Method for amplifying or detecting target substances using nanoparticles

Abstract

The present application provides nanoparticles comprising a plurality of partially double-stranded probes, and compositions, arrays, kits, devices, and / or methods utilizing same for amplifying, detecting, quantifying, identifying, and / or visualizing a target substance. The nanoparticles can comprise (1) a template strand that includes (i) a target cloning site that includes a complementary sequence to a target oligonucleotide and (ii) a target binding site that includes a complementary sequence to a portion of a target nucleic acid fragment, and (2) a target-cloning strand that includes a sequence identical to the target oligonucleotide and hybridizes to the target cloning site of the template strand.

Description

[Technical Field]

[0001] Mutual citation with related applications This application claims priority rights based on Republic of Korea Patent Application No. 10-2021-0151779 dated November 5, 2021, and Republic of Korea Patent Application No. 10-2021-0159555 dated November 18, 2021, and all content disclosed in the documents of said Korean patent applications is incorporated herein by reference.

[0002] This application provides nanoparticles comprising a plurality of partially double-stranded probes, and compositions, arrays, kits, apparatus, and / or methods utilizing the same for amplification, detection, quantification, identification, and / or visualization of target substances. [Background technology]

[0003] Onsite sensing and diagnostic methods offer advantages in that they provide a rapid, inexpensive, and direct means of identifying sources of acute diseases, infections, or biological and chemical weapons (warfare agents) with little to no special equipment, and are particularly useful on portable platforms. Among such methods, colorimetric methods are powerful and widely applicable because they allow targets to be detected with the naked eye without expertise or equipment. However, in most cases, nanoparticle-based colorimetric assays rely solely on the optical properties of the nanomaterials, which limits their sensitivity, reliability, and quantification capabilities. Therefore, colorimetric analysis generally has a narrow practical range, low reliability, and is not suitable for analyses requiring high sensitivity. Generally, ultra-high sensitivity detection methods are based on polymerase chain reaction (PCR) or highly sophisticated amplification, sensing, and imaging techniques.

[0004] Therefore, there is a need for the development of simpler yet highly sensitive biological detection techniques. [Overview of the project] [Problems that the invention aims to solve]

[0005] To provide a highly sensitive, rapid, inexpensive, and simple means of detecting target substances, this application provides nanoparticles having a large number of partially double-stranded probes and compositions, arrays, kits, apparatus, and / or methods for amplification, detection, quantification, confirmation, and / or visualization of target substances using the same.

[0006] More specifically, one example provides a partially double-stranded oligonucleotide, which includes the following: (1)(i) A target-cloning site capable of hybridizing with a target oligonucleotide, and (ii) A target-binding site that can hybridize with a portion of the target oligonucleotide or is complementary to it. The first chain (template chain) including; and (2) A second strand containing the same sequence as the target oligonucleotide (target-cloning strand or target-amplification strand).

[0007] Another example provides nanoparticles containing two or more of the aforementioned partially double-stranded oligonucleotides on their surface.

[0008] Other examples include (a) substrate particles comprising a target-trapping chain partially hybridizing with a target oligonucleotide on its surface, and / or (b) compositions, arrays, kits, and / or apparatus for amplification, detection, quantification, confirmation, and / or visualization of a target substance comprising the nanoparticles.

[0009] Another example provides a method for amplifying, detecting, quantifying, confirming and / or visualizing a target substance using (a) a substrate particle comprising on the surface thereof a target-capture strand that partially hybridizes with a target oligonucleotide, and / or (b) said nanoparticles.

[0010] Another example provides use of (a) a substrate particle comprising on the surface thereof a target-capture strand that partially hybridizes with a target oligonucleotide, and / or (b) said nanoparticles for amplifying, detecting, quantifying, confirming and / or visualizing a target substance. Means for Solving the Problems

[0011] Provided herein are nanoparticles having a specific structure and a "Nanoparticle Chain Reaction (NCR)" technique using the same. The NCR technique is one type of nucleic acid molecule amplification method, and is differentiated from existing polymerase chain reaction (PCR) in that it is an enzyme-free technique that uses nanoparticles having a specific structure instead of polymerase; while being simpler than existing techniques, it has significantly higher amplification efficiency, and thus can be very usefully used for detecting nucleic acid molecules and various target substances that can be amplified via nucleic acid molecules.

