WO2003083478A1 - Face de capteur pour biopuce porteuse de nanoparticules de polyethylene glycolate - Google Patents
Face de capteur pour biopuce porteuse de nanoparticules de polyethylene glycolate Download PDFInfo
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- WO2003083478A1 WO2003083478A1 PCT/JP2003/003504 JP0303504W WO03083478A1 WO 2003083478 A1 WO2003083478 A1 WO 2003083478A1 JP 0303504 W JP0303504 W JP 0303504W WO 03083478 A1 WO03083478 A1 WO 03083478A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
Definitions
- the present invention relates to the technical field of bioassay, and more specifically, to reduce or prevent nonspecific adsorption or binding by contaminants other than the analyte contained in a biological fluid or the like, or to increase the detection sensitivity of the analyte.
- the present invention relates to a biosensor system that can be enhanced, and an Atsie method using the biosensor system.
- biosensors having various detection modes have been proposed.
- sensors using surface plasmon resonance (hereinafter also referred to as SPR) are sensitive to changes in the refractive index at and near the surface of the metal thin film (for example, A Szabo et al., Curr. Opin. Strnct. Bio 1.5 (1995) 699-1705).
- SPR is capable of observation of the processes occurring between the surface and the complex raw Monogaku solution-situ (i n situ), and for example, data from the test body in real time without the use of labels
- it is one of the sensors that has attracted attention because it is suitable for obtaining kinetic and thermodynamic parameters.
- a typical example of a biosensor chip having such a surface is BIACORE TM available from Amersham Pharmacia Biotech Co., Ltd.
- BIACORE the matrix of dextran with a carboxylated end is fixed on a gold thin film in a translucent state.
- R is a hydrocarbon chain having a chain length of more than 10 atoms and optionally interrupted by heteroatoms, wherein Y is a ligand or biocompatible
- An organic molecule represented by the formula (1), which is an active group for covalently bonding a porous matrix is bonded to the surface of a thin film of a free electron metal such as gold or silver via its thiol (or mercapto) group.
- the surface is covered with a tightly packed monolayer, and then, as a biocompatible porous matrix, agarose, dextran, polyethylene glycol, etc. which may have a functional group for binding a ligand.
- a biosensor chip having a surface covalently bonded to a hydrogel consisting of (for example, see US Pat. No. 5,763,191).
- a biosensor chip having a surface covalently bonded to a hydrogel consisting of (for example, see US Pat. No. 5,763,191).
- a sulfur atom mercapto group
- a support to prevent non-specific adsorption of contaminants present in a biological fluid or the like to the sensor chip surface.
- a hydrophilic linker a linear molecule having a chain length of 4 to 15 atoms
- a solid-phase reactant A sensor chip having a surface to which covalently bound biotin derivative residues are sequentially provided (see, for example, US Pat. No. 3,071,823).
- HS Spacer molecule (alkylene chain with 11 carbon atoms) —Hydrophilic linker (chain consisting of 3 or 6 ethylene oxide units)
- Sensor chips having a monolayer self-assembled on a gold surface have also been provided (see, eg, Roberts et al., J. Am. Chem. Soc. 1998, 120, 6548-6555). Further, there has been proposed a sensor chip having a surface carrying a heteroteremeric polymer having an ethylene oxide unit in the range of 5 to 10,000 (see WO 01/86301 A1).
- a sensor chip having such a surface also achieves a constant amplification of the SPR signal.
- colloidal gold Au
- Au colloidal gold
- a number of sensors are also provided that are used in combination with a sensor chip having no polyethylene glycol layer.
- the advantages of such a system include a shift with a large plasmon angle, a wide plasmon resonance, and a significant increase in the minimum reflectance compared to a system without colloidal gold.
- the surface of the sensor chip of a system that uses these gold colloids or gold nanoparticles to amplify or enhance SPR has a common property of alkanethiol (eg, 3-mercaptopro- (Pionic acid or 3-mercaptoethylamine), and then covalently bind biotin or avidin or streptavidin, antibody, etc.
