WO2021193108A1 - Particules de billes biologiques et leur procédé de fabrication - Google Patents

Particules de billes biologiques et leur procédé de fabrication Download PDF

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WO2021193108A1
WO2021193108A1 PCT/JP2021/009814 JP2021009814W WO2021193108A1 WO 2021193108 A1 WO2021193108 A1 WO 2021193108A1 JP 2021009814 W JP2021009814 W JP 2021009814W WO 2021193108 A1 WO2021193108 A1 WO 2021193108A1
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particles
polymer
containing polymer
amino group
group
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PCT/JP2021/009814
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English (en)
Japanese (ja)
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竹林　恭志
亮 森下
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日本軽金属株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin

Definitions

  • the present invention uses particles for biobeads for immobilizing bio-related substances such as proteins, nucleic acids, peptide derivatives, sugar chains and their derivatives, natural products, and small molecule compounds as probes, a method for producing the same, and the like. Regarding biobeads.
  • Patent Document 1 discloses a biochip in which a reactive active group such as an amino group, an aldehyde group, an epoxy group, or a carboxylic acid group is added to the surface of a substrate made of glass, ceramics, or the like. Then, the DNA fragment having a reactive active group is spotted on the substrate surface to form a covalent bond between the reactive active group on the substrate surface and the reactive active group of the DNA fragment, thereby fixing the DNA fragment on the substrate surface. doing.
  • a reactive active group such as an amino group, an aldehyde group, an epoxy group, or a carboxylic acid group
  • the entire substrate is usually used for operations such as immersion and cleaning. Therefore, for example, even when it is desired to examine a single combination of a certain detection object and a probe by changing various conditions such as reaction and washing, a biochip having the probe immobilized therein. It was necessary to use the entire sheet, which led to an increase in cost.
  • Patent Document 2 discloses biobeads for DNA immobilization made of amorphous carbon, and as a method for immobilizing DNA, a reactive group is introduced on the surface of a substrate and a functional group modified at the DNA terminal is used. A method of binding DNA by a chemical reaction is disclosed.
  • Patent Document 2 the only immobilization method specifically disclosed in Patent Document 2 is a method of immobilizing DNA as a reactive group via an amide bond. Further, Patent Document 2 only discloses that the beads have good durability and fluidity.
  • An object of the present invention is to provide particles for biobeads, which can independently detect an object to be detected via a probe and increase the amount of immobilized probe.
  • the inventors of the present application bond a polymer containing a specific reactive group to a polymer containing a reactive group by covalently immobilizing a polymer containing a specific reactive group on the surface of particles whose surface is at least carbon.
  • the present invention has been completed by finding that it is possible to independently detect an object to be detected via the probe and to provide particles for biobeads in which the amount of immobilized probe is increased.
  • At least one polymer selected from the group consisting of an amino group-containing polymer, a carboxyl group-containing polymer, an aldehyde group-containing polymer, and an epoxy group-containing polymer is shared on the surface of particles whose surface is made of carbon at least.
  • biobead particles immobilized by binding are provided.
  • the present invention also provides biobeads in which a bio-related substance is bound to the amino group of the biobead particles of the present invention by a covalent bond or a non-covalent bond.
  • the present invention comprises applying the amino group-containing polymer to the surface of the particles and then irradiating the surface of the particles with light in a reduced pressure atmosphere or an inert gas atmosphere.
  • the present invention provides the method for producing the particles for biobeads of the present invention, which comprises irradiating the surface of the particles with plasma or light, and then applying the amino group-containing polymer to the particles. Furthermore, the present invention provides the use of the particles of the present invention as particles of biobeads.
  • Particles for beads can be provided.
  • FIG. It is a figure which shows the fluorescence image of the biobead particle 1 produced in Example 1.
  • FIG. It is a figure which shows the SEM image of the biobead particle 1 produced in Example 1.
  • FIG. It is a figure which shows the result of having performed the mapping of the nitrogen atom by EPMA about the biobead particle 1 produced in Example 1.
  • FIG. It is a figure which shows the SEM image of the biobead particle 2 produced in Example 2.
  • the biobead particles of the present embodiment (hereinafter, may be simply abbreviated as "particles”) have an amino group-containing polymer, a carboxyl group-containing polymer, an aldehyde group-containing polymer, and an aldehyde group-containing polymer on the surface of particles whose surface is at least carbon. It is characterized in that at least one polymer selected from the group consisting of epoxy group-containing polymers is immobilized by covalent bonds.
