopolymer and the target biopolymer Based on the correspondence between the vibration change (binding signal) that occurs when the binding portion is bound and the position of the binding portion (array position), the target biopolymer (single type) present in the sample alone ( The amount of protein or the like, or the amount of the plurality of target biopolymers (protein or the like) present in the sample can be detected and grasped collectively. For this reason, the biopolymer detection device or disease diagnosis apparatus of the present invention can function and function suitably as a protein chip.
[0061]
According to the biopolymer detection device or the disease diagnosis apparatus, for example, diabetes can be diagnosed in more detail than before. In the above-mentioned diabetes, when hepatocytes switch intracellular glycogen metabolism according to the insulin accepting state, a part or all of the interaction network of proteins from the insulin receptor to the glycogenase is reduced or promoted. As a result, the population of the series of proteins, including post-translational modifications such as phosphorylation and glycosylation, can be grasped by using the biopolymer detection device or disease diagnosis device. It is possible to grasp the protein causing the diabetes. As a result, it is possible to reliably detect the causative protein of dysfunction or abnormality and perform more appropriate treatment instead of diagnosing the diabetes generally based only on the manifestation symptoms as in the past. Such a detailed diagnosis can be performed.
[0062]
【Example】
Examples of the present invention will be specifically described below, but the present invention is not limited to these examples.
[0063]
Example 1
The biopolymer detection apparatus of Example 1 will be described with reference to FIGS. As shown in FIG. 1, the biopolymer detection apparatus 1 of Example 1 includes a silicon substrate 10, an anti-CEA antibody Fab fragment 20, a carbon nanotube 40, a base electrode 60a, a vibration induction electrode 80, and an AC power source. 82, a base electrode 60b, a vibration detection electrode 90, and an ammeter 92.
[0064]
The silicon substrate 10 is a support for fixing each member of the biopolymer detection device. Although the silicon substrate is used in the first embodiment, a substrate made of another material may be used as the support.
[0065]
On the silicon substrate 10, a base electrode 60b and a base electrode 60a are stacked in this order. In Example 1, the base electrode 60b and the base electrode 60a are made of titanium. A catalyst layer 50 is provided on the base electrode 60a. In Example 1, the catalyst layer 50 is made of iron, but may be made of nickel, cobalt, or the like. Carbon nanotubes 40 were formed on the catalyst layer 50 as follows. That is, in a state where a DC voltage is applied in a direction orthogonal to the silicon substrate 10, the temperature of the silicon substrate 10 is set to 500 ° C. or higher, methane gas is supplied as a raw material gas, and a substantially linear carbon is formed on the catalyst layer 50 by thermal CVD. Nanotubes 40 were grown on the silicon substrate 10 in a substantially vertical direction. In Example 1, the carbon nanotube 40 functions as the sensitive part.
Next, the anti-CEA antibody Fab fragment was bound to the tip of the carbon nanotube 40 as follows. That is, the dangling bond 30 was formed by ashing the carbon nanotube 40 with oxygen plasma and opening the five-membered ring present at the tip of the carbon nanotube 40. Then, the anti-CEA antibody Fab fragment dissolved in 10 mM ammonium carbonate pH buffer (pH 7) was atomized and introduced into a vacuum system in which the carbon nanotubes 40 having the dangling bonds 30 were arranged. Then, moisture is frozen and sublimated by adiabatic expansion to produce freeze-dried powder of the anti-CEA antibody Fab fragment, and the freeze-dried powder of the anti-CEA antibody Fab fragment reacts with the dangling bond 30 of the carbon nanotube 40. Then, the anti-CEA antibody Fab fragment 20 is fixed to the tip of the carbon nanotube 40. In Example 1, the anti-CEA antibody Fab fragment 20 functions as the binding portion. The anti-CEA antibody Fab fragment 20 and the carbon nanotube 40 function as the binding means.
[0066]
Further, on the silicon substrate 10, a vibration inducing electrode mounting convex portion 70 is provided in the vicinity of the carbon nanotube 40, and the vibration inducing electrode 80 is disposed on the vibration inducing electrode mounting electrode 70. The vibration induction electrode 80 and the base electrode 60a are connected to each other through an AC power source 82 so as to be conductive. The AC power source 82, the vibration induction electrode 80 and the base electrode 60a that are connected so as to be conductive with each other function as the vibration induction means. In Example 1, the vibration induction electrode 80 is made of aluminum.
