JP5371302B2 - Single probe molecular device and single probe molecule fixed metal fine particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biomolecule detection element capable of detecting biomolecules on a single molecule level with high accuracy and high reproducibility. <P>SOLUTION: Metal particles having a single probe molecule immobilized on each surface are manufactured, and a single probe molecule element formed by immobilizing metal particles on the carrier substrate surface is manufactured. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a single probe molecular element for detecting a biomolecule and a biomolecule detection method using the same.

  In recent years, various biomolecule detection elements have been researched and developed as methods for specifically and comprehensively analyzing biomolecules such as nucleic acids and proteins. An important example of such a biomolecule detection element is a probe molecule-fixed fine particle in which a probe molecule for detecting a biomolecule is fixed on the surface of a nanometer to micrometer-size fine particle. So far, various biomolecule detection methods using probe molecule fixed fine particles have been reported. In particular, probe molecule-immobilized metal microparticles, in which probe molecules such as nucleic acids, proteins, and sugar chains are immobilized on the surface of nanometer-sized metal microparticles, can be prepared and detected relatively easily. Examples have been reported.

On the other hand, research on nanotechnology aimed at the development of highly functional materials has progressed, and many nanometer-sized molecules having a characteristic geometric structure have been developed. For example, dendrimer, a nanometer-sized molecule that can be artificially controlled in shape and size, has been reported. Dendrimer is a monodisperse spherical polymer, and its size is about several nanometers to several tens of nanometers. The dendrimer has a branched structure from the core in the center of the molecule toward the outside. By repeatedly bonding monomer molecules outward from the core, the number of branches and the molecular size increase exponentially. The number of repetitions of monomer molecule bonding is called generation. FIG. 1 shows the structure of a polyamidoamine dendrimer (abbreviated as PAMAM dendrimer), which is a typical dendrimer. The PAMAM dendrimer shown in Fig. 1 is a second generation (Generation 2, abbreviated as G2) in which the monomer methyl acrylate is bonded twice to the ethylenediamine (Ethylenediamine) portion corresponding to the core. And 16 terminal amino groups on the outermost surface. In addition, a radius of gyration (abbreviation symbol R _G ) is used as a physical quantity representing the molecular size of a spherical polymer such as dendron. The turning radius of a spherical polymer can be measured by the light scattering method, small-angle neutron scattering method, and small-angle X-ray scattering method. is there. In addition, the radius of rotation of the spherical polymer can be estimated by molecular dynamics simulation. FIG. 2 shows the relationship between the generation of PAMAM dendrimer, the number of terminal amino groups, and the radius of rotation. Note that the radius of rotation of the PAMAM dendrimer shown in FIG. 2 is a value in a neutral pH solution reported by Lee et al. (Lee, I. et. Al., Macromolecules, Vol. 35, p. 4510 ( 2000)).

  In Patent Document 1 and Non-Patent Document 1, Dendron, which is a molecule having a hemispherical structure in which a PAMAM dendrimer having a cystamine having a disulfide bond as a core is used as a starting material and the PAMAM dendrimer is divided into two equal parts. A method of synthesizing is disclosed. In addition, a method is disclosed in which the dendron and DNA (Deoxylibonucleotide, deoxyribonucleic acid) are reacted to synthesize a conjugate in which dendron and DNA are bound at a ratio of 1: 1.

U.S. Patent No. 6,020,457 Nano Letters Vol.4, No.5, p.771 (2004)

  In order to quantitatively detect biomolecules using the probe molecule-fixed metal fine particles, it is very important to control the number of probe molecules immobilized on the surface of the metal fine particles. In particular, in order to realize quantitative detection of a biomolecule at a single molecule level, it is indispensable to use metal fine particles having a single probe molecule immobilized on the surface. Probe molecule-fixed metal fine particles used in various conventional examples are manufactured by mixing and reacting metal fine particles and probe molecules having terminal functional groups that strongly bond to the metal fine particles at a predetermined mixing ratio. There are many. Accordingly, the probe molecule-fixed metal fine particles produced by this method become a mixture of metal fine particles having a distribution in the number of probe molecules immobilized on the surface.

  An object of the present invention is to dramatically improve the detection accuracy and reproducibility of biomolecules by utilizing metal microparticles with a single probe molecule fixed on the surface in a biomolecule detection element that detects biomolecules at a single molecule level. Is to improve. The problem to achieve this object is to produce metal fine particles with a single probe molecule fixed on the surface in high yield, and to use the metal fine particles on the surface of the biomolecule detection element to be analyzed. Capturing biomolecules one by one with high recovery.

  In the present invention, in order to fix the probe molecule and the metal fine particle used for biomolecule detection 1: 1, a linker molecule having the characteristics shown in the following (1) to (3) is used, and the probe molecule: linker molecule: metal The problem is solved by producing single probe molecule-fixed metal fine particles with fine particles = 1: 1: 1 in high yield. The characteristics that the linker molecule should have are the following (1) to (3).

