KR20200141575A - Method for detecting target material by SERS using assembly of metal nanocubes with hierarchical structure - Google Patents

Abstract

The present invention relates to: a method for detecting a target material by surface-enhanced Raman scattering using electric field enhancement by forming a hierarchical structure assembly, wherein metal nanoparticles modified with DNA of a complementary sequence with a controlled length are spaced at nanometer intervals; and a kit for detecting a target material for implementing the method.

Description

A method for detecting target material by SERS using assembly of metal nanocubes with hierarchical structure using a hierarchical structure of metal nanocubes formed by DNA hybridization.

The present invention is a method for detecting a target material by surface-enhanced Raman scattering using electric field enhancement by forming an assembly of a hierarchical structure, spaced apart at nanometer intervals of metal nanoparticles modified with DNA of a complementary sequence of a controlled length, and It relates to a kit for detecting a target substance for implementing this.

Recently, several high-sensitivity technologies have been developed for the detection of biomolecules such as DNA and proteins, leading to remarkable developments in clinical diagnosis, disease treatment, and drug discovery. These technologies include polymerase chain reaction (PCR), fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), electrochemistry (EC), and colorimetry. ), and surface-enhanced Raman scattering (SERS). PCR is the most preferred technique due to its high-speed, high-volume screening ability and high sensitivity, but it is relatively expensive and time-consuming, and has disadvantages such as polymerase-induced mutations or unwanted amplification of non-target DNA sequences. Can be derived. In addition, other techniques including FRET, SPR, EC, and colorimetric methods allow easy and fast analysis in biodetection, but have limitations in ultra-sensitive detection.

Accordingly, SERS, which is based on an electric field enhancement induced by localized surface plasmon resonance on a noble metal surface such as gold or silver, is attracting attention in the field of chemical and biological detection due to its ultrahigh sensitivity. Electric field enhancement, a major factor in increasing the sensitivity of SERS, is dependent on the shape of the precious metal surface used. By increasing the electric field, the sensitivity of the SERS technique can be improved to the single-molecule level. In order to improve and control SERS activity, various types of plasmonic nanostructures, such as branched, nanoporous, and anisotropic nanostructures, as well as materials having roughened surfaces and core/shell types, have been prepared. In addition, the electric field is significantly affected by plasmon coupling within nanometer-sized gaps between plasmonic nanostructures. In this regard, various nanogap-engineering-based synthesis to prepare intermolecular- and intramolecular-nanogaps by lithography, wet chemical synthesis, and assembly of nanostructures for enhancing SERS activity The strategy is being studied. However, accurate gap engineering to fabricate reproducible and reliable SERS-active substrates remains challenging.

The present inventors discovered nanostructures capable of remarkably and uniformly enhancing the SERS effect, and as a result of diligent research efforts to provide a DNA detection method with reproducibility and/or sensitivity based on this, and as a result of hybridization with target DNA To form a magnetic microbead of a relatively large size modified with a DNA fragment having a sequence complementary to a part of the target DNA and a first metal nano-modified with a DNA fragment having a sequence complementary to another part of the target DNA After binding the particles, a plurality of second metal nanoparticles modified with a DNA fragment of a complementary sequence capable of specifically binding to the DNA fragment bound to the first metal nanoparticle reacts, and the nanoparticles by DNA hybridization In the case of forming an assembly but using a cube-shaped particle as the nanoparticle, the assembly formation speed is improved by DNA hybridization, and since it is located face-to-face adjacent, it can exhibit a uniform electric field enhancement effect over the entire surface. Therefore, by labeling the Raman active material on the DNA modified on the first metal nanoparticle, and adjusting the distance between the first metal nanoparticle and the second metal nanoparticle to the length of the DNA fragment of the complementary sequence modified Since the SERS effect can be exhibited, it was confirmed that a trace amount of target DNA present in the sample can be detected more sensitively and reproducibly, and the present invention was completed.

As one aspect for achieving the above object, the present invention is a magnetic microbead labeled with a first DNA comprising a first sequence complementary to a part of the target DNA to be detected, and complementary to the rest of the target DNA to be detected. By reacting the first metal nanocube labeled with the second DNA containing the second sequence with the sample predicted to contain the target DNA to be detected, in the presence of the target DNA in the sample, one or more magnetic microbeads are formed by DNA hybridization. A first step of forming a first composite in which the first metal nanocubes are combined; Magnetic microbeads of the first metal nanocube by reacting the first complex with a second metal nanocube modified with a third DNA comprising a third sequence complementary to the second DNA labeled on the first metal nanocube A second step of forming a second composite in which a second metal nanocube is bonded to the other surface not bonded to the other; And a third step of measuring a surface enhanced Raman scattering (SERS) signal from the second complex, comprising: a method for detecting a target DNA, wherein the second DNA modified in the first metal nanocube is Raman It is labeled as an active material, so that the Raman active material is located between the first metal nanocube and the second metal nanocube to show an improved Raman signal by the enhancement of the electric field between the first metal nanocube and the second metal nanocube. It provides a method that is characteristic.

The present invention exhibits the SERS effect by enhancing the electric field at adjacent points of two or more nanostructures spaced at a predetermined interval, and according to the specificity of DNA hybridization and the number of nucleic acids due to the formation of a double helix structure. It was designed to discover a DNA detection method that is sensitive, reproducible, and capable of quantitative analysis using the uniformity of the length. Specifically, it binds to the first metal nanocube that becomes a seed through hybridization with the target DNA from magnetic microbeads of relatively large size, and then introduces the second and third metal nanocube layers by additional DNA hybridization therefrom. By expanding the assembly within a predetermined size in a controlled manner, a remarkably enhanced SERS effect can be exhibited, but reproducible results can be obtained. Furthermore, the detection limit is improved to the sub-femtomol level and a DNA detection method capable of even quantitative analysis was discovered. It is a feature.

The DNA detection method of the present invention may further include a 1-1 step of removing the first metal nanocube not bound to the magnetic microbead by applying a magnetic field from the outside and washing between the first step and the second step. have. The first metal nanocube that is not bound to the magnetic microbead through step 1-1, that is, has not hybridized with the target DNA, forms an assembly through a later step, thereby preventing generation of a false-positive signal. I can.

The first metal nanocube and the second metal nanocube may each independently use cube-shaped particles of a metal selected from the group consisting of gold, silver, platinum, palladium, and copper, but are not limited thereto.

At this time, the first metal nanocube and the second metal nanocube are particles having an edge length of 10 to 300 nm, and the deviation of the edge lengths of the first metal nanocube and the second metal nanocube does not exceed ±20%. You can choose not to, but is not limited thereto.

In the present invention, the term "cube (type)" does not mean only a cube having the same length in the three directions of horizontal, vertical, and height, but a hexahedron capable of growing an assembly by being adjacent to it face-to-face May include without limitation.

For example, the magnetic microbeads used in the detection method of the present invention may be particles having an average diameter of 0.5 to 10 μm, but are not limited thereto. The use of the magnetic microbead not only makes it possible to more easily perform the 1-1 step for removing the first metal nanocube that did not participate in the reaction, but also the surface thereof due to the low curvature due to the relatively large size. By providing an environment similar to forming an assembly on a flat surface rather than a curved surface when manufacturing an assembly formed by the first metal nanocube and the second metal nanocube, the random growth of the assembly is blocked and magnetic microparticles It can be controlled so that it can only grow in the direction of the height adjacent to and higher. Based on this controlled growth, a uniform signal enhancement can be achieved to enable quantitative analysis.

In the detection method of the present invention, the first to third sequences may each independently consist of 6 to 30 nucleic acids. In particular, the number of nucleic acids constituting the second sequence and the third sequence included in the second DNA and the third DNA labeled on the first metal nanocube and the second metal nanocube, respectively, is the first metal in the state of forming an assembly. Since the distance between the nanocube and the second metal nanocube is determined, it has a decisive effect on signal enhancement of the Raman active material, which is labeled on the second DNA and located between the first metal nanocube and the second metal nanocube. Therefore, it is desirable to adjust the number of nucleic acids to maintain a distance of 1 to 10 nm, which is a range in which the SERS effect can be maximized. However, when the number of nucleic acids is less than 6, incomplete binding may be caused and/or may be easily dissociated, and it may be difficult to stably maintain the assembly structure. Meanwhile, the number of nucleic acids constituting the first sequence and the second sequence included in the first DNA and the second DNA labeled on the magnetic microbead and the first metal nanocube, respectively, is the distance between the magnetic microbead and the metal nanocube assembly. To decide. At this time, when the distance between the magnetic microbead and the first metal nanocube that becomes the seed of is longer than the first metal nanocube and/or the second metal nanocube, the magnetic microbead of the first metal nanocube becomes a seed. The assembly can grow to the bottom of the side adjacent to the. Since this is performed in a relatively narrow space, it is possible to induce non-uniform and uncontrolled arbitrary growth, and as described above, it may adversely affect quantitative analysis by uniform signal enhancement based on controlled growth.