[0067]
In addition, a vibration detection electrode 90 is disposed in the vicinity of the carbon nanotube 40, and the vibration detection electrode 90 and the base electrode 60b are connected to each other through an ammeter 92 so as to be conductive. The ammeter 92, the vibration detection electrode 90 and the base electrode 60b that are connected so as to be conductive with each other function as the detection means. In the first embodiment, the vibration detection electrode 90 is made of aluminum.
[0068]
In the biopolymer detection device 1 of Example 1, when an AC voltage is applied from the AC power source 82, an AC electric field is applied between the vibration inducing electrode 80 and the base electrode 60a, and the conductive carbon nanotubes 40 are formed. According to the frequency of the alternating electric field, as shown in FIG. The anti-CEA antibody Fab fragment 20 bound to the tip of the carbon nanotube 40 also resonated with the carbon nanotube 40.
In Example 1, the vibration frequency of the resonating carbon nanotube 40 was 1 to 10 MHz, and the amplitude was several nm to several tens of μm.
[0069]
Since the anti-CEA antibody Fab fragment 20 fixed to the tip of the carbon nanotube 40 can bind to the CEA protein 100, which is a cancer marker as the target biopolymer, by an antigen-antibody reaction, the anti-CEA antibody Fab fragment 20 is When placed in a sample solution in which CEA protein 100, which is a cancer marker as the target biopolymer, is present, anti-CEA antibody Fab fragment 20 was bound to CEA protein 100 by an antigen-antibody reaction. At this time, the anti-CEA antibody Fab fragment 20 and the CEA protein 100 were not in a state of being bound, but were bound or dissociated according to the dissociation constant of the anti-CEA antibody Fab fragment 20.
[0070]
The state in which the carbon nanotube 40 is resonating toward and away from the vibration-inducing electrode 80 includes a vibration detection electrode 90 disposed in the vicinity of the carbon nanotube 40 and a base electrode 60b provided at the end of the carbon nanotube 40. Was measured (monitored) by an ammeter 92 as a conduction intermittent pattern of current flowing between them. In Example 1, conduction of current flowing between the vibration detection electrode 90 and the base electrode 60b in a state where a DC voltage of 1 V is applied to the electric circuit formed by the vibration detection electrode 90 and the base electrode 60b. The intermittent pattern was measured (monitored).
[0071]
When the anti-CEA antibody Fab fragment 20 binds to the CEA protein 100, the tip mass of the carbon nanotube 40 increases, so the resonance frequency of the carbon nanotube 40 changes, and the amplitude of the carbon nanotube 40 increases as shown in FIG. Along with this, the conduction intermittent pattern of the current flowing between the vibration detection electrode 90 and the base electrode 60b also changed. By detecting the change in the conduction intermittent pattern of the current, the presence of the CEA protein 100 in the sample solution can be detected, and the amount of the CEA protein 100 present in the sample, the anti-CEA antibody Fab fragment 20 and According to the dissociation constant between, the amount of CEA protein 100 in the sample could be quantified.
[0072]
(Example 2)
A biopolymer detection apparatus of Example 2 will be described with reference to FIGS. As shown in FIG. 4, the biopolymer detection device 1 according to the fourth embodiment has a configuration in which the position of the vibration detection electrode 90 is disposed in the immediate vicinity of the carbon nanotube 40. Is different. In the biopolymer detection apparatus 1 of Example 4, before the anti-CEA antibody Fab fragment 20 binds to the CEA protein 100, the carbon nanotubes 40 vibrate as shown in FIG. However, when the anti-CEA antibody Fab fragment 20 binds to the CEA protein 100, as shown in FIG. 6, the amplitude of the carbon nanotubes 40 increases and comes into contact with the vibration detection electrode 90, and the vibration detection electrode 90 and the base electrode Current directly conducted to 60b. The presence of CEA protein 100 in the sample solution can be detected by detecting a change in the direct conduction pattern of the current, and the amount of CEA protein 100 present in the sample and the anti-CEA antibody Fab According to the dissociation constant with fragment 20, the amount of CEA protein 100 in the sample could be quantified.
[0073]
The biopolymer detection apparatus 1 in the first and second embodiments will be described with reference to a block configuration diagram as shown in FIG. 7. On the silicon substrate 10, the vibration inducing unit 2, the detection unit 3, and the sensitive unit 4. And the coupling part 5 are arranged. These are arranged in a sample container 9 in which a sample solution is accommodated.