(1) Have only one functional group that can bind to the functional group of the probe molecule
(2) Having two or more functional groups that can bind to the surface of the metal fine particles
(3) When the shape of the linker molecule is approximated by a sphere, the radius of rotation is R ₁ and the relationship with the radius of rotation R ₂ of the metal fine particles fixing the probe molecule is 0.1 ≦ (R ₁ / R ₂ ) ≦ 1.5. Fulfill

The feature shown in (3) above will be described. When the rotation radius R ₁ of the linker molecule in the present invention is very small relative to the rotation radius R ₂ of the metal fine particle, the probability that two or more linker molecules are bonded to the surface of one metal fine particle via a chemical bond. And the object of the present invention cannot be achieved. Therefore, it is necessary that the rotation radius R ₁ of the linker molecule is not too small with respect to the rotation radius R ₂ of the metal fine particles, and specifically, it is desirable that the relational expression 0.1 ≦ (R ₁ / R ₂ ) is satisfied. On the other hand, when the rotation radius R ₁ of the linker molecule is very large relative to the rotation radius R ₂ of the metal fine particle, two or more metal fine particles are bonded to the terminal functional group of one linker molecule through a chemical bond. The probability of fixing becomes high and the object of the present invention cannot be achieved. Therefore, it is necessary that the rotation radius R ₁ of the linker molecule is not too large with respect to the rotation radius R ₂ of the metal fine particle, and specifically, it is desirable that the relational expression (R ₁ / R ₂ ) ≦ 1.5 is satisfied. . Summarizing the above conditions, in order to achieve the object of the present invention, the rotation radius R ₁ of the linker molecule satisfies the relational expression 0.1 ≦ (R ₁ / R ₂ ) ≦ 1.5 with respect to the particle radius R ₂ of the metal fine particles. It is desirable to satisfy.

  By using a linker molecule that satisfies the above conditions (1) to (3), a probe molecule: linker molecule: metal fine particle = 1: 1: 1 conjugate, that is, a single probe molecule-fixed metal fine particle is obtained with high yield. It becomes possible to produce at a rate.

  A single probe molecular device according to the present invention includes a substrate, a plurality of metal fine particles fixed on the surface of the substrate, a linker molecule fixed on the surface of one metal fine particle, each via a chemical bond, and a linker. One probe molecule bonded to each molecule via a chemical bond.

  The single probe molecule-fixed metal fine particle according to the present invention includes a metal fine particle, one linker molecule fixed to the surface of the metal fine particle via a chemical bond, and 1 bonded to the linker molecule via a chemical bond. Probe molecules.

  The linker molecule has only one first terminal functional group that can chemically bond the probe molecule, and two or more second terminal functional groups that can be fixed to the metal fine particle. preferable.

  According to the present invention, it is possible to provide a single probe molecule-fixed metal fine particle for detecting a biomolecule at a single molecule level with high accuracy and high reproducibility, and a single probe molecule element using the same. . In this single probe molecule element, single probe molecule-fixed metal fine particles are fixed on the surface of the carrier substrate. Therefore, by using the single probe molecule, the biomolecules to be analyzed are placed on the carrier substrate surface one molecule at a time. It is possible to capture at a high recovery rate.

Embodiments of the present invention will be described below with reference to the drawings.
FIG. 3A shows an example of single probe molecule-fixed metal fine particles according to the present invention. A single probe molecule 301 for capturing only one biomolecule to be analyzed binds to the terminal functional group X of the linker molecule 303 via the terminal functional group Z. That is, the probe molecule and the linker molecule are bound at 1: 1. The linker molecule 303 to which the single probe molecule 301 is bonded is fixed to the surface of the metal fine particle 306 via a plurality of terminal functional groups Y. In FIG. 3A, a linker molecule 303 to which a single probe molecule 301 is bonded is fixed to the surface of the metal fine particle 306 by eight terminal functional groups Y. Further, an adsorption inhibiting molecule W for inhibiting nonspecific adsorption of various molecules is bound to a portion of the surface of the metal fine particle 306 that is not covered with the linker molecule 303.

  3A shows spherical fine particles as the metal fine particles, the shape of the metal fine particles 306 may be a cylinder, a cone, a prism, a pyramid, or the like. As the material for the metal fine particles, any of noble metals such as gold, silver, platinum, palladium, rhodium, iridium, ruthenium, osmium, or an alloy thereof can be used. Alternatively, fine particles obtained by coating fine particles made of these noble metals with other noble metals, for example, fine particles obtained by coating silver on gold fine particles may be used. The size of the metal fine particles in the present invention is preferably 10 nm or more in order to ensure the stability of fixing the metal fine particles to the substrate. Further, in order to fix a single probe molecule on the surface of the metal fine particle with high efficiency, the size of the metal fine particle is preferably 100 nm or less. Accordingly, the size of the metal fine particles in the present invention is preferably from 10 nm to 100 nm.

  As shown in FIG. 3 (b), when a single probe molecule and a linker molecule are bound, the functional group A that binds to the terminal functional group X of the linker molecule and the terminal functional group Z of the single probe molecule are bound. Alternatively, a bifunctional compound 312 having both functional groups B to be used may be used. Further, as shown in FIG. 3C, when the linker molecule to which the single probe molecule is bonded is fixed to the surface of the metal fine particle, the functional group C bonded to the terminal functional group Y of the linker molecule 313, and the metal fine particle Alternatively, a bifunctional compound 317 having a functional group D that binds to the surface may be used.

  FIG. 4 shows an example of a single probe molecular device using single probe molecule-fixed metal fine particles according to the present invention. Single probe molecule-fixed metal fine particles 402 are fixed to the surface of the carrier substrate 401 by chemical bonding. As the carrier substrate, a glass substrate, a quartz substrate, a plastic substrate, a metal coating substrate, or the like can be used.

  In this example, a dendron synthesized using a PAMAM dendrimer (generation is any one of G0, G1, G2, G3, G4, G5, and G6) having cystamine as a core was used as a linker molecule. In addition, a single-stranded DNA was selected as a probe molecule, and the probe molecule and the linker molecule were combined at a ratio of 1: 1 to synthesize a linker-bound probe molecule, that is, a dendron-bound probe DNA. The process of synthesizing the dendron-bound probe DNA consists of the following processes.

