Patent Description:
<CIT> discloses a surface enhanced Raman scattering (SERS) active particle which comprises a metal-containing particle and a cationic coating provided on the metal-containing particle and carries a positive electric charge. <CIT> also discloses a SERS active particle which includes a metal-containing particle and a non-metal molecule, in which the metal-containing particle is derivatized with the non-metal molecule. <CIT> also discloses a method in which an analyte is contacted with SERS active particles to capture the analyte on the SERS active particles, the SERS active particles each having the analyte captured thereon are agglutinated, and a signal of a SERS spectrum is detected.

The highly sensitive detection of an analyte has been required in various fields. For example, in a clinical test, it has been required to detect an amino acid in a protein with high sensitivity. In <CIT>, detection sensitivity is improved by enhancing a signal of a SERS spectrum using a SERS active particle. However, further enhancement of the signal is still required.

An object of the present invention is to provide: a measurement sample preparation method whereby the sensitivity of detection of an analyte can be improved; an analysis method; a reagent for preparing a measurement sample; and a reagent kit.

The present invention relates to a preparation method for preparing a measurement sample comprising an aggregate of metal nanoparticles having an analyte bound thereto, the preparation method comprising: contacting the analyte with a linker to bind the analyte to the linker; and contacting the linker that has been bound to the analyte with the metal nanoparticles to bind the linker to the metal nanoparticles, wherein the analyte has a functional group, and the linker has a reactive group capable of reacting with the functional group, and wherein the functional group comprises at least one group selected from the group consisting of an amino group, a carboxyl group and a hydroxyl group, and wherein in case the functional groups is the amino group or the hydroxyl group, the reactive group comprises at least one group selected from the group consisting of an N- hydroxysuccinimide ester group, an isothiocyanate group, an isocyanate group, an acyl azide group, a sulfonyl chloride group, an aldehyde group, an imide ester group, a fluorobenzene group, an epoxide group, a carbodiimide group, a carbonate group, and a fluorophenyl ester group, and wherein in case the functional group is a carboxyl group, the reactive group comprises at least one group selected from the group consisting of a hydroxyl group and an amino group.

It becomes possible to prepare a measurement sample whereby an analyte contained therein can be detected with high sensitivity.

The present invention relates to an analysis method including: obtaining an optical spectrum from the measurement sample prepared by the preparation method; and outputting information about the analyte on the basis of the obtained optical spectrum. It becomes possible to detect the analyte with high sensitivity.

The present invention relates to the use of a reagent in the preparation of a measurement sample by the above-mentioned preparation method, the reagent including a linker that is not bound to a metal nanoparticle.

The present invention relates to the use of a reagent kit in the preparation of a measurement sample by the above-mentioned preparation method, the reagent kit including: the reagent; and metal nanoparticles that are packed separately from a linker.

According to the present invention, the sensitivity of detection of an analyte can be improved.

According to the preparation method, it becomes possible to prepare a measurement sample including an aggregate of metal nanoparticles bonded to an analyte. As shown in <FIG>, the preparation method includes (i) contacting the analyte with the linker to bind the analyte to the linker and (ii) contacting the linker that has been bound to the analyte with the metal nanoparticles to bind the linker to the metal nanoparticles. The method may further include (iii) adding an inorganic salt or an acid, subsequent to the contact of the linker that has been bound to the analyte with the metal nanoparticles, depending on the type of the analyte.

In the preparation method, the analyte and the linker are reacted with each other in advance, and subsequently a complex of the analyte and the liker is reacted with the metal nanoparticles. As a result, the sensitivity of the detection of the analyte can be improved.

The measurement sample is preferably a sample which can be subjected to a spectroscopic analysis of the analyte. Examples of the spectroscopic analysis include fluorescence spectroscopy, surface plasmon resonance (SPR) and infrared spectroscopy. The spectroscopic analysis is preferably Raman scattering spectroscopy, more preferably surface enhanced Raman scattering (SERS) spectroscopy.

The analyte can include at least one component selected from the group consisting of a nucleic acid (e.g., RNA, DNA), an amino acid, a polypeptide, a catecholamine, a polyamine, an organic acid, an extracellular vesicle and a virus. The term "polypeptide" as used herein refers to a compound having a structure that two or more amino acid residues are linked to each other through a peptide bond. Examples of the polypeptide include a dipeptide, an oligopeptide and a protein.

The analyte has at least one functional group selected from an amino group, a carboxyl group and a hydroxyl group. The analyte binds to the below-mentioned linker by utilizing the functional group.