The vibration inducing portion 2 includes a vibration inducing electrode placement convex portion 70, a vibration inducing electrode 80, an AC power source 82, and a base electrode 60a. The detection unit 3 includes a base electrode 60b, a vibration detection electrode 90, and an ammeter 92. The binding portion 5 is composed of an anti-CEA antibody Fab fragment 20. The measuring unit 6 is connected to the vibration inducing unit 2, the detecting unit 3, and the sensitive unit 4, and measures (monitors) changes in current in these. The measurement unit 6 is connected to the data processing unit 7, compares the measurement data with the calibration value data 7a and the array arrangement data 7b, and calculates the amount of the CEA protein 100 in the sample solution.
[0074]
8 is different from the biopolymer detection apparatus 1 in the first and second embodiments in that the piezoelectric element is used as the vibration inducing unit 2, and the detection unit 3, the sensitive unit 4, and the coupling unit in the silicon substrate 10. 5 is a biopolymer detection device arranged on the side opposite to the side on which 5 is arranged. This biopolymer detection device also exhibits the same effects as the biopolymer detection device 1 in the first and second embodiments.
[0075]
Here, it will be as follows if the preferable aspect of this invention is appended.
(Supplementary Note 1) Vibration inducing means for inducing vibration, coupling means capable of resonating by vibration induced by the vibration inducing means, and capable of binding to or interacting with a target biopolymer; A biopolymer detection device comprising detection means for detecting whether or not the biopolymer has bound or interacted with the biopolymer.
(Supplementary note 2) The biopolymer detection device according to Supplementary note 1, wherein the vibration inducing means is capable of inducing vibration by at least one selected from current having a frequency, ultrasonic waves, magnetism, light, and mechanical stimulation.
(Supplementary Note 3) The vibration inducing means is connected to the base electrode provided at one end of the coupling means, the vibration inducing electrode disposed in the vicinity of the coupling means, and the base electrode and the vibration inducing electrode so as to be conductive. The biopolymer detection device according to appendix 1 or 2, comprising an AC power supply capable of applying an AC voltage.
(Supplementary note 4) The biopolymer detection according to Supplementary note 3, wherein the vibration inducing means is configured such that the base electrode is fixed, the coupling means is disposed upright, and the vibration inducing electrode is disposed in the vicinity of the peripheral side surface of the coupling means. device.
(Supplementary note 5) The biopolymer detection device according to Supplementary note 1 or 2, wherein the vibration inducing means is a piezoelectric element.
(Appendix 6) The biopolymer detection device according to Appendix 1 or 2, wherein the vibration inducing means is an ultrasonic oscillator.
(Additional remark 7) The biopolymer detection device in any one of the said additional remarks 1-6 whose frequency of vibration is 1-10 MHz.
(Supplementary note 8) The biopolymer detection device according to any one of Supplementary notes 1 to 7, wherein an amplitude of vibration is 0.1 nm to 10 µm.
(Supplementary Note 9) The supplementary note 1 is described in any one of the supplementary notes 1 to 8, wherein the coupling unit includes a sensitive unit that can resonate by vibration applied by the vibration inducing unit, and a coupling unit that can bind to or interact with the target biopolymer. Biopolymer detection device.
(Supplementary note 10) The biopolymer detection device according to supplementary note 9, wherein the sensitive part is flexible.
(Appendix 11) The biopolymer detection device according to Appendix 9 or 10, wherein the sensitive part is conductive.
(Supplementary note 12) The biopolymer detection device according to any one of Supplementary notes 9 to 11, wherein the sensitive part is any one of a silicon thin film and a crystal oscillator.
(Supplementary note 13) The biopolymer detection device according to any one of Supplementary notes 9 to 11, wherein the sensitive part is a carbon nanotube.
(Supplementary note 14) The biopolymer detection device according to supplementary note 13, wherein the carbon nanotube has a single wall structure.
(Supplementary note 15) The biopolymer detection device according to Supplementary note 14 or 14, wherein the carbon nanotube has a substantially linear axis.
(Supplementary note 16) The biopolymer detection device according to any one of Supplementary notes 13 to 15, wherein the carbon nanotube is produced by a chemical vapor deposition method under a direct current electric field.
(Additional remark 17) The biopolymer detection device in any one of said additional remark 13 to 16 manufactured using the structure in which a carbon nanotube has a substantially linear cavity part.