(1) Introduction of carboxyl group into dendrimer (2) Cleavage of disulfide bond of dendrimer (3) Introduction of maleimide group into single-stranded DNA (4) Binding of dendron and single-stranded DNA (5) By gel electrophoresis Separation and purification of dendron-bound probe DNA (6) Separation and purification of dendron-bound probe DNA using nucleic acid extraction reagents (7) Introduction of terminal amino groups into dendron-bound probe DNA (8) Measurement of ultraviolet and visible absorption spectra Of dendron-bound probe DNA by HPLC (9) Immobilization of dendron-bound probe DNA to metal particles (10) Binding of adsorption-inhibiting molecules to metal particles

  Hereinafter, the process of synthesizing a dendron-bound probe DNA will be described using a G2-PAMAM dendrimer having cystamine as a core shown in FIG. 5 as a starting material. The G2-PAMAM dendrimer shown in FIG. 5 has a disulfide bond 501 in the cystamine core and 16 terminal amino groups 502 on the outermost surface. In FIGS. 6, 8, 10, and 11, the PAMAM structure is simplified. For example, in FIG. 6, a G2-PAMAM dendrimer having cystamine as a core is simplified as 601. Details of each process will be described below.

(1) Introduction of carboxyl group into dendrimer (Fig. 6)
After dissolving 100 nmol of G2-PAMAM dendrimer 601 (Sigma) in 1 × PBS (Phoshphate buffered saline, pH 10.0), the number of amino groups 602 on the outermost surface of G2-PAMAM dendrimer 601 (1 of G2-PAMAM dendrimer 601) 16 μmol of succinic anhydride 603 corresponding to 10 times the amount of 16 molecules per molecule) was added. This solution was reacted by shaking at 25 ° C. for 16 hours. After completion of the reaction, the reaction solution was treated with a molecular weight cut-off filter (MWCO filter, molecular weight 10 k, Millipore) to remove unreacted succinic anhydride 603. By this reaction, a carboxyl group 604 was introduced on the outermost surface of the G2-PAMAM dendrimer. In this example, succinic anhydride was used as a compound for converting the terminal amino group of the dendrimer into a carboxyl group, but in addition, a functional group capable of binding to an amino group and a molecule having a terminal carboxyl group are used. It is possible. For example, SCN-Bzl-DTPA (official name 2- (4-Isothiocyanatobenzyl) -diethylenetriaminepentaacetic acid) shown in FIG. 7 has one isothiocyanate group 701 that reacts with an amino group and five terminal carboxyl groups 702. Therefore, when this molecule is used, it is possible to introduce 5 terminal carboxyl groups for each terminal amino group of the dendrimer to increase the radius of rotation of the dendrimer and the number of terminal functional groups.

(2) Breaking the disulfide bond of dendrimers (Figure 8)
After dissolving the carboxyl group-type G2-PAMAM dendrimer 801 (amount of 100 nmol) prepared in (1) above in 1 × PBS (pH 7.0) that has been degassed and nitrogen bubbling, the carboxyl group-type G2-PAMAM dendrimer 801 10 Dithiothlaytol (abbreviated as DTT) 802 (amount of 1 μmol) corresponding to the double amount was added. The reaction was allowed to stand for 2 hours in an incubator kept at 37 ° C. By this reaction, the disulfide bond 803 of the carboxyl group-type G2-PAMAM dendrimer 801 was cleaved to prepare G2-PAMAM dendron 804 having a thiol group 805 at the terminal. In this reaction, G2-PAMAM dendron 804 is cleaved at the disulfide bond portion of the cystamine core of G2-PAMAM dendrimer 801. Therefore, the turning radius of the G2-PAMAM dendron 804 is half that of the G2-PAMAM dendrimer 801. After completion of the reaction, the reaction solution was purified with a gel filtration purification column (NAP-5 Column, GE Healthcare) at 25 ° C., and unreacted dithiothreitol 802 was removed from the reaction solution.

(3) Introduction of maleimide group into single-stranded DNA (Fig. 9)
7.5 nmol of single-stranded DNA 901 (base number 10 to 100, Sigma Genosys) having an amino group 902 at the 3 ′ end was dissolved in 1 × PBS (pH 7.0). Although FIG. 9 shows single-stranded DNA in which the fluorescent dye Cy3 is bound to the 5 ′ end, the fluorescent dye at the 5 ′ end is not limited to Cy3, and Cy5, Alexa, or the like may be used. Alternatively, single-stranded DNA having no fluorescent dye bonded to the end may be used. In a reaction solution in which single-stranded DNA 901 is dissolved, Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (abbreviated as sulfo-SMCC, Pierce, FIG. 9) corresponding to 100 times the amount of the single-stranded DNA. 750 nmol (shown by 904) was added, and the mixture was reacted at 25 ° C. for 2 hours while being shielded from light and shaken. By this reaction, a maleimide group 905 was introduced on the 3 ′ end side of the single-stranded DNA 901. After completion of the reaction, the reaction solution was purified with a gel filtration purification column (NAP-5 Column, GE Healthcare) to remove unreacted sulfo-SMCC (904).

(4) Binding of dendron and single-stranded DNA (Figure 10)
A solution of the G2-PAMAM dendron 1001 prepared in (1) and (2) above and the maleimide group-modified single-stranded DNA 1003 prepared in (3) above was mixed in 1 × PBS (pH 7.0). At this time, the molar ratio of the compounds in the reaction solution was mixed so that G2-PAMAM dendron: single-stranded DNA = 3: 1. The reaction solution was shielded from light and shaken, and reacted at 25 ° C. for 16 hours. By this reaction, the terminal thiol group 1002 of G2-PAMAM dendron 1001 and the terminal maleimide group 1004 of maleimide group-modified single-stranded DNA 1003 reacted to generate G2 dendron-binding probe DNA 1005.