  • an amino group, a carboxyl group, an aldehyde group, and an epoxy group may be collectively referred to as a "reactive group”.
  • the amino group-containing polymer containing these reactive groups, the carboxyl group-containing polymer, the aldehyde group-containing polymer, and the epoxy group-containing polymer may be collectively referred to as a “polymer”.
  • ⁇ Particles> particles having at least a carbon surface are used. Since the surface is made of carbon, radicals are likely to be generated by light irradiation or plasma irradiation described later, and the polymer is easily covalently bonded.
  • the carbon amorphous carbon and diamond-like carbon are preferable. Among them, amorphous carbon is particularly preferable in that the amount of polymer immobilization increases as the number of fixed points increases, and the detection sensitivity and the amount of adsorption are improved.
  • the material of the particle body is not limited at all.
  • the entire particle can be made of carbon such as amorphous carbon, or a carbon layer such as amorphous carbon can be formed on the particle body.
  • the particle body can be formed of, for example, any one of carbon, metal, glass, ceramics, and plastic, or a composite thereof, and when the particle body is other than carbon, a carbon layer is formed on the surface thereof. After the treatment for providing, the polymer described later can be immobilized. Of these, it is preferable to form the entire particle with carbon, particularly amorphous carbon, because it has no chemical resistance, heat resistance, and self-emission.
  • thermosetting resin examples include urea resin, benzoguanamine resin, alkyd resin, unsaturated polyester resin, vinyl ester resin, diallyl phthalate resin, epoxy resin, melamine resin, urethane resin, phenol resin, furan resin, ketone resin, and xylene resin.
  • examples thereof include polyimide resins, polycarbodiimide resins, styrylpyridine resins, and triazine resins.
  • epoxy resin polyimide resin
  • phenol resin is particularly preferable.
  • LPS registered trademark
  • series / spherical phenol resin manufactured by Lignite, Marilyn series / true spherical phenol resin manufactured by Gunei Chemical Co., Ltd. which are commercially available as phenol resin particles, can be used.
  • metal when metal is used as the particle body, aluminum, stainless steel, steel, copper, etc. can be used as the particle material.
  • Aluminum can be used as a particle material after being subjected to a surface treatment such as nickel-phosphorus plating in order to improve corrosion resistance and surface hardness, and after the carbon layer is further provided on the surface.
  • carbon or diamond-like carbon can be formed on the surface of the particle body made of metal by a treatment method such as sputtering, CVD, PVD, etc.
  • a layer can be provided to form a particle material.
  • the lower limit of the average particle size of the particles is usually 0.5 ⁇ m or more, preferably 1 ⁇ m or more, more preferably 5 ⁇ m or more, and particularly preferably 10 ⁇ m or more. Further, if the average particle size of the particles is too large, for example, when the particles are dispersed in a solution in which the pro-part molecule is dissolved for fixing the probe, the particles are likely to settle, and the association between the probe molecule and the particle is reduced, so that the probe is used. The fixed amount decreases.
  • the upper limit of the average particle size of the particles is usually 500 ⁇ m or less, preferably 200 ⁇ m or less, more preferably 100 ⁇ m or less, and particularly preferably 50 ⁇ m or less.
  • At least one polymer selected from the group consisting of an amino group-containing polymer, a carboxyl group-containing polymer, an aldehyde group-containing polymer, and an epoxy group-containing polymer is shared on the surface of particles whose surface is made of carbon at least. It is fixed by binding.
  • These polymers are compounds formed by polymerizing a plurality of monomers containing reactive groups. That is, the polymer has a large number of reactive groups derived from the monomer.
  • Bonding of the polymer to the particles is confirmed by binding the reactive groups of the polymer and the fluorescent molecules having the reactivity to the particles and observing the fluorescent image of the particles for biobeads using a fluorescent scanner to see if there is a fluorescent signal. be able to.
  • it can be confirmed from the increase in the fluorescence intensity by measuring the fluorescence intensity of the carbon beads to which the polymer is not fixed using a fluorescence scanner and the fluorescence intensity of the particles for biobeads.
  • the binding of the polymer to the particles can be confirmed from the change in the surface texture of the particles by observing the SEM image of the particles for biobeads using an SEM (Scanning Electron Microscope).
  • EPMA Electro Probe Micro Analyzer
  • EPMA Electro Probe Micro Analyzer
  • a nitrogen atom is used in the case of the amino group-containing polymer.
  • an oxygen atom is used in the case of an aldehyde group-containing polymer.
