WO2015159535A1

WO2015159535A1 - Detection solution, method for preparing same, and biosensing method

Info

Publication number: WO2015159535A1
Application number: PCT/JP2015/002062
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: JPWO2015159535A1

Abstract

[Problem] To improve detection accuracy in biosensing using precious metal nanoparticles as a marker. [Solution] This detection solution (1) is made by dispersing first nanoparticle probes (P1) and second nanoparticle probes (P2) in a buffer aqueous solution (40). Each first nanoparticle probe (P1) is made by fixing, to the surface of a first precious metal nanoparticle (10A), a first probe (20A) including a biological substance that specifically binds to a first section (50A) of a substance to be detected (50). Each second nanoparticle probe (P2) is made by fixing, to the surface of a second precious metal nanoparticle (10B), a second probe (20B) including a biological substance that specifically binds to a second section (50B) of the substance to be detected (50). The first probe (20A) and the second probe (20B) are fixed to the first precious metal nanoparticle (10A) and the second precious metal nanoparticle (10B), respectively, such that the respective base ends (20Ab (20Bb)) of the first probe (20A) and the second probe (20B) specifically bind, respectively, to the respective leading ends (50At (50Bt)) of the first section (50A) and the second section (50B) on the side where the two section are proximal to one another.

Description

Detection solution and its preparation method, biosensing method

The present invention relates to a detection solution used for biosensing using noble metal nanoparticles, a method for preparing the same, 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 for detecting changes in optical signals includes a method of detecting genetic materials such as DNA, RNA, peptide nucleic acids, oligonucleotides, etc. using fluorescent materials or noble metal nanoparticles as labels.

The label with a fluorescent substance has a weak signal when the concentration of a substance to be detected (for example, a target gene) is low, and it is difficult to detect with high sensitivity. High-sensitivity detection is possible by methods using amplification such as surface-enhanced Raman (SERS), but it requires many amplification processes and is controlled by chance. Not suitable for measurement.

Since the late 1990s, in biosensing for detecting a nucleic acid fragment (oligonucleotide) having a specific sequence, when the base sequences of single-stranded nucleic acids are complementary (specific), complementary base pairs A method using noble metal nanoparticles as a label for a hybridization method for forming a double-stranded nucleic acid by hydrogen bonding between them has been energetically studied (

Patent Documents

1 and 2, Non-Patent Documents 1 to 4). In such a method, noble metal nanoparticles carrying a biological material that forms a complementary strand with a nucleic acid fragment to be detected on a surface are used as a probe.

For example, Patent Document 1 and Patent Document 2 describe the result of using a gold nanoparticle attached with an oligonucleotide having a sequence complementary to a part of the nucleic acid sequence as a probe and hybridizing the oligonucleotide to the nucleic acid. A genetic diagnosis method for detecting a change in coloration of gold nanoparticles obtained by shifting the local plasmon resonance wavelength of gold nanoparticles by a long wavelength is described.

However, since these methods are also visual detections, the detection sensitivity is equivalent to the fluorescence method, and the detection limit remains at a level of several tens of nM.

For the detection of the presence or absence of hybridization using noble metal nanoparticles, a cross-linking agglutination reaction system promoted by Mirkin et al. And a non-cross-linking agglutination reaction system promoted by Maeda et al. There is. Non-Patent Document 1 discloses that in a non-crosslinking type agglutination reaction system, surface plasmon is used to accelerate aggregation by suppressing charge repulsion by adding a high concentration of salt (NaCl), and to increase detection sensitivity. A method using a resonance sensor is described.

Further, Maeda et al., In Non-Patent Document 2, in the high-concentration salt addition system described in Non-Patent Document 1, the speeding up and simplification of the aggregation process, and the end group on the side opposite to the bonding part with noble metal particles The mismatch states that for sensitive detection, factors such as the size of the gold nanoparticles, the type of target gene, and the pH of the buffered aqueous solution need to be individually optimized.

In Non-Patent Document 3, for the purpose of improving detection sensitivity, the presence or absence of hybridization, that is, the presence or absence of a target gene is optically detected by measuring the particle size of gold nanoparticles by the dynamic light scattering (DLS) method. Advocates how to do. This is based on the fact that when the target gene is present, two gold nanoparticles having their complementary strands are combined by hybridization to increase the apparent particle size. Claims that detection is possible.

Non-Patent Document 4 describes a method using a resonance light scattering correlation spectroscopy (RLSCS) method in order to make it possible to detect light scattering of gold nanoparticles having a small particle diameter with high sensitivity in a non-crosslinking type agglutination reaction system. ing.

Special table 2004-515208 gazette Japanese Patent No. 4347049

In biosensing using the above precious metal nanoparticles as a label, a method of detecting one target gene by hybridization using a plurality of probes is suitable for detecting a target gene having a relatively large number of bases. However, the length (number of bases) of the target gene to be detected is generally not constant, and FIG. 10 (FIG. 40 of Patent Document 2) applies to any of the methods described in the above-mentioned patent documents and non-patent documents. As shown, the gold nanoparticles have different intervals depending on the type of the target gene, and those having a large number of bases are separated from each other even if they are hybridized. For example, when the target DNA is as short as 30 bases (about 10 nm) with respect to the gold nanoparticle diameter φ = 30 nm, it becomes an “integrated particle” of two hybridized gold nanoparticles. When measuring a long target gene or the like, it becomes two single particles even when it is hybridized.

In addition, since the structure of the genetic material is not strong, it is twisted or bent during the measurement, so even when the same target gene is used, the interval between the gold nanoparticles is difficult to be constant.