The analyte is contained in a solvent such as water and a buffer solution. Alternatively, the analyte is contained in a liquid biological sample such as blood, serum, plasma, saliva, ascitic fluid, pleural effusion, cerebrospinal fluid, interstitial fluid and urine.

The linker has a reactive group capable of reacting with the functional group. The linker is not particularly limited, as long as the linker can bind to the below-mentioned metal nanoparticles. For example, the linker is represented by general formula (I).

R<NUM>-O-CO-R<NUM>-S-S-R<NUM>-CO-O-R<NUM>     (I).

(In the formula, R<NUM> and R<NUM> may be the same as or different from each other, and independently represent an alkylene group having <NUM> to <NUM> carbon atoms; and
R<NUM> and R<NUM> may be the same as or different from each other, and independently represent a reactive group.

It is preferred that the number of carbon atoms constituting each of R<NUM> and R<NUM> is <NUM> to <NUM>. It is more preferred that R<NUM> and R<NUM> are the same as each other.

Each of R<NUM> and R<NUM> represents a reactive group capable of reacting with the functional group in the analyte. It is preferred that R<NUM> and R<NUM> are the same as each other. When the functional group in the analyte is an amino group, the reactive group comprises at least one group selected from an N-hydroxysuccinimide ester group, an isothiocyanate group, an isocyanate group, an acyl azide group, a sulfonyl chloride group, an aldehyde group, an imide ester group, a fluorobenzene group, an epoxide group, a carbodiimide group, a carbonate group, and a fluorophenyl ester group. It is preferred that the reactive group is an N-hydroxysuccinimide ester group.

When the reactive group is an N-hydroxysuccinimide ester group, the linker preferably includes at least one component selected from the group consisting of dithiobis(succinimidyl propionate), dithiobis(succinimidyl undecanoate), dithiobis(succinimidyl octanoate) and dithiobis(succinimidyl hexanoate).

When the functional group in the analyte is a carboxyl group, the reactive group comprises at least one group selected from a hydroxyl group and an amino group.

When the functional group in the analyte is a hydroxyl group, the reactive group comprises at least one group selected from an N-hydroxysuccinimide ester group, an isothiocyanate group, an isocyanate group, an acyl azide group, a sulfonyl chloride group, an aldehyde group, an imide ester group, a fluorobenzene group, an epoxide group, a carbodiimide group, a carbonate group, and a fluorophenyl ester group.

A single type of linker may be used, or a mixture of two or more types of linkers may be used.

In the sample containing the analyte, it is preferred to contain the analyte in an amount of, for example, about <NUM> to about <NUM>. However, in a sample containing an analyte, the content of the analyte may be unknown. In this case, the requirement of the above-mentioned analyte content is not necessarily applied. It is preferred that the pH value of the sample containing the analyte is, for example, about <NUM> to about <NUM>. Therefore, when the analyte is not a liquid sample, it is preferred to dissolve the analyte in a buffer solution having a pH value of about <NUM> to about <NUM>, such as PBS.

It is preferred that the linker is dissolved in an organic solvent, such as dimethyl sulfoamide and dimethyl sulfoxide, at a concentration of about <NUM> to about <NUM>.

About <NUM>µL to about <NUM> of a sample containing an analyte is mixed with about <NUM>µL to about <NUM> of a linker solution, and the resultant mixture is left to stand or is stirred at about <NUM> to about <NUM> for about <NUM> hour to about <NUM> hours, preferably about <NUM> hour to about <NUM> hours. In this manner, the analyte can contact with the linker to bind the analyte to the linker.

It is preferred that the pH value of the reaction solution in which the analyte is to be contacted with the linker is about <NUM> to about <NUM>. This pH value is close to that in a biological environment. Therefore, a liquid sample collected from a living body can be used without any modification in the preparation of a measurement sample.

As the metal nanoparticles, nanoparticles of at least one metal selected from the group consisting of gold, silver, platinum, copper and palladium can be used. The particle diameter of each of the metal nanoparticles is, for example, equal or greater than <NUM> and equal or smaller than <NUM>. The particle diameter is preferably <NUM> to <NUM>, more preferably <NUM> or <NUM>. The shape of each of the metal nanoparticles is not limited. Metal nanoparticles each having a spherical, rod-like, shell-like, cube-like, triangular plate-like, star-like or wire-like shape can be used. Preferably used are spherical metal nanoparticles. The metal nanoparticles are commercially available from, for example, BBI Solutions or the like, and the commercially available products can be used. The method for measuring the particle diameter or the particle shape is performed in accordance with the instructions provided by a manufacturer. The particle diameter is a volume-based median diameter which is measured using a particle size distribution measurement device by a laser diffraction/scattering method. As the particle size distribution measurement device, "Microtrac MT3000II" manufactured by Nikkiso Co. and the like can be used. The term "particle diameter" as used herein refers to a diameter.