For example, in the DNA detection method of the present invention, the target DNA to be detected may be composed of 10 to 100 nucleic acids. The target DNA refers to a sequence that substantially participates in detection, and the DNA present in an actual sample may be a DNA composed of a longer sequence including this, for example, a DNA composed of tens to tens of thousands of nucleic acids. In the detection method of the present invention, when the number of nucleic acids constituting the target DNA is less than 10, it is difficult to bind firmly, and even after forming the complex, it can be easily separated from the complex by temperature conditions, and thus sensitive detection may be difficult. On the other hand, if it exceeds 100, not only does it take a long time for DNA hybridization, but also because the distance between the magnetic microparticles and the metal nanocube assembly is long, it is difficult to control the degree of formation of the assembly. It can be disadvantageous.

As described above, it is assumed that the DNA is positioned perpendicular to the surface of the structure in order to constantly adjust the spacing of the structure to be bound by adjusting the number of nucleic acids that bind complementarily. For example, if this is not guaranteed, the DNA hybridization reaction rate cannot be guaranteed, and a uniform reaction is also difficult to expect. Accordingly, the first DNA to the third DNA modified on the magnetic microbead, the first metal nanocube, and the second metal nanocube may further include a spacer sequence in a direction in contact with these particles. The spacer sequence exhibits a higher interaction with the surface of the particle and covers the surface, thereby revealing the possibility that nucleic acids constituting the first to third sequences involved in DNA hybridization are lying close to the surface or attached to the surface to delay DNA hybridization. It is desirable to be able to block it. Substances other than nucleic acids may be included for this purpose. For example, in the present invention, A ₁₀ -PEG was used as a spacer sequence, but the scope of the present invention is not limited thereto.

Meanwhile, the first to third DNAs may be bonded to each particle through a linker compound. The linker compound refers to a compound linked to the 3'or 5'end of the DNA to attach the first to the third DNA to the surface of each particle. The type of linker compound is not limited as long as it produces small aggregated nanoparticles that will not precipitate in solution. Methods for cross-linking particles to DNA via linker compounds are known in the art (see Feldheim, The Electrochemical Society Interface, Fall, 2001, pp. 22-25). For example, one end of the linker compound holds a reactive group attached to the surface of the core particle, preferably a sulfur-containing group such as a thiol group or a sulfhydryl group (HS). The reactor may be a compound having the formula of RSH containing sulfur as a derivative of alcohol and phenol. Alternatively, the reactor may be a thiol ester or a dithiol ester having a formula of RSSR' or RSR', respectively. Alternatively, the reactor may be an amino group (-NH ₂ ). Additionally, the linker compound may be connected with various reactive groups such as -NH ₂ , -COOH, -CHO, -NCO, and an epoxide group, and the sulfur-containing group may bind to a metal such as a gold surface. A variety of such reactive groups are known in the art and can be used in the present method.

In the present invention, the term "Raman active material" is a material detectable by Raman spectroscopy, and the Raman active material is an organic or inorganic molecule, atom, complex or synthetic molecule, dye, naturally occurring dye (phycoerythrin, etc.), C60 And organic nanostructures, such as buckyballs, carbon nanotubes, quantum dots, organic fluorescent molecules, and the like. Specifically, as examples of Raman active substances, mercaptobenzoic acid, aminothiophenol, FAM, Dabcyl, TRITC (tetramethyl rhodamine-5-isothiocyanate), MGITC (malakit green isothiocyanate), XRITC ( X-rhodamine-5-isothiocyanate), DTDC (3,3-diethylthiadicarbocyanine iodide), TRIT (tetramethyl rhodamine isothiol), NBD (7-nitrobenz-2-1 ,3-diazole), phthalic acid, terephthalic acid, isophthalic acid, para-aminobenzoic acid, erythrosine, biotin, digoxigenin, 5-carboxy-4',5'-dichloro-2',7' -Dimethoxy, fluorescein, 5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethyl aminophthalocyanine, azomethine, cyanine (Cy3, Cy3.5, Cy5), xanthine, succinylfluorescein, aminoacridine, quantum dot, carbon allotrope, cyanide, thiol, chlorine, bromine, Methyl, phosphorus, sulfur, etc., but are not limited thereto, and the Raman active material used should exhibit a distinct Raman spectrum. Preferably, it is an organic fluorescent molecule containing a fluorescent molecule of the Cy3, Cy3.5, Cy5 or FAM, Dabcyl, Rhodamine series of fluorescent organic molecules of siamine, or non-fluorescent Raman active substances containing mercaptobenzoic acid and aminothiophenol. However, it is not limited thereto. These organic fluorescent molecules resonate with the excitation laser wavelength used in the Raman analysis and thus have the advantage of being able to detect higher Raman signals, but the scope of the present invention is not limited thereto.

In addition, in the detection method of the present invention, the second complex is reacted with the first metal nanocube modified with a second DNA comprising a second sequence complementary to the third DNA labeled on the second metal nanocube. The 2-nth step of forming a 2-n complex in which additional first metal nanocubes are bonded to the remaining surfaces of the second metal nanocube, which is not combined with the first metal nanocube and does not face the magnetic microbead. (More than n is an integer of 1 or more) may be further included.

Further, step 2-n and optionally step 2-(n+1) performed in the same manner as in step 2 may be sequentially repeated. At this time, in the additionally performed step, the size of the assembly consisting of the first metal nanocube and the second metal nanocube, excluding the magnetic microbeads, from the resulting 2-n-th composite or the 2-(n+1) composite It can be repeatedly performed by adjusting the number of times to form within the range of the average radius of 30 nm to 900 nm.

Specifically, the detection method of the present invention maintains the interface between the first metal nanocube and the second metal nanocube labeled with the Raman active material at several nm level so as to maximize the SERS effect, and in a controlled manner. The purpose of this is to detect a trace amount of target DNA by measuring the increased Raman signal by increasing the number while forming an assembly of a hierarchical structure. Accordingly, by alternately forming layers of the first metal nanocube and the second metal nanocube, the number of interfaces between the first metal nanocube and the second metal nanocube can be rapidly increased. However, if the size of the finally formed assembly becomes larger than the wavelength irradiated to excite the Raman active molecule, penetration into the assembly may become impossible and the Raman signal itself may not be generated.Therefore, the size of the finally formed assembly does not exceed this. It is necessary to adjust it so that it is not. Therefore, considering that the wavelength of the light source used for excitation of the Raman active material is ultraviolet or near-infrared of 1000 nm or less, the first metal nanocube and the second metal nanocube are maintained until the radius of the assembly is maintained at 900 nm or less. The layer may be repeatedly introduced, but is not limited thereto, and as described above, it may be determined in consideration of the wavelength to be used for excitation of the Raman active material.

In the detection method of the present invention, the signal for the second complex measured from the third step may provide a signal that is augmented by 100 times or more compared to the signal measured for the first complex at the same wavelength. Specifically, in the first complex, a first metal nanocube labeled with a Raman active material is bound to a magnetic microbead, and a signal measured therefrom may be a signal from the Raman active material itself. However, since a plurality of Raman active materials are labeled on one first metal nanocube, and multiple first metal nanocubes may be attached on one magnetic microbead, the total Raman active material is not a signal of a single Raman active material. It may be a number that increases proportionally to the number. On the other hand, the first complex includes a second metal nanocube bonded to five surfaces of the first metal nanocube at intervals of several nm, thereby exerting the SERS effect at these interfaces, so that the signal of a simple Raman active material It may appear as a sum of signals synergistically enhanced by an enhanced electric field at the interface rather than the sum, and the resulting signal may be enhanced by 100 times or more compared to the value measured by the first complex.

In another aspect, on the first side, a first biomaterial that specifically binds to another part of the substance to be detected is included, and on the other 5, a first'metal nanocube labeled with the first' DNA is prepared. The first step; Magnetic microbeads labeled with one or more first biomaterials that specifically bind to a part of the substance to be detected, and a first surface prepared from the first step, which specifically binds to another part of the substance to be detected. It contains a biological material, and on the other 5 sides, the first'metal nanocube labeled with the first'DNA is reacted with a sample predicted to contain the substance to be detected, so that specific binding is achieved in the presence of the target substance in the sample. A second step of forming a first'composite in which at least one first'metal nanocube is coupled to a magnetic microbead; The first'complex is reacted with a second'DNA modified 2'metal nanocube containing a sequence complementary to the first'DNA labeled on the first' metal nanocube, and the first metal nanocube A third step of forming a second'composite in which a second'metal nanocube is bonded' on the other side of the cube that is not combined with the magnetic microbeads; And a fourth step of measuring a surface-enhanced Raman scattering signal from the second' complex, comprising: a method for detecting a target material, wherein the first'DNA modified in the first'metal nanocube is labeled with a Raman active material. The Raman active material is located between the first'metal nanocube and the second' metal nanocube to show an improved Raman signal by the enhancement of the electric field between the first'metallic nanocube and the second' metal nanocube. It provides a method that is characteristic.