- alkanethiol eg, 3-mercaptopro- (Pionic acid or 3-mercaptoethylamine)
- Gold colloids or gold nanoparticles bind proteins (including chemical adsorption) with or without the above-mentioned alkane thiols, and produce biotin-streptavidin, antigen-antibody. By forming a biologically specific binding pair as described above, it is bound to the sensor-chip surface.
- the surface of the metal particles used as a label in the bioassay is made of a water-soluble and aqueous solution such as polyethylene glycol (or polyethylene oxide).
- a method of modifying the dispersion stability of the particles by modifying with a single polymer chain having high mobility in a medium W. Pwuelfine et al., J. Am. Chem. Soc. 120 (48), 12696-12697 (1998) )
- PEG modified Polyethylene glycol having a residue of an acidic compound
- Metal particles, semiconductor particles or magnetic particles are also known to exhibit dispersion stability in an aqueous medium (Otsuka et al., J. Am. Chem. So 2001, 123, 8226-8230); See Japanese Patent Application Laid-Open No. 2000-80903. ). It has also been proposed to use semiconductor nanoparticles surrounded by a polymer (eg, diacetylene, styrene, etc.) as a probe for detecting biological substances (eg, US Pat. No. 6,072,392). No.).
- a polymer eg, diacetylene, styrene, etc.
- the sensitizing effect differs depending on the distance between the gold nanoparticles and the surface of the gold thin film of the biosensor chip or the bonding mode between the particles and the surface. (See, eg, N. Nath et al., Supra.) Therefore, even when the system using the gold nanoparticles and the biosensor chip as described above is applied to the system described in U.S. Pat.No. 5,763,191, the sensitivity is reduced, or nonspecific adsorption of contaminants occurs. Can not be prevented.
- the present inventors have developed a BI AC ORE® sensor chip, a sensor chip with a polyethylene glycol-modified surface, and polyethylene glycol-modified metal particles that are mainly provided to improve the dispersion stability in aqueous media.
- semiconductor particles when semiconductor particles are used in combination, the sensitivity of the bioassay by the corresponding sensor chip can be increased, and non-specific adsorption by contaminants can be prevented or suppressed.
- the present invention has been completed based on such findings. According to the present invention, (A) structural formula I:
- X represents a functional group or a functional moiety capable of binding to the biosensor chip surface
- Y is one selected from the group consisting of Ci one C 6 alkyl group, X functional group or protected functional group, which may also be, and X the same or different that the functional moiety can be used to form the functional moiety
- L represents a group or a binding moiety that binds to PCL;
- W 1 and W 2 represent a single bond or the same or different linking group
- PEG is an ethylene oxide unit: (one CH 2 CH 20 —). (Where n is an integer from 5 to 10,000).
- X and y are independently integers greater than or equal to 1 and together represent polyethylene glycol-modified nanoparticles of P EG chains representing an integer sufficient to coat the surface of the PCL in an aqueous medium;
- (B) Including a set with a biosensor chip that allows the particles of (A) to bind through X and has a surface made of a dielectric material such as glass or a material responsible for the PCL material.
- a biosensor system for use in a bioassay is provided.
- X in the structural formula I is a member residue forming a biological specific binding pair
- Y is a residue forming a biological specific binding pair.
- X + y is the surface 1 nm 2 equivalents or 0.1 0.5 PCL, preferably a number of one corresponding to 0.25- 0.40, (xZx + y) xl OO 1 one 99, preferably Polyethylene glycol-modified nanoparticles are also provided that are integers between 20-65 and have an average PCL cross-sectional dimension of 1-1500 nm, preferably 5-500 nm.
- the set of the particles of (A) and the chip of the biosensor of (B) is formed by bonding (covalent bond or non-covalent bond (for example, hydrophobic bond found in specific biological bond, When ionic bonds, chemisorption, etc.) are formed, for example, resonance Raman scattering is increased by the surface effect.
- the biosensor chip surface of (B) coated with the particles of (A) can be prepared, for example, even if the chip surface is not modified with a dextran layer or polyethylene glycol as described above. Nonspecific adsorption of proteins and the like present in a biological fluid can be significantly suppressed.
- FIG. 1 is a conceptual diagram showing a relationship between a PEG-modified gold nanoparticle according to the present invention and a sensor chip surface.