  • an epoxy group-containing polymer an oxygen atom is used.
  • the above-mentioned method for confirming the bond of the polymer to the particles can confirm the bond of the polymer to the particles by using one method, but it is possible to confirm by combining a plurality of methods of 2 or more or 3 or more. Preferred for increased certainty.
  • the amino group-containing polymer is not particularly limited as long as it is a polymer containing an amino group.
  • the "amino group” means a primary amino group, that is, -NH 2 .
  • the amino group-containing polymer is preferably 50% or more, more preferably 90% or more, still more preferably 99% or more of the constituent units constituting the amino group-containing polymer before being immobilized on the particle surface.
  • each has at least one amino group. Since such an amino group-containing polymer has a large number of amino groups in one molecule, the amino groups are uniformly and densely bonded on the particle surface.
  • the amino group-containing polymer is preferably formed by addition polymerization of a vinyl-based monomer having an amino group, and polyallylamine (PAA) is particularly preferable.
  • PAA polyallylamine
  • the amino group-containing polymer does not come off even after washing in pure water with shaking for 1 hour.
  • the amino group can be quantified by a method of quantifying the free bromide ion by bromoacetylation treatment of the amino group and then treatment with mercaptoethanol. It is also possible to directly confirm the presence or absence of covalent bonds by X-ray photoelectron spectroscopy (XPS).
  • the lower limit of the average molecular weight (weight average molecular weight) of the amino group-containing polymer is usually 1000 or more, preferably 1500 or more, preferably 2000 or more, in terms of polyallylamine (PAA), from the viewpoint of better achieving the effects of the present invention. More preferably, 2500 or more is further preferable.
  • PAA conversion means conversion based on the number of amino groups in one molecule (the same applies hereinafter).
  • the upper limit of the average molecular weight (weight average molecular weight) of the amino group-containing polymer is not particularly limited as long as there is no problem in handleability such as solubility and stability of the coating solution, and is usually 60,000 or less. It is preferably 10000 or less, more preferably 8000 or less, further preferably 6000 or less, and particularly preferably 5000 or less.
  • the carboxyl group-containing polymer is not particularly limited as long as it is a polymer containing a carboxyl group.
  • an acrylic polymer is preferable, and among them, polyacrylic acid, polymethacrylic acid, or a copolymer containing acrylic acid or methacrylic acid is more preferable, and polyacrylic acid is particularly preferable.
  • the copolymerization component other than acrylic acid or methacrylic acid is not particularly limited, and for example, poly (acrylic acid-co-styrene), poly (acrylic acid-co-). Methacrylic acid) and poly (acrylic acid-co-ethylene) can be mentioned.
  • the copolymer may be any of a random copolymer, a block copolymer, and a graft copolymer.
  • the content of the acrylic acid or methacrylic acid component in the copolymer is usually about 20 to 99%, preferably about 50 to 99% on a molar basis.
  • the lower limit of the average molecular weight (weight average molecular weight) of the carboxyl group-containing polymer is not particularly limited, but is usually 1000 or more, preferably 2000 or more, and more preferably 2000 or more from the viewpoint of better achieving the effects of the present invention. It is 2500 or more, more preferably 3000 or more, and particularly preferably 3500 or more.
  • the upper limit of the average molecular weight (weight average molecular weight) of the carboxyl group-containing polymer is not particularly limited as long as there is no problem in handleability such as solubility and stability of the coating solution, and is usually 60,000 or less. It is preferably 10000 or less, more preferably 8000 or less, and further preferably 6000 or less.
  • ⁇ Polymer immobilization method> In order to covalently bond the carbon on the particle surface and the polymer, the first method of applying the polymer to the particle surface and then covalently bonding the carbon surface and the polymer by generating radicals by light irradiation, and the carbon on the particle surface. There is a second method in which radicals are generated by subjecting them to plasma treatment or light irradiation, and a polymer is applied to the radicals to cause them to react and bond them.
  • the first method is a method in which a polymer solution is applied to the particle surface and then the carbon on the particle surface and the polymer are covalently bonded by radicals generated by light irradiation.
  • a general method can be used for coating on the particle surface, and the method is not particularly limited as long as the method can control the coating amount of the polymer.
  • it can be carried out by selecting from a spray coating method in which the polymer solution is sprayed onto the particles, a dipping method in which the particles are immersed in the polymer solution, and the like. Among them, particles having a high polymer immobilization rate and a uniform immobilization density can be obtained.
