WO2015159534A1

WO2015159534A1 - Detection solution and biosensing method using same

Info

Publication number: WO2015159534A1
Application number: PCT/JP2015/002061
Authority: WO
Inventors: 大塚　尚; 敏治斎木; 恵子江刺家
Original assignee: 富士フイルム株式会社
Priority date: 2014-04-16
Filing date: 2015-04-14
Publication date: 2015-10-22
Also published as: JPWO2015159534A1

Abstract

[Problem] To improve specific binding reaction efficiency and detection accuracy in biosensing using precious metal nanoparticles as a marker. [Solution] This detection solution (1) is a detection solution (1) used for biosensing that detects, by using precious metal nanoparticles (10) as a marker, a substance to be detected (R) consisting of a biological substance and included in a sample to be measured (2), wherein the precious metal nanoparticles (10) are dispersed in a buffer aqueous solution (40), each precious metal nanoparticle having, fixed to the surface (10s) thereof: a probe (20) including a hydrophilic biological substance which specifically binds to the substance to be detected (R); and an amphiphilic SAM (30) having a hydrophobic part (31) and a hydrophilic part (32). The side of the amphiphilic SAM (30) that is fixed to the particle (10) is the hydrophobic part (31). The probe (20) is fixed to the surface (10s) by a chemical bond (Bc).

Description

Detection liquid and biosensing method using the same

The present invention relates to a detection solution used for biosensing using noble metal nanoparticles and a biosensing method using the detection solution.

Biosensing is performed by detecting a specific binding (specific binding) of a biological substance using a change in an optical or electrical signal. Biosensing that detects a change in an optical signal includes a method of detecting a substance to be detected using a radioisotope, a fluorescent substance, a noble metal nanoparticle, or the like as a label.

Biosensing using precious metal nanoparticles as a label has been attempted in fields such as immunodiagnostics and gene analysis, as a large-scale measuring device is not required and simple sensing is possible. For example, in Patent Document 1 and Non-Patent Document 1, in biosensing for detecting a nucleic acid fragment having a specific sequence, it has complementarity when the base sequences of single-stranded nucleic acids are complementary (specific). A method of using noble metal nanoparticles as a label for a hybridization method in which a double-stranded nucleic acid is formed by hydrogen bonding between base pairs is described.

Patent Document 1 uses a noble metal particle to which an oligonucleotide having a sequence complementary to part of a nucleic acid sequence is attached as a probe, and detects a change in color resulting from the hybridization of the oligonucleotide to a nucleic acid. How to do is described.

Non-Patent Document 1 describes that the localized plasmon resonance wavelength of gold nanoparticles is shifted by a long wavelength due to the hybridization that occurs when the gold nanoparticles carrying brushes on their surfaces approach each other. In addition, a genetic diagnosis method using the change in coloration of gold nanoparticles is described. In FIG. 1 of Non-Patent Document 1, two types of DNA probes that form complementary strands with a DNA target (usually complementary to different portions (approximately half each) of the DNA target) are supported on precious metal nanoparticles, respectively. A measurement method is also shown. In this case, the substance to be detected is a DNA target not supported on the noble metal nanoparticles.

JP 2004-515208 A

However, in the methods of Patent Document 1 and Non-Patent Document 1, for example, in detection using gel electrophoresis, detection of specific binding (hybridization) such as detection of a plurality of bands in a state before hybridization. There is a problem that the accuracy is low, and the specific binding reaction efficiency is also low.

The present invention has been made in view of the above circumstances, and has a high specific binding reaction efficiency in biosensing for detecting a detection target substance made of a biological substance contained in a measurement sample using noble metal nanoparticles as a label. It is an object of the present invention to provide a biosensing method with good detection accuracy and a detection liquid that enables the biosensing method.

The detection liquid of the present invention is a detection liquid used for biosensing for detecting a detection target substance composed of a biological substance, contained in a measurement target sample, using noble metal nanoparticles as a label.
Noble metal nanoparticles with a probe containing a hydrophilic biological substance that specifically binds to the substance to be detected and an amphiphilic self-assembled monolayer with a hydrophobic part and a hydrophilic part are buffered. Dispersed in an aqueous solution,
In the self-assembled monolayer, the side fixed to the particle is a hydrophobic part, and the functional group at the end of the hydrophilic part has either a positive or negative charge, and the probe is It is fixed to the surface of the noble metal nanoparticles by chemical bonds.

In this specification, the buffered aqueous solution means an aqueous solution generally used for microbial culture, chemical substances, biological material storage, separation, etc., and is used for external factors (such as carbon dioxide in the atmosphere) or internal factors ( It means an aqueous solution in which the pH hardly varies depending on the metabolite of the microorganism itself.
A chemical bond is an intermolecular force (inter-ion interaction, hydrogen bond, van der Waals force, hydrophobic interaction, physical adsorption) that is a weak interaction between molecules. It means binding by strong interaction due to electron sharing. Specifically, it means a covalent bond, a coordination bond, an intermetallic bond, and a noble metal-sulfur bond.

In the detection solution of the present invention, it is preferable that the charge of the functional group at the end of the hydrophilic portion and the charge of the hydrophilic biological substance are the same type.

The hydrophilic part of the self-assembled monolayer is preferably a polyethylene glycol chain or a sugar chain.