[0012] Hereinafter, the present application is described in more detail:

[0013] Definition of Terms As used herein, the term "target material" refers to a substance to be ultimately amplified, detected, quantified, confirmed and / or visualized by the compositions, arrays, kits, devices and / or methods for amplification, detection, quantification, confirmation and / or visualization provided herein, and may be selected from all substances having biological activity. In one example, the target material is an organism (various eukaryotic cells or prokaryotic cells; for example, viruses, bacteria, fungi, etc.) or biologically active substance to be amplified, detected, quantified, confirmed and / or visualized, such as nucleic acid molecules (e.g., whole genomic genomes (DNA or RNA), genomic fragments, genes, gene fragments, siRNA, miRNA, biomarkers, other DNA or RNA fragments, etc.), proteins, peptides, macromolecules, small-molecule chemical substances (e.g., chemical drugs comprising one or more selected from the group consisting of various metal ions, organic compounds, etc.), and may be one or more selected from the group consisting of, but not limited to, the above. In one example, the bioactive substances such as the nucleic acid molecules, proteins, and peptides may be derived from the aforementioned organisms (e.g., viruses, bacteria, fungi, etc.), but are not limited thereto, and may be recombinantly or chemically synthesized.

[0014] As used herein, the term "sample" may refer to any biological and / or environmental sample for which the presence, absence and / or level of a target material is to be measured. For example, the biological sample may be selected from the group consisting of cells, tissues, blood, lymph, saliva, sputum, nasal discharge, urine, feces, other body fluids isolated from a subject (patient), but is not limited thereto. The subject includes, but is not limited to, humans, livestock (e.g., cattle, pigs, horses, sheep, goats, dogs, cats, etc.), birds (chickens, ducks, geese, turkeys, ostriches, quails, etc.), etc.

[0015] In this specification, the term "target oligonucleotide" refers to an oligonucleotide that is directly amplified by the chain reaction provided herein, and is also referred to as "target strand." The two terms are interchangeable and have equivalent meanings. For example, the term target oligonucleotide is: -If the target substance is a nucleic acid molecule, then the whole (if the nucleic acid molecule is a DNA fragment and / or an RNA fragment) or a part thereof (the sequence to be amplified; for example, a portion specific to the nucleic acid molecule), -If the target substance is a protein, peptide, macromolecule, or small molecule chemical, it can be understood to be the same as the target-cloning chain described later.

[0016] In this specification, the term “template strand” means a single-stranded oligonucleotide used as a template for target oligonucleotide amplification in the NCR technology provided herein, which may include a target-cloning site having a nucleic acid sequence complementary to the whole genome sequence of the target oligonucleotide and a target-binding site having a nucleic acid sequence complementary to a portion of the target oligonucleotide. The template strand may be, but is not limited to, oligonucleotides of 10-1000 nt, 10-500 nt, 10-200 nt, 10-100 nt, 10-90 nt, 10-80 nt, 10-70 nt, 10-60 nt, 10-55 nt, 10-50 nt, or 10-45 nt in length.

[0017] In this specification, the term "target-cloning strand" means a single-stranded oligonucleotide having the same nucleic acid sequence as the target oligonucleotide or a target sequence of a portion of the target that can be amplified, and which is capable of hybridizing (complementarily binding) with the target-cloning site of the template strand.

[0018] In this specification, the term “target-capturing strand” means a single-stranded oligonucleotide that functions to capture a target oligonucleotide onto a substrate particle and has a nucleic acid sequence complementary to a portion of the target oligonucleotide's template strand that is complementary to the target-binding site.

[0019] In this specification, the phrase "having a specific nucleic acid sequence or amino acid sequence" of a nucleic acid molecule or protein may mean that it includes or essentially consists of such sequence.