  • the dipping method is preferable.
  • the polymer solution is applied by immersing the particles in the polymer solution under stirring, holding the particles under stirring for a predetermined time, and then vacuum-drying the particles obtained by solid-liquid separation.
  • the polymer coating amount is an amount that achieves the desired polymer immobilization amount.
  • the concentration of the polymer solution may be any concentration that can achieve the required polymer coating amount and can be uniformly applied, and is preferably about 0.5 to 3% by mass. Such a coating operation may be performed a plurality of times before the next light irradiation.
  • ultraviolet rays having a wavelength of about 150 nm to 260 nm, for example, a wavelength of 185 nm or 254 nm can be used.
  • Light of this wavelength breaks the CC, CO, and CH bonds to generate radicals.
  • oxygen molecules and water molecules in the air are also decomposed to generate oxygen radicals and ozone, and oxidative decomposition of the particle material carbon and the polymer also occurs at the same time, which causes inhibition of covalent bond formation.
  • the depressurization is carried out at a degree of vacuum of ⁇ 0.05 MPa or less, more preferably ⁇ 0.08 MPa or less, based on atmospheric pressure (0 MPa).
  • a noble gas element such as argon or helium that is hard to be radicalized even when irradiated with light is used.
  • the amount of light irradiation may be any amount necessary for covalent bonding of the polymer, and the amount of energy is usually about 1 to 6 joules, preferably about 2 to 4 joules per 1 cm 2 of the particle surface. For example, 18.5 mW of ultraviolet light per 1 cm 2 of particle surface is usually irradiated for 1 to 5 minutes, preferably 2 to 4 minutes.
  • the first method it is possible to immobilize the polymer on the particle surface only by performing the operation of applying the polymer and the operation of irradiating the light once, respectively, but the operation of applying the polymer and the operation of irradiating the light are performed. It is preferable to perform the operation a plurality of times, for example, 2 to 4 times. At this time, when performing the operation of performing the light irradiation a plurality of times, it is preferable to perform the operation of changing the direction of the light irradiated to the particles during each light irradiation operation. Such an operation can be performed, for example, by stirring or shaking the container containing the particles.
  • the irradiated surface By performing such a plurality of coatings and light irradiation, it is possible to make the irradiated surface to be irradiated with light not only one surface but also a plurality of surfaces (preferably the whole) on the surface of the particles, and the polymer is immobilized.
  • the amount can be increased. Further, it is not necessary to increase the concentration of the polymer solution used in one coating operation in order to increase the amount of polymer immobilization, and a low concentration polymer solution can be used.
  • the polymer can be evenly immobilized on a plurality of surfaces of the particles, preferably the entire surface of the particles, even if the total amount of immobilization is the same. ..
  • the polymer and the bio-related substance are less likely to be peeled off from the particles, and when used as biobeads, the substance to be detected is bound to the beads to facilitate detection, and the detection sensitivity is improved.
  • the second method is a method in which the surface of the particles is irradiated with plasma or light to generate radicals, and the particles are immediately coated with a polymer solution to react the radicals generated on the surface of the particles with the polymer.
  • the wavelength of the light to be irradiated by the light irradiation in the second method can be the same as that in the light irradiation in the first method.
  • plasma irradiation or light irradiation may be performed a plurality of times.
  • Plasma irradiation can be carried out under reduced pressure or atmospheric pressure, but it can also be carried out as reactive plasma by appropriately introducing argon gas, carbon dioxide gas, ammonia gas, water vapor or the like.
  • the plasma irradiation amount is high frequency output 1 to 100 W, reaction gas flow rate 5 to 20 cm 3 / min, irradiation time 10 to 120 seconds, more preferably 1 to 10 W, reaction gas flow rate 5 to 15 cm 3 / min, irradiation time 20. ⁇ 90 seconds.
  • the amount of light irradiation may be any amount necessary for covalent bonding of the polymer, and the amount of energy is usually about 3 to 35 joules, preferably about 5 to 16 joules per 1 cm 2 of the particle surface. For example, 18.5 mW of ultraviolet rays per 1 cm 2 of particle surface is usually irradiated for 3 to 30 minutes, preferably 5 to 15 minutes. Light irradiation can be performed under atmospheric pressure.
  • the same operations and conditions as the application operation in the first method can be used.
  • it can be carried out by selecting from a spray coating, a dipping method and the like, and among them, the dipping method is preferable in that particles having a high polymer immobilization rate and a uniform immobilization density can be obtained.