The detection solution of the present invention is suitable when the biological substance is an antibody, and more preferably when it is a fragmented antibody. In the case of an antibody in which the biological material is fragmented, the chemical bond with the noble metal nanoparticle may be a bond between the sulfur atom of the disulfide group or thiol group of the fragmented antibody and the noble metal atom on the surface of the noble metal nanoparticle. preferable.

The biosensing method of the present invention uses precious metal nanoparticles as a label, and in biosensing for detecting a detection substance made of a biological substance contained in a measurement sample,
A detection liquid preparation step for preparing two or more kinds of detection liquids having different specific bindings and having the same kind of functional group charge,
A reaction solution preparation step of preparing a reaction solution by mixing two or more kinds of detection solutions prepared in a buffered aqueous solution containing a sample to be measured;
A reaction step for specifically binding the biological substance and the substance to be detected of all the probes in the reaction solution;
It has a detection step of detecting the presence or absence of a specifically bound detection target substance in the sample to be measured by detecting the label using the reaction solution after completion of the reaction step.

In the detection step, the presence / absence of the substance to be detected is preferably detected by gel electrophoresis or an optical detection method described later.

The detection liquid of the present invention comprises an amphiphilic self-assembled monolayer (SAM) having a probe containing a hydrophilic biological substance that specifically binds to a target substance made of a biological substance, and a hydrophobic part and a hydrophilic part. ) And noble metal nanoparticles fixed on the surface are dispersed in a buffered aqueous solution. In the amphiphilic SAM, the side fixed to the noble metal nanoparticles is a hydrophobic part, and the end group of the hydrophilic part is Has either a positive or negative charge, and the probe is fixed to the surface of the noble metal nanoparticle by a chemical bond. In such a configuration, a SAM having either positive or negative charge at the end is provided on the surface of the noble metal nanoparticle, so that nonspecific aggregation due to factors other than specific binding to the detected substance is suppressed. Can do. In addition, since the probe is strongly fixed to the surface of the noble metal nanoparticle by a chemical bond, there is no fear that the detection limit is deteriorated due to the probe being detached from the surface of the noble metal nanoparticle with time or physical force. Therefore, according to the present invention, biosensing with high specific binding reaction efficiency and good detection accuracy can be performed.

Further, in the detection solution of the present invention, the charged state of the terminal functional group (end group) on the side not fixed to the SAM particle is the charged state of the probe containing a hydrophilic biological substance in the buffered aqueous solution. In the same embodiment, since the end group of SAM and the hydrophilic biological substance repel each other, it is possible to obtain a more effective non-specific aggregation suppressing effect and a specific binding reaction efficiency improving effect, Highly accurate biosensing can be performed.

The left figure is a schematic sectional view showing the structure of the detection liquid of the present invention, and the right figure is a schematic diagram showing the structure of the sample to be measured. The schematic cross-sectional schematic diagram which shows the suitable aspect of the noble metal nanoparticle contained in the detection liquid of this invention. FIG. 1B is a schematic enlarged view showing an embodiment of the surface of the noble metal nanoparticle of FIG. 1B. Diagram showing the structure of the antibody and its fragmentation treatment FIGS. I to IV are schematic diagrams showing an embodiment in which an antibody and SAM are bonded to the surface of a noble metal, and show how the binding state changes with time when the antibody is bonded by normal physical bonding. Schematic cross-sectional schematic diagram showing a preferred embodiment of the detection liquid preparation step of the biosensing method of the present invention Schematic cross-sectional schematic diagram showing a preferred embodiment of the mixing step of the biosensing method of the present invention Schematic cross-sectional schematic diagram showing a preferred embodiment of the reaction step and the binding step of the biosensing method of the present invention Schematic configuration schematic diagram of the device used for detection by optical polarimetry Figure showing detection example (correlation between particle shape and anisotropic index AI) using anisotropic index Monomer and dimer autocorrelation functions obtained by light scattering correlation spectroscopy. The figure which shows the detection result by the gel electrophoresis method of Example 1 and Comparative Examples 1 and 2.

(Detection solution)
A detection liquid and a preparation method thereof according to an embodiment of the present invention will be described with reference to the drawings. 1A is a schematic cross-sectional schematic diagram showing the configuration of the detection liquid of the present embodiment, the right figure is a schematic diagram showing the configuration of a sample to be measured (sample liquid), and FIG. 1B is a diagram of noble metal nanoparticles contained in the detection liquid. It is a schematic cross-sectional schematic diagram which shows a suitable aspect. FIG. 1C is an enlarged schematic schematic view showing an embodiment of the surface of the noble metal nanoparticle shown in FIG. 1B. In the schematic diagram and schematic diagram of the present specification, the scale of each part is appropriately changed for easy visual recognition.

In the biosensing using the noble metal nanoparticles carrying the biological material in the shape of a brush on the surface, the inventor mainly reduces the detection accuracy and the specific binding reaction efficiency described in the “Background” section. It has been found that the cause is that the noble metal nanoparticles aggregate nonspecifically even when no specific bond is formed with the substance to be detected. Therefore, the present inventor provides a surface modification having either a positive or negative charge at the terminal, so that the surface of the noble metal nanoparticle is positively or negatively charged. It was thought that the non-specific aggregation could be suppressed. The surface modification of the noble metal nanoparticle surface with either positive or negative charge at the terminal is a self-assembled monolayer (SAM), which makes a monomolecular-size ultrathin film (monolayer) a noble metal. It can be easily formed on the nanoparticle surface.