- a) is a schematic diagram of a high-sensitivity system in which PEG-modified gold nanoparticles are immobilized on a metal surface, (i) represents a PEG chain that suppresses nonspecific adsorption, ( ⁇ ⁇ ⁇ ⁇ ) represents a ligand molecule, And (ffi) represents gold particles that enhance the SPR response.
- b) is a schematic diagram of the competing Atsui system.
- FIG. 2 is a sensorgram of the results of an experiment performed to confirm the specific binding between 1 ac65 and lectin.
- Figure 3 is a graph showing the relationship between lactose density on the surface of the PEG-modified gold nanoparticles and the response to the lectin-immobilized surface.
- Fig. 4 is a gram of the sensor showing the dissociation state due to galactose competition between the PEG-modified gold nanoparticles bound via lactose-lectin and the sensor chip.
- Figure 5 is a graph showing the measured potential of the PEG-modified gold nanoparticles having an amino group at the non-bonded terminal (the curve marked with a letter) and the same measurement of the gold nanoparticles having a non-bonded terminal acetalized formyl group. It is a graph (curve with a triangle) showing the result.
- FIG. 6 is a graph showing the results of measuring the amount of protein carried on PEG-modified gold nanoparticles having a biotin residue at the non-bonded terminal.
- FIG. 7 is a graph showing the adsorptivity of various proteins on the surface where PEG-modified gold nanoparticles having an amino group at the non-bonded terminal are directly adsorbed on the gold chip surface.
- FIG. 8 is a graph showing the protein adsorption on the surface of the gold chip used in FIG. 7 bound to the gold chip surface using SPDP. Specific description of the invention
- a particularly focused use of the biosensor system according to the present invention is directed to bioassays utilizing surface plasmon resonance (SPR), but the present invention refers to traces other than SPR. Includes those that use changes in signals, radioactivity, contact angles of various electromagnetic waves, sedimentation, ultraviolet spectroscopy, Raman scattering, etc.
- the biological molecule to be detected by the bioassay according to the present invention is a so-called biological specific binding pair (for example, the biological molecules are formed by a hydrophobic bond, an ionic bond, etc.), more specifically.
- ligands and receptors such as antigens or haptens and antibodies, sugars and lectins, substrates and enzymes, hormones and their receptors, oligonucleotides and their complementary strands, and biotin and avidin
- ligands and receptors such as antigens or haptens and antibodies, sugars and lectins, substrates and enzymes, hormones and their receptors, oligonucleotides and their complementary strands, and biotin and avidin
- it can be, but is not limited to, any member that forms a binding pair, such as streptavidin.
- biosensor system means each element or an assembly or combination thereof that can be used for performing the above-mentioned assay.
- fine particle and “nanoparticle” are used interchangeably, and are not limited to nano-order, sub-nano or several micrometer, unless otherwise specified. Used to include
- PCL includes free-electron metals (eg, gold, silver, platinum, aluminum, copper, etc.), semiconductors (eg, CdS, ZnS, CdSe, InAs, etc.) and metal oxides (eg, Ti 0 4 , Cr 2 0 3 etc.) Microparticles made of such materials. Without limitation, those having an average cross-sectional dimension of 1 to 500 nm can be conveniently utilized.
- free-electron metals eg, gold, silver, platinum, aluminum, copper, etc.
- semiconductors eg, CdS, ZnS, CdSe, InAs, etc.
- metal oxides eg, Ti 0 4 , Cr 2 0 3 etc.
- L is a bond via a group or a moiety which can be bonded to the particle surface (chemical bond or chemisorption, and in the case of metal oxide, a covalent bond via surface OH group generated by hydroxylation). Anything may be used as long as it meets the purpose of the above. And preferably, the following equations (i)-(M) and (ffi)
- n represents an integer from 2 to 12; RR 2 and R 3 independently represent a d—C 6 alkyl group; and m represents an integer of 2 to 500, preferably 5 to 100.
- a bond is, for example, a group or a portion (or segment) of (i) and (iii), it may be formed between the above-mentioned particle surface and (ii) When it is a group or a moiety, it may be a bond formed by a dealcoholization reaction between one OH and a silanol group on the surface of the hydroxylated metal oxide.