  • the polymer solution is applied by immersing the particles in the polymer solution under stirring, holding the particles under stirring for a predetermined time, and then vacuum-drying the particles obtained by solid-liquid separation. be able to.
  • the polymer coating amount is an amount that achieves the desired polymer immobilization amount.
  • the concentration of the polymer solution may be any concentration that can achieve the required polymer coating amount and can be uniformly applied, and is preferably about 0.5 to 3% by mass. Such a coating operation may be performed a plurality of times. Further, when the polymer is immobilized only by the second method, it is necessary to promptly apply the polymer solution to the particles before the generated radicals disappear.
  • either the first method or the second method described above may be performed, but the first method is performed after the second method is performed by combining these.
  • the particle surface may be irradiated with plasma or light, then the polymer solution may be applied to the particles, and then light irradiation may be performed. In this case, even if the radicals generated by the second method are reduced or eliminated, there is no problem because further light irradiation is performed after the application of the polymer solution.
  • the operation of applying the polymer to the particles and the operation of subsequently irradiating the light are performed only once.
  • Such an operation can be performed, for example, by stirring or shaking the container containing the particles.
  • the surface to be irradiated with light can be not only one surface but also a plurality of surfaces (preferably the whole) on the surface of the particles, and the amount of polymer immobilization can be increased. Can be made to. Further, it is not necessary to increase the concentration of the polymer solution used in one coating operation in order to increase the amount of polymer immobilization, and a low concentration polymer solution can be used.
  • the polymer can be evenly immobilized on a plurality of surfaces of the particles, preferably the entire surface of the particles, even if the total amount of immobilization is the same. ..
  • the polymer and the bio-related substance are less likely to be peeled off from the particles, and when used as biobeads, the substance to be detected is bound to the beads to facilitate detection, and the detection sensitivity is improved.
  • Surface modification can be performed by subjecting the particles to light irradiation or plasma treatment.
  • Surface modification produces polar groups such as carboxyl groups, hydroxyl groups, and aldehyde groups from the carbon surface of the particles. This polar group improves the affinity for polymer solutions with protic solvents. That is, by modifying the surface of the particles before immobilization, the parent coating liquid property of the particles can be improved. This makes it possible to increase the amount of polymer immobilized on the particles.
  • ultraviolet rays having a wavelength of about 150 nm to 260 nm, for example, a wavelength of 185 nm or 254 nm can be used. Light of this wavelength breaks the CC, CO, and CH bonds and reacts with oxygen and water to generate polar groups such as carboxyl groups, hydroxyl groups, and aldehyde groups. Light irradiation is preferably carried out in the atmosphere.
  • the amount of light irradiation may be any amount necessary to generate polar groups such as carboxyl groups, hydroxyl groups, and aldehyde groups, and the amount of energy is usually about 1 to 6 joules, preferably about 1 cm 2 per 1 cm 2 of the particle surface. It is about 2 to 4 joules. For example, 18.5 mW of ultraviolet light per 1 cm 2 of particle surface is usually irradiated for 1 to 5 minutes, preferably 2 to 4 minutes.
  • Plasma irradiation is preferably carried out in the atmosphere.
  • the amount of plasma irradiation may be any amount necessary to generate polar groups such as carboxyl groups, hydroxyl groups, and aldehyde groups, and has a high frequency output of 1 to 100 W, a reaction gas flow rate of 5 to 20 cm, 3 / min, and an irradiation time of 10 to. It is 120 seconds, more preferably 1 to 10 W, a reaction gas flow rate of 5 to 15 cm 3 / min, and an irradiation time of 20 to 90 seconds.
  • the biobead particles of the present embodiment obtained as described above have a reactive group such as an amino group, a carboxyl group, an aldehyde group, and an epoxy group on the surface of the particles.
  • a reactive group such as an amino group, a carboxyl group, an aldehyde group, and an epoxy group on the surface of the particles.
  • biobeads can be constructed by binding a bio-related substance to particles.
  • the bio-related substance may be any substance conventionally used as a probe for a detection system, and any polypeptide (including natural or synthetic proteins and oligopeptides), nucleic acid (DNA and RNA, and artificial). Nucleic acids are included), sugars, lipids, complexes thereof (glycoproteins, etc.) and derivatives (modified proteins, nucleic acids, etc.).
  • the probe refers to a substance that binds to a specific substance to be detected.
  • the biobead particles can detect a substance that acts on the bio-related substance by using a bio-related substance immobilized on the particle as a probe. In this way, the biobead particles and biobeads can be used for testing substances that act with bio-related substances.