(Supplementary note 18) The biopolymer detection device according to supplementary note 17, wherein the structure having a substantially linear cavity is anodized alumina.
(Supplementary note 19) The coupling means is formed by coupling the coupling portion to the tip of the carbon nanotube, and forming the coupling portion in the dangling bond at the tip of the carbon nanotube generated by processing the carbon nanotube in an oxygen plasma atmosphere. 19. The biopolymer detection device according to any one of Supplementary notes 13 to 18, which is manufactured by reacting and binding a material to be processed.
(Supplementary note 20) In any one of the supplementary notes 9 to 19, wherein the binding part is at least one selected from a substance, an antibody, and an antibody fragment that can bind to or interact with a target biopolymer under physiological conditions. The biopolymer detection device described.
(Supplementary note 21) The biopolymer detection device according to supplementary note 20, wherein the antibody is an anti-target biopolymer IgG antibody, and the antibody fragment is a Fab fragment of the anti-target biopolymer IgG antibody and a fragment thereof.
(Additional remark 22) A detection means has a measurement part which detects a vibration change, This measurement part detects this vibration change, and detects that the coupling | bonding means couple | bonded thru | or interacted with the said target biopolymer. The biopolymer detection device according to any one of appendices 1 to 21.
(Supplementary note 23) The biopolymer detection device according to supplementary note 22, wherein the vibration change is a natural vibration change.
(Supplementary Note 24) The detection means has a measurement unit that detects the presence / absence of energization, the measurement unit detects that there is energization, and the binding means has bound or interacted with the target biopolymer. 22. The biopolymer detection device according to any one of appendices 1 to 21, which detects
(Supplementary Note 25) The detection means has a measurement unit that captures the vibration state of the coupling unit, detects the amplitude change of the coupling unit, and detects the vibration change, and the measurement unit indicates that the amplitude change has occurred. 22. The biopolymer detection device according to any one of appendices 1 to 21, wherein the biopolymer detection device detects and detects that a binding unit has bound or interacted with the target biopolymer.
(Additional remark 26) The data processing part which a detection means calculates content of the said target biopolymer in a sample based on the detection result which the measurement part detected, and the dissociation constant of a target biopolymer and a coupling | bond part. The biopolymer detection device according to any one of Supplementary Notes 22 to 25, which has the above.
(Supplementary note 27) The biopolymer detection device according to any one of Supplementary notes 1 to 26, wherein the binding means is disposed in the sample solution.
(Supplementary note 28) The biopolymer detection device according to any one of Supplementary notes 1 to 27, which is used for diagnosis of a disease.
(Supplementary Note 29) A disease is a disease that develops due to a decrease or increase in at least one protein from a required amount in a biological reaction involving a plurality of proteins,
29. The biopolymer detection device according to appendix 28, wherein there are a plurality of the binding portions and the target biopolymers that can be bound or interacted are different for each of the fractionated binding portions.
(Supplementary Note 30) A vibration inducing step for inducing vibration in a binding means capable of binding or interacting with a target biopolymer, and detecting a vibration change of the binding means when the binding means is bound to the target biopolymer. A biopolymer detection method comprising: a detection step.
(Additional remark 31) The dangling of the front-end | tip of this carbon nanotube which has the coupling | bond part which can be couple | bonded with a target biopolymer at the front-end | tip of a carbon nanotube, and was produced | generated by processing the said carbon nanotube in oxygen plasma atmosphere A carbon nanotube structure produced by reacting and bonding a material forming the bonding portion to a bond.
(Appendix 32) A disease diagnosis apparatus comprising the biopolymer detection device according to any one of Appendices 1 to 29.
(Supplementary note 33) A disease diagnostic apparatus for diagnosing the presence or absence of a disease caused by at least one protein being reduced or increased from a required amount in a biological reaction involving a plurality of proteins,
33. The disease diagnosis apparatus according to appendix 32, wherein a binding part in the biopolymer detection device is divided by the number of the plurality of proteins, and a target biopolymer that can be bound or interacted is different for each binding part.
(Supplementary note 34) A disease diagnostic apparatus for diagnosing the presence or absence of a disease caused by at least one protein being reduced or increased from a required amount in a biological reaction involving a plurality of proteins,
33. The disease diagnosis apparatus according to appendix 32, wherein the biopolymer detection device has the same number of the plurality of proteins and the target biopolymers that can be bound or interacted are different for each binding portion.