The second and third steps are carried out in reverse order, first combining the first'metal nanocube with the second' metal nanocube, and reacting with the magnetic microbeads and samples labeled with the first biomaterial. Phosphorus, a method of detecting a target substance.

In another aspect, the present invention provides a magnetic microbead labeled with at least one first biomaterial that specifically binds to a part of a target substance to be detected; On the first side, a first biomaterial that specifically binds to another part of the substance to be detected is included, and on the other side, a first metal nanocube labeled with a first'DNA'; And it provides a kit for detection of a target substance by SERS comprising a second'DNA-modified 2'metal nanocube containing a sequence complementary to the first'DNA labeled on the first'metal nanocube. .

Specifically, in order to detect DNA as a target material, the magnetic microbeads included in the kit of the present invention are labeled with a first DNA comprising a first sequence complementary to a part of the target DNA to be detected, and the first'metal nanoparticles' The cube contains a second sequence that is complementary to the rest of the target DNA to be detected and is labeled with a second DNA to which a Raman active molecule is bound, and the second'metal nanocube includes the second DNA labeled on the first metal nanocube. 3DNA including a complementary third sequence may be modified.

For example, the kit for detecting target DNA according to the present invention may be provided with a buffer solution to provide an environment suitable for DNA hybridization, but is not limited thereto. In this case, the buffer may be a buffer solution widely used in the art, for example, a phosphate-based buffer such as PBS or PBSS, but is not limited thereto.

Furthermore, the kit for target DNA detection according to the present invention is sequence-specific and provides a remarkably enhanced Raman signal by sequential DNA hybridization, forming an assembly of hierarchical and controlled size and/or spacing nanocubes By doing so, it can be used to detect target DNA with high sensitivity.

Detection of the target DNA using the kit of the present invention may be performed according to the method for detecting the target DNA of the present invention, but is not limited thereto.

In the formation of a complex by complementary binding with the target DNA of the present invention and the formation of a hierarchical, sized and/or spaced nanocube assembly comprising an increased number of nanocubes using the complementary binding of additional DNA The principle of detecting the target DNA with high sensitivity achieved by the method, that is, the present invention is not limited to the detection of DNA, and magnetic microbeads and metals modified with ligands capable of specifically binding to the target substance instead of DNA By using a nanocube, it can be applied without limitation to detection of a target analyte using binding between antigen-antibody or binding between ligand-receptor, forming mutually specific binding.

For example, prior to labeling DNA on the surface of a metal nanocube for manufacturing a nanocube assembly, the surface of the metal nanocube is modified with SDS, etc. to have a negative charge, thereby functionalized with APTES ((3-aminopropyl)triethoxysilane), etc. It can be induced to be electrostatically attached to the glass or silicon wafer substrate through one side of the cube, and then by labeling the DNA, DNA can be selectively labeled on the remaining 5 sides except the side in contact with the substrate. The DNA-labeled metal nanocube can be detached from the substrate by applying physical stimulation such as ultrasonic treatment. Therefore, by using metal nanoparticles selectively labeled with DNA on 5 sides except 1 side prepared as described above, a material capable of specifically binding to the target material to be detected on 1 side where DNA is not labeled Since it can be introduced, the metal nanocube assembly of the present invention can be used to detect target substances based on SERS through complementary binding of DNA as well as antigen-antibody reactions that specifically bind to each other, and reactions between ligands and receptors. Can be applied.

In the detection method of the present invention, by using a metal nanocube instead of using a conventional spherical metal nanoparticle, not only can it be assembled at a faster bonding speed by a face-to-face interaction over the entire area, but also Since it exhibits an even electric field enhancement effect over the span, uniform signal enhancement can be implemented, and thus a trace amount of target DNA in the range of 100 aM to 10 pM can be sensitively and quantitatively detected within a faster time.

1 is a hierarchical-nanocube-assembly-based surface enhanced Raman scattering (H-Cube-SERS) assay for DNA detection according to an embodiment of the present invention It is a diagram schematically showing the principle. In the presence of target DNA (target DNA, hepatitis A virus; HAV) strands, target-capturing Au nanocube (TC-AuNC) and DNA-modified magnetic microparticles (DNA) -MMP) forms a "TC-AuNC"-"MMP" complex (NC-complex-1) through sandwich-hybridization (target-capturing step), so Raman on TC-AuNC The surface-enhanced Raman scattering signal can be provided from the molecule (Cy3). However, the SERS signal intensity of NC-complex-1 is too weak to detect a low concentration of target DNA due to the absence of plasmonic coupling. When forming a plasmonic AuNC cluster (NC-complex- 2 ) assembled by hybridization with a signal-amplifying Au nanocube (SA-AuNC), the SERS signal strength is the assembled region of the AuNC cluster. The height was enhanced by the strong plasmonic coupling in (signal amplifying step). Therefore, the sensitivity to the target DNA was significantly improved, leading to a low limit of detection (LOD).
Figure 2 is a standard curve of the fluorescence (Cy3) signal intensity of the known concentration of the Cy3-labeled DNA strand (blue dot) for quantitative analysis of the DNA loading capacity of AuNC and AuNS of 50 nm size (Standard curve, red line) Is a diagram showing. The intensity of the DNA fluorescence signal detached from AuNC (black dot) and AuNS (red dot) is shown on a standard curve.
3 is a diagram showing a TEM image of an assembled AuNC cluster according to an embodiment of the present invention. The scale bar is 50 nm.
Figure 4 is a diagram showing (a to c) the synthesis of AuNC and AuNS and their spectroscopic properties, and (d) the preparation of the AuNC assembly by DNA sequence specific hybridization using AuNC and their properties. (a and b) are TEM images of the synthesized nanoparticles, AuNC and AuNS, respectively. AuNC (edge length) and AuNS (length) of 50 nm size were uniformly synthesized with a high yield of 95% or more. (c) shows the UV-vis spectrum of the synthesized nanoparticles. Sharp extinction peaks appeared at 563 nm and 529 nm for AuNC and AuNS, respectively. (d-g) show the DNA-sequence-specific hybridization ability for the selective capture of the target DNA strand and SA-AuNC. (d and f) show changes in the UV-vis spectrum of the assay solution recovered using an external magnetic field after the target capture step and after the signal amplification step, respectively. The inset represents the color of the assay solution under a magnetic field after DNA hybridization. (e and g) are TEM images of sequence specific sandwich hybridization complexes formed by different SA-AuNCs during the signal amplification step, each formed by different linker DNA during the target capture step. In the presence of the complementary target DNA or SA-AuNC, the assay solution turned colorless, and the UV-vis extinction peak intensity rapidly decreased. This represents the formation of DNA-sequence-specific NC-complex-1 or NC-complex-2, respectively. The AuNC-MMP complex formed by DNA-sequence-specific hybridization can be clearly identified on the TEM image.
Figure 5 shows the form of DNA-modified nanoparticles (curvature) It is a diagram showing the effect-induced binding and dissociation properties thereof. (a) is the morphology (curvature) of hybridization between DNA-modified Au nanoparticles It is a diagram schematically showing the effect. Due to structural properties, in the case of AuNC (flat surface), the number of DNA strands (referred to as "effective binding DNA strands") participating in hybridization is higher than in the case of AuNS (convex surface), which is the morphology (curvature) effect. Indicates that The number of "effective binding DNA strands" influences the binding and dissociation properties of the DNA-modified nanoparticles. That is, a higher number of "effective binding DNA strands" leads to a faster binding rate and a higher melting transition temperature. (b and c) show the structure-dependent DNA binding kinetics during the target-capture and signal-amplification steps shown in FIG. 1, respectively. When the TC-probe hybridizes to the MMP (b, target-capture step), the binding kinetics of each TC-probe is independent of the shape of the nano-probe, due to the excessive binding sites due to the rough surface of the micron-sized MMP. similar. Conversely, cube-shaped nanoprobes (SA-AuNC) have a higher binding rate compared to spherical nanoprobes (SA-AuNS), due to the presence of a higher number of "effective binding DNA strands" for DNA hybridization between nanoprobes. (C, signal-amplification step). (d) shows the structure-dependent dissociation of nanoprobe aggregates. AuNC aggregates, due to the morphology (curvature) effect, exhibited a higher melting point ( T _m ) and a narrower melting transition temperature range than AuNS aggregates. The inset shows the first derivative of the dissociation (denaturation) curve of AuNC and AuNS aggregates.
6 is a diagram showing the UV-vis spectrum of an assay solution according to the binding of MMP and gold nanocubes by DNA hybridization according to an embodiment of the present invention as a function of hybridization time.
7 is a diagram showing a change in solution color and UV-vis spectrum according to the temperature of the agglomerated AuNC according to an embodiment of the present invention.
8 is a diagram showing the detection of SERS-based supersensitive DNA using 3D-assembled plasmonic nanoprobe cluster. (a) is the concentration of the target DNA (HAV, 100 aM to 10 pM) and the non-complementary target DNA (HBV, 10 pM) of the AuNC-MMP complex (NC-complex-1 and NC-complex-2) Shows the SERS signal intensity plot. The SERS signal intensity of the formed AuNC cluster was highly enhanced (red dot) by strong plasmonic coupling in the assembled portion of the cluster, indicating an improved LOD. Compared to the control experiments (blue and green dots), the LODs for NC-Complex-1 and NC-Complex-2 are 1 pM and 100 aM, respectively. (b) shows the SERS spectrum of the AuNC-MMP complex (NC-complex-1 and NC-complex-2) according to the concentration of the target DNA strand. (c) is the AuNC- and AuNS-MMP complexes (NC-complex-2 and NS-complexes according to the concentration of the target DNA strand (HAV, 100 aM to 10 pM) and non-complementary target DNA (HBV, 10 pM) -2) shows the SERS signal intensity plot. In the case of NS-complex-2, due to inefficient binding (hybridization) and non-uniform plasmonic coupling, compared to NS-complex-2, the SERS signal intensity was lower and showed a higher standard deviation. All spectra were obtained with an acquisition time of 10 seconds using an excitation laser of 633 nm (190 μW), and recorded for a wavenumber of 500 to 1,800 cm ^-1 .
9 shows the results of a boundary element method (BEM) simulation of an electrical near field of a single plasmonic nanoparticle and a 3D-assembled nanoparticle cluster. (a-c) show 2D (top) and 3D (bottom) plots of the electrical near-field distribution for a single AuNC, 3D-assembled AuNC cluster, and (c) 3D-assembled AuNS cluster, respectively. The inset shows the 3D structural model used in the BEM simulation. The blue-shaded area represents the plane where the electric field was evaluated ( y - z plane). In all cases, x - linearly polarized in the direction of light (linearly polarized light) (wavelength 633 nm) is z - were examined in a direction. In the case of the 3D-assembled AuNC cluster shown in (b), the electric field is in the assembled part of the AuNC cluster due to the strong plasmonic coupling at the face-to-face interface, unlike the case of the single AuNC shown in (a). It appeared noticeably stronger. In addition, unlike plasmonic coupling at the surface-to-surface interface that induces a strong electric field over a large area, the point-to-point contact formed in the case of AuNS is due to the convex surface of the AuNS, and thus (c) As shown, electric field enhancement was induced only at narrow local points. (d-i) show 2D plots of the electrical near-field distribution for 3D-assembled clusters. Specifically, (d to f) are results for AuNC, and (g to i) are results for AuNS. The blue shaded area represents the plane of the electric field being evaluated: (d and g) are y - z planes, (e and h) are x - z planes, and (f and i) are x - y planes. In all cases, light polarized linearly in the x -direction (wavelength 633 nm) was irradiated in the z -direction.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