Therefore, since the measurement results differ depending on the interval of the gold nanoparticles, the method of Patent Document and

Non-Patent Documents

1 and 2 is ambiguous in color change due to the agglutination reaction, and the method of

Non-Patent Documents

3 and 4 However, high measurement accuracy cannot be obtained. Non-Patent Document 2 describes that optimization is performed depending on the type of target gene in a high-concentration salt addition system. However, in a high-concentration salt addition system, aggregation is accelerated while dispersibility is improved. Decrease and accompanying aggregation due to non-specific binding are likely to occur, resulting in low detection sensitivity.

The same applies to the method of detecting the presence or absence of hybridization by gel electrophoresis using an agarose gel or the like. Since the apparent particle size differs even in the same dimer, there is a difference in mobility in the gel matrix. And the distinction between bands becomes unclear.

The present invention has been made in view of the above circumstances, and has excellent detection sensitivity and accuracy in biosensing for detecting a detection target substance composed of a biological substance contained in a measurement sample using noble metal nanoparticles as a label. An object of the present invention is to provide a novel biosensing method and a detection solution that enables it.

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,
A first nanoparticle probe in which a first probe containing a biological substance that specifically binds to a first site of a substance to be detected is immobilized on the surface of the first noble metal nanoparticle;
A second nanoparticle probe in which a second probe containing a biological substance that specifically binds to the second site of the substance to be detected is immobilized on the surface of the second noble metal nanoparticle is dispersed in a buffered aqueous solution. ,
The first part and the second part of the substance to be detected, the ends of these parts close to each other;
In order for the first probe and the proximal end of the second probe to specifically bind,
The first probe and the second probe are fixed to the first noble metal nanoparticles and the second noble metal nanoparticles, respectively.

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.

The proximal ends of the first probe and the second probe mean the ends of the probes that are bonded to the noble metal nanoparticles.

As a preferred embodiment of the detection liquid of the present invention, the first nanoparticle probe and the second nanoparticle probe are fixed with an amphiphilic surface modification having a hydrophobic portion and a hydrophilic portion on the surface. An embodiment in which the side fixed to the amphiphilic surface-modified noble metal particles is a hydrophobic portion can be mentioned.

In such an embodiment, it is preferable that the charged state of the functional group at the terminal on the side not fixed to the surface-modified first noble metal nanoparticles and the second noble metal nanoparticles is the same as the charged state of the biological material.

In this embodiment in which an amphiphilic surface modification having a hydrophobic part and a hydrophilic part is immobilized on the surface, the first probe and the second probe are composed of the first noble metal nanoparticle and the second probe. An embodiment in which the noble metal nanoparticles are fixed via a buffer unit that inhibits specific binding to the substance to be detected by amphiphilic surface modification is more preferable. It is preferable that the buffer part has a hydrophilic part at least in part.

The surface modification and the hydrophilic part in the buffer part are preferably a polyethylene glycol chain or a sugar chain, and preferably have a base sequence that does not produce a complementary chain with respect to the first and second probes.

The amphiphilic surface modification is preferably a self-assembled monolayer having water solubility.

The present invention can be preferably applied when the specific binding is hybridization of a nucleic acid.

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 step (A) of preparing the detection solution of the present invention;
The sample to be measured and the detection liquid containing the substance to be detected having the first part and the second part are mixed under the condition that the substance to be detected and the first probe and the second probe can form a specific bond. Step (B) to perform,
And a step (C) of detecting the presence or absence of a specifically bound detection target substance in the sample to be measured by detecting the label of the noble metal nanoparticles using the solution obtained by the mixing.

In step (C), the presence / absence of the substance to be detected can be detected by gel electrophoresis, a method of detecting by the polarization anisotropic index of scattered light, a method of detecting by local plasmon resonance wavelength shift of noble metal nanoparticles, or a light scattering correlation. It is preferable to carry out by spectroscopic methods.

The detection liquid of the present invention comprises a first nanoparticle probe in which a first probe containing a biological substance that specifically binds to a first site of a target substance is fixed to the surface of the first noble metal nanoparticle, A second probe comprising a second probe containing a biological substance that specifically binds to a second site of the detection substance and being immobilized on the surface of the second noble metal nanoparticle, and being dispersed in a buffered aqueous solution;
The first part and the second part of the substance to be detected are first bonded so that the ends of the parts close to each other and the base ends of the first probe and the second probe are specifically bound. The probe and the second probe are fixed to the first noble metal nanoparticles and the second noble metal nanoparticles, respectively. According to such a detection solution, the first probe and the second probe are specifically bound to the substance to be detected so that the first noble metal nanoparticle and the second noble metal nanoparticle are close to each other. To suppress the detection of particles and second precious metal nanoparticles as individual particles, and to minimize the difference in the distance between gold nanoparticles caused by the length (number of bases) of the substance to be detected it can. Therefore, according to the present invention, biosensing with good detection sensitivity and accuracy is performed in biosensing for detecting a substance to be detected, which is a biological substance contained in a sample to be measured, using noble metal nanoparticles as a label. Can do.

In addition, the first nanoparticle probe and the second nanoparticle probe are fixed on the surface with an amphiphilic surface modification having a hydrophobic portion and a hydrophilic portion. In a mode in which the side fixed to the modified noble metal particle is a hydrophobic part, the probe is separated from the surface of the noble metal nanoparticle because the surface of the noble metal nanoparticle surface is hydrophobic, and the probe is located near the surface of the noble metal nanoparticle. Non-specific aggregation caused by entanglement can be suppressed. Therefore, biosensing with high specific binding reaction efficiency and better detection accuracy can be performed.