The contact between the linker having the analyte bound thereto and the metal nanoparticles is not particularly limited, as long as the contact is performed under the conditions where the linker can be bound to the surface of each of the metal nanoparticles.

For example, about <NUM>µL to about <NUM> of the reaction solution that has been prepared in (i) by contacting the analyte with the linker is diluted with the same volume of water (preferably ultrapure water), and then about <NUM>µL to about <NUM> of a metal nanoparticle solution (about <NUM>×<NUM><NUM> to <NUM>×<NUM><NUM> particles/mL) is added to the diluted solution. This mixed solution is left to stand or is stirred at about <NUM> to about <NUM> for about <NUM> minutes to about <NUM> minutes, preferably about <NUM> minutes to about <NUM> minutes. In this manner, the linker that has been bound to the analyte is contacted with the metal nanoparticles. As a result, an aggregate of the linker that has been bound to the analyte and the metal nanoparticles can be formed. It is preferred to perform the mixing of the linker that has been bound to the analyte with the metal nanoparticles on a glass-bottomed plate.

As a substance for promoting the formation of the aggregate, an inorganic salt or an acid may be added to the reaction system. An example of the inorganic salt is sodium chloride. An example of the acid is trifluoroacetic acid. The concentration of the inorganic salt is not particularly limited, as long as the effect to promote the formation of the aggregate can be exerted. For example, the final concentration of the inorganic salt may be <NUM> to <NUM>. The concentration of the acid is not particularly limited, as long as the effect to promote the formation of the aggregate can be exerted. For example, the final concentration of the acid may be <NUM> to <NUM>. After the mixing of the inorganic salt or the acid, the reaction system is left to stand or is stirred at <NUM> to <NUM> for <NUM> hour to <NUM> hours. In this manner, an aggregate of the metal nanoparticles can be formed.

Hereinafter, an analysis device for analyzing a measurement sample prepared by the preparation method mentioned in <NUM>, and an analysis method which the analysis device performs are described.

A schematic illustration of the configuration of the analysis device <NUM> is shown in <FIG>. The analysis device <NUM> is provided with: a microscope <NUM> for observing aggregates; a laser light source <NUM> for supplying excitation light that is emitted to a measurement sample by the microscope <NUM>; a control unit <NUM> for controlling the microscope <NUM> and the laser light source <NUM>; and an output unit <NUM> connected to the control unit <NUM>. Examples of a device equipped with the microscope <NUM> and the laser light source <NUM> include a slit-scanning confocal Raman microscope and a multi-focus Raman microscope. The control unit <NUM> may be a general-purpose computer.

The control unit <NUM> controls in such a manner that the laser light source <NUM> can emit excitation light, and the control unit <NUM> obtains an image from the microscope <NUM>.

When the microscope <NUM> is a slit-scanning confocal Raman microscope, the control unit <NUM> performs analysis under, for example, the following conditions.

These conditions may be adjusted appropriately depending on the type of the analyte, the material for the metal nanoparticles, and the shape of the metal nanoparticles.

Under these conditions, the laser irradiation is performed in a line irradiation mode. However, the laser irradiation may be performed in a spot irradiation mode.

The control unit <NUM> obtains an optical spectrum from the measurement sample, and the control unit <NUM> outputs information about the analyte to the output unit <NUM> on the basis of the obtained optical spectrum.

The term "information about an analyte" refers to information about the type of the analyte. Information about an optical spectrum characteristic to each of various substances is recorded in database previously. The information about the spectra is compared with that of a spectrum of the analyte, and the information about the analyte can be obtained from information about the similar spectra.

With respect to the spectrum, a single spectrum obtained from a single position in a measurement sample may be used, or spectra obtained from a plurality of positions in a measurement sample may be used.

A reagent for use in the preparation of the measurement sample mentioned in <NUM> above contains a linker that is not bound to a metal nanoparticle.

A reagent kit for use in the preparation of the measurement sample mentioned in <NUM> above includes: a linker that is not bound to a metal nanoparticle; and metal nanoparticles that are packed separately from the linker.