- PEG is an ethylene oxide unit: (one CH 2 CH 20 —) n (where n is 5 to 10,000, preferably 10 to 10,000, more preferably an integer of any of 20 to 2500. ).
- X represents a functional group or a functional moiety capable of binding to the biosensor chip surface.
- the functional group or functional moiety may be a group or a moiety represented by the formulas (i), (ii) and (m) exemplified as those capable of forming the above L. It can be a residue of one member capable of forming a chemical specific binding pair, or a residue of a protein that does not affect bioassay.
- the members of the specific binding pair those having a small molecular weight, for example, residues derived from hapten, sugar, substrate, hormone, oligonucleotide, and biotin are preferred.
- Y is d-C e alkyl group, a group or a moiety in another group or functional moiety, or a protected form the moiety or X in the group or a functional moiety or a protected form as defined for X There can be.
- Representative groups or moieties other than X include the following formulas (iv), (V) and (vi)
- R a independently represents a hydrogen atom or C, —C e alkyl
- R b independently represents C, -C 6 alkyloxy or two R b taken together as oxy or d—Ce represents an atomic group necessary for forming a optionally substituted ethylene group with alkyl.
- Preferred examples of Y include the above formulas (), (V) and (vi), and groups or moieties selected from the group consisting of formula (i) and a C, 1Ce alkyl group defined for X. be able to.
- W 1 and W 2 are independently a linking group, for example, a single bond, d—C 6 alkylene, —COO— (attached to the methylene group of the ethylene oxide unit via an oxygen atom), It can be a group selected from the group consisting of S—, 1- (d-Ce alkylene) -1 COO—, 1- (Ct—C e alkylene) -1 0— and 1- (d—C 6 alkylene) -1 S— .
- the polymers represented by can be the same as can be understood from the definitions of X and Y above, but are preferably different.
- W 2 —PEG—W′—L in these formulas can be the same or different.
- one or more integers (corresponding to the integers of X and y, respectively) are independently combined on a single PCL surface. .
- These x + y are present in integers sufficient to cover the PCL surface with PEG chains. This number can suppress the adsorption of non-specific proteins and the like in an aqueous medium on the surface of the polyethylene glycolated particles (when the surface of the sensor chip is coated with such particles, according to the present invention). Will be a number.
- non-specific protein adsorption means, for example, when X is a member capable of forming a biological specific binding pair, for example, when it is an antigen, It refers to adsorption other than through specific binding to which antibodies bind.
- a specific number of x + y is any number corresponding to a number of 0.1-0.5, preferably 0.25-0.40 per 1 nm 2 of PCL surface.
- the proportion of X and y can be arbitrary, as described above, so that the polymers represented by formula ( ⁇ ) and formula (DI) can be the same.
- the ratio of X to the total number of X and y can be 1-99, preferably 20-65.
- the polymer of the formula ( ⁇ ) (X is a member forming a biological specific binding pair) and a group of the formula (ID) (Y is different from X and does not bind to X) Is a part.)) can be placed on the PCL. Particles in this form are useful for performing quick and sensitive assays.
- Typical examples of the above-mentioned polyethylene glycolated particles are described in otsuka et al., J. Am. Chem. So 2001, 123, 8226-8230, JP-A-2001-200050, JP-A-2002-80903. And can be manufactured according to these descriptions. It can also be used to form the particles.
- the heterotelechelic polymer can be easily prepared by those skilled in the art by referring to the functionalization of the ⁇ , ⁇ -terminal of the block copolymer described in WO 96/32434 and WO 96/33233 WO 97Z06202, which was proposed by a part of the present invention. It could be manufactured in a short time.
- d-Ce alkyl, C, - C 6 alkyl Okishi, d-C 6 Arukiren are used for different groups or moieties Even if they do, they have a common meaning.
- C 8 alkyl denotes methyl, Echiru, n- propyl, iso- propyl, n- heptyl, se C.- heptyl, to n- and hexyl.
- Examples of d-C e al Kiruokishi, the C -! C 6 Each corresponding alkyloxy of alkyl is exemplified.
- C! -C 6 -alkylene is methylene, ethylene, propylene, 1,3-trimethylene, 1,6-hexamethylene and the like.