Further, in the aspect in which the charged state of the terminal functional group (terminal group) on the side not fixed to the surface-modified noble metal nanoparticles is the same as the charged state of the first and second probes in the buffer solution. Since the end group of the surface modification and the first and second probes repel each other, more effective non-specific aggregation suppression effect and specific binding reaction efficiency improvement effect can be obtained.

Schematic cross-sectional schematic diagram showing the configuration of the detection liquid of the present invention The schematic diagram which shows the structure of 1st Embodiment of the nanoparticle probe contained in the detection liquid of this invention. The schematic diagram which shows the structure of 2nd Embodiment of the nanoparticle probe contained in the detection liquid of this invention. 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. 2A is a schematic enlarged view showing an embodiment of the surface of the noble metal nanoparticle. Schematic cross-sectional schematic diagram showing a preferred embodiment of step (A) of the biosensing method of the present invention Schematic cross-sectional schematic diagram showing a preferred embodiment of the step (B) of the biosensing method of the present invention Schematic cross-sectional schematic diagram showing another preferred embodiment of step (B) of the biosensing method of the present invention The schematic diagram which shows an example of the detection result at the time of implementing the detection of a process (C) by gel electrophoresis Schematic configuration schematic diagram of the device used for detection by optical polarimetry Illustration of polarization rotation of scattered light by gold nanoparticle dimer The figure which shows an example of the measurement result obtained using the apparatus of FIG. 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. Example of detection by light scattering correlation spectroscopy (decay time of simple substance and dimer) FIG. 40 of Patent Document 2

"Detection solution"
A detection solution and a biosensing method using the same according to an embodiment of the present invention will be described with reference to the drawings. FIG. 1A is a schematic cross-sectional schematic diagram showing the configuration of the detection liquid 1 of the present embodiment. 2A is a schematic cross-sectional schematic diagram showing a preferred embodiment of the first and second noble metal nanoparticle probes P1 and P2 included in the detection liquid 1, and FIG. 2B is an enlarged schematic schematic diagram of the surface of the noble metal nanoparticle in FIG. 2A. It is. In the schematic diagram and schematic diagram of the present specification, the scale of each part is appropriately changed for easy visual recognition.

Biosensing using precious metal nanoparticles as a label is suitable for detection of target genes with a relatively large number of bases, and is conventionally bound to the precious metal nanoparticles even in detection methods based on hybridization of multiple probes. The probe end (base end) is configured to be specifically bound to both ends of the substance to be detected.

As described in the section of “Background Art”, the present inventors have found that the conventional noble metal nanoparticle probe has a variation in the interval between the gold nanoparticles depending on the number of bases (length) of the target gene, In addition, in the detection of target genes with a large number of bases, the fact that the first noble metal nanoparticles and the second noble metal nanoparticles are easily detected as individual particles is found to be a factor in reducing measurement accuracy and detection sensitivity. The present invention has been completed.

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 -Lectin binding and the like, and the present invention is applicable to any specific binding.

In the present invention, the noble metal nanoparticles are not particularly limited, and it is preferable to select a noble metal having good coloration characteristics at the wavelength of light used. Among them, gold can be preferably used because it exhibits a red color that is easily visible in natural light.

Hereinafter, a case where the noble metal nanoparticles are gold nanoparticles, the hydrophilic biological substance (probe) is a nucleic acid fragment, and the specific bond is a complementary bond H (hybridization) of a nucleic acid will be described as an example.

As shown in FIG. 1A, the detection liquid 1 is a detection liquid used for biosensing for detecting a detection target substance made of a biological substance contained in a measurement target sample using noble metal nanoparticles as a label. A first nanoparticle probe P1 in which a first probe 20A containing a biological substance that specifically binds to the first portion 50A of the target substance 50 shown in FIG. 5 is immobilized on the surface of the first noble metal nanoparticle 10A;
A second nanoparticle probe P2 in which a second probe 20B containing a biological substance that specifically binds to the second portion 50B of the detection target substance 50 is immobilized on the surface of the second noble metal nanoparticle 10B is buffered aqueous solution. 40 is distributed.

As shown in FIG. 1B or FIG. 1C, the first and second nanoparticle probes P1 and P2 are close to each other in the first part 50A and the second part 50B of the detection target substance 50. Side ends 50At, 50Bt;
The base ends 20Ab and 20Bb of the first probe 20A and the second probe 20B are specifically bound.
The first probe 20A and the second probe 20B are fixed to the first noble metal nanoparticles 10A and the second noble metal nanoparticles 10B, respectively.

Moreover, in this embodiment, in the first nanoparticle probe P1 and the second nanoparticle probe P2, an amphiphilic surface modification 30 having a hydrophobic portion and a hydrophilic portion on the surface of the

noble metal nanoparticles

10A and 10B. Is fixed to the surface.

In the first embodiment of the nanoparticle probes P1 and P2 shown in FIG. 1B, the first site 50A and the second site 50B where the nanoparticle probe P1 and the second nanoparticle probe P2 of the detected substance 50 are specifically bound. And are adjacent to each other. The nanoparticle probes P1 and P2 are not limited to such an embodiment. As shown in FIG. 1C, the first portion 50A and the second portion 50B are not adjacent to each other, and some of them are in between. An embodiment comprising a base sequence may be used (second embodiment).

Further, FIGS. 1B and 1C show a mode in which the first nanoparticle probe P1 and the second nanoparticle probe P2 that specifically bind to both ends of the detected substance 50 are specifically bound. The specific binding site in is not particularly limited.