The present invention is described in detail hereinafter by way of examples. However, the present invention is not intended to be limited to these examples.

As the analytes, <NUM> amino acids, <NUM> dipeptides and <NUM> amyloid-β (Aβ) were used. As the linker, DSP (Dithiobis (Succinimidyl Propionate)) (Dojindo Laboratories, Product code: D629) was used. As the metal nanoparticles, gold nanoparticles ϕ40nm (<NUM>×<NUM><NUM> particles/aqueous Ml solution; BBI, catalogue No.: EMGC40) and the like were used.

The scheme of the sample preparation method according to examples is shown in <FIG>.

The same materials as those in Examples were used.

For obtaining an SERS spectrum, a slit-scanning confocal Raman microscope was used. The laser irradiation was performed by a line irradiation method. The conditions for measurement were as follows.

In order to verify as to whether or not a peak of an analyte-specific spectrum could be obtained by Examples, phenylalanine (Phe) was used as an analyte and the difference between a case where a linker was present and a case where the linker was not present was verified. Phe was added in such a manner that the final concentration of Phe in a sample became <NUM>. The results are shown in <FIG> to3 D. <FIG> shows SERS spectra of measurement samples which ware prepared in accordance with the procedure of Examples. <FIG> shows SERS spectra of measurement samples which ware prepared in accordance with the procedure of Examples except that no analyte was added. <FIG> shows SERS spectra of measurement samples which ware prepared in accordance with the procedure of Examples except that DSP was not added. <FIG> shows SERS spectra of negative control samples in which any analyte and DSP were not added.

Each spectrum shows an average spectrum of SERS spectrum obtained at <NUM> points.

As a result, in only measurement samples prepared in accordance with the procedure of Examples, peaks of analyte-dependent spectra were obtained (in <FIG>, points indicated by arrows were peaks).

Next, it was verified as to whether or not a peak of an analyte-specific SERS spectrum could be obtained by using each of <NUM> amino acids as an analyte. The results are shown in <FIG>. The symbol "a" in the drawings indicates an average spectrum obtained when an analyte was added, and the symbol "b" in the drawings indicates an average spectrum of a negative control which was obtained when the analyte was not added. In each of the amino acids, a peak of an analyte-dependent spectrum was obtained (in <FIG>, positions indicated by arrow heads are peaks).

Next, it was verified as to whether or not a peak of an analyte-specific spectrum could be obtained by using a dipeptide as an analyte. The dipeptides used are shown in <FIG>. For example, with respect to a dipeptide composed of different amino acid residues (e.g., Phe-Ala, Ala-Phe), dipeptides in which the positions of the two amino acid residues were changed were prepared and an average spectrum was obtained with respect to each of the dipeptides. An average spectrum of a negative control in which no dipeptide was added was also obtained. In <FIG>, the symbol "c" indicates a negative control. In each of the dipeptides used in the experiment, a peak of a characteristic spectrum was obtained (in <FIG>, positions indicated by arrow heads are peaks). With respect to dipeptides composed of different amino acid residues, even when the positions of the two amino acid residues were changed, a same spectrum peak was obtained.

Next, it was verified as to whether or not a peak of an analyte-specific SERS spectrum could be obtained by using Aβ42, Aβ40 or Aβ38. The results are shown in <FIG>. <FIG> shows an average spectrum obtained when Aβ42 was added as an analyte, and also shows an average spectrum of a negative control which was obtained when the analyte was not added. <FIG> shows an average spectrum obtained when Aβ40 was added as an analyte, and also shows an average spectrum of a negative control in which the analyte was not added. <FIG> shows an average spectrum obtained when Aβ38 was added as an analyte, and also shows an average spectrum of a negative control in which the analyte was not added. <FIG> show bright-field images of aggregates of gold nanoparticles in measurement samples used for obtaining average spectrums shown in <FIG>, respectively. <FIG> shows a bright-field image of aggregates of gold nanoparticles in a measurement sample that was a negative control. In each of Aβ42, Aβ40 and Aβ38, a peak of a specific spectrum was obtained (in each of <FIG>, positions indicated by arrow heads are peaks).

The effect of Examples was verified using the measurement samples prepared by the sample preparation method employed in Examples and measurement samples prepared by the sample preparation method employed in Comparative Examples.