(5) Separation and purification of dendron-bound probe DNA by gel electrophoresis The reaction solution containing the G2 dendron-bound probe DNA 1005 generated in (4) above was subjected to molecular weight cut-off filter (MWCO filter, for molecular weight of 10k, Millipore) The concentration of G2 dendron-bound probe DNA 1005 was adjusted to approximately 100 μM. Methylene blue, which is a DNA staining dye, was added to this concentrated solution, and then it was injected into a 10% polyacrylamide gel and subjected to gel electrophoresis (applied voltage: 100 V, migration time: 1 hour). After completion of electrophoresis, the position of the electrophoresis band of G2 dendron-binding probe DNA 1005 was visually confirmed.

(6) Separation and purification of dendron-bound probe DNA using nucleic acid extraction reagent From the polyacrylamide gel after completion of electrophoresis shown in (5) above, a migration band (several mm) corresponding to the desired G2 dendron-bound probe DNA A corner gel piece) was cut out. The gel pieces were finely ground and G2 dendron-bound probe DNA was extracted using a Mermaid-Spin kit (Q-Bio Gene), which is a DNA extraction reagent.

(7) Introduction of terminal amino group into dendron-binding probe DNA (FIG. 11)
The G2 dendron-bound probe DNA 1101 extracted in (6) above was dissolved in 0.2 M sodium phosphate buffer (pH 7.5). To this reaction solution, a solution of Ethylenediamine 1103, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, abbreviation EDC, concentration 2 mg / ml) was added. This reaction solution was reacted at 25 ° C. for 2 hours while being shielded from light and shaken. By this reaction, ethylenediamine 1103 was bonded to the terminal carboxyl group 1102 of the G2 dendron-bound probe DNA 1101 to introduce a terminal amino group 1105, and a G2 dendron-bound probe DNA 1104 having a terminal amino group was generated. After completion of the reaction, the reaction solution was purified with a gel filtration purification column (NAP-5 Column, GE Healthcare) to remove unreacted ethylenediamine 1103.

  In this example, ethylenediamine having amino groups at both ends was used as a molecule that binds to the terminal carboxyl group of the dendron. In addition, it is possible to use a molecule having both a functional group bonded to the terminal carboxyl group of the dendron and a functional group bonded to the surface of the metal fine particle. For example, the following molecules (a) to (c) can be used.

(A) Molecules having amino groups at both ends (FIG. 12)
Ethylenediamine (Fig. 12 (a), molecular weight 60)
・ 3,3'-Iminobispropylamine (Fig. 12 (b), molecular weight 131)
1,6-Diaminohexane (FIG. 12 (c), molecular weight 116)
・ Jeffamine EDR-148 (Fig. 12 (d), molecular weight 148)
(B) Linear polyethylene glycol having amino groups at both ends (Polyethleneglycol, abbreviated PEG)
・ SUNBRIGHT DE-010PA (Molecular weight 1,000)
・ SUNBRIGHT DE-020PA (Molecular weight 2,000)
・ SUNBRIGHT DE-034PA (Molecular weight 3,400)
・ SUNBRIGHT DE-100PA (molecular weight 10,000)
・ SUNBRIGHT DE-200PA (molecular weight 20,000)
・ SUNBRIGHT DE-300PA (molecular weight 30,000)
These molecules are sold by Nippon Oil. A general structural formula of these molecules is shown in FIG.

(C) Branched polyethylene glycol having four terminal amino groups (Polyethleneglycol, abbreviated PEG)
・ SUNBRIGHT PTE-100PA (Molecular weight 10,000)
・ SUNBRIGHT PTE-200PA (molecular weight 20,000)
These molecules are sold by Nippon Oil. The general structural formula of these molecules is shown in FIG.

  In addition, after introducing an amino group at the end of the dendron using the above molecules (a) to (c), by using the molecule listed in (d) below, a 2-pyridyldithiol group ( It is also possible to introduce a 2-pyridyldithiol group). In these molecules, the NHS (N-hydroxysuccinimide) ester group reacts with the amino group at the end of the dendron. Further, when the 2-pyridyldithiol group reacts with the metal surface, it releases pyridine-2-thione, and the sulfur atom forms a coordinate bond on the metal surface. Therefore, by using the molecule listed in (d), it is possible to increase the rotational radius of the dendron having an amino group at the terminal and introduce a 2-pyridyldithiol group that strongly binds to the metal fine particles.

(D) Molecule having NHS ester group and 2-pyridyldithiol group (FIG. 15)
SPDP (N-Succinimidyl-3- (2-pyridyldithio) propionate (Fig. 15 (a), molecular weight 312.4)
LC-SPDP (Fig. 15 (b), molecular weight 425.52)
・ Sulfo-LC-SPDP (FIG. 15 (c), molecular weight 527.56)
These molecules are sold by Thermo Fisher.

(8) Quantification of dendron-bound probe DNA by ultraviolet / visible absorption spectrum measurement Measure UV / visible absorption spectrum of the reaction solution prepared in (7) above, containing G2 dendron-bound probe DNA 1104 having a terminal amino group. The concentration of the G2 dendron-bound probe DNA having a terminal amino group was determined from the absorbance at 260 nm, which is the maximum absorption wavelength of nucleic acid.

(9) Immobilization of dendron-bound probe DNA on metal microparticles G2 dendron-bound probe DNA having a terminal amino group prepared according to the above method is bound to the surface of metal microparticles. In this example, gold nanoparticles (particle diameter: 5 to 100 nm) were used as the metal fine particles. Add bis- (p- (sulfonatophenyl) phenylphosphine, concentration 1 mg / ml) to the citric acid solution of gold nanoparticles, and add 4 mg while shaking and shaking at 25 ° C. After completion of the reaction, G2 dendron-bound probe DNA and sodium chloride (NaCl, concentration 10 mM) were added to the reaction solution, and the molar amount of the dendron-bound probe DNA with respect to the gold nanoparticles in the reaction solution. The reaction mixture was reacted for 20 hours while being shielded from light and shaken at 25 ° C. This reaction allowed the G2 dendron-bound probe DNA to bind to the surface of the gold nanoparticle via multiple terminal amino groups. did.