Further, as shown in FIG. 2A, in this embodiment, in the buffered aqueous solution 40, the first and second nanoparticle probes P1 and P2 are

noble metal nanoparticles

10A and 10B having an amphiphilic surface modification 30. The end on the side not fixed to the electrode has a negative charge, and the first and

second probes

20A and 20B also have a negative charge.

As shown in FIG. 2B, the amphiphilic surface modification 30 has a hydrophobic portion 31 and a hydrophilic portion 32 that are fixed to the gold nanoparticles 10 </ b> A and 10 </ b> B. In FIG. 2B, the hydrophobic part 31 which consists of the alkyl group (carbon number 11) whose functional group of the base end 30a is a thiol group, and the hydrophilic part which consists of polyethylene glycol (carbon number 6) whose end group 30e is a carboxyl group A surface modification 30 formed by ester bonding with 32 is illustrated as an example. In FIG. 2B, but is shown as a carboxyl group, in a buffered aqueous solution COO ^- present as.

In FIG. 2B, the first probe 20A and the second probe 20B are connected to the first gold nanoparticle 10A and the second gold nanoparticle 10B by the amphiphilic surface modification 30 and the detection target substance 50. Both the base end 30a of the surface modification 30 and the base end 21a of the buffer part 21 fixed to the gold nanoparticle 10A (10B), which are fixed through the buffer part 21 that suppresses inhibition of specific binding of In the embodiment in which is a thiol group, it is already bound to gold. The base end 20Ab (20Bb) of each probe coupled to the buffer portion 21 is the end 50At, 50Bt of the first part 50A and the second part 50B of the detected substance 50 on the side where these parts are close to each other. Specific binding.

The size of the gold 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. Further, when optical detection is performed, the particle diameter is preferably 30 nm or more.

The concentration of the gold nanoparticle 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.

FIG. 2B shows a mode in which the buffer part 21 is provided on the base end 20Ab (20Bb) side of the first and second probes 20A (20B), and the buffer part 21 is provided with a thiol group and fixed to the gold nanoparticle 10. Although it is shown, the functional group on the side bonded to the gold nanoparticle 10 is not particularly limited as long as it is a functional group capable of binding to the metal of the gold nanoparticle 10, and other than the thiol group, there are a disulfide group, a mercapto group, and the like. Can be mentioned. The method for fixing these functional groups is not particularly limited, and existing techniques can be used.

The inventor of the present invention, when generating complementary binding H (hybridization) in a close state due to steric hindrance depending on the particle diameter of the gold nanoparticle 10, the proximal end of the probe on the side fixed to the gold nanoparticle 10 It has been found that about 10 to 20 bases of (20Ab, 20Bb) cannot contribute to hybridization, and this may lead to a decrease in hybridization efficiency with the detection target substance 50 and a deterioration in detection limit.

In order to reduce steric hindrance, it is preferable that the particle size is small, but the amount of scattered light of gold nanoparticles having a particle size equal to or smaller than the wavelength of light, that is, the amount of signal light in optical detection is determined by the Rayleigh scattering law. Therefore, if the particle size is too small, the signal light is buried in noise, and the optical detection sensitivity is greatly deteriorated (see Non-Patent Document 3). Therefore, when performing optical detection, it is preferable that each particle size of the gold nanoparticle 10 (10A, 10B) is 30 nm or more.

On the other hand, when the gold nanoparticle 10 has a particle size (for example, φ ≧ 30 nm) at which a sufficient optical signal amount can be obtained, not only steric hindrance becomes a problem, but also the surface area of the particle with respect to the probe 20 is increased. Since it becomes large, the probe 20 is easily adsorbed on the surface of the gold nanoparticle 10, and the reaction efficiency is greatly lowered. Further, there arises a problem that the detection sensitivity is deteriorated due to a nonspecific reaction caused by a plurality of probes being entangled with each other.

As shown in the present embodiment, the present inventors provide the surface of the gold nanoparticle 10 with an amphiphilic surface modification 30 so that the probe 20 stands up in a brush shape without being adsorbed on the surface. 20 is immobilized on the surface of the gold nanoparticle 10 via the buffer portion 21, thereby preventing the amphiphilic surface modification 30 from inhibiting the specific binding with the substance 50 to be detected, and the nonspecific reaction. And it succeeded in suppressing the deterioration of detection sensitivity and reaction efficiency due to steric hindrance.

The buffer part 21 arrange | positions the probe part complementary to the to-be-detected substance 50 in the place which is hard to receive the steric hindrance of the gold nanoparticle outside surface modification (30), and a specific binding with the to-be-detected substance 50 is inhibited. In order to prevent this, it is preferable to have a hydrophilic portion at least partially so as not to be buried in the hydrophobic portion 31 in contact with the gold nanoparticle 10 of the surface modification 30.

As such a hydrophilic part, it is preferable that the buffer part 21 has a hydrophilic part at least in part. The hydrophilic part is not particularly limited as long as it can improve dispersibility (water solubility) in a buffered aqueous solution, but is complementary to a polyethylene glycol (PEG) chain, a sugar chain, a hydrophilic polyester, and a probe. Examples thereof include a base sequence that does not generate a chain (for example, T; repeating thymine and enumeration).

Moreover, it is preferable that the length of the buffer part 21 is longer than the thickness of the surface modification 30, and the base ends 20Ab (20Bb) of the first and second probes 20 (A, B) coupled to the buffer part 21 are More preferably, the length is not buried in the surface modification 30. It is preferable that the buffer 21 is appropriately designed in consideration of the particle size of the gold nanoparticles 10A (10B) and the probe length.