That is, the detection liquid 1 is used for biosensing to detect a detection target substance R made of a biological substance contained in the sample 2 to be detected, using the noble metal nanoparticles 10 as a label. The noble metal nanoparticle 10 in which the probe 20 containing the hydrophilic biological material and the amphiphilic SAM 30 are immobilized on the surface 10 s is dispersed in the buffered aqueous solution 40, and the SAM 30 is immobilized on the noble metal nanoparticle 10. The hydrophobic portion 31 and the functional group at the end 30e of the hydrophilic portion 32 has one of positive and negative charges, and the probe 20 is attached to the surface 10s of the noble metal nanoparticle 10 It is fixed by chemical bond Bc. In the present embodiment, as shown in FIGS. 1B and 1C, the probe 20 also has a negative charge.

In the detection liquid 1, the noble metal nanoparticles 10 are not particularly limited, and it is preferable to select a noble metal having a good coloration property at the wavelength of light used. Among these, gold can be preferably used because it exhibits a red color that is easily visible in natural light and can form a strong bond with a thiol group or a disulfide group.

The probe 20 containing a hydrophilic biological substance is not particularly limited as long as it is capable of specifically binding to the substance R to be detected and can be bound to the surface 10s of the noble metal nanoparticle 10 by the chemical bond Bc. Specific binding of biological substances includes antigen-antibody binding, DNA and RNA, complementary binding (hybridization) of nucleic acids such as PNA (Peptide Nucleic Acid, peptide nucleic acid), biotin-avidin binding, antigen-aptamer binding, sugar chain -There is lectin binding and the like, and the detection solution 1 can be applied to any specific binding.

In the amphiphilic SAM 30, the constituent atom (sulfur atom) of the functional group at the base end 30 a is bonded to the noble metal nanoparticle 10 and the noble metal atom of the noble metal nanoparticle 10. The side to which 10 is fixed is the hydrophobic portion 31, and the functional group at the end 30 e of the hydrophilic portion 32 has one of positive and negative charges.

The hydrophobic part 31 preferably has an alkyl group that is a functional group capable of binding to the noble metal nanoparticles 10 at the base end 30a. Examples of the functional group that can easily form a strong bond with the noble metal atom of the noble metal nanoparticle 10 include a thiol group, a disulfide group, and a mercapto group. It is known that the functional groups of these sulfur compounds are spontaneously adsorbed on the surface of a noble metal such as gold, and alkanethiols and alkane disulfides can be SAMs that are ultrathin films of a single molecular size. The method for fixing these functional groups to the surface 10s is not particularly limited, and existing techniques can be used.

The hydrophilic portion 32 improves the decrease in dispersibility in the buffered aqueous solution 40 when the surface 10 s of the noble metal nanoparticle 10 is covered with the hydrophobic portion 31. In the case of using a highly hydrophobic SAM, the water solubility of the noble metal nanoparticles 10 is low, so in order to disperse well in the buffering aqueous solution, once through a highly compatible solvent such as alcohol. Need to be distributed. However, by performing such treatment, biological substances such as antibodies and nucleic acids are damaged and denatured, and non-specific aggregation is likely to increase, making it difficult to perform good sensing. An increase in non-specific aggregation also causes inactivation of the specific binding reaction, reducing the reaction efficiency of the reaction. In particular, when the alkyl chain of the hydrophobic part has 7 or more carbon atoms, the water solubility (dispersibility) becomes very low due to the high hydrophobicity.

The inventor has a hydrophilic portion 32 at the end opposite to the fixing side (base end 30a) of the gold nanoparticle 10 of the hydrophobic portion 31 and has either positive or negative charge at the end 30e. By using SAM30, which maintains the effect of suppressing the non-specific aggregation of precious metal nanoparticles, detection with good dispersibility in buffered aqueous solution and almost no damage to biological materials I found it to be liquid.

The hydrophilic part 32 is not particularly limited as long as it can improve dispersibility (water solubility) in a buffered aqueous solution, and examples thereof include polyethylene glycol (PEG) chains, sugar chains, and hydrophilic polyesters.

The end group 30e of the hydrophilic portion 32 is not particularly limited, and examples of the acidic group (negative charge) include a hydroxyl group, a carboxyl group, a phosphoric acid group, and a sulfonic acid group. As the basic group (positive charge), Examples thereof include an amino group, a quaternary ammonium group, an imidazole group, and a guanidinium group. In any case, a functional group having a high ionization tendency is preferable. When the probe 20 is charged with a positive or negative charge in the buffered aqueous solution 40, the charge of the end group 30e of the hydrophilic portion 32 is in the same charged state so as to repel the probe 20. It is preferably a functional group that can be obtained.