- the shape and size of the biosensor chip are not limited as long as the surface can be bonded to the X group or the part of the PEG-modified nanoparticles described in (A) above and can be used for bioassay.
- the surface is of the same or the same class as the material forming the PCL used in the set (eg, gold and gold, gold and silver for free electron metals; C d S for semiconductors). It is preferable to select CdS, CdS and InAs) in order to enhance the above-mentioned signal obtained from the particles of (A) and the sensor chip.
- the signal is detected using a visible-UV spectrometer, it can be a crystal or glass that transmits these lights.
- the surface can usually be a thin film on which the corresponding material has been deposited.
- Such a surface must be modified so as to promote the formation of a bond with the group or moiety of X in the particles of (A) above, or, if X is a protein, the material itself.
- Such modifications should be made with an organic compound having at least one terminal having a group or a moiety capable of binding to PCL as described for L in formula (I).
- a chip with a surface made of gold, silver, or semiconductor may be modified with alkanethiol (for example, 3-mercaptopropionic acid or 2-mercaptoamine) and then free carboxy or
- alkanethiol for example, 3-mercaptopropionic acid or 2-mercaptoamine
- the amino group can be used to covalently bind members (for X members) that can form a biologically specific binding pair to complete the desired surface.
- biosensor chip having such a surface those described in the aforementioned LA Lyon et al., ⁇ . Hutter et al., JP-A-2002-267669, and JP-A-2000-55920 are described. I can show you an example.
- a BI ACORE (trademark) sensor chip having a dextran layer on the surface and a sensor chip having a surface coated with a heterotelechelic polymer having a poly (oxyethylene) chain in the middle (for example, see WO 01/86301 A1) Can also be used in the present invention.
- Typical heterotelechlipp polymers that can be used to modify the PCL surface and sensor chip surface described above can be manufactured according to the following reaction scheme.
- CH 2 CH-CH 2 0CH 2 CH— (0CH 2 CH 2 ) n — 0 ⁇ M®
- M represents potassium, sodium, and lithium.
- the above-mentioned riving polymerization step can be carried out under reaction conditions known per se (for example, see WO 96/32434, WO 97/06202, etc.). Others can be carried out according to Examples described later or by modifying the conditions described.
- the free electron metal fine particles, metal oxide fine particles, or semiconductor fine particles constituting PCL in Structural Formula I are either commercially available or used, respectively.
- Fine particles obtained by forming corresponding colloids can be used.
- the PEG-modified nanoparticles according to the present invention form, for example, corresponding fine particles (for example, 0.5 ⁇ -1; « ⁇ , preferably an average particle diameter of lnm to 200 nm).
- the chips are used, and in particular, they are used in combination.
- the surface in which the particles of (A) cover the surface of the sensor chip of (B) is formed by the action of the polymer represented by the formulas ( ⁇ ) and (DI) on the surface of the particles of (A).
- the effect of amplifying various signals by the particles can be obtained.
- a set of particles (A) and one sensor chip (B) can be used in a manner as shown in FIG. 1 schematically illustrating the concept of (1).
- the triangles in the figure represent, for example, sugars, biotins, antigens or haptens, hormones, oligonucleotides, etc., and the triangles are fitted with lectins, avidin or streptavidin, antibodies, receptors, respectively.
- the wavy lines associated with these marks can be poly (ethylene oxide) segments.
- the bioassay method represented by such a conceptual diagram is also one embodiment of the present invention.
- the change in the degree of coupling between the particles in (a) and the sensor chip in (b) is caused by a change in the surface plasmon resonance spectrum, for example, a shift in the plasmon angle and a change in the minimum reflectance. It is preferred that Such an Atsey method is theoretically applicable to any aqueous liquid sample suspected of containing a member (analyte) that forms a biologically specific binding pair. It is intended to be applied to biological fluids, such as serum, plasma, urine, saliva, etc., or their concentrated or diluted solutions.
- a quick and highly sensitive assay can be performed.
- Production Example 1 Production method of PEG-modified gold fine particles (Part 1):
- Acetal-PEG-SH was produced as follows. In a receiver purged with argon, add 20 ml of distilled tetrahydrofuran (THF) and 0.2 mmol (0.032 ml) of 3,3-diethoxyquin-l-propanol as initiator, add an equivalent amount of potassium naphthalene, and stir for 15 minutes. Metalization was done by doing. Thereafter, 22.7 mmo 1 (1,135 ml) of ethylene oxide was added, and the mixture was stirred at room temperature for 2 days to polymerize.