In the detection liquid 1, the surface modification 30 is for suppressing detection of non-specific binding that occurs when the first and second probes 20 </ b> A (20 </ b> B) are entangled near the surface 10 s of the gold nanoparticle 10. Therefore, it is preferable that the length of one molecule is shorter than the length of the first and second probes 20A (20B).

When the first and second probes 20A (20B) are 20-base oligo DNA, the chain length is about 7 nm. On the other hand, the thickness (the distance from the surface 10s to the terminal) of the surface modification 30 that provides a good effect is preferably about 3.0 nm.

In the surface modification 30, the hydrophobic part 31 is preferably an alkyl group whose base end 30 a is a functional group capable of binding to the gold nanoparticle 10. Examples of the functional group at the base end 30a include sulfur compounds such as thiols and disulfides, as in the buffer section 21.

A hydrophilic part 32 is provided at the end of the hydrophobic part 31 opposite to the fixed side of the gold nanoparticles 10. The hydrophilic part 32 improves the decrease in dispersibility in the buffered aqueous solution 40 by being covered with the hydrophobic part 31.

Since the gold nanoparticles covered with the highly hydrophobic surface modification 30 and the hydrophobic part 31 have low water solubility, in order to disperse well in the buffering aqueous solution, compatibility with alcohol or the like is required. Although it is necessary to disperse once through a high solvent, there is a high possibility that it will be difficult to perform good sensing due to damage and denaturation of biological substances such as nucleic acids, increasing non-specific aggregation. 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 of the present invention uses the surface modification 30 provided with the hydrophilic portion 32 at the end opposite to the fixing side (base end) of the gold nanoparticle 10 of the hydrophobic portion 31, so that the first and the second It has been found that the detection solution has good dispersibility in a buffered aqueous solution and hardly damages a biological material while maintaining the effect of suppressing the entanglement between the probes 20A (20B).

According to the present embodiment, biological substances that are entangled in the vicinity of the surface of the gold nanoparticle 10s can be peeled off from the surface to increase the specific reaction efficiency and suppress aggregation due to non-specific binding.

The hydrophilic portion 32 is not particularly limited as long as it can improve the dispersibility (water solubility) in a buffered aqueous solution. However, the hydrophilic portion 32 is not limited to a polyethylene glycol (PEG) chain, a sugar chain, a hydrophilic polyester, or a probe. Examples include a base sequence that does not generate a complementary strand (for example, T; thymine repeats / enumeration), and the like, and those that impart water solubility and that the surface modification 30 can be a self-assembled monolayer are preferable.

The end group 30e of the hydrophilic portion 32 is not particularly limited, and examples thereof include a hydroxyl group, a carboxyl group, a phosphoric acid group, and a sulfonic acid group. Even if the terminal group 30e is a functional group that is not an acidic functional group such as a hydroxyl group, it is possible to peel the biological material that is entangled in the vicinity of the surface 10s of the gold nanoparticle, but the first and second probes 20A (20B) ) Cannot be fixed in a brush shape while standing up from the surface.

2A and 2B, when the end group 30e of the hydrophilic portion 32 is the same as the charged state in the buffered aqueous solution of the nucleic acid fragment 20, the surface (30e) of the surface modification 30 and the nucleic acid fragment 20 Repel each other, and the nucleic acid fragment 20 rises further away from the gold nanoparticle surface 10s, so that it is fixed in a brush shape. As a result, nonspecific aggregation of only the same sequence can be more effectively suppressed.

Since the charged state of the first and second probes 20A (20B) in the buffered aqueous solution is negative, the end group 30e is a functional group having a high ionization tendency to become a negative ion in the buffered aqueous solution, that is, acidic. It is preferably a functional group. In the case of PEG, since it is a hydroxyl group and not an acidic functional group, it is preferably used after being substituted with the illustrated carboxyl group, sulfonic acid group or the like. The method for fixing these functional groups is not particularly limited, and existing techniques can be used.

As described above, the surface modification 30 has a functional group of a sulfur compound such as thiols and disulfides at the terminal to be fixed to the gold nanoparticle 10. The functional group of these sulfur compounds is adsorbed spontaneously on the surface of a noble metal such as gold, and alkanethiols and alkane disulfides give a monomolecular-sized ultrathin film (monomolecular film). In addition, the aggregate is called a self-assembled monomolecular film because it shows an arrangement depending on the crystal state of the particles and the molecular structure of the adsorbed molecule, and is preferable because it can be easily fixed on the surface of the noble metal.

Suitable surface modification 30 includes 20- (11-mercaptoundecanyloxy) -3,6,9,12,15,18-hexaoxaeicosanoic acid (English name: 20- (11-Mercaptoundecanyloxy) -3, 6,9,12,15,18-hexaoxaeicosanoic acid)). The length of this single molecule is about 2.5 nm to 3.0 nm, and the length of the hydrophobic portion 31 is about 1.2 nm.

The concentration of the surface modification 30 in the detection solution 1 (the concentration of a single molecule) is not particularly limited as long as sensitive detection is possible, but a range of 10 nmol / l or more and 1000 nmol / l or less is preferable. It is preferable to optimize depending on the particle size and the type of biological material.