(10) Immobilization of adsorption-inhibiting molecules on metal fine particles An adsorption-inhibiting molecule is immobilized on a portion of the surface of the metal fine particles that is not coated with the G2 dendron-bound probe DNA. In this example, 1-mercaptohexanol or 2-mercaptohexanol having both a thiol group that forms a strong coordination bond with a metal surface and a hydrophilic hydroxyl group as an adsorption-inhibiting molecule. It was used. An aqueous solution of mercaptohexanol was added to the solution of gold nanoparticles prepared in (9) above with G2 dendron-bound probe DNA immobilized on the surface, and reacted for 0.5 to 10 hours while being shielded from light and shaken at 25 ° C.

  In this example, the dendron-binding probe DNA was immobilized on the surface of the metal fine particles and then the adsorption-inhibiting molecule was bound. However, the dendron-binding probe DNA and the adsorption-inhibiting molecule may be immobilized simultaneously. That is, a solution in which both the dendron-bound probe DNA and the adsorption-inhibiting molecule are dissolved may be prepared, and this solution may be reacted with the metal fine particles.

  In this example, a method for evaluating metal fine particles in which a dendron-bound probe DNA having a terminal amino group produced by the method disclosed in Example 1 is bound to the surface is shown.

  In this example, gold nanoparticles (particle diameters of 15 nm and 50 nm) were used as the metal fine particles. In addition, according to the method shown in Example 1, dendron-coupled probe DNAs with dendron generation numbers G3 and G6 were prepared. As the probe DNA, a 50-base synthetic oligo DNA having a fluorescent dye Cy3 at the 5 'end was used.

  Bis- (p- (sulfonatophenyl) phenylphosphine) and reacted gold nanoparticles (particle diameter 15 nm or 50 nm) and G3 or G6 dendron-bound probe DNA were mixed, and the molar ratio in the reaction solution was Gold nanoparticles: Dendron-coupled probe DNA was prepared to have a ratio of 1:10, and this reaction solution was reacted for 20 hours while being shielded from light and shaken at 25 ° C. By this reaction, the following four types of gold nanoparticles were obtained. Was prepared.

(A1) Gold nanoparticles with a G3 dendron-binding probe DNA bound to the surface (particle size 15 nm)
(A2) Gold nanoparticles with a G3 dendron-binding probe DNA bound to the surface (particle diameter 50 nm)
(A3) Gold nanoparticles with a G6 dendron-binding probe DNA bound to the surface (particle size 15 nm)
(A4) Gold nanoparticles with a G6 dendron-binding probe DNA bound to the surface (particle diameter 50 nm)

  On the other hand, as a control sample, a solution of gold nanoparticles (particle size 15 nm or 50 nm) reacted with bis- (p- (sulfonatophenyl) phenylphosphine), a thiol group at the 3 ′ end, and a fluorescent dye Cy3 at the 5 ′ end (The number of bases is 50 and the base sequence is the same as the probe DNA having the fluorescent dye Cy3 at the 5 ′ end used for the synthesis of the above-mentioned dendron-bound probe DNA). The molar ratio in the reaction solution was adjusted to gold nanoparticle: thiol-modified probe DNA = 1: 10, and this reaction solution was reacted for 20 hours while being shielded and shaken at 25 ° C. By this reaction, the following two types were prepared. Of gold nanoparticles were prepared.

(A5) Gold nanoparticles (particle size 15 nm) with thiol-modified probe DNA bound to the surface
(A6) Gold nanoparticles with thiol-modified probe DNA bound to the surface (particle size 50 nm)

  Each of the six types of gold nanoparticle dispersions (A1) to (A6) was dropped onto a quartz substrate, sealed with a cover glass, and installed in the fluorescence detection system shown in FIG. In this system, excitation laser light 1601 is incident from a back surface of a carrier substrate 1602 such as a quartz substrate through a quartz prism 1603 under total reflection conditions. Among the excitation laser light incident under the total reflection condition, dendron-binding probe DNA or thiol group-modified probe DNA is obtained by evanescent light that has oozed out on the surface on which the gold nanoparticles 1604 to be analyzed in this example are arranged. The fluorescent molecule Cy3 1607 immobilized on the gold nanoparticle 1604 is excited through the via. The fluorescence 1608 emitted from the fluorescent molecule Cy3 is measured from the upper surface of the substrate by the high sensitivity CCD camera 1609. Using this system, each gold nanoparticle 1604 was irradiated with excitation laser light, and the fluorescence generated at this time was received by a high-sensitivity CCD camera, and the temporal change in the fluorescence intensity of Cy3 was tracked. In the figure, 1605 is a single probe DNA, and 1606 is a Cy3-linked nucleotide.

  FIG. 17 shows an example of temporal change in fluorescence intensity emitted by the gold nanoparticles to which the dendron-bound probe DNA or thiol group-modified probe DNA is bound. In each of the four graphs shown in FIGS. 17A to 17D, the vertical axis represents fluorescence intensity (arbitrary unit) and the horizontal axis represents time (unit: second). The fluorescence intensity emitted by the gold nanoparticles to which the dendron-bound probe DNA or thiol group-modified probe DNA was immobilized decreased stepwise with time. This corresponds to the process of quenching after the fluorescent molecule Cy3 bound to the end of the probe DNA is photoexcited and emits fluorescence. The quenching process of one fluorescent molecule corresponds to one step of the stepwise decrease in fluorescence intensity. On the surface of the gold nanoparticle where the steps of decrease in the fluorescence intensity are 0, 1, 2, 3, 4,..., N, the probe DNA having the fluorescent molecule Cy3 bound to the end is 0, 1, 2, 3, 4, ..., N is determined to be fixed. That is, from FIG. 17A, it is determined that one probe DNA is immobilized on the surface of the gold nanoparticle. Similarly, from FIGS. 17B, 17C, and 17D, it is determined that two, three, and four probe DNAs are immobilized on the surface of the gold nanoparticle, respectively.