As the amphiphilic SAM 30, for example, the sulfur atom of the disulfide group at the base end 30a is bonded to the noble metal atom of the noble metal nanoparticle 10, and the hydrophobic group 31 composed of an alkyl group (11 carbon atoms), the terminal group Monomolecular film (20- (11-mercaptoundecanyloxy) -3,6,9,12,15) in which the hydrophilic part 32 made of polyethylene glycol (carbon number 6) 30e is a carboxyl group is ester-bonded , 18-hexaoxaeicosanoic acid (English name: 20- (11-Mercaptoundecanyloxy) -3,6,9,12,15,18-hexaoxaeicosanoic acid)). In the case of such SAM 30, the length of a single molecule is about 2.5 nm to 3.0 nm, and the length of the hydrophobic portion 31 is about 1.2 nm. For clarity, only one molecule is shown in FIG. 1C. Although shown as a carboxyl group in FIG. 1C, it exists as COO 2 ⁻ in the buffered aqueous solution 40.

The fixing temperature of the SAM 30 to the noble metal nanoparticle 10 and the reaction time required for the fixing may be appropriately set according to the material of the monomolecular film. 20- (11-mercaptoundecanyloxy) -3,6,9, In the case of 12,15,18-hexaoxaeicosanoic acid, it can be formed by reacting at about 50 ° C. for about 3 hours.

The concentration of SAM30 in the detection solution 1 (single molecule concentration) is not particularly limited as long as sensitive detection is possible, but is preferably in the range of 10 nmol / l or more and 1000 nmol / l or less. It is preferable to optimize depending on the diameter and the type of biological material.

The present inventor initially made the noble metal nanoparticle surface 10s charged with the SAM 30 having either a positive or negative charge at the terminal, so that the noble metal nanoparticle surface 10s was charged. It was thought that nonspecific aggregation between particles could be suppressed and an effect of improving the reaction efficiency of specific binding could be obtained. However, it has been found that there is a specific binding in which sufficient effects cannot be obtained only by forming such SAM30.

In biochemical analysis in which specific binding is detected, it is known that there is a method in which the binding between the probe 20 and the noble metal nanoparticle 10 is usually suitable for each of the substances. As a result of the inventor's investigation, when the binding to the noble metal nanoparticle 10 is carried out by a conventional method, such as when the probe 20 is an antibody, the non-specific binding is sufficiently suppressed in the specific binding where the binding becomes physical adsorption. Although obtained, it was found that the detection limit deteriorates with time.

In biochemical analysis by antigen-antibody reaction, immobilization of antibodies on noble metal nanoparticles has been performed in various ways. The fixation of the antibody to the metal is described in paragraph [0037] of Japanese Patent Application Laid-Open No. 2008-197038 because the hydrophobic part of the terminal part of the Fc region is more easily bound on the metal film than the other terminal part. As described above, it is usually performed by a method in which noble metal nanoparticles and an antibody are mixed in a buffer solution and then physically adsorbed.

However, when the Fc end is bound to the noble metal nanoparticles by ordinary physical adsorption, as shown in Comparative Example 2 described later, a specific binding reaction efficiency is high in the measurement immediately after the preparation of the detection solution, and the detection accuracy is good. Sensing can be performed, but when similar detection was performed after lapse of several hours or the like, specific binding to be detected could not be detected.

Therefore, the present inventors have examined the mechanism of such a phenomenon, and as a result, even when a substance that is physically adsorbed to the noble metal nanoparticles 10 is used as the probe 20, noble metal nano-particles are used. It has been found that by making the bond to the particle 10 a chemical bond, the non-specific aggregation of the particles can be suppressed without deteriorating the detection limit over time, and the effect of improving the specific binding reaction efficiency can be obtained. It was.

The inventor believes that the cause of the deterioration of the detection limit with time in the probe 20 bonded by physical adsorption is based on the mechanism of SAM self-organization.
The formation of SAM is described in the non-patent literature “Kenfumi Tanaka et al., Surface Science Vol. 25, No. 10, pp. 650-655, 2004” and the like, as described in noble metal atoms (gold atoms) and sulfur atoms. It is composed of two steps, ie, the coupling that occurs at a very early stage followed by the dense arrangement (rearrangement) of hydrocarbon chains, and the latter rearrangement involves the van der Waals force and the entropy term related to the enthalpy term. It is known that this occurs due to crystallization of hydrocarbon groups with the passage of time (over time) due to the solvent elimination by hydrophobization related to.
According to the idea of the present inventors, the antibody (probe) bound to the noble metal nanoparticle 10 is released by the rearrangement in the two-step self-assembly mechanism of the SAM and the binding due to the physical adsorption having a weak binding force is released. ) Since the absolute amount of 20 is reduced, the detection limit is deteriorated. FIG. 3 is a schematic diagram showing stepwise a process for preparing the detection solution 1 by adding a SAM-forming solution to the noble metal nanoparticles 10 to which the antibody 20 is bound by an ordinary method based on the above inference. is there. In FIG. 3, I is immediately after addition of the SAM forming solution, II is immediately after completion of preparation of the detection solution, III is at the start of SAM rearrangement, and IV is after completion of SAM rearrangement.

3 are the bonding steps of the gold atom and sulfur atom described above. In II immediately after the completion of the preparation of the detection liquid 1, the surface of the gold nanoparticle 10 is rearranged by negatively charged SAM. Without being joined. In this case, since the gold nanoparticles 10 are negatively charged, the gold nanoparticles 10 can be detected satisfactorily without causing aggregation due to non-specific binding due to repulsion due to surface charges.
However, when the rearrangement of the second stage is started (FIG. 3III), the bond due to weak physical adsorption between the antibody 20 and the gold nanoparticle surface 10s is broken by the van der Waals force and the solvent exclusion force due to the SAM rearrangement. Thus, the antibody is peeled off from the gold nanoparticle surface 10s. As a result, after the rearrangement is completed, the number of antibodies bound to the gold nanoparticles 10 is reduced, and the detection limit is greatly deteriorated.