<Reagents and substances>

Magnetic Microparticles (Dynabeads ^® MyOne™ Carboxylic Acid, average diameter 1 mm) was purchased from Invitrogen Dynal AS, Oslo, Norway. HPLC-purified oligonucleotides were purchased from IDT (IDT, Inc., Coralville, IA, USA). Gold(III) chloride trihydrate (HAuCl ₄ ·3H ₂ O, ≥99.9%), cetyltrimethylammonium bromide (CTAB), benzyldimethylhexadecylammonium chloride (BDAC), L-ascorbic acid (AA, ≥99.9%), DL-dithiothreitol (DTT, ≥99.0%), sodium dodecyl sulfate (SDS, ≥98.5%), 2- (N - Dimorpholino Reno) ethanesulfonic acid (2- (N -morpholino) ethanesulfonic acid ; MES, ≥99.0%), N - (3- dimethylaminopropyl) - N '- ethylcarbodiimide hydrochloride (N - ( 3-dimethylaminopropyl) - N '-ethylcarbodiimide hydrochloride; EDC, commercial grade), ethylenediaminetetraacetic acid (ethylenediaminetetraacetic acid; EDTA, 99.4-100.06% ), potassium cyanide (potassium cyanide; KCN, ≥96.0% ), and tween-20 (Tween ^® -20) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cetyltrimethylammonium chloride (CTAC) was purchased from Chemical Industry Co., Ltd., Tokyo, Japan. Sodium borohydride (NaBH ₄ , 97.0%) and sodium bromide (NaBr, ≥99.0%) are DAEJUNG Chemicals & Metals Co., Siheung, Gyeonggi, Korea, respectively, and Samjeon Pure Water. It was purchased from a chemical company (Samchun Pure Chemical Co., Pyungtack, Gyeonggi, Korea). All chemicals were used without further purification. NANO pure water (Millipore, Milli-Q 18.2 MQ.cm) was used for all experiments.

<Characteristic analysis method>

The morphology of the synthesized nano-probe and AuNC-assembled cluster was confirmed using a TEM system (H-7600, Hitachi). UV-vis spectra were obtained using a UV-vis spectrophotometer (HP 8453, Agilent Technologies). Fluorescence measurement for quantification of DNA-loading capacity was performed using a fluorophotometer (FP-8300, JASCO Inc.).

Manufacturing example 1: 50 nm Size Au Nanosphere (AuNS) synthesis

Gold nanospheres (AuNS) were synthesized using a modified seed-mediated growth method (Zheng, Y. et al. , Part. Part. Syst. Charact., 2014, 31: 266-273). Specifically, i) synthesized CTAB-capped Au seeds, ii) synthesized 10 nm sized AuNS, iii) grown into 50 nm sized AuNS, and performed the following three-step reaction to obtain 50 nm sized AuNS. AuNS was synthesized.

First, in a 50 mL round bottom flask, an aqueous solution of CTAB (200 mM, 5 mL) and an aqueous solution of HAuCl ₄ (0.5 mM, 5 mL) were mixed with stirring (300 rpm) at 27°C. Immediately prepared NaBH ₄ aqueous solution (10 mM, 0.6 mL) was rapidly added to the flask, after which the mixture immediately turned brown. The reaction mixture was stirred at 300 rpm for 3 minutes, and then incubated at 27° C. for 3 hours without agitation to synthesize CTAB-capped Au seeds.

Next, 50 μL of the obtained CTAB-capped Au seeds were added to a mixture of CTAC solution (200 mM, 2 mL) and AA solution (100 mM, 1.5 mL). Subsequently, an aqueous HAuCl ₄ solution (0.5 mM, 2 mL) was rapidly injected into the reaction mixture, followed by stirring at 27° C. at 300 rpm for 15 minutes. After centrifugation at 20,000 × g for 30 minutes, the product was washed once with distilled water, thereby synthesizing AuNS having a size of 10 nm. For later use, it was dispersed in CTAC solution (20 mM, 1 mL).

Finally, using a syringe pump at an injection rate of 2 mL/h, CTAC solution (100 mM, 2 mL), AA solution (10 mM, 130 μL), and the obtained 10 nm-sized AuNS (6 μL ) To the mixture of HAuCl ₄ aqueous solution (0.5 mM, 2 mL) was added. While the HAuCl ₄ solution was added, the reaction mixture was continuously stirred at 27° C. at 500 rpm. The resulting solution was centrifuged at 4,000 × g for 5 minutes and washed twice with distilled water to obtain AuNS having a size of 50 nm.

Manufacturing example 2: 50 nm Size Au Nano Cube (AuNC) synthesis

To synthesize a 50 nm-sized gold nanocube (AuNC), CTAC aqueous solution (100 mM, 6 mL), NaBr aqueous solution (20 mM, 30 μL), and AA aqueous solution (10 mM, 390 mL) in a 20 mL vial. μL) was mixed while stirring at 27° C. at 500 rpm. 10 μL of AuNS (3.9 OD) of 10 nm size prepared as in Preparation Example 1 was added to the mixture. Then HAuCl ₄ aqueous solution (0.5 mM, 6 mL) was added. The reaction mixture was stirred at 500 rpm for 25 minutes, and then left undisturbed. The product was washed twice by centrifugation at 4,000 × g for 5 minutes, and the final volume was adjusted to 1 mL. In order to obtain AuNC in high yield, a purification step was performed according to a known method (Park, K. et al. , Nano Lett., 2010, 10: 1433-1439). First, the synthesized 50 nm-sized AuNC was dispersed in the CTAB solution so that the final concentration was 1 mM. Subsequently, 100 μL of the dispersion of AuNC dispersed in CTAB solution was mixed with 100 μL of BDAC solution (180 mM), and the resulting mixture was allowed to stand overnight at 30° C. without stirring. Finally, the supernatant was carefully removed and centrifuged at 4,000 × g for 5 minutes to wash the pellet twice with distilled water.