  • Bio-related substances and reactive groups can be bound by covalent or non-covalent bonds.
  • the biorelated substance and the reactive group can be covalently bonded directly or can be covalently bonded via a desired linker.
  • the bio-related substance and the reactive group can be directly non-covalently bonded or can be non-covalently bonded via a desired linker.
  • the bio-related substance and the reactive group are usually non-covalently bonded via a linker.
  • the covalent bond between the biological substance and the reactive group can be easily carried out by a well-known conventional method.
  • the bio-related substance When the bio-related substance has a carboxyl group or the like that binds to an amino group, such as a protein, it can be directly bonded, and if it does not have such a functional group, or if desired, a linker can be used. It is also possible to combine via. Linkers are also well known, and for example, those having a carboxyl group at one end and a maleimide group at the other end are widely used.
  • the non-covalent bond between the bio-related substance and the reactive group can be carried out by, for example, a hydrogen bond, an electrostatic interaction, a hydrophobic interaction, a van der Waals interaction, or the like. These may be used individually by 1 type, and may be used in combination of 2 or more type.
  • non-covalent bonds specific or non-specific interactions found between proteins, sugar chains, lipids, etc. can be utilized.
  • the linker may be introduced on the reactive group side, on the bio-related substance side, or on both the reactive group side and the bio-related substance side.
  • a linker may be bound to the reactive group side to non-covalently bind the linker to the bio-related substance.
  • a set of linkers that can be non-covalently bonded to each other may be bound to both the reactive group side and the bio-related substance side, and these linkers may be non-covalently bonded to each other.
  • Examples of such a set of linkers include biotin / avidin, glutathione / glutathione-S-transferase, histidine / metal and the like.
  • the conventionally used biochip is discarded once it is used. Therefore, even if you want to consider the detection of the detection target by the coupling between one probe immobilized on the biochip and the detection target, use the entire biochip on which the probe is immobilized. It was necessary to dispose of it just by doing it, which led to an increase in cost.
  • many kinds of probes are fixed in an array on a biochip, and it is possible to detect an object to be detected by many kinds of probes with one operation. Considering the detection of an object to be detected by only one probe immobilized on the biochip for such a biochip does not effectively use the residual probe immobilized on the biochip. It was unproductive because it was wasted.
  • the particles for biobeads of the present embodiment for example, by dispensing the biobeads into each well of the well plate, the conditions such as reaction and washing of the beads on which the probe is immobilized can be met. It can be used for a variety of different independent operations. Therefore, it can be used for inspection of small lots or small types of detection objects. That is, if it is a conventional biochip, it is necessary to use a whole biochip, which leads to an increase in cost.
  • the detection of the object to be detected via the probe is individually performed in relation to the one type of probe bound to the polymer immobilized on the particles and the object to be detected. Can be done independently. Further, before the inspection of the target bio-related substance is performed using the biochip, a preliminary inspection can be performed in advance by using the biobeads of the present embodiment.
  • the biobead particles or biobeads of the present embodiment are used for an adsorption treatment for removing unnecessary components contained in a sample sample, which causes an increase in background when performing an immunoprecipitation method or a biochip test.
  • the detection system includes substances used for the detection system of particles for biobeads, substrates such as substrates for biochips; biological substances such as immunoglobulins and blocking materials; Non-specific adsorbates such as proteins that are easily adsorbed non-specifically to the substance may be contained in a sample sample such as serum.
  • the non-specific adsorbent and the biobead particles or biobeads can be adsorbed by mixing the nonspecific adsorbent with the biobead particles or biobeads to be adsorbed with the sample sample. Further, the non-specific adsorbent can be precipitated together with the biobead particles or biobeads, and then the supernatant separated by centrifugation can be separated. Then, the obtained supernatant can be used as a sample for the subsequent detection operation. In this way, it is possible to carry out the adsorption treatment of the particles for biobeads or the non-specific adsorbent using the biobeads.
  • any particles that adsorb the non-specific adsorbent can be appropriately used.
  • the non-specific adsorbent is adsorbed on the biobead particles
  • the biobead particles to which the bio-related substance as a probe is not bound can be used.
  • biobeads to which immunoglobulins having no specificity for the protein to be detected and having specificity for non-specific adsorbents can be used.
  • an immobilization method for immobilizing a probe on a biochip via a reactive group has been known, but in such a method, the reactive group formed on the biochip is directly or directly or.