Based on this consideration, in the detection solution 1, the antibody 20 is firmly bonded to the gold nanoparticle surface 10 s by chemical bonding, thereby suppressing deterioration of the detection limit due to SAM rearrangement and preparing the detection solution. Not only immediately after that, biosensing with high specific binding reaction efficiency and good detection accuracy was made possible (see Examples below).

As a method of chemically bonding an antibody to a noble metal nanoparticle by chemical bonding, it may be functionalized by adding a thiol group, a disulfide group or the like to the Fc terminal, or the terminal part formed by fragmentation treatment of the antibody. Alternatively, a fragmented antibody having a disulfide group or a thiol group may be used. FIG. 1C shows that fragmented antibody F (ab ′) was used as probe 20 and 20- (11-mercaptoundecanyloxy) -3,6,9,12,15,18-hexaoxaeicosanoic acid was used as SAM30. The case is an enlarged schematic illustration of the surface 10s of the noble metal nanoparticle. At the end of the fragmented antibody F (ab '), there is a paratope Bs that specifically binds to an epitope of the antigen (Ant).

FIG. 2 is a diagram schematically showing the structure of the antibody and its fragmentation treatment. As shown in the left diagram of FIG. 2, the antibody has a Y-shaped four-chain structure as a basic structure, and consists of two heavy chains and two light chains. The heavy chain and the light chain are connected by a disulfide bond to form a heterodimer, and the heterodimers are further connected by a hinge part including two disulfide bonds to form a Y-shaped heterotetramer. The V-shaped part of the upper half of the Y-shape is called the Fab region (Fab fragment), and the straight part of the lower half is called the Fc region (Fc fragment), and has a paratope Bs at the end of the Fab region.

An appropriately fragmented antibody is preferable because it has a disulfide bond and / or a thiol group exposed and can be directly bonded to the surface of the noble metal nanoparticle 10s without using a SAM or the like. As shown in FIG. 2, a fragmented antibody is digested into two Fab fragments and an Fc fragment by treatment with the proteolytic enzyme papain, and two Fab fragments by treatment with the proteolytic enzyme pepsin. Fragments are broken down into F (ab ′) ₂ fragments linked by disulfide bonds and fragmented Fc fragments. By further treating the F (ab ′) ₂ fragment with a reducing agent such as 2-mercaptoethylamine (2-MEA), the F (ab ′) 2 fragment is decomposed into F (ab ′) fragments, and at the same time, a thiol group is exposed at the terminal.
In addition to these, enzymes that produce antibody fragments include ficin, lysyl endopeptidase, V8 protease, bromelain, clostripain, metalloendopeptidase, and activated papain obtained by activating papain.
In addition to the fragmentation using the enzymes described above, fragmentation by chemical treatment or genetic engineering techniques can also be employed.

Antibody fragmentation can be performed by a known method such as K.L. Brogan et al. Analytica Chimica Acta 496 (2003) 73-80.

As shown in FIG. 1C, when the probe 20 is an antibody, the probe 20 is a fragmented antibody F (ab ′), and the sulfur atom of the thiol group and the noble metal atom on the surface 10s of the noble metal nanoparticle are chemically bonded. It is bound by Bc. By adopting such an embodiment, as shown in Example 1 described later, nonspecific aggregation between particles can be suppressed without causing deterioration in detection limit over time, and an effect of improving the reaction efficiency of specific binding can be obtained. it can.

As described above, the detection liquid 1 is an amphiphilic substance having the probe 20 including the hydrophilic biological material that specifically binds to the detection target substance R made of the biological material, the hydrophobic portion 31, and the hydrophilic portion 32. A noble metal nanoparticle 10 having a self-assembled monolayer (SAM) 30 fixed on the surface 10s is dispersed in a buffered aqueous solution 40. The amphiphilic SAM 30 is hydrophobic on the side fixed to the noble metal nanoparticle 10. The end portion 30 e of the hydrophilic portion 32 has a positive or negative charge, and the probe 20 is fixed to the surface 10 s of the noble metal nanoparticle 10 by chemical bonding. In such a configuration, the SAM30 having either positive or negative charge at the terminal is provided on the surface 10s of the noble metal nanoparticle, so that nonspecific aggregation due to factors other than specific binding to the detection target R is suppressed. can do. In addition, since the probe 20 is strongly fixed to the noble metal nanoparticle surface 10s by chemical bonding, there is no fear that the detection limit will deteriorate due to the probe coming off the noble metal nanoparticle surface 10s with time or physical force. Therefore, according to the detection liquid 1, biosensing with high specific binding reaction efficiency and good detection accuracy can be performed.

(Biosensing method)
The biosensing method of the present invention to be performed using the detection solution of the present invention will be described with reference to FIGS. 4A to 4C. 4A to 4C show examples of configurations in which the amount of the substance R to be detected is detected by labeling with the noble metal nanoparticles 10 by the sandwich method.