Manufacturing example 3: Preparation of DNA-modified magnetic microparticles (DNA-MMP)

DNA-modified magnetic microparticles (DNA-MMP) were prepared according to the manufacturer's instructions. An amine group-modified oligonucleotide at the 5'end (SEQ ID NO: 1: 5'-NH _2- A ₁₀ -PEG ₆ -AGAAAGAGGAGTTAA-3') was coated with a carboxyl functional group on the MMP with EDC (1-Ethyl-3-( 3-dimethylaminopropyl)carbodiimide) was attached through coupling. Specifically, 200 μL of MMP solution (10 mg/mL) was washed twice with 100 mM MES buffer (pH 4.8), and redispersed in 20 μL of MES buffer. Thereafter, 10 μL of amine-modified oligonucleotide (1 mM) and 10 μL of 1 M EDC solution (MES buffer) were added to the MMP dispersion. The mixture was shaken at room temperature for 4 hours. The thus prepared DNA-modified MMP was washed three times with a washing buffer (250 mM, Tris pH 8.0, 0.01% Tween ^® -20), and 200 μL of Tris-EDTA buffer (10 mM, Tris pH 8.0, 1 mM EDTA).

Manufacturing example 4: DNA-modified AuNC And AuNS Produce

Target capturing (TC)-gold nanocubes (TC-AuNC) and gold nanospheres (TC-AuNS) as probes, slightly modified salt-aging method (J. Am. Chem. Soc., 2000, 122: 9071-9077), a DNA strand (SEQ ID NO: 2: 5'-TCCATGCAACTCTAA-A ₁₀ -Cy3-SH-3') labeled with a fluorescent substance Cy3 on the surface of 50 nm-sized AuNC and AuNS. Attached. Prior to introducing the DNA strand onto AuNC and AuNS, the synthesized 50 nm-sized AuNC and AuNS were washed three times with 0.1% (wt/vol) SDS solution. First, the disulfide-modified oligonucleotide was reduced with a 0.1 M DTT solution prepared with 0.17 M phosphate buffer (pH 8.0), and desalted NAP-5 column (Sephadex™ G-25 DNA Grade, GE Healthcare, UK). To a dispersion of 50 nm-sized nanoparticles (AuNC and AuNS), an excess of purified oligonucleotides (2×10 ⁵ times the number of nanoparticles) was added, and the mixture was incubated overnight at room temperature. The solution was adjusted to a final phosphoric acid concentration of 10 mM (pH 7.4) and an SDS concentration of 0.1% (wt/vol). Thereafter, the solution was slowly stirred at room temperature for 1 hour. To the mixture, 6 aliquots of a chlorinated solution (2 M NaCl, 10 mM PB, 0.1% SDS) were added every hour to obtain a 0.3 M NaCl solution. During the salt aging process, the mixture was incubated in a water bath at 60° C. to minimize non-specific interactions of DNA on the gold surface. After the above chlorination step, the mixture was incubated overnight at room temperature. The mixture was then centrifuged at 4,000 × g for 5 minutes, and the supernatant was carefully removed. The precipitate was redispersed in 0.3 M phosphate-buffered saline solution (PBSS, 10 mM PB, 0.3 M NaCl, 0.01% SDS, pH 7.4). The washing step was repeated twice. In the same manner as TC-AuNC and TC-AuNS, except that the DNA strand is replaced with the DNA strand of SEQ ID NO: 3 (TTAGAGTTGCATGGA-A ₁₀ -SH) that does not contain a phosphor, signal amplifying (SA)- Probes such as SA-AuNC and SA-AuNS were prepared.

Example 1: gold Nanocube Used For SERS Nano probe Preparation of aggregates and their structure-dependent DNA binding kinetics

A complex of DNA-modified magnetic microparticles (DNA-MMP) and DNA-modified AuNC and AuNS corresponding to the target-capturing step of FIG. 1 (NC-complex-1 and NS-complex, respectively -1) in order to confirm the structure-dependent DNA binding rate in the formation of DNA-MMP (10 mg/mL, 1 μL) target DNA (HAV DNA, SEQ ID NO: 4: 5'-TTAGAGTTGCATGGATTAACTCCTCTTTCT-) 3′) solution (1 nM, 100 μL) was added and incubated for 1 hour. Specifically, six reaction samples were prepared to monitor the number of unbound nanoprobes as a function of hybridization time. HAV DNA strands that did not bind under magnetic force were removed, and the resulting solution was washed three times with 0.3 M PBSS buffer. Next, a previously prepared TC-probe solution (3 pM, 60 μL) was added to each reaction sample, and the mixture was incubated for different hybridization times. At certain hybridization times (0, 5, 10, 15, 20, and 25 minutes), NC-composite-1 and NS-composite-1 were recovered by magnet. Immediately, the number of unbound TC-probes was monitored by measuring the extinction spectrum of the supernatant, including the unbound TC-probe, recovered from each reaction sample.

Further, corresponding to the signal-amplifying step of FIG. 1, NC-complex-1 and SA-AuNC and NS-complex-1 and SA-AuNS (NC-complex-2 and NS-complex- respectively) 2) in order to confirm the structure-dependent DNA binding rate in formation, the method of monitoring the binding rate of the TC-probe was modified. Briefly, NC-complex-1 and NS-complex-1 using 100 μL of HAV DNA (1 nM), 1 μL of DNA-MMP (10 mg/mL), and 60 μL of TC-probe (2 pM). Prepared. The obtained complexes were washed 3 times with 0.3 M PBSS buffer. Subsequently, SA-AuNC and SA-AuNS (6 pM, 60 μL) solutions were added to the prepared NC-complex-1 and NS-complex-1 solutions, respectively. Extinction spectra of unbound SA-probes were measured at different hybridization times (0, 10, 20, 30, 40, 50, and 60 minutes).

Comparative example 1: gold Nanospheres Used Nano probe Preparation of aggregate

A nanoprobe aggregate was prepared in the same manner as in Example 1, except that the gold nanospheres of Preparation Example 1 were used instead of the gold nanocubes of Preparation Example 2.

Experimental example 1: 50 nm Size AuNC And AuNS Quantitative analysis of DNA-loading capacity for

In order to determine the number of DNA strands bound on the surface of the Au nanoprobe, TC-AuNC and TC-AuNS prepared using Cy3-labeled DNA strands were used. The KCN solution was mixed with a dispersion of TC-AuNC and TC-AuNS, and the mixture was heated at 90° C. for 5 minutes to dissolve the TC-probe; The final concentration of KCN was 100 mM, and the particle concentration was 11.7 and 10.2 pM for TC-AuNC and TC-AuNS, respectively. The fluorescence emission intensity of the separated and released Cy3-labeled DNA strand was measured using a fluorophotometer (λ _excitation = 546 nm and λ _emission = 560 nm). The number of attached DNA strands was calculated from a standard curve obtained using the same DNA of a known concentration (FIG. 2).

Experimental example 2: Nano probe For the evaluation of the structure-dependent dissociation of aggregates Melting transition (melting transition) measurement

To prepare a nanoprobe aggregate, 3 pM of TC-probe and SA-probe (2 mL) were allowed to hybridize at room temperature for 4 hours. As the two probes hybridized, the resulting compound turned colorless, indicating the formation of nanoprobe aggregates. Temperature-induced dissociation (melting transitions) of the cluster was confirmed by monitoring the change of the extinction peak of the dissociated nanoprobe as the temperature of the solution increased from 25°C to 85°C. The melting temperatures ( T _m ) of the AuNC and AuNS aggregates were determined from the temperature corresponding to the maximum value of the first derivative of the dissociation (denaturation) curve.