[0076]
【The invention's effect】
According to the present invention, the above-mentioned problems can be solved in response to the above-mentioned demand, and the abundance of a plurality of target biopolymers having a series of functionally close relationships existing in a sample can be easily and reliably obtained. In addition, the biopolymer detection device and the biopolymer detection method applicable to the array chip technology, which can be easily detected, can efficiently diagnose a disease, and the like, a carbon nanotube structure used therefor, and A disease diagnosis apparatus can be provided.
[Brief description of the drawings]
FIG. 1 is a schematic explanatory view showing one embodiment of the biopolymer detection method of the present invention using the biopolymer detection apparatus of the present invention.
FIG. 2 is a schematic explanatory diagram showing an example of the operation of the biopolymer detection apparatus in FIG. 1;
FIG. 3 is a schematic explanatory diagram showing an example of detection by the biopolymer detection apparatus in FIG. 1;
FIG. 4 is a schematic explanatory view showing another embodiment of the biopolymer detection method of the present invention using the biopolymer detection apparatus of the present invention.
FIG. 5 is a schematic explanatory diagram showing an example of the operation of the biopolymer detection apparatus in FIG. 4;
6 is a schematic explanatory diagram showing an example of detection by the biopolymer detection device in FIG. 4. FIG.
FIG. 7 is a block diagram showing an example of a biopolymer detection apparatus of the present invention.
FIG. 8 is a block diagram showing another example of the biopolymer detection apparatus of the present invention.
[Explanation of symbols]
1 Biopolymer detector
2 Vibration induction part
3 detector
4 sensitive parts
5 joints
6 Measurement unit
7 Data processing section
7a Calibration value data
7b Array layout data
8 Display section
9 Sample container
10 Silicon substrate
20 Anti-CEA antibody Fab fragment
30 dangling bonds
40 carbon nanotubes
50 catalyst layer
60a Base electrode
60b Base electrode
70 Convex part for placing vibration induction electrode
80 Vibration-inducing electrode
82 AC power supply
90 Vibration detection electrode
92 Ammeter
100 CEA protein

Claims (10)

Vibration inducing means for inducing vibration,
A binding means formed of carbon nanotubes, capable of resonating by vibration induced by the vibration inducing means, and capable of binding to or interacting with a target biopolymer;
A biopolymer detection device comprising: detection means for detecting whether or not the binding means has bound or interacted with the target biopolymer.   The vibration inducing means is connected to the base electrode provided at one end of the coupling means, the vibration inducing electrode disposed in the vicinity of the coupling means, the base electrode and the vibration inducing electrode in a conductive manner, and an AC voltage is applied. The biopolymer detection device according to claim 1, comprising a possible AC power source.   The biopolymer detection device according to claim 1 or 2, wherein the vibration inducing means is a piezoelectric element.   The biopolymer detection according to any one of claims 1 to 3, wherein the coupling means includes a sensitive part capable of resonating by vibration applied by the vibration inducing means and a coupling part capable of binding to or interacting with the target biopolymer. device.   The biopolymer detection device according to claim 4, wherein the sensitive part is a carbon nanotube.   The bonding means is formed by bonding the bonding portion to the tip of the carbon nanotube, and reacts the material forming the bonding portion with the dangling bond at the tip of the carbon nanotube generated by processing the carbon nanotube in an oxygen plasma atmosphere. The biopolymer detection device according to any one of claims 3 to 5, wherein the biopolymer detection device is manufactured by bonding them together.   The living body height according to any one of claims 4 to 6, wherein the binding portion is at least one selected from a substance, an antibody, and an antibody fragment that can bind to or interact with a target biopolymer under physiological conditions. Molecular detection device. A vibration inducing step capable of binding or interacting with the target biopolymer and inducing vibration in the binding means formed of carbon nanotubes; and vibration of the binding means when the binding means is bound to the target biopolymer. A biopolymer detection method comprising: a detection step of detecting a change.   The carbon nanotube has a bond portion that can bind to or interact with the target biopolymer, and the bond portion is formed in a dangling bond at the carbon nanotube tip generated by processing the carbon nanotube. A carbon nanotube structure produced by reacting and bonding materials.   A disease diagnosis apparatus comprising the biopolymer detection device according to any one of claims 1 to 7.

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