  • the probe was bound via a linker. That is, since the bond between the biochip, the reactive group, and the probe is one, the amount of the probe immobilized on the biochip is limited.
  • the polymer having a reactive group is immobilized on the particles by a covalent bond.
  • the polymer immobilized on the particles modifies the plurality of reactive groups branched from the polymer into the particles. That is, a plurality of reactive groups are presented by the polymer immobilized on the particles, and a large number of polymers can be further immobilized through the plurality of reactive groups. Therefore, the particles for biobeads of the present embodiment have an increased amount of probe immobilization as compared with the conventional immobilization method.
  • the probe immobilization rate improves and the immobilization density becomes uniform. Furthermore, by using the particles for biobeads of the present embodiment, it is possible to provide biobeads capable of inspecting a detection object with high detection sensitivity and high reproducibility.
  • the particles for biobeads of the present embodiment have a larger surface area than the flat plate-shaped substrate because they are granular, the amount of polymer or probe immobilized on the surface of the particles can be increased. As a result, the detection sensitivity can be improved when used as biobeads. In addition, the amount of adsorption of unnecessary components can be increased during the above-mentioned adsorption treatment.
  • the complex formed by binding the detection target to the probe on the particle surface is granular, which makes it easier to handle.
  • the particle body is made of a magnetic material and the surface of the particle body is a magnetic particle made of carbon, the complex can be easily separated, washed and recovered by using a magnetic force or the like. can.
  • the particles for biobeads of the present embodiment use at least particles whose surface is made of carbon, autofluorescence is less than that of the case where the surface is made of glass or plastic. Therefore, it is possible to provide biobeads capable of accurately detecting an object to be detected even in a measurement using a fluorescent label, which has become widely used in recent years. Further, since the particles for biobeads of the present embodiment use at least particles whose surface is made of carbon, non-specific adsorption of proteins is less than in the case where the surface is made of glass or plastic. Therefore, it is possible to provide biobeads capable of detecting an object to be detected with high detection sensitivity and reproducibility by improving the S / N ratio by suppressing the signal caused by non-specific adsorption. can.
  • Example 1 Particles for biobeads on which an amino group-containing polymer is immobilized
  • the carbon beads were placed in a quartz cell with a lid, and after the lid was closed, the cell was turned sideways and the cell was shaken so that the carbon beads were evenly distributed in the cell.
  • the carbon beads were irradiated with ultraviolet rays (18.5 mW / cm 2 , 254 nm) for 2 minutes under atmospheric pressure. Further, the cells were shaken to stir the carbon beads, and then the carbon beads were again irradiated with ultraviolet rays (18.5 mW / cm 2 , 254 nm) under atmospheric pressure for 2 minutes.
  • a coating solution was prepared by adjusting the concentration of polyallylamine (PAA: average molecular weight 3000) to 1% by mass with ethanol. Under room temperature conditions, carbon beads irradiated with ultraviolet rays under stirring were immersed in 20 ml of a coating solution, and the beads were kept under stirring for 15 minutes. Then, the carbon beads and the coating liquid were solid-liquid separated by vacuum filtration using a filter paper, and the obtained carbon beads were vacuum-dried for 30 minutes (vacuum degree ⁇ 0.09 MPa) (polymer solution coating operation).
  • PAA polyallylamine
  • the dried carbon beads are placed in a quartz cell with a lid, the cell is shaken so that the carbon beads are evenly distributed in the cell, and then the cell is subjected to ultraviolet rays under vacuum (vacuum degree ⁇ 0.09 MPa) for 2 minutes. irradiation (18.5mW / cm 2, 254nm) was. After opening the cell to atmospheric pressure, the cell is shaken to stir so that the carbon beads are evenly distributed in the cell, and then the second ultraviolet irradiation is performed under vacuum (vacuum degree ⁇ 0.09 MPa) for 2 minutes. (Polymer immobilization operation). Next, the carbon beads were immersed in pure water and washed under stirring. Further, the carbon beads and water were solid-liquid separated by vacuum filtration using a filter paper, and the obtained carbon beads were vacuum-dried for 30 minutes (vacuum degree ⁇ 0.09 MPa) to obtain particles 1 for biobeads.
  • Example 2 Particles for biobeads on which an amino group-containing polymer is immobilized (multiple treatments)
  • the particles 2 for biobeads were similarly applied except that the polymer solution coating operation and the two ultraviolet irradiations were performed, and then the polymer solution coating operation and the two ultraviolet irradiations were repeated.