Example 2: 3D -Assembled- Plasmon Castle - AuNC -Cluster-based DNA detection Assay

For the DNA detection assay, a sandwich-hybridization method using DNA-MMP, target DNA strand, TC-AuNC, and SA-AuNC was performed. First, a target DNA (hepatitis A virus; HAV, SEQ ID NO: 4: 5'-TTAGAGTTGCATGGATTAACTCCTCTTTCT-3') solution was prepared at different concentrations ranging from 100 aM to 10 pM using a 0.3 M PBSS buffer. In addition, a 10 pM concentration of non-complementary DNA (hepatitis B virus; HBV, SEQ ID NO: 5: 5'-TTGGCTTTCAGTTATATGGATGATGTGGTA-3') solution was prepared and used as a control. Next, 1 μL of the DNA-MMP solution was added to 100 μL of the diluted target DNA solution, and the mixture was incubated for 1 hour at room temperature while stirring. Thereafter, 20 μL of TC-AuNC solution (10 pM) was added to a target DNA-capturing MMP solution, and the mixture was incubated for 30 minutes at room temperature while stirring. During the incubation process, corresponding to the target-capturing step of FIG. 1, a sandwich hybridization complex, NC-complex-1 was formed. The obtained NC-complex-1 solution was washed 3 times with 0.3 M PBSS buffer, and the volume of the solution was adjusted to 10 μL. In order to form AuNC clusters, 20 μL of SA-AuNC solution (50 pM) was added to the prepared NC-complex-1 solution, and the mixture was incubated for 30 minutes at room temperature while stirring. During the incubation process, corresponding to the signal-amplification step of FIG. 1, an assembled AuNC cluster-MMP hybridization complex (NC-complex-2) was formed. Finally, the NC-complex-2 solution was washed three times with 0.3 M PBSS buffer, and concentrated to a volume of 10 μL. NS-complex-2 was prepared in the same manner as above, except that TC-AuNS and SA-AuNS were used instead of TC-AuNC and SA-AuNC.

Example 3: SERS -Based DNA detection The assay For micro-Raman measurements

For Raman analysis, 5 μL of the synthesized complex solution was poured onto a cover glass, and concentrated using a magnet placed under the cover glass. The residual solvent was removed by drying under atmospheric conditions. The dry cover glass was turned over, and the focus was adjusted to be on the back side of the sample. All Raman measurements were performed with a 633 nm excitation laser (190 μW), a standard charge-coupled device (CCD) array detector (576 pixels x 384 pixels; Peltier; cooled to -70°C) and a 50× objective (NA = 0.75, Leica) using a Renishaw inVia Raman microscope system. SERS spectra were acquired with 10 second acquisition time, and recorded for wavenumbers of 500 to 1,800 cm ^-1 .

Example 4: electrical Electric near-field Theoretical calculation

A boundary element method (BEM) simulation was performed using the MNPBEM toolbox. The edge length and radius of curvature at the corner of AuNC were 50 nm and 6 nm, respectively. The diameter of AuNS was 50 nm. Based on the transmission electron microscopy (TEM) image, the gap size between AuNCs in the assembled AuNC cluster was 2 nm (FIG. 3). The surrounding environment was regarded as air (n=1), and Johnson and Christy's data were used as the dielectric function of gold. The excitation wavelength was 633 nm.

<Conclusion>

To prepare a plasmonic nanoprobe for a SERS-based DNA detection assay, two types of nanoparticles, a cube (AuNC) and a sphere (AuNS), were used using a seed-mediated growth method. Was synthesized first. In particular, the synthesized AuNC was purified by depletion-induced flocculation in a surfactant micelle solution. The synthesized AuNC and AuNS showed excellent uniformity to the target structure, and showed a high yield of 95% or more (FIGS. 4a and 4b). In addition, their average sizes (edge length for the AuNC and diameter for AuNS) were similar, showing a narrow size distribution of 50.7 ± 0.6 nm for AuNC and 50.6 ± 0.7 nm for AuNS. UV-vis spectra of AuNC and AuNS showed sharp extinction peaks at 563 nm and 529 nm, respectively (Fig. 4c). The optical properties of plasmonic nanoparticles, in particular the electric field enhancement for SERS, are highly morphologically dependent. Therefore, the structural uniformity and narrow size distribution ensure low variability in the SERS signal from the product, make it reliable and reproducible, and are therefore suitable for SERS-based DNA detection.

Thereafter, the surfaces of AuNC and AuNS were functionalized with target DNA strand-capturing oligonucleotides as Raman probes for SERS-based DNA detection. DNA-modified plasmonic nanoparticles were accompanied by sequence-specific hybridization upon DNA detection (FIGS. 4D to 4G ). During the target-capturing step (Fig. 1), in the presence of complementary DNA strands (hepatitis A virus; HAV), the assay solution collected using a magnet turned colorless, and the intensity of the extinction peak rapidly decreased (Fig. 4d). This indicates that TC-AuNC captured the target DNA strand and hybridized with DNA-MMP to form a sandwich hybridization complex (NC-Complex-1 in Fig. 1) (Fig. 4e). right). However, when the linker DNA was replaced with a non-complementary DNA strand (hepatitis B virus; HBV), the color of the solution as well as the extinction spectrum did not change, indicating that NC-complex-1 was not formed ( 4d and 4e left). This DNA-sequence-specific hybridization process also occurred during the signal-amplification step. When sequence-complementary SA-AuNC was added to the NC-complex-1 solution, the assay solution became transparent under an external magnetic field, and the intensity of the extinction peak was due to the formation of the hybridization complex (NC-complex- 2 of 2). It was significantly reduced (Figure 4f and 4g right). On the other hand, in the case of non-complementary SA-AuNC, the solution color and extinction spectrum did not change (Figs. 4F and 4G left).

Next, the DNA-loading capacity of individual nanoparticles, that is, the total amount of DNA attached on the surface of the nanoparticles, was measured. In order to quantify the DNA-loading capacity of a single nanoparticle, a DNA-modified nanoparticle was dissolved using potassium cyanide (KCN), and a fluorescent signal of a fluorescent substance (Cy3)-labeled DNA strand was measured. The total number of DNA strands attached on the surface of individual nanoparticles was calculated from a standard curve obtained using the same DNA of a known concentration (Fig. 2). The DNA-loading capacity of a single AuNC (5004 ± 9) was higher compared to the value for a single AuNS (2934 ± 21) due to the larger surface area despite having the same size. However, the DNA-loading densities of the two types of nanoparticles, that is, the number of attached DNA strands per unit surface area of the nanoparticles, were similar to 0.325 nm ^-2 for AuNC and 0.365 nm ^-2 for AuNS. Although the DNA-loading density of AuNC is similar or lower than that of AuNS, the number of "effective binding DNA strands" that can actually participate in hybridization is more due to the structural properties of AuNC. It was high (Fig. 5A). When spherical nanoparticles form an assembly, the number of effective binding DNA strands is low because they bind only at narrow sites (point junctions) due to their convex surface structure.

Structure-dependent binding kinetics during DNA hybridization of various target capture nanoprobes (TC-probes; TC-AuNC and TC-AuNS) were comparatively studied (target capture step in FIG. 1). The target capture MMP and the complementary target DNA strand (HAV) were mixed for 1 hour and washed to remove unbound DNA strands. Then, the TC-probe was added to the washed MMP solution. During the DNA hybridization process, the decrease in the extinction peak intensity of the assay solution was monitored, indicating consumption of the nanoprobe due to the formation of the sandwich hybridization complex (NC- or NS-complex-1) (FIG. 6 ). At a specific time ( A _t ), the extinction intensity was divided by the initial intensity ( A ₀ ), and the relative extinction intensity ( A _t / A ₀ ) was plotted against the hybridization time ( t ). The A _t / A ₀ vs. t plot could be optimized using an exponential decay function: A _t / A ₀ = C exp(- k t) + b ( k is the decay constant). Unlike expected, as shown in Figure 5b, the amount of TC-probe consumed by the target capture MMP during DNA hybridization was similar in the two constructs, which was similar attenuation constants ( k _{TC - AuNC} = 0.1203 and k _{TC - AuNS} = 0.1257 min ^-1 ) and half-life ( t _1/2 ; t _{1/2, TC - AuNC} = 5.76 and t _{1/2, TC - AuNS} = 5.51 min) (Table 1). These results were contrary to the hypothesis that binding of a higher number of "effective binding DNA strands" due to the presence of distinct structural properties leads to higher binding (attenuation) rates. There are several reasons for this. First, since MMP contains a large number of binding sites on its micron-sized surface, the binding potential of TC-AuNC and TC-AuNS to MMP is probably similar. Second, the morphology (curvature) effect in relation to DNA hybridization is negligible due to the rough surface of the MMP (Fig. 4e). Thirdly, the TC-probe was directly captured by MMP instead of hybridizing with similar types of nanoparticles, so the concept of "effective binding DNA strand" induced by the structure (curvature) effect was not applied to the present invention ( Target capture step of Figure 1). For this reason, in the case of AuNC and AuNS, despite the different number of "effective binding DNA strands", the morphology (curvature) was considered to have no effective effect on binding potential or efficiency.