In the biosensing method of the present embodiment, in the biosensing for detecting the detection target substance R made of a biological substance contained in the measurement target sample 2 using the noble metal nanoparticles 10 as a label,
A detection liquid preparation step of preparing two or more detection liquids (1a, 1b) having different specific bindings and having the same type of functional group charge;
A reaction solution preparation step of preparing a reaction solution 3 by mixing the sample 2 to be measured and two or more kinds of prepared detection solutions (1a, 1b) in a buffered aqueous solution;
A reaction step of specifically binding the biological substances of all the probes 20 (A, B) in the reaction solution 3 and the substance R to be detected;
It has a detection step of detecting the presence or absence of the specifically bound detection target substance R in the sample 2 to be measured by detecting the label using the reaction solution 3 after completion of the reaction step.

Here, the case where the substance R to be detected is an antigen and the two types of antibodies 20 (A) and 20 (B) that specifically bind to the antigen are probes will be described as an example.

<Detection solution preparation process>
As shown in FIG. 4A, the detection liquid 1 is manufactured by performing the detection liquid preparation steps (A-1) to (A-3) and (B-1) to (B-3). Can do. Since (A-1) to (A-3) and (B-1) to (B-3) have the same process except for the types of probes, (A-1) to (A-3) Only will be described.

First, a predetermined amount of the buffering aqueous solution 40 is injected into a container such as a beaker, and a plurality of noble metal nanoparticles 10 are added therein (A-1). (A-1) may be carried out without heating.
The size of the noble metal nanoparticle 10 is not particularly limited as long as it is a size capable of inducing localized plasmon by light irradiation, and each particle size is preferably less than or equal to half of the wavelength of light, and is 10 nm or more and 100 nm. The following is more preferable.

The concentration of the noble metal nanoparticles 10 in the detection liquid 1 is not particularly limited as long as sensitive detection is possible, but is appropriately 0.1 pmol / l or more depending on the concentration (quantitative region) of the non-detection substance to be detected. It is preferable to adjust to the range of 10000 pmol / l or less.

Next, the antibody 20 (A) is immobilized on the surface 10 s of the noble metal nanoparticle 10 by chemical bonding (A-2). The functional group on the side bonded to the noble metal nanoparticle 10 is not particularly limited as long as it is a functional group capable of binding to the metal of the noble metal nanoparticle 10, and examples thereof include a disulfide group and a mercapto group other than the thiol group. By using a fragmented antibody as the antibody 20 (A), it can be easily bound by the thiol group and disulfide group possessed by the fragmented antibody. The method for fixing these functional groups is not particularly limited, and existing techniques can be used.

Next, an amphiphilic SAM 30 having a hydrophobic portion 31 and a hydrophilic portion 32 is formed on the surface 10 s of the noble metal nanoparticle 10 on which the antibody 20 (A) is fixed, and a proximal end 30 a on the hydrophobic portion 31 side is provided. A detection liquid 1a is obtained which is immobilized on the surface 10s of the gold nanoparticle 10 and in which the label A is dispersed in the buffered aqueous solution 40 (A-3). In the same process, the detection liquid 1b in which the label B is dispersed in the buffered aqueous solution 40 is obtained.

<Reaction solution preparation process>
Next, as shown in FIG. 4B, a sample 2 to be measured in which a substance to be detected (antigen) R is dispersed in a buffered aqueous solution S is prepared, and the prepared two (or more) detection liquids (1a, 1b) is mixed to prepare a reaction solution 3 (reaction solution preparation step).

<Reaction process>
Next, as shown in the left diagram of FIG. 4C, the obtained reaction solution 3 is specifically bound to the biological material of all the probes 20 (A, B) in the reaction solution 3 and the antigen R which is the detection target. Let The reaction conditions are appropriately set according to the type of antigen-antibody reaction. Thus, by detecting the antigen with the two types of antibodies by the sandwich method, the noble metal nanoparticle 10 becomes a dimer, and the presence or absence of the substance to be detected (antigen) R can be accurately detected in the subsequent detection step. Can do.

<Detection process>
In the detection step, the presence or absence of specific binding is detected by detecting the presence or absence of a noble metal nanoparticle label that has become a dimer using the reaction solution 3 after the reaction in the reaction step. This is a step of detecting the presence or absence of the detection substance R.
Although the detection method in the detection step is not particularly limited, the right diagram in FIG. 4C shows a schematic configuration of the gel electrophoresis apparatus 100 with respect to an embodiment in which the presence / absence of the substance to be detected (antigen) R is detected by gel electrophoresis. A schematic top view showing an example of detection of band D is shown.

The gel electrophoresis apparatus 100 includes a gel matrix 102 installed in a container, and includes a well 101 for dispensing a sample to be detected and a voltage applying unit.

In this embodiment, the reaction solution 3 after the reaction in the reaction step is dispensed into the well 101, and electrophoresis is performed by applying a voltage so that the well 101 side is negative. The direction of voltage application is determined by the charged state of the substance to be detected.

In electrophoresis, when a substance to be detected or a label moves in a gel matrix, the mobility changes depending on its molecular weight. Since the single strand moves farther, a band colored in red by irradiating the noble metal nanoparticle 10 with light having a wavelength capable of inducing plasmon after applying a voltage for a certain time. It becomes D and the presence or absence of specific binding (antigen-antibody reaction), that is, the presence or absence of the substance to be detected (antigen) R can be detected.