- THF distilled tetrahydrofuran
- 0.2 mmol (0.032 ml) of 3,3-diethoxyquin-l-propanol as initiator
- Metalization was done by doing. Thereafter, 22.7 mmo 1 (1,135 ml) of ethylene oxide was added, and the mixture was stirred at room temperature for 2 days to polymerize.
- N-succinimidyl 3- (2-pyridylthio) propionate (SPDP) 0.4 mmo 1 (0.125 g) is dissolved in a small amount of distilled THF as a terminator, and the above polymerization reaction solution is dropped to this solution under an equal pressure. The mixture was dropped in a funnel under ice cooling. After stirring overnight to carry out a termination reaction, the polymer was recovered through washing with saturated saline, extraction with black-mouthed form, reprecipitation with ether, and freeze-drying with benzene.
- the structure of the recovered polymer was confirmed by 1 H-NMR, and the amount of SPDP residue introduced at the terminal was confirmed by UV absorption of 2-thiopyridone released by reacting with 2-mercaptoethanol.
- PEG- SS- Py 2.0x 10 - 2 mmo 1 (l O Omg) was dissolved in distilled water 4 m 1 and further 5 fold mo 1 of Jichiotorei tall 0.1 m mo 1 a (15.42mg) was added, 30 at room temperature For a minute.
- a polymer hereinafter abbreviated as PEG 5000
- the structure of the recovered polymer was confirmed by 1 H-N MR, and the terminal SH group was quantified by reaction with 2-pyridyl disulfide (2-PDS).
- a heterobifunctional PEG having an acetal group and a methylsulfonyl group is synthesized by anion polymerization using 3,3-methoxy-1-propanol as an initiator and methylsulfonyl chloride as a terminator. did. Furthermore, a polymer in which a methylsulfonyl group was converted into an ethyldithiocarbonate group was obtained by reacting potassium tetraethyldithiocarbonate with tetrahydrofuran (THF) at room temperature for 3 hours.
- THF tetraethyldithiocarbonate
- the UV spectrum of the unmodified gold particles was calculated from the UV-vis spectrum obtained when the particles were redispersed in a THF solution after centrifugation. It was confirmed that a large absorption peak was observed at 600 nm or more based on the aggregation of. Gold particles treated with Acetal—PE 0—OH (comparative example) did not have a large peak at 600 nm or more like the UV spectrum of unmodified gold particles, but the overall peak was high. It was confirmed that the dispersion was shifted to the wavelength side and dispersion of the fine particles was somewhat unstable.
- Table 1 shows the measured data. Zeta potential of acetal PEG-SHZ gold (Au) particles
- the polymer used (Acetal—PEG—PAMA):
- the UV-Vis spectrum was measured at predetermined time intervals. As a result, the peak at 540 nm derived from the fine gold particles gradually increased, and a gold colloid particle (fine particle) dispersion was formed without adding a reducing agent. It was confirmed that.
- This solution was measured by light scattering (DLS: dynamic light scattering), and it was confirmed that monodisperse colloid particles having an average particle size of 12 nm were formed.
- DDB 1,2-diamino-4,5 dimethoxy dihydrochloride
- the solution was dialyzed with a dialysis membrane having a molecular weight cutoff of 500, and the fluorescence was measured at an excitation wavelength of 269 nm. As a result, strong fluorescence was observed at 410 nm. From these results, it was confirmed that the terminal acetal group of the Acetal-PEG / PAMA block copolymer was converted to an aldehyde group on the prepared gold particle surface and reacted efficiently with DDB. . The aldehyde group thus formed Various functional parts can be provided via the.
- Production Example 4 Production of PEG-modified semiconductor fine particles:
- Example 1 Immobilization of PEG-modified fine particles on the sensor chip surface:
- the value of 0.25 was calculated from the Tg of the PEGylated gold particles.) Thereafter, Na BH 4 was added and the mixture was stirred for 3 days. After centrifugal purification, a solvent replacement solution was prepared with 10 mM PBS (pH 6.4). The chip having the gold surface prepared in (1) was immersed in the thus obtained solution of the biotinylated PEG-modified gold fine particles, whereby the PEG-modified gold fine particles were fixed on the surface.