Damping constant
( k , min ^-1 ) Half-life
( t _1/2 , min) Average decay rate up to t _1/2
( R _{half -life} , amole/min) TC-probe TC-AuNC 0.1203±0.0110 5.76±0.53 13.51±1.26 TC-AuNS 0.1257±0.0130 5.51±0.58 13.79±1.47 SA-probe SA-AuNC 0.0428±0.0038 16.21±1.45 7.54±0.68 SA-AuNS 0.0367±0.0036 18.87±1.88 4.35±0.44

Conversely, the effect of the shape (curvature) on the binding kinetics was clear when the signal amplification nanoprobes (SA-probes; SA-AuNC and SA-AuNS) were bound to Complex-1 (signal amplification step in FIG. 1). . SA-AuNC was consumed by Complex-1 more rapidly than SA-AuNS, which indicates that the binding (hybridization) rate of the cube-shaped nanoprobe (SA-AuNC) is high (FIG. 5C). As a result, as shown in Table 1 above, the binding kinetics of SA-AuNC is more than that of SA-AuNS ( k _SA-AuNS = 0.0367 min ^-1 , t _{1/2, SA-AuNS} = 18.87 min). It _{showed a} high attenuation constant and a shorter half _- life ( k _SA-AuNC = 0.0428 min ^-1 , t _{1/2, SA- AuNC} = 16.21 min). Furthermore, the average decay rates up to t _1/2 (defined as the number of moles of nanoprobes consumed to half-life per unit half-life) were calculated as 7.54 and 4.35 attomoles/min for SA-AuNC and SA-AuNS, respectively. Interestingly, as opposed to the binding between the nanoprobe and the MMP (target capture step in Figure 1), the cube-shaped nanoprobe has an increased binding (hybridization), which has a binding rate of about 73% higher than that of the spherical nanoprobe. It showed the efficiency (signal amplification step in Fig. 1). It was thought that the presence of a higher number of "effective binding DNA strands" by morphology (curvature) effect leads to an increased binding rate. Unlike the case of the binding between the nanoprobe and the MMP, the SA-probe should bind to the surface of the TC-probe instead of binding to the MMP, and thus, the hybridization efficiency between the nanoprobes was determined by their surface morphology (Fig. ). The presence of a higher number of "effective binding DNA strands" between the cube-shaped nanoprobes is available and of increased binding rate, which can lead to higher binding rates (ie, higher attenuation constants). Induce a seat. Thus, the increased binding (hybridization) kinetics associated with morphology (curvature) effects can shorten the assay time of DNA hybridization for SERS-based DNA detection.

Next, a hypothesis was tested that if the number of "effective binding DNA strands" is high, the assembled nanoprobes bind to each other more tightly. It was assumed that the number of "effective binding DNA strands" by morphology (curvature) effect determines the binding strength of the assembled clusters. To demonstrate this, the cluster was tested for temperature-induced dissociation. In particular, in order to accurately determine the structure-dependent bonding strength between nanoprobes and to prevent errors that may occur due to hybridization between the nanoprobes and MMP, AuNC or AuNS aggregates without an MMP support were used. When the nanoprobes were agglomerated in the solution, the interparticle distance was reduced than the average particle size, resulting in a change in the color of the solution and the UV-vis spectrum accompanying it (FIG. 7). As the temperature of the solution increased, the nanoprobe aggregates began to dissociate at a temperature close to the melting temperature ( T _m ) of DNA. Accordingly, as the temperature of the solution increased from 25°C to 85°C, the change in intensity of the extinction peak of the dissociating nanoprobe was monitored. AuNC aggregates were found to have a higher melting point (74.8 ℃) than AuNS aggregates (71.2 ℃) (Fig. 5d). Furthermore, the dissociation (denaturation) curve for the AuNC aggregate was more steep than that for the AuNS aggregate (FIG. 5D); The full width at half maximum (FWHM) obtained from the first-order derivatives of the dissociation (denaturation) curve was 2.8°C and 4.0°C for the AuNC aggregates and AuNS aggregates, respectively (Fig. 5d, inset ). In general, aggregates formed by DNA-modified Au nanoparticles are of course much more rapid than free double-stranded DNA of the same nucleotide sequence by greater cooperation, i.e., narrow FWHM. It showed a higher melting point ( T _m ). The effect of this collaboration on the melting transition of DNA is being studied experimentally and theoretically and is highly dependent on conditions such as surface DNA density, salt concentration, interparticle distance and particle. In the present invention, by studying the nanoparticle-structure-dependent melting transition of DNA, the only difference between the aggregates formed by AuNC and AuNS with respect to the DNA melting transition is the difference in the number of "effectively bound DNA strands" of nanoparticles. It was confirmed that it was in the shape of the surface. As the temperature of the solution increased, some of the "effective binding DNA strands"dissociated; The UV-vis spectrum did not change before the nanoprobes were detached from the aggregates by complete dissociation of the "effective binding DNA strand". Thus, the presence of a small number of "effective binding DNA strands" induced a relatively easily occurring dissociation process, indicating a change in the UV-vis spectrum at low temperatures as well as a wide transition temperature range. On the other hand, the presence of a large number of "effective binding DNA strands" is relative to aggregates having a UV-vis spectrum that changes rapidly by sudden dissociation of the aggregates near the complete dissociation point of the "effective binding DNA strand". To keep it intact even at high temperatures. Similarly, dissociation of AuNS clusters is relatively easier, at lower temperatures (lower T _m ), and over a wide transition range (wide FWHM), due to the lack of “effective binding DNA strands” between AuNCs. Occurred. In contrast, the presence of a higher number of "effective binding DNA strands" in the case of AuNC aggregates is at a higher temperature (higher temperature), accompanied by dissociation of the nanoprobe that occurs over a very narrow range of transition temperatures (narrow FWHM). T _m ) to maintain the structure. Thus, the higher melting point and narrower FWHM of AuNC aggregates indicate that the presence of a higher number of “effective binding DNA strands” induces stronger binding between assembled clusters by morphology (curvature) effects. This effect makes the assembled clusters long-lasting and stable, suppressing the loss of the SERS signal during the assay or measurement process.

Finally, 3D-assembled-plasmonic-AuNC-cluster-based ultrasensitive DNA detection was performed using the SERS technique. To detect target DNA (HAV), a typical sandwich-hybridization assay was performed using DNA-MMP and TC-AuNC to form NC-Complex-1. In order to enhance the SERS signal strength from NC-complex-1, SA-AuNC was introduced into NC-complex-1 to form a plasmonic AuNC cluster (NC-complex-2) assembled on MMP. Figure 8 shows the change in the SERS signal intensity of the NC-complex as a function of target DNA (HAV) concentration. The sequence specific hybridization ability of the proposed system (Figs.4D-4G) is the SERS of NC-Complex-1 and NC-Complex-2 during control experiments, where HAV DNA was replaced with non-complementary HBV DNA. The signal intensity was meant to be negligibly low (blue and green dots in FIG. 8A; blue and green lines in FIG. 8B). In the case of NC-complex-1, the SERS signal intensity (black dot in FIG. 8A; black line in FIG. 8B) was discriminable (distinguishable), but as opposed to during the control experiment, the limit of detection (LOD) was It was only about 1 pM. On the other hand, the SERS signal intensity of the assembled plasmonic AuNC cluster (NC-complex-2) was enhanced high, and the LOD was higher at about 100 aM (red dot in FIG. 8A; red line in FIG. 8B). As described above, strong plasmonic coupling in the assembled region of the AuNC cluster induced a stronger electric field near the Raman molecule, resulting in an enhanced SERS signal and improved sensitivity to DNA detection by about 10000 times. Next, the characteristics of SERS-based DNA detection using nanoprobes having different morphologies were compared (Fig. 8c). In the case of 3D-assembled plasmonic clusters (NS-complex-2) formed by AuNS, the SERS signal strength is due to insufficient and slower binding (hybridization) kinetics between AuNSs due to morphology (curvature) effect, NC-complex It was lower compared to that of -2. Thus, the LOD was limited to about 10 fM. Furthermore, in comparison with NC-complex-2, NS-complex-2 exhibited a larger variance (i.e., higher standard deviation) in its signal intensity during repeated experiments, which is relatively poorer reproducibility for DNA detection. And reliability. Due to the convex surface structure of the AuNS, the electric field was enhanced only at the local center of the assembled part of NS-composite-2, and the degree of plasmonic coupling decreased sharply as it deviated from the center of the assembled part (point-to-point. -Point-to-point coupling). Unlike face-to-facet coupling, which occurs between AuNC-based clusters, which induces stable and uniform plasmonic coupling over a large area of the assembled part, AuNS-based The point-to-point coupling between clusters induced unstable, non-uniform and non-reproducible SERS signals. From these results, it was confirmed that AuNC is a promising nanostructure for use in SERS-based analytical applications that require stable, highly sensitive, reproducible and reliable results.