Identification of each band in band D can be performed by cutting out the band and observing it with a TEM. FIG. 8 is a top view photograph of bands detected in Example 1 and Comparative Examples 1 and 2 which will be described later. As shown in the figure, one noble metal nanoparticle 10 in which no specific bond is formed is observed in the downstream band having a higher mobility, and two or three gold nanoparticles 10 are observed as going upstream. The presence of the antigen R can be confirmed.

Since detection by gel electrophoresis requires a sample with a relatively high concentration, detection of a very small amount of sample requires detection by a more sensitive detection method. The present inventors use the optical anisotropy detection apparatus 200 whose schematic configuration is shown in FIG. 5, whereby two substantially isotropic noble metal nanoparticles each bind specifically to an antigen to form a dimer. It has been found that highly sensitive detection is possible by detecting the presence or absence of an antigen by detecting the anisotropy that appears.

The optical anisotropy detection apparatus 200 includes a transparent sample stage 210 on which a sample (mixed solution) 3 is placed, a laser 230, an irradiation light receiving optical system 220 including a beam splitter 221 and an objective lens 222, and a λ / 2 plate. 240, a polarization beam splitter 250 that separates into P-polarized light and S-polarized light,

photodiodes

261 and 262 that detect each polarized light, and an oscilloscope 270 that displays a detection signal.

Specifically, the measurement is performed by configuring the detection apparatus shown in FIG. 5 using an inverted microscope (for example, manufactured by Nikon). First, the semiconductor laser 230 is condensed in the sample 2 to be measured on the sample stage 210 by the objective lens 222 to form a micro spot, and the monomer and dimer particles are formed while the micro spot is rotated by Brownian motion. Scattered light generated by passing through is collected by the objective lens 222, and the collected light is separated into orthogonal polarization components by the polarization beam splitter 250. The separated scattered light is received by the

photodiodes

261 and 262, respectively, and the time trace of the intensity is displayed on the oscilloscope 270, which is collated with the time trace of the polarization anisotropic index AI expressed by the following formula (1). By doing so, the optical anisotropy of the noble metal nanoparticles in the sample is quantified to detect the presence or absence of specific binding (antigen).
AI = (Ix−Iy) / (Ix + Iy) (1)
(Ix is the scattered light intensity in the x direction, Iy is the scattered light intensity orthogonal to the x direction, and the x direction is the polarization direction of the incident light)

When passing through the microspot, the monomer and dimer particles rotate a sufficient number of times, so that any arrangement can be taken with respect to the polarization direction of the light. Therefore, paying attention to the maximum value of the polarization anisotropy index AI, the presence or absence of a dimer, that is, the presence or absence of specific binding, is detected from each polarization intensity and its phase shift at the time when the AI value is given.

FIG. 6 shows the result of calculating the polarization anisotropy index AI by measuring the polarization intensity of the actual monomer and dimer by the above method.
As shown in FIG. 6, the monomer and the dimer can be clearly distinguished on the boundary of the polarization anisotropic index AI = 1.3.

In addition to the above-mentioned method, a trace amount of analyte can be detected in a sample solution of noble metal nanoparticles by light scattering correlation spectroscopy using strong scattered light due to plasmon resonance of noble metal nanoparticles instead of fluorescence in fluorescence correlation spectroscopy. It is also possible to detect from the time constant of the rotational motion at.

The autocorrelation functions of the monomer and dimer obtained by light scattering correlation spectroscopy are shown in FIG. 7 (monomer (single) on the left side and dimer on the right side). The rotational diffusion rate of the dimer is theoretically predicted to be about 4 times that of the monomer, and the difference in the decay time of the autocorrelation function shown in FIG. 7 is considered to reflect that difference. It is done.

Furthermore, the detection liquid 1 and the biosensing method of the present invention are also suitable for an embodiment in which the presence or absence of the substance to be detected R is detected by the local plasmon resonance wavelength shift of the noble metal nanoparticles in the step (D).

Since the biosensing method of the present invention performs sensing using the detection liquid of the present invention, the same effect as the detection liquid of the present invention is exhibited.

Hereinafter, examples and comparative examples of the present invention will be described. The present inventor believes that it is necessary to stably obtain both non-specific aggregation suppressing effect and specific binding reaction efficiency improving effect as a detection solution used for biochemical analysis, and about these effects Evaluation was performed immediately after the preparation of the detection liquid 1 and after 24 hours (next day).

Example 1
Two reaction containers in which a gold nanocolloid solution having a diameter of 40 nm (0.17 pM gold colloid solution manufactured by Tanaka Kikinzoku) was stored were prepared. In addition, using anti-hCG monoclonal IgG antibody (Anti-hCG 5008 SP-5, Medix Biochemica) as a primary antibody, pepsin and mercaptomethylamine in a sodium acetate buffered aqueous solution in the same manner as KLBrogan et al. After fragmentation by IgG and fragmentation treatment of IgG, the end groups were thiolated, added to a reaction vessel in which the gold nanocolloid solution was stored so as to be 50 pM, and reacted at a temperature of 52 ° C. for about 1 hour. Next, 20- (11-mercaptoundecanyloxy) -3,6,9,12,15,18-hexaoxaeicosanoic acid reagent (manufactured by Dojindo) Was added to a concentration of 50 nM and reacted at a temperature of 52 ° C. for about 3 hours to prepare Detection Solution 1a. Similarly, an anti-hCG monoclonal IgG antibody (Anti-Alpha subunit 6601 SPR-5, Medix Biochemica) was used as a secondary antibody, and a detection solution 1b was similarly prepared.