- the surface immobilized with the PEG-modified gold microparticles prepared in (2) was immersed in 0.1 lmgZm 1 ⁇ cysteine albumin (BSA) in PBS (pH 6.4) for 1 hour, and the amount of adsorption on the surface was determined by SPR.
- BSA cysteine albumin
- Example 2 Technique by SPR using PEG-modified gold nanoparticles
- the flow rate of SPR is always 10 L / min, and the set temperature is 25 ° C. All buffers were degassed after passing through a 0.22 ⁇ m filter. Another channel was a control channel in which lectin was not immobilized.
- a buffer containing lOOmgZmL of galactose was used as the regenerating solution for dissociating all the gold nanoparticles bound to the lectin on the sensor-one chip surface.
- RCA 12 n 50 g / mL; solvent is acetate buffer solution of pH 5.0
- 1M ethanolamide hydrochloride was finally injected at 70 // L, and the remaining was injected.
- Figure 2 shows the sensorgram of 1 ac 65.
- the same concentration of free lactate-PEG-S-S-PEG-lactose showed little increase in RU, whereas 1ac65 gave 5200 RU increase.
- 7000 RU Injecting 5 OO ja gZmL of micelle onto the sensor chip to which the lectin binds a response of ⁇ 1400 RU was obtained, but the particle size of the gold nanoparticles used in this experiment was the same as this micelle.
- the response was four times higher at 1/10 concentration, it can be said that the response was greatly increased by the gold nanoparticles.
- One reason that such a large response was obtained was that the specific gravity of the gold nanoparticles was so large that the dielectric constant on the chip surface increased significantly, and that the gold substrate of the chip and the gold nanoparticles And the interaction between surface plasmons.
- Gold nanoparticles (1 ac 0, I acl 0, 1 ac 20, 1 ac 30 lac 40, lac 50, lac 65) with lactose densities of 0-65% are 40, 10, 1, and 0.
- a 300 / L injection was performed at a concentration of 1 gZmL, and the amount of binding was measured.
- a large ligand density binds a large number of ligands to lectins, resulting in strong binding, while a small ligand density binds only a small number of ligands, such as a one-to-one ligand-lectin binding. May not be able to participate.
- Figure 4 (a) shows the sensorgram when galactose was injected at 0.1 zg / mL.
- lac 30 and lac 40 gradually dissociated over time, but lac 65 and lac 50 hardly dissociated, so the relationship between ligand density and dissociation amount was determined.
- Fig. 4 (b) shows the relationship between ligand density and dissociation amount.
- a significant difference was seen between 40% and 50%. That is, the amount of dissociation of 1 ac50 and 1 ac65 was considerably smaller than the amount of dissociation of lac30 and 1 ac40. This may be related to the large difference between the 40% and 50% increase in NIA in UV experiments. Probably, this ligand density changed the valence of multivalent bonds.
- Figure 5 shows the measurement results. From the figure, the negative potential at low pH was positive and amination was confirmed.
- Example 3 Method of Immobilizing Gold Nanoparticles Directly on SPR Chip Surface and Pegating PEG Terminals An ozone-washed gold chip (15 minutes with an ozone washing machine) was prepared. Separately, PEG-OH containing PEG-containing gold nanoparticles with amino groups (PEG4500 particle size 5 nm, 0.76 10 ° mo1 / ml) was prepared separately. (2000) Dipped in 1 mg / m 1 (overnight) (Sample 1).
- sample was attached to a BiaCore 3000, and sulfosuccinimidyl D-biotin (0.1 mgZm, 1 flow rate, 20 ⁇ 1 / min) was passed for 10 minutes to biotinylate the PEG terminal amino group (sample 2).
- sulfosuccinimidyl D-biotin 0.1 mgZm, 1 flow rate, 20 ⁇ 1 / min
- acetic anhydride flow rate: 10 H1Z
- Example 4 An active ester is introduced into the surface of one SPR sensor chip by SP DP, and PEGylated gold nanoparticles having amino groups are immobilized. Prepare an ozone-washed gold chip (15 minutes with an ozone washer). did. This was immersed in an ethanol solution containing ImMSPDP and 2 mM DTT for 30 minutes.