  • the polymer solution coating operation and the two ultraviolet irradiations were repeated.
  • the fluorescence image and the fluorescence intensity of the biobead particles 2 were measured in the same manner as in Example 1.
  • the fluorescence image of the biobead particles 2 is shown in FIG.
  • the fluorescence intensity was 2068 LAU / mm 2 .
  • the surface texture of the beads of the biobead particles 2 was evaluated by SEM and the nitrogen atoms were mapped by EPMA.
  • the SEM image of the biobead particles 2 is shown in FIG.
  • the mapping result of N atom of the biobead particle 2 is shown in FIG.
  • ⁇ Comparative example 1> (Untreated carbon beads)
  • the fluorescence image and fluorescence intensity of the untreated carbon beads formed from amorphous carbon used as the particle material in Example 1 before irradiation with ultraviolet rays were measured by the same method as in Example 1.
  • the fluorescence image of the carbon beads is shown in FIG.
  • the fluorescence intensity was 86 LAU / mm 2 .
  • the surface texture of the carbon beads was evaluated by SEM, and the nitrogen atom was mapped and the strength was measured by EPMA.
  • the SEM image of the carbon beads is shown in FIG.
  • the mapping result of N atoms of carbon beads is shown in FIG.
  • the strength of the N atom of the carbon beads is shown in FIG.
  • "unmodified-2" to "unmodified-4" in FIG. 11 indicate the analyzed locations, respectively.
  • Example 5 in which the application operation of the polymer solution and the irradiation with ultraviolet rays were repeated twice, the fluorescence signal was emitted more densely than the fluorescence image of Example 1 (FIG. 1). , Showed strong fluorescence intensity. Furthermore, the fluorescence of the fluorescence intensity (268LAU / mm 2) is much higher than the fluorescence intensity of Comparative Example 1 (86LAU / mm 2), also in Example 2 biobeads particles bio beads for particles of Example 1 The intensity (2068 LAU / mm 2 ) is even higher than the fluorescence intensity of Example 1, and it can be seen that the amount of the amino group-containing polymer immobilized is large.

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Abstract

La présente invention concerne des particules de billes biologiques qui peuvent détecter indépendamment un objet à détecter par l'intermédiaire d'une sonde et avec lesquelles la quantité de la sonde immobilisée est augmentée.　Les particules de billes biologiques sont obtenues en immobilisant, par liaison covalente, au moins un polymère, choisi dans le groupe constitué par un polymère contenant un groupe amino, un polymère contenant un groupe carboxyle, un polymère contenant un groupe aldéhyde et un polymère contenant un groupe époxy, sur les surfaces des particules dont au moins les surfaces sont composées de carbone. Dans ces billes biologiques, une substance biologique est liée à un groupe amino des particules de billes biologiques par une liaison covalente ou une liaison non covalente. Les particules de billes biologiques peuvent être produites par une combinaison de : revêtement de la surface de la particule avec un polymère ; et irradiation de la surface de la particule avec du plasma ou de la lumière.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04346830A (ja) * 1991-05-23 1992-12-02 Tonen Corp クロマトグラフィー用充填剤
JP2009513798A (ja) * 2005-10-27 2009-04-02 クレムソン・ユニヴァーシティ 蛍光性の炭素ナノ粒子
US20110177619A1 (en) * 2008-07-14 2011-07-21 Andrew Metters In vitro diagnostic markers comprising carbon nanoparticles and kits
US20120238725A1 (en) * 2009-09-04 2012-09-20 Northwestern University Primary carbon nanoparticles
JP2015513107A (ja) * 2012-04-13 2015-04-30 ビバクタ、リミテッドＶｉｖａｃｔａ Ｌｉｍｉｔｅｄ アッセイ標識

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04346830A (ja) * 1991-05-23 1992-12-02 Tonen Corp クロマトグラフィー用充填剤
JP2009513798A (ja) * 2005-10-27 2009-04-02 クレムソン・ユニヴァーシティ 蛍光性の炭素ナノ粒子
US20110177619A1 (en) * 2008-07-14 2011-07-21 Andrew Metters In vitro diagnostic markers comprising carbon nanoparticles and kits
US20120238725A1 (en) * 2009-09-04 2012-09-20 Northwestern University Primary carbon nanoparticles
JP2015513107A (ja) * 2012-04-13 2015-04-30 ビバクタ、リミテッドＶｉｖａｃｔａ Ｌｉｍｉｔｅｄ アッセイ標識

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