As described above, the detection liquid 1 includes the first probe 20A containing a biological substance that specifically binds to the first portion 50A of the detection target substance 50 and is fixed to the surface of the first noble metal nanoparticle 10A. 1st nanoparticle probe P1 and 2nd probe 20B containing the biological material which bind | bond | couples specifically with 2nd site | part 50B of a to-be-detected substance are fixed to the surface of 2nd noble metal nanoparticle 10B, and the 2nd nanoparticle Probe P2 is dispersed in buffered aqueous solution 40,
The first part 50A and the second part 50B of the detection target substance are specific to the ends 50At and 50Bt on the side where these parts are close to each other and the base ends 20Ab and 20Bb of the first probe and the second probe The first probe 20A and the second probe 20B are respectively fixed to the first noble metal nanoparticle 10A and the second noble metal nanoparticle 10B so as to be coupled. According to this configuration, the first probe 20A and the second probe 20B are specifically bound to the substance to be detected so that the first noble metal nanoparticle 10A and the second noble metal nanoparticle 10B are close to each other. The noble metal nanoparticles 10A and the second noble metal nanoparticles 10B are suppressed from being detected as individual particles, and the difference in the distance between the gold nanoparticles caused by the length (number of bases) of the substance to be detected is minimized. Can be limited.

Therefore, by using the detection liquid 1, biosensing with good detection sensitivity and accuracy is performed in biosensing for detecting a target gene made of a biological substance contained in a sample to be measured, using noble metal nanoparticles as a label. be able to.

"Biosensing method"
With reference to drawings, the biosensing method of one Embodiment concerning this invention is demonstrated. FIG. 3A, FIG. 3B, and FIG. 3C are schematic schematic diagrams showing preferred embodiments of steps (A) to (C) of the biosensing method of the present invention.

Here, the case where the substance to be detected is the nucleic acid fragment 50 that binds complementarily to the nucleic acid fragment and the noble metal nanoparticle 10 is a gold nanoparticle will be described as an example.

<Process (A)>
As shown in FIG. 3A, in the step (A), the detection solution 1 of the present invention is prepared. The detection liquid 1 includes a first nanoparticle probe P1 and a second nanoparticle probe P2. Therefore, first, the detection liquid 1 is obtained by preparing each nanoparticle probe and mixing them.

First, a predetermined amount of the buffer aqueous solution 40 is injected into a container such as a beaker, and a plurality of noble metal nanoparticles 10A are added therein (A-1). This step may be performed without heating.

Next, the first probe 20A is fixed to the surface of the gold nanoparticle 10A (A-2). At this time, the end 50At of the first part 50A of the detected substance 50 on the side close to the second part 50B and the base end 20Ab on the side bound to the gold nanoparticle 10A of the first probe are unique. In order to bond, the proximal end 20Ab of the first probe is functionalized. The functional group of the base end 20Ab is as described above, and when the first probe 20A is fixed to the gold nanoparticle 10A via the buffer part 21, it is fixed to the gold nanoparticle 10A of the buffer part 21. Keep the side functionalized. A known method may be used as a functionalization method.

Next, an amphiphilic surface modification 30 having a hydrophobic portion 31 and a hydrophilic portion 32 is applied to the surface of the gold nanoparticle 10A to which the first probe 20A is fixed, and a base end 30a on the hydrophobic portion 31 side. Is fixed to the surface 10s of the gold nanoparticle 10 to obtain the first nanoparticle probe P1 (A-3).

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

Next, a second nanoparticle probe P2 is prepared in the same manner as the first nanoparticle probe P1, except that it specifically binds to the second portion 50B of the substance 50 to be detected.

Finally, the first nanoparticle probe P1 and the second nanoparticle probe P2 are mixed, and the first nanoparticle probe P1 and the second nanoparticle probe P2 are dispersed in the buffered aqueous solution 40. Detection liquid 1 is obtained (A-4).

<Process (B)>
In the step (B), a sample to be measured is mixed in the detection liquid 1, and complementary binding between the substance to be detected 50 in the sample to be measured and the first and

second probes

20A and 20B in the detection liquid 1 is performed. This is a step of carrying out H formation (specific binding) reaction.

It is known that the formation of a complementary strand (hybridization) between nucleic acids is formed at a temperature slightly lower than the temperature at which a nucleic acid fragment (single strand) can exist as a single strand (melting temperature). In the step (B), the conditions for the specific binding reaction (hybridization) are appropriately set within the range where there is no damage to the nucleic acid fragments that adversely affect the formation of complementary strands and the deterioration of the mode of fixation of each nucleic acid fragment to the gold nanoparticles. can do.

FIG. 3B and FIG. 3C show an example of a detection system when a measurement target material containing the target substance 50 is mixed in the detection liquid 1. FIG. 3B is a detection system using the nanoparticle probes P1 and P2 of the first embodiment shown in FIG. 1B, and the nanoparticle probe P1 and the second nanoparticle probe P2 of the detection target substance 50 are specifically bound. The first part 50A and the second part 50B are adjacent to each other. FIG. 3C is a detection system when the nanoparticle probes P1 and P2 of the second embodiment shown in FIG. 1C are used, and the first part 50A and the second part 50B are not adjacent to each other. Is a mode comprising several base sequences.

As shown in FIGS. 3B and 3C, according to the detection liquid 1 of the present invention, the first portion 50A and the second portion 50B of the substance to be detected, the end 50At, on the side where these portions are close to each other, 50Bt and the base ends 20Ab and 20Bb of the first probe and the second probe are specifically bound, so that the first gold nanoparticle 10A and the second gold nanoparticle 10B are closest to each other in the complementary strand. It is formed.

Therefore, the formation of complementary strands enables the presence of two gold nanoparticles in the form closest to the dimer, and detection of complementary strand formation with good detection sensitivity and detection accuracy in the subsequent step (C). can do.