In order to prove that a strong and uniform electric field is formed within the gap region of the AuNC cluster, a simulation using the boundary element method (BEM) and the MNPBEM toolbox was performed (FIG. 9). During the BEM simulation, when linearly polarized light (wavelength 633 nm) was irradiated onto a single AuNC, a weak electric field was formed only at the edge of the AuNC due to the absence of plasmonic coupling (FIG. 9A). On the other hand, a significantly stronger electric field was formed in the gap region of the AuNC cluster due to the strong plasmonic coupling (FIG. 9B). In particular, the strong electric field was distributed over a large area of the assembled AuNC cluster due to the face-to-face coupling. This phenomenon is quite different from that observed in the case of AuNS clusters, in which an electric field is formed only at small local points due to the convex surface shape (point-to-point coupling) (Fig. 9c). Therefore, unlike AuNS clusters that amplify the electric field only in a specific gap region with a plasmonic coupling direction parallel to the incident light, AuNC clusters strongly amplify the electric field for all different coupling directions within the cluster over a large area of the assembled plane. (Fig. 9d to 9i). Due to the planar metal/insulator/metal geometry, the AuNC assembly involved a transverse gap plasmon (TGP), a surface plasmon running along the metal/insulator/metal interface. Upon light irradiation, the TGP was excited in a plane perpendicular to the incident polarization. When reaching the edge, the excited TGP proceeded to the adjacent plane parallel to the incident polarization. Therefore, unlike the AuNS assembly, the enhanced electric field was distributed across all nanogaps in the AuNC assembly, and Raman molecules could exist in the hot spot with a much higher probability, thus creating a strong and uniformly enhanced electric field. Considering that hierarchically assembled clusters are randomly formed in various orientations on the surface of spherical magnetic microparticles, cube-shaped-plasmonic-nanoparticles that uniformly and strongly enhance electric field on all assembled surfaces regardless of the direction of incident light The -based cluster can provide a more uniform, reproducible, and quantitative signal for SERS-based DNA detection, compared to the spherical-plasmonic-nanoparticle-based cluster. In conclusion, the H-cube-SERS strategy according to the present invention can provide a stable and ultra-sensitive SERS platform for obtaining reproducible and reliable results when applied analytically.

<110> Seoul National University R&DB Foundation BioNano Health Guard Research Center <120> Method for detecting target material by SERS using assembly of metal nanocubes with hierarchical structure <130> KPA180358-KR <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> DNA attached on MMP <400> 1 agaaagagga gttaa 15 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> DNA attached on 1st AuNP <400> 2 tccatgcaac tctaa 15 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> DNA attached on 2nd AuNP <400> 3 ttagagttgc atgga 15 <210> 4 <211> 30 <212> DNA <213> Hepatitis A virus <400> 4 ttagagttgc atggattaac tcctctttct 30 <210> 5 <211> 30 <212> DNA <213> Hepatitis B virus <400> 5 ttggctttca gttatatgga tgatgtggta 30

Claims (16)

Magnetic microbeads labeled with a first DNA comprising a first sequence complementary to a part of the target DNA to be detected, and a first metal labeled with a second DNA comprising a second sequence complementary to the rest of the target DNA to be detected By reacting the nanocube with a sample predicted to contain the target DNA to be detected, DNA hybridization in the presence of the target DNA in the sample forms a first complex in which at least one first metal nanocube is bound to a magnetic microbead. The first step;
Magnetic microbeads of the first metal nanocube by reacting the first complex with a second metal nanocube modified with a third DNA comprising a third sequence complementary to the second DNA labeled on the first metal nanocube A second step of forming a second composite in which a second metal nanocube is bonded to the other surface not bonded to the other; And
A third step of measuring a surface enhanced Raman scattering (SERS) signal from the second complex; comprising, a method of detecting a target DNA,
Since the second DNA modified in the first metal nanocube is labeled with a Raman active material, the Raman active material is located between the first metal nanocube and the second metal nanocube, so that the first metal nanocube and the second metal nanocube The method characterized by showing an improved Raman signal by the enhancement of the electric field between.
The method of claim 1,
The method further comprising a step 1-1 of removing the first metal nanocube not coupled to the magnetic microbead by applying a magnetic field from the outside and washing between the first step and the second step.
The method of claim 1,
The first metal nanocube and the second metal nanocube are each independently made of a metal selected from the group consisting of gold, silver, platinum, palladium, and copper.
The method of claim 1,
The first metal nanocube and the second metal nanocube are particles having an edge length of 10 to 300 nm, and the deviation of the edge lengths of the first metal nanocube and the second metal nanocube does not exceed ±20%. Being, the way.
The method of claim 1,
The method, wherein the magnetic microbeads have a diameter of 0.5 to 10 μm.
The method of claim 1,
The first to third sequences are each independently composed of 6 to 30 nucleic acids.
The method of claim 1,
The target DNA to be detected is composed of 10 to 100 nucleic acids.
The method of claim 1,
The Raman active material is a cyanine-based fluorescent organic molecule including Cy3, Cy3.5, and Cy5, or an organic fluorescent molecule of FAM, Dabcyl or rhodamine, or a non-fluorescent Raman activity including mercaptobenzoic acid and aminothiophenol. Material.
The method of claim 1,
By reacting the second complex with the first metal nanocube modified with a second DNA comprising a second sequence complementary to the third DNA labeled on the second metal nanocube,
The 2-n-th complex forming a 2-n complex in which additional first metal nanocubes are bonded to the remaining surfaces of the second metal nanocube, which is not combined with the first metal nanocube and does not face the magnetic microbead. The method further comprising the step (more than n is an integer of 1 or more).
The method of claim 9,
As a method of sequentially repeating the 2-n-th step and optionally the 2-(n+1) step, which is performed in the same manner as the second step, wherein the additionally performed step is the final 2-n-th complex or Excluding the magnetic microbeads from the 2-(n+1) complex, the size of the assembly consisting of the first metal nanocube and the second metal nanocube is repeatedly performed within a range of 30 nm to 900 nm in average radius. , Way.
The method of claim 1,
The method, wherein the signal for the second complex measured from the third step is augmented by 100 times or more compared to the signal measured for the first complex at the same wavelength.
A first step of preparing a first biomaterial that specifically binds to another part of the substance to be detected on one side, and a first'metal nanocube labeled with the first' DNA on the other 5 side;
Magnetic microbeads labeled with one or more first biomaterials that specifically bind to a part of the substance to be detected, and a first surface prepared from the first step, which specifically binds to another part of the substance to be detected. It contains a biological material, and on the other 5 sides, the first'metal nanocube labeled with the first'DNA is reacted with a sample predicted to contain the substance to be detected, so that specific binding is achieved in the presence of the target substance in the sample. A second step of forming a first'composite in which at least one first'metal nanocube is coupled to a magnetic microbead;
The first'complex is reacted with a second'DNA modified 2'metal nanocube containing a sequence complementary to the first'DNA labeled on the first' metal nanocube, and the first metal nanocube A third step of forming a second'composite in which a second'metal nanocube is bonded' on the other side of the cube that is not combined with the magnetic microbeads; And
A fourth step of measuring a surface-enhanced Raman scattering signal from the second'complex, comprising: a method for detecting a target substance,
The first'DNA modified in the first'metal nanocube is labeled with a Raman active material, so that the Raman active material is located between the first'metal nanocube and the second' metal nanocube, so that the first'metal nanocube The method characterized in that the Raman signal is enhanced by the enhancement of the electric field between the and the second'metal nanocube.
The method of claim 12,
The second and third steps are carried out in reverse order, first combining the first'metal nanocube with the second' metal nanocube, and reacting with the magnetic microbeads and samples labeled with the first biomaterial. Phosphorus, a method of detecting a target substance.
Magnetic microbeads labeled with one or more first biomaterials that specifically bind to a part of the target material to be detected;
On the first side, a first biomaterial that specifically binds to another part of the substance to be detected is included, and on the other side, a first metal nanocube labeled with a first'DNA'; And
A kit for detection of a target substance by SERS, comprising a modified 2′ metal nanocube in which the 2′ DNA containing a sequence complementary to the 1′ DNA labeled on the 1′ metal nanocube.
The method of claim 14,
For detection of DNA as a target material, the magnetic microbeads are labeled with a first DNA comprising a first sequence complementary to a part of the target DNA to be detected, and the first'metal nanocube A second DNA containing a complementary second sequence and bound to a Raman active molecule is labeled, and the second'metal nanocube includes a third DNA that is complementary to the second DNA labeled on the first metal nanocube. Modified one, a kit for detection of a target substance by SERS.
The method of claim 14,
The kit further comprising a buffer.

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