  With respect to the six types of gold nanoparticles (A1) to (A6) prepared above, the gold nanoparticles whose fluorescence intensity is quenched in one step are measured by measuring the time change of the fluorescence intensity of 1,000 gold nanoparticles. FIG. 18 shows the ratio of gold nanoparticles that are quenched at the step or higher.

  For example, in the case of a gold nanoparticle having a particle diameter of 15 nm, when reacted with a thiol group-modified probe DNA, the ratio of the gold nanoparticle exhibiting one-step quenching was 5%. Accordingly, it is shown that 5% of gold nanoparticles having one fluorescent molecule Cy3, that is, one thiol group-modified probe DNA immobilized on the surface are present. On the other hand, when reacted with G6 dendron-bound probe DNA, the percentage of gold nanoparticles showing one-step quenching was 95%. Therefore, it is shown that 95% of gold nanoparticles to which one fluorescent molecule Cy3, that is, one G6 dendron-binding probe DNA is immobilized, are present. Therefore, the results shown in FIG. 18 indicate that the gold nanoparticle having a single probe DNA immobilized on its surface can be obtained by appropriately combining the particle diameter of the gold nanoparticle and the size of the dendron-bound probe DNA, that is, the radius of rotation of the dendron portion. It shows that the particles can be produced in high yield.

  In this example, the molar ratio of the gold nanoparticles to the dendron-bound probe DNA in the solution was reacted at gold nanoparticles: dendron-bound probe DNA = 1: 10, but this molar ratio is adjusted appropriately. As a result, the yield of gold nanoparticles having a single probe DNA immobilized on the surface can be further increased.

  In this example, a manufacturing process of a single probe molecular element (an example of which is shown in FIG. 19) in which the dendron-bound probe DNA-immobilized gold nanoparticles produced in Example 2 are immobilized on the surface of a carrier substrate will be described. The manufacturing process of a single probe molecular device in which dendron-bound probe DNA-immobilized gold nanoparticles are immobilized on the surface of a carrier substrate includes the following processes.

(1) Formation of a linker molecular layer for immobilizing gold nanoparticles on the surface of a carrier substrate (2) Immobilization of dendron-binding probe DNA-immobilized gold nanoparticles on the surface of a carrier substrate (3) Formation of an adsorption-inhibiting molecular layer on the surface of a carrier substrate Each process will be described using poly T20 (PolyT20) in which 20 thymines (abbreviated symbol T) are bound in succession as probe DNA.

(1) Formation of linker molecule layer for fixing gold nanoparticles on carrier substrate surface The washed quartz substrate was immersed in a methanol solution of 3-aminopropyltrimethoxysilane (3-Aminoprooyltrimethoxysilane) as a silane coupling agent for 5 minutes. Subsequently, the substrate was washed with methanol and then annealed at 80 ° C. for 2 hours. By this process, amino groups for fixing gold nanoparticles were introduced on the surface of the quartz substrate.

(2) Immobilization of dendron-bound probe DNA-immobilized gold nanoparticles on the surface of a carrier substrate A solution of dendron-bound polyT20-immobilized gold nanoparticles is dropped on the quartz substrate surface into which amino groups have been introduced, and reacted at 25 ° C for 16 hours. did. The density of the dendron-bonded PolyT20-fixed gold nanoparticles fixed on the quartz substrate surface was adjusted by adjusting the concentration of the dendron-bonded PolyT20-fixed gold nanoparticle solution dropped onto the substrate to 0.01 to 100 nM.

(3) Formation of adsorption-inhibiting molecular layer on the surface of carrier substrate Polyethylene having NHS (N-Hydroxysuccinimide) ester as an adsorption-inhibiting molecule in the region where dendron-bound PolyT20-immobilized gold nanoparticles are not immobilized on the carrier substrate Glycol (hereinafter abbreviated as NHS-PEG) was conjugated. Specifically, the support substrate was immersed in NHS-PEG solution prepared to 4 mM with triethanolamine (pH 8.0), and reacted at 25 ° C. for 1 hour. After completion of the reaction, the carrier substrate was sufficiently washed with pure water.

  In this example, when a single probe molecular element is manufactured, after fixing the dendron-binding probe DNA to the metal fine particles, the metal fine particles are fixed to the substrate surface, and then the portion other than the portion where the metal fine particles are fixed. The method of immobilizing adsorption-inhibiting molecules on the surface of the substrate was used. On the other hand, after fixing the metal fine particles to the substrate, it is possible to use a method in which the adsorption-inhibiting molecules are fixed to the surface of the substrate other than the portion where the metal fine particles are fixed, and then the dendron-binding probe DNA is fixed to the surface of the metal fine particles. good.

  In this example, an example in which DNA sequencing is performed using the single probe molecular element produced in Example 3 will be described with reference to FIGS. 19, 20, 21, and 22. FIG. FIG. 19 shows an example of a single probe molecular device fabricated in Example 3. Single DNA 1904 to be sequenced was hybridized to a single dendron-bound PolyT20 (1903) immobilized on the surface of gold nanoparticles 1902 immobilized on a quartz substrate 1901. That is, the DNA solution for DNA sequencing (concentration 1 nM to 1 μM) with 5 × SSC (citrate buffer, standard saline citrate) and 0.5% SDS (sodium dodecyl sulfate). Prepared. This solution was dropped on the gold nanoparticle fixed part on the surface of the single probe molecular element, and hybridization was performed at 25 ° C. In this example, DNA having adenine (abbreviated symbol A) sequence continuously from the 5 'end was hybridized as a single DNA to be sequenced. The base sequence of this single DNA molecule 1904 is shown from the 5 'end to the 3' end.