- sample was attached to a Bia Core 3000, and sulfosuccinimidyl D-piotin (0.1 mg / m1 at a flow rate of 20 m1 / min) was flowed for 10 minutes to biotinylate the PEG terminal amino group (sample 5).
- a 10% aqueous solution of acetic anhydride flow rate: 10 a for 1 minute was flowed for 20 minutes to acetylate unreacted amino groups (sample 6).
- a protein adsorption test was performed on the surface of the SPR sensor chip thus prepared.
- Fig. 6 shows the results. Run 1 in Fig. 6 was obtained using sample 1, and Run 2 was obtained using acetal-PEG gold nanoparticles instead of sample 1. As a result, Run 3 shows the result using sample 4, and Ru 4 shows the result using acetal-PEG gold nanoparticles instead of sample 4. From FIG. 6, it can be seen that the amount of the PEGylated gold nanoparticles having an amino group carried on one SPR sensor chip surface is large. Table 3 below shows the response change at each stage of surface preparation. Table 3: Changes in response at each step Step Immobilization by SPDP by direct adsorption 11 (1) Immobilization 11 (2) (X 10-40 ) (X 10 " 40 )
- Fig. 7 shows the results of observation of specific non-specific adsorption on the surface (1) by direct adsorption prepared by immersing the aminated gold particles directly on the surface of the gold chip (in Fig. 7, the top line is streptavidin. The middle line is BSA (amino residue), the bottom line is the adsorption of BSA, the bar graph is streptavidin in sample 1 from left to right, BSA in sample 2 and sample 3 in order from left to right.
- Fig. 8 shows the results of specific and non-specific adsorption observed on the surface (2) where the aminated gold particles were immobilized using SPDP on the gold chip surface.
- a bioassay sensor system which suppresses nonspecific adsorption of contaminating proteins and the like when detecting an analyte in a biological fluid and has high sensitivity. Therefore, the present invention can be used in the manufacturing industry of various diagnostic devices and in the diagnostic industry.
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KR10-2004-7015647A KR20040105839A (ko) | 2002-04-03 | 2003-03-24 | 폴리에틸렌글리콜화 나노 입자를 담지하는 바이오센서 칩표면 |
EP03712842A EP1496363A4 (en) | 2002-04-03 | 2003-03-24 | POLYETHYLENE GLYCOLATED NANOPARTICLES WEARING BIOCHIPSENSOR SURFACE |
US10/509,576 US20050106570A1 (en) | 2002-04-03 | 2003-03-24 | Biochip sensor surface carrying polyethylene glycolated nanoparticles |
CA002480770A CA2480770A1 (en) | 2002-04-03 | 2003-03-24 | Biochip sensor surface carrying polyethylene glycolated nanoparticles |
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US (1) | US20050106570A1 (ja) |
EP (1) | EP1496363A4 (ja) |
KR (1) | KR20040105839A (ja) |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008100161A1 (en) * | 2007-02-12 | 2008-08-21 | The New Zealand Institute For Plant And Food Research Limited | Surface plasmon resonance assay for steroids |
US9566604B2 (en) | 2010-08-24 | 2017-02-14 | Cline Scientific Ab | Method for preparing a surface with a controlled coverage of nanograde particles |
US10274415B2 (en) | 2010-08-24 | 2019-04-30 | Cline Scientific Ab | Method for preparing a surface with a controlled coverage of nanograde particles |
JP2015522830A (ja) * | 2012-07-26 | 2015-08-06 | サントル ナショナル ドゥ ラ ルシェルシュ シアンティフィク | 試料を観察し、化学種または生物学的種を検出または計量するための光学的方法 |
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CA2480770A1 (en) | 2003-10-09 |
KR20040105839A (ko) | 2004-12-16 |
US20050106570A1 (en) | 2005-05-19 |
EP1496363A4 (en) | 2005-05-18 |
EP1496363A1 (en) | 2005-01-12 |
CN1646912A (zh) | 2005-07-27 |
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