In the system shown in FIG. 3B, in the subsequent step (C), it is possible to detect particles of approximately the same size as a dimer when forming complementary strands. In the system shown in FIG. Enables detection close to a mass. In the conventional detection solution, when the number of bases is long, the distance between the first gold nanoparticle 10A and the second gold nanoparticle 10B after the formation of the complementary strand is too large, and the detection sensitivity and detection accuracy are greatly reduced. However, according to the present invention, it is possible to suppress detection of the first gold nanoparticles 10A and the second gold nanoparticles 10B as individual particles, and to minimize the decrease thereof.

Further, as the detection liquid 1, the first nanoparticle probe P1 and the second nanoparticle probe P2 have an amphiphilic surface modification 30 having a hydrophobic portion 31 and a hydrophilic portion 32 on the surface. The aspect in which the side fixed to the

noble metal nanoparticles

10A and 10B of the amphiphilic surface modification 30 is the hydrophobic portion 31 has been described. In this aspect, since the vicinity of the surface of the noble metal nanoparticle is hydrophobic, it is possible to suppress nonspecific aggregation caused by the probe being separated from the surface of the noble metal nanoparticle and entangled with the probe near the surface of the noble metal nanoparticle. . Therefore, biosensing with high specific binding reaction efficiency and better detection accuracy can be performed.

Furthermore, in the above description, the charged state of the functional group at the end 30e on the side not fixed to the noble metal nanoparticles of the surface modification 30 is the charge of the ends of the first and

second probes

20A and 20B in the buffered aqueous solution 40. It was set as the aspect which is the same as a state. In this embodiment, since the end group 30e of the surface modification 30 and the first and

second probes

20A and 20B are repelled, more effective non-specific aggregation suppression effect and specific binding reaction efficiency improvement effect Can be obtained.

<Process (C)>
In the step (C), by using the mixed solution mixed in the step (B), a gold nanoparticle label is detected to detect complementary strand formation (specific binding) H, and a substance to be detected in a sample to be measured This is a step of detecting the presence or absence of 50.

The detection method in step (C) is not particularly limited, and is a method of detecting by gel electrophoresis, polarization anisotropic index of scattered light, a method of detecting by local plasmon resonance wavelength shift of noble metal nanoparticles, or light scattering correlation spectroscopy. It is preferable to implement by.

FIG. 4A shows a schematic configuration of the gel electrophoresis apparatus 100 and a schematic top view showing an example of detection of band D in a mode in which the presence / absence of the target substance 50 is detected by gel electrophoresis.

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 mixed solution prepared in the step (B) 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 nucleic acid fragment or a nucleic acid complementary strand moves in a gel matrix, the mobility changes depending on the molecular weight. Therefore, the heavier one, that is, the one that forms a complementary strand, has a smaller amount of movement and the front side (well Since the single strand moves farther, a band colored in red by irradiating the gold nanoparticles 10 with light having a wavelength capable of inducing plasmons after applying a voltage for a certain period of time. It becomes D, and the presence or absence of specific binding (hybridization), that is, the presence or absence of the substance to be detected can be detected.

Identification of each band in band D can be performed by cutting out the band and observing it with a TEM. One gold nanoparticle 10 in which no complementary strand is formed is observed in the most downstream band having higher mobility, and two or three gold nanoparticles 10 are combined as they go upstream, that is, Complementary strand formation is 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 have used the optical anisotropy detection device 200 whose schematic configuration is shown in FIG. 4B, and anisotropy that appears when substantially isotropic noble metal nanoparticles are hybridized to form a dimer. It has been found that highly sensitive detection is possible by detecting the presence or absence of hybridization.

The optical anisotropy detection apparatus 200 includes a transparent sample stage 210 on which a sample (mixed solution) 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.

The detection principle by the optical anisotropy detection apparatus 200 will be described with reference to FIG. As shown in FIG. 5, when the linearly polarized light E0 is incident on the sample containing the specimen, the scattered light generated by the linearly polarized light E0 irradiated to the isotropic particles has a polarization plane substantially similar to the linearly polarized light E0. have. On the other hand, scattered light generated by linearly polarized light E0 irradiated to a multimer such as a dimer having anisotropy (Plong ≠ Pshort) is induced in a major axis (Plong direction) direction and a minor axis direction (Pshort direction). Polarization rotation occurs due to different dipole moments. Utilizing this phenomenon, the presence or absence of hybridization can be detected by detecting the presence or absence of rotation of the polarization plane.

Specifically, the detection apparatus shown in FIG. 4B is configured and measured using an inverted microscope (for example, manufactured by Nikon). First, the semiconductor laser 230 is condensed into the sample S on the sample stage 210 by the objective lens 222 to form a micro spot, and the monomer and dimer particles pass through the micro spot while rotating by the Brownian motion. The scattered light generated by this is condensed 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). Thus, the presence or absence of hybridization is detected by quantifying the optical anisotropy of the noble metal nanoparticles in the sample.

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, hybridization is detected from each polarization intensity and its phase shift at the time when the AI value is given.

FIG. 6 shows an example of a detection result obtained by the above method when a dimer is present. The upper diagram in FIG. 6 shows the time trace of each polarization intensity detected by the oscilloscope, the lower diagram shows the time change of the AI value, and the time axes of both diagrams match.