Next, an antigen hCG (Human chorionic gonadotropin: one of female hormones) dispersed in a sodium acetate buffered aqueous solution is prepared as a measurement sample, and the measurement sample, the detection solution 1a and the detection solution 1b are mixed, and room temperature is obtained. For 8 hours to obtain a reaction solution.

(Comparative Example 1)
As a primary antibody and a secondary antibody, IgG that was not fragmented was used, and in the other steps, a reaction solution was prepared in the same manner as in Example 1.

(Comparative Example 2)
Using IgG without fragmentation as the primary antibody and the secondary antibody, it was added to the same gold nanocolloid solution as in Example 1 and allowed to react at a temperature of 52 ° C. for about 1 hour. The antibody was physically adsorbed. Next, a 1% polyethylene glycol + 2% BSA (Bovine serum albumin) aqueous solution was added to the reaction vessel containing the detection solution and the measurement sample and stirred to prepare a detection solution. Subsequent steps were performed in the same manner as in Example 1 to prepare a reaction solution. A reaction solution was prepared in the same manner as in Example 1 except that undecyl mercaptan (English name: Undecyl Mercaptan) was used as the SAM reagent.

(Evaluation)
About the reaction liquid of each example of Example 1 and Comparative Examples 1 and 2, it dispensed to the agarose gel electrophoresis apparatus, the band was observed with natural light after performing electrophoresis for 15 minutes at the voltage of 175V. The result is shown in FIG.

In FIG. 8, for easy understanding, the band position of the monomer of gold nanoparticles and the band position of the dimer are indicated by ● in the right column of each example. In Example 1, on the day of detection solution preparation (preparation) and on the next day after the lapse of 24 hours, no dimer band was detected without antigen, and nonspecific binding was not detected. In addition, even in the presence of antigen, it was confirmed that a dimer band could be clearly detected in both cases, and highly accurate sensing could be performed.

On the other hand, in Comparative Example 1 in which a negatively charged SAM was arranged, it was confirmed that nonspecific binding was well suppressed both on the day of preparation of the detection solution and on the next day. On the other hand, in the case where the antigen was present, the result was that the dimer of the antigen was well detected on the day of preparation of the detection solution, but not detected the next day. In Comparative Example 2, a plurality of bands were detected even without an antigen, causing aggregation due to non-specific binding, resulting in low-precision sensing.
As described above, the effectiveness of the present invention was confirmed.

Claims

A detection liquid used for biosensing for detecting a detection target substance made of a biological substance contained in a sample to be measured, using noble metal nanoparticles as a label,
The noble metal nanoparticles having a probe containing a hydrophilic biological substance that specifically binds to the substance to be detected and an amphiphilic self-assembled monolayer having a hydrophobic part and a hydrophilic part fixed on the surface Dispersed in a buffered aqueous solution,
In the self-assembled monolayer, the side fixed to the particle is the hydrophobic part, and
The functional group at the end of the hydrophilic part has either a positive or negative charge,
A detection solution in which the probe is fixed to the surface by chemical bonding.
The detection liquid according to claim 1, wherein the charge of the functional group at the end of the hydrophilic part is the same as the charge of the hydrophilic biological substance.
The detection liquid according to claim 1, wherein the hydrophilic part is a polyethylene glycol chain.
The detection liquid according to claim 1, wherein the hydrophilic part is a sugar chain.
The detection liquid according to any one of claims 1 to 4, wherein the biological substance is an antibody.
The detection solution according to any one of claims 1 to 4, wherein the biological material is a fragmented antibody.
The detection solution according to any one of claims 1 to 6, wherein the chemical bond is a bond between a sulfur atom of a disulfide group or a thiol group of the fragmented antibody and a noble metal atom on the surface.
In biosensing for detecting a detection target substance made of a biological substance contained in a sample to be measured, using noble metal nanoparticles as a label,
A detection liquid preparation step for preparing two or more detection liquids according to any one of claims 1 to 7, wherein the specific bonds are different from each other, and the functional groups have the same charge.
A reaction solution preparation step of preparing a reaction solution by mixing the two or more detection solutions in a buffered aqueous solution containing the sample to be measured;
A reaction step of specifically binding the biological substance of all the probes in the reaction solution and the substance to be detected;
A biosensing method comprising a detection step of detecting the presence or absence of the specifically bound substance to be detected in the sample to be measured by detecting the label using the reaction solution after the reaction step.
The biosensing method according to claim 8, wherein in the detection step, the presence or absence of the substance to be detected is detected by gel electrophoresis.
The biosensing method according to claim 8, wherein in the detection step, the presence or absence of the substance to be detected is detected by a polarization anisotropic index of scattered light.
The biosensing method according to claim 8, wherein in the detection step, the presence or absence of the substance to be detected is detected by a light scattering correlation spectroscopy method.