Average spectra were obtained using phenylalanine, triptophan and tyrosine as analytes. With respect to negative controls, average spectra were also obtained in the same manner. The results are shown in <FIG>. With respect to each of the measurement samples prepared by the preparation method employed in Examples, a peak of an analyte-specific peak was confirmed when any one of phenylalanine, triptophan and tyrosine was used (in <FIG>, positions indicated by arrow heads are peaks). Meanwhile, with respect to the measurement samples prepared by the preparation method employed in Comparative Examples, a peak of an amino acid-specific spectrum was not confirmed for each of the amino acids. From these results, it was demonstrated that the detection sensitivity could be improved by the sample preparation method of Examples compared with those of Comparative Examples.

Next, the effect of Examples was verified using the measurement samples prepared by the sample preparation method employed in Examples and measurement samples prepared by the sample preparation method employed in Comparative Examples, in which dipeptides (Phe-Trp, Gly-Phe) and Phe-(Tyr)<NUM> and Aβ38 were used as analytes. Phe-(Tyr)<NUM> represents a peptide in which eight tyrosine residues are linked, followed by a phenylalanine residue. With respect to negative controls, average spectra were also obtained in the same manner. The results are shown in <FIG>. With respect to each of the measurement samples prepared by the preparation method employed in Examples, a peak of an analyte-specific spectrum was confirmed when any one of the dipeptides, Phe-(Tyr)<NUM> and Aβ38 was used (in <FIG>, positions indicated by arrow heads are peaks). Meanwhile, with respect to the measurement samples prepared by the preparation method employed in Comparative Examples, a peak of an amino acid-specific spectrum was not confirmed for each of the amino acids. From these results, it was demonstrated that the detection sensitivity could be improved by the measurement sample preparation method of Examples compared with those of Comparative Examples whatever the level of the molecular weight of the analyte might be (i.e., regardless of the use of a low-molecular weight substance or a high-molecular-weight substance (e.g., Aβ)).

Next, the reason why the detection sensitivity was enhanced in Examples was examined. A measurement sample composed of only gold nanoparticles and a measurement sample prepared by mixing only gold nanoparticles and DSP with each other were prepared, and an absorption spectrum of each of the measurement samples was obtained. The results are shown in <FIG>. In <FIG>, the symbol "a" indicates a spectrum of a measurement sample composed of only gold nanoparticles, and the symbol "b" indicates a spectrum of a measurement sample prepared by mixing only gold nanoparticles with DSP. The measurement sample composed of only gold nanoparticles showed one peak of an absorption spectrum at about <NUM>, while the measurement sample prepared by mixing only gold nanoparticles with DSP showed an absorption in a range from about <NUM> to about <NUM>. It was considered that this absorption was attributed to aggregates of gold nanoparticles which were formed as the result of the addition of DSP. It was considered that, in the conventional method, because the metal nanoparticles and the linker were mixed with each other in advance, the gold nanoparticles were aggregated before the binding of the analyte to the linker occurred, and therefore the analyte did not enter into the aggregates satisfactorily.

In order to verify the influence of the chain length of a linker on detection sensitivity, measurement samples were prepared using DSP, DSH (dithiobis (succinimidyl hexanoate)) and DSU (dithiobis (succinimidyl undecanoate)), and average spectra of SERS spectra of the measurement samples were obtained. The results are shown in <FIG>. When each of the linkers was used, an analyte-specific spectrum peak was confirmed. From the results, it was demonstrated that the preparation method of Examples could be employed regardless of the types of the linkers.

Claim 1:
A preparation method for preparing a measurement sample comprising an aggregate of metal nanoparticles having an analyte bound thereto,
the preparation method comprising:
contacting the analyte with a linker to bind the analyte to the linker; and
contacting the linker that has been bound to the analyte with the metal nanoparticles to bind the linker to the metal nanoparticles, wherein
the analyte has a functional group, and the linker has a reactive group capable of reacting with the functional group, and wherein
the functional group comprises at least one group selected from the group consisting of an amino group, a carboxyl group and a hydroxyl group, and wherein
in case the functional groups is the amino group or the hydroxyl group, the reactive group comprises at least one group selected from the group consisting of an N-hydroxysuccinimide ester group, an isothiocyanate group, an isocyanate group, an acyl azide group, a sulfonyl chloride group, an aldehyde group, an imide ester group, a fluorobenzene group, an epoxide group, a carbodiimide group, a carbonate group, and a fluorophenyl ester group, and wherein
in case the functional group is a carboxyl group, the reactive group comprises at least one group selected from the group consisting of a hydroxyl group and an amino group.