  AAAAAAAAAAAAAAAAAAAAAGTCGAGCGGTAGCACAGAGAGCTTGCTCTCGGGTGACGAGCGGCGGACG (70 bases) (SEQ ID NO: 1)

Next, single DNA (1904) was sequenced using probe DNA, dendron-bound PolyT20 (1903) as a primer. Single probe molecule element hybridized with DNA to be sequenced, adenine (Adenine, abbreviation A), and thymine (Thymine), which are four nucleotides having a fluorescent molecule Cy3 at the phosphate group end of nucleotide 3-phosphate Abbreviated symbol T), guanine (abbreviated symbol G), and cytosine (abbreviated symbol C) were reacted by polymerase reaction. The reaction solution used here was prepared with a solution of 10 mM Tris-HCl (Tris-HCl) and 5 mM magnesium chloride (MgCl ₂ ), and four kinds of Cy3-linked nucleotides and DNA polymerase were dissolved. A Cy3-binding nucleotide having a base complementary to the base sequence of a single DNA to be sequenced (1904) is bound to the 5 ′ end of the dendron-binding PolyT20 (1903) as a probe DNA by the polymerase reaction. In this example, when a solution of Cy3-linked nucleotide C (1905) is reacted with a single probe molecular element, Cy3-linked nucleotide C is bound to the 5 ′ end of dendron-bound PolyT20 (1903). This is because in the single DNA 1904 to be sequenced, nucleotide C complementary to nucleotide G adjacent to the PolyA sequence at the 5 ′ end binds.

  A fluorescence image during the polymerase reaction is acquired using the fluorescence detection system shown in FIG. The fluorescence intensity was measured with a high sensitivity CCD camera 2011 from the upper surface of the quartz substrate. By irradiating the excitation light under total reflection conditions, fluorescence from the gold nanoparticle 2004 to which the nucleotide C to which the fluorescent molecule Cy3 (2009) is bound is bound is detected. After detecting fluorescence, the phosphate group of nucleotide C bound to dendron-bound PolyT20 (2005) is cleaved. As a result, the fluorescent molecule Cy3 (2009) bound to the end of the phosphate group is removed from the dendron-bound PolyT20 (2005). Next, when the single probe molecular element is reacted with a solution of Cy3 binding type nucleotide A, Cy3 binding type nucleotide A binds to T adjacent to nucleotide G of single DNA (1904) to be sequenced. At this time, fluorescence of Cy3 is detected from the gold nanoparticles. By repeating the above process, it was possible to read the base sequence of a single DNA to be sequenced in each gold nanoparticle fixed on the surface of a single probe molecular element.

For comparison with a single probe molecular device fabricated using dendron-bonded PolyT20-fixed gold nanoparticles, a quartz substrate with PolyT20 bonded directly to the surface was fabricated. FIG. 21 (a) shows a conceptual diagram of a single probe molecular device manufactured in this example using a dendron-coupled PolyT20 fixed gold nanoparticle 2101 on a quartz substrate 2102, and PolyT20 (2103) on the surface. A conceptual diagram of the quartz substrate 2104 directly fixed is shown in FIG. In order to perform single DNA sequencing using these substrates, an extension reaction of one base contained in the DNA to be sequenced was performed. A histogram of the fluorescence intensity of the fluorescent bright spots on each substrate obtained at this time is shown in FIG. About 3,000 for each of the single-probe molecular device (FIG. 21 (a)) fabricated using the dendron-bonded PolyT20 fixed gold nanoparticles and the quartz substrate (FIG. 21 (b)) directly bonded to the surface of PolyT20. The intensity of the fluorescent bright spot included in the area of μm ² was quantified. As a result, the total number of fluorescent bright spots in the quartz substrate with PolyT20 directly bonded to the surface was about 1,200 (data shown as “no gold nanoparticles” in FIG. 22), whereas dendron-coupled PolyT20 fixed gold. About 3,000 fluorescent bright spots were detected in the single probe molecular device fabricated using nanoparticles (data indicated by “with gold nanoparticles” in FIG. 22).

  Compared to a quartz substrate with PolyT20 bonded directly to the surface, the single probe molecular device fabricated using dendron-bonded PolyT20 fixed gold nanoparticles has an average fluorescence intensity approximately three times higher than fluorescence. The standard deviation of intensity was also halved. In the quartz substrate with PolyT20 directly fixed on the surface, PolyT20 was randomly fixed on the substrate surface, the distance between adjacent PolyT20 was not constant, and the variation in fluorescence intensity was large. On the other hand, in a single probe molecular device fabricated using dendron-bonded PolyT20 fixed gold nanoparticles, one dendron-bonded PolyT20 fixed on the surface of the gold nanoparticle is placed on the substrate surface, and the distance is sufficiently large. The variation in the fluorescence intensity was reduced because it was placed under more homogeneous reaction conditions. Furthermore, it was also confirmed that the fluorescence intensity increased due to the fluorescence enhancement effect by the gold nanoparticles. Therefore, it was shown that DNA sequencing can be performed with high accuracy and high reproducibility in the single probe molecular device using the dendron-binding DNA-immobilized gold nanoparticles in this example.