In the lower diagram of FIG. 6, the maximum value of AI is surrounded by a broken line. The spectrum in the upper graph at the peak time shows that the intensity Ix in the x direction is a convex portion and the concave portion is Iy (the deflection intensity in the y direction is reduced when the deflection intensity in the x direction is large). Has been. This is due to the fact that when the dimer is present, the polarization direction also follows and rotates due to the rotation, so that the phase is reversed.
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)

FIG. 7 shows the result of calculating the polarization anisotropy index AI after measuring the polarization intensity of the actual monomer and dimer by the above method.

As shown in FIG. 7, the monomer and the dimer can be clearly distinguished on the boundary of the 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. 8 (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 four times that of the monomer, and the difference in the decay time of the autocorrelation function shown in FIG. 8 is considered to reflect that difference. It is done.

FIG. 9 shows the results of measuring the polarization intensity of the actual monomer and dimer by light scattering correlation spectroscopy. As shown in FIG. 9, the boundary between the monomer and the dimer is unclear compared to the method using the anisotropic index, but the monomer and the dimer can be distinguished. .

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 50 is detected by the local plasmon resonance wavelength shift of the noble metal nanoparticles in the step (C).

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.

(Design changes)
In the said embodiment, the 1st nanoparticle probe and the 2nd nanoparticle probe which are the aspects from which detection sensitivity and detection accuracy become more favorable are the amphipathic which has a hydrophobic part and a hydrophilic part on the surface. Although the embodiment in which the surface modification is fixed on the surface has been described, even in the embodiment without such a surface modification, the difference in the distance between the gold nanoparticles is minimized depending on the length (number of bases) of the substance to be detected. Biosensing with good detection sensitivity and accuracy can be implemented.

Furthermore, in the above-described embodiment, the configuration including the buffer portion has been described. However, even in an embodiment in which the buffer portion is not provided, the difference in the interval between the gold nanoparticles is determined depending on the length (base number) of the substance to be detected. Biosensing with good detection sensitivity and accuracy can be implemented.

In the above embodiment, the case where the substance to be detected is a nucleic acid fragment and the specific binding is hybridization of a nucleic acid has been described as an example. However, the present invention is not limited to the configuration, and has already been described. It can be applied to sensing the presence or absence of various specific bonds such as antigen-antibody binding and avidin-biotin binding.

Although the case where the terminal group of the detected substance is acidic has been described, when the terminal group is basic, examples of the terminal group 30e of the surface modification 30 include an amino group, a quaternary ammonium group, an imidazole group, and a guanidinium group. Is mentioned.

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,
A first nanoparticle probe in which a first probe containing a biological substance that specifically binds to a first site of the substance to be detected is immobilized on the surface of the first noble metal nanoparticle;
A second probe comprising a second probe containing a biological substance that specifically binds to the second portion of the substance to be detected is immobilized on the surface of the second noble metal nanoparticle and dispersed in a buffered aqueous solution. And
The ends of the first part and the second part on the side where the parts are close to each other;
So that the base ends of the first probe and the second probe are specifically bound,
A detection liquid in which the first probe and the second probe are fixed to the first noble metal nanoparticles and the second noble metal nanoparticles, respectively.
In the first nanoparticle probe and the second nanoparticle probe, an amphiphilic surface modification having a hydrophobic part and a hydrophilic part is fixed to the surface, and the amphiphilic surface modification is performed. The detection liquid according to claim 1, wherein the side fixed to the particles is the hydrophobic part.
The charged state of the functional group on the terminal that is not fixed to the first noble metal nanoparticle and the second noble metal nanoparticle of the surface modification is the same as the charged state of the biological material. Detection liquid.
The first probe and the second probe are fixed to the first noble metal nanoparticle and the second noble metal nanoparticle through a buffer unit that inhibits the specific binding from being inhibited by the surface modification. The detection liquid according to any one of claims 1 to 3, wherein the detection liquid is formed.
The detection liquid according to any one of claims 2 to 4, wherein the hydrophilic part is a polyethylene glycol chain.
The detection solution according to any one of claims 2 to 4, wherein the hydrophilic part is a sugar chain.
The detection solution according to any one of claims 2 to 4, wherein the hydrophilic portion has a base sequence that does not generate a complementary strand with respect to the first and second probes.
The detection solution according to any one of claims 2 to 7, wherein the amphiphilic surface modification is a self-assembled monolayer having water solubility.
The detection solution according to any one of claims 4 to 8, wherein the buffer part has a hydrophilic part.
The detection liquid according to claim 9, wherein the hydrophilic part of the buffer part is a polyethylene glycol chain.
The detection solution according to claim 9, wherein the hydrophilic part of the buffer part is a sugar chain.
10. The detection solution according to claim 9, wherein the hydrophilic part of the buffer part has a base sequence that does not generate a complementary strand with respect to the first and second probes.
The detection solution according to any one of claims 1 to 12, wherein the specific binding is hybridization of a nucleic acid.
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,
The step (A) of preparing the detection solution according to any one of claims 1 to 13;
A measurement comprising the substance to be detected having the first part and the second part on the condition that the substance to be detected, the first probe, and the second probe can form the specific bond. A step (B) of mixing the sample and the detection liquid;
A step (C) 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 solution obtained by the mixing. Sensing method.
The biosensing method according to claim 14, wherein in the step (C), the presence or absence of the detection target substance is detected by gel electrophoresis.
The biosensing method according to claim 14, wherein in the step (C), 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 14, wherein in the step (C), the presence or absence of the substance to be detected is detected by a light scattering correlation spectroscopy method.
The biosensing method according to claim 14, wherein in the step (C), the presence or absence of the substance to be detected is detected by a local plasmon resonance wavelength shift of the noble metal nanoparticles.