  The single probe molecular element of the present invention is a biomolecule for performing highly accurate and reproducible detection at the single molecule level in DNA microarrays for comprehensive quantitative analysis of nucleic acids and DNA sequencers for decoding nucleic acid base sequences. It can be applied as a detection element. The single probe molecular element can be used as a detection element for detecting biomolecules such as proteins and sugar chains and various chemical substances with high accuracy and high reproducibility.

The figure which shows the structure of the 2nd generation polyamidoamine dendrimer (G2-PAMAM dendrimer) which has a core of ethylenediamine. The figure which shows the relationship of the generation of the PAMAM dendrimer which has a core of ethylenediamine, the number of terminal amino groups, and a turning radius. (A) Schematic diagram showing a single probe molecule-fixed metal fine particle, (b) a diagram showing a modified example of a structure for binding a single probe molecule and a linker molecule, (c) a structure for fixing the linker molecule on the surface of the metal fine particle FIG. The schematic diagram which shows the single probe molecular element using a single probe molecule fixed metal microparticle. The figure which shows the structure of G2-PAMAM dendrimer which has a cystamine core. The schematic diagram which shows the introduction process of the carboxyl group to a dendrimer. The figure which shows the structure of SCN-Bzl-DTPA. The schematic diagram which shows the cutting process of the disulfide bond of a dendrimer. The schematic diagram which shows the introduction | transduction process of the maleimide group to single stranded probe DNA. Schematic diagram showing the binding process between dendron and single-stranded probe DNA. The schematic diagram which shows the introduction process of the amino group to a dendron bond type probe DNA. The figure which shows the structure of the molecule | numerator which has an amino group in both ends. The figure which shows the structure of the linear polyethyleneglycol molecule | numerator which has an amino group at both ends. The figure which shows the structure of the branched polyethylene glycol molecule | numerator which has four terminal amino groups. The figure which shows the structure of the molecule | numerator which has NHS ester group and 2-pyridyl dithiol group. The schematic diagram which shows a fluorescence detection system. The figure which shows the example of the time change of the fluorescence intensity which a probe DNA binding type gold nanoparticle emits. The figure which shows the ratio which the fluorescence intensity of a probe DNA binding type gold nanoparticle quenches in one step, and the ratio which quenches in multiple steps. The schematic diagram which shows a single probe molecular element. The schematic diagram which shows a fluorescence detection system. (A) Schematic diagram showing a single probe molecular device fabricated using dendron-bonded probe DNA-immobilized gold nanoparticles, and (b) a quartz substrate on which probe DNA is directly immobilized on the surface. Histogram of fluorescence intensity of fluorescent spots on the substrate detected during single-base extension reaction of DNA sequencing.

Explanation of symbols

301: Single probe molecule, 303: Linker molecule, 306: Metal fine particle, 312: Bifunctional compound, 313: Linker molecule, 317: Bifunctional compound, 401: Support substrate, 402: Single probe molecule fixed metal fine particle 501: disulfide bond, 502: terminal amino group, 601: G2-PAMAM dendrimer, 602: amino group, 603: succinic anhydride, 604: carboxyl group, 701: isothiocyanate group, 702: terminal carboxyl group, 801: G2-PAMAM dendrimer, 802: dithiothreitol, 803: disulfide bond, 804: G2-PAMAM dendron having a thiol group at the terminal, 805: thiol group, 901: 1 single-stranded DNA, 902: amino group, 904: sulfo- SMCC, 905: maleimide group, 1001: G2-PAMAM dendron, 1002: terminal thiol Group: 1003: maleimide group-modified single-stranded DNA, 1004: terminal maleimide group, 1005: G2 dendron-binding probe DNA, 1101: G2 dendron-binding probe DNA, 1102: terminal carboxyl group, 1103: ethylenediamine, 1104: terminal amino Group-containing G2 dendron-binding probe DNA, 1105: terminal amino group, 1601: excitation laser beam, 1602: carrier substrate, 1603: quartz prism, 1604: gold nanoparticle, 1605: single probe DNA, 1606: Cy3 binding type Nucleotides, 1607: Fluorescent molecule Cy3, 1608: Fluorescence emitted from fluorescent molecule Cy3, 1609: High-sensitivity CCD camera, 1901: Quartz substrate, 1902: Gold nanoparticles, 1903: Dendron-binding PolyT20, 1904: Sequencing target Single DNA, 1905: Cy3-linked nucleotide C: 2001: Excitation laser light, 2002: Quartz substrate, 2003: Quartz prism, 2004: Gold nanoparticle, 2005: Dendron-binding PolyT20, 2006: Single DNA to be sequenced, 2007: DNA polymerase, 2008: Nucleotide 2009: fluorescent molecule Cy3, 2010: fluorescence emitted from fluorescent molecule Cy3, 2011: high sensitivity CCD camera, 2101: dendron-coupled PolyT20 fixed gold nanoparticles, 2102: quartz substrate, 2103: PolyT20, 2104: quartz substrate.

Claims (4)

A substrate,
A plurality of fine metal particles fixed to the surface of the substrate;
A linker molecule fixed on the surface of each of the metal fine particles, one by one through a chemical bond;
A probe molecule bonded to the linker molecule one by one through a chemical bond,
A single probe molecular device, wherein the linker molecule is a dendron type molecule. 2. The single probe molecular device according to claim 1 , wherein the dendron type molecule is a dendron type molecule having a polyamidoamine structure. Metal fine particles,
One linker molecule fixed to the surface of the metal fine particle through a chemical bond;
A probe molecule bonded to the linker molecule via a chemical bond,
The linker molecule is a dendron type molecule, and the single probe molecule-fixed metal fine particle. 4. The single probe molecule-fixed metal fine particle according to claim 3 , wherein the dendron type molecule is a dendron type molecule having a polyamidoamine structure.

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