[0020] Partially double-stranded oligonucleotide One example provides a partially double-stranded oligonucleotide containing the following: (1) The first chain (long (extended) chain; hereafter also referred to as the template strand) includes the following: (i) A target-cloning site that can hybridize with the target oligonucleotide (complementary site), and (ii) A target-binding site that is hybridizable or complementary to a portion of the target oligonucleotide, wherein the target-cloning site and the target-binding site are linked to each other (for example, the 3' end of the target-cloning site may be linked to the 5' end of the target-binding site, or the 5' end of the target-cloning site may be linked to the 3' end of the target-binding site); and (2) A second strand (a shorter strand than the first strand) containing the same sequence as the target oligonucleotide (i.e., containing a sequence complementary to (hybridizable with) the target-cloning site of the first strand) (hereinafter also referred to as the target-cloning strand or target-amplification strand, and the two terms are interchangeable in meaning), At this time, the target-cloning strand hybridizes (complementarily binds) with the target-cloning site of the template strand to form a double-stranded structure (i.e., the double-stranded portion (site) of a partially double-stranded oligonucleotide), and the target-binding site of the template strand is exposed in a single-stranded form.

[0021] The template chain may be, but is not limited to, oligonucleotides of 10-1000 nt, 10-500 nt, 10-200 nt, 10-100 nt, 10-90 nt, 10-80 nt, 10-70 nt, 10-60 nt, 10-55 nt, 10-50 nt, or 10-45 nt in length. The target-cloning chain may be an oligonucleotide of a length that hybridizes with the target-cloning site of the first chain and exposes the target-binding site, and may, for example, be identical to the target oligonucleotide.

[0022] Nanoparticles (NCR probes) Another example is a nanoparticle having one or more (e.g., two or more) partially double-stranded oligonucleotides on its surface. The partially double-stranded oligonucleotide is as described above. For example, the partially double-stranded oligonucleotide may be immobilized (attached) to the nanoparticle surface, either directly or via a linker (first linker), at one end of a template chain (e.g., one of the two ends of the target-cloning site in the template chain, different from the end linked to the target-binding site).

[0023] In other words, the nanoparticles have a surface on which (1) A template chain comprising: (i) a target-cloning site fixed directly to the surface or via a linker (first linker); and (ii) a target-binding site linked to one end of the target-cloning site (e.g., a different end from the end fixed to the nanoparticle surface); and (2) A target-cloning chain that is hybridizable (complementary) to the target-cloning site of the template chain. This may include the following: In this case, the target-cloning site of the template chain (first chain) and the target-cloning chain (second chain) hybridize to form a partially double-stranded oligonucleotide having a partially double-stranded structure, and the target-binding site of the template chain may be in an exposed form.

[0024] The number of partially double-stranded oligonucleotides fixed (attached) to the surface of each nanoparticle may be 1 or more, 2 or more, 10 or more, 20 or more, 50 or more, 70 or more, 100 or more, 120 or more, 150 or more, or 170 or more (there is no special limit on the upper limit, and it may be the maximum number of oligonucleotides that can be attached to the surface of the nanoparticle (which can be appropriately determined based on the surface area of ​​the nanoparticle), for example, 10,000, 5,000, 2,500, 1,000, 750, 500, 250, or 200, but is not limited to these).

[0025] For example, the nanoparticles may be made of a material that is visible (e.g., naked-eye-detectable) and / or biomolecule (e.g., nucleic acid molecules, antibodies, etc.) to which biomolecules can be bound, and may be made of materials such as metals such as gold (e.g., gold nanoparticles), silver (e.g., silver nanoparticles), platinum, copper, alloys (e.g., including two or more selected from the group consisting of gold, silver, platinum, copper, etc.), silica, hydrogels, carbon nanotubes, and quantum dots, but are not limited thereto. In another example, the nanoparticles may be made visible by a fluorescent label contained in an oligonucleotide bound to their surface. The size (average diameter) of the nanoparticles may be 1-1000 nm, 1-500 nm, 1-200 nm, 1-100 nm, 10-1000 nm, 10-500 nm, 10-200 nm, or 10-100 nm, but are not limited thereto. The aforementioned nanoparticles are not limited in their structure and / or morphology, and may have, for example, a solid structure, a core-shell structure, a halo structure, etc., and / or circular, elliptical, tubular, polygonal structures, etc. In some cases, the type of nanoparticle can be used to adjust the level of biomolecular binding, amplification, and / or visibility.

[0026] For example, the nanoparticles can be used as probes (NCR probes) for detecting or amplifying target oligonucleotides.

[0027] As described above, partially double-stranded oligonucleotides or nanoparticles with them attached to their surface contain a site (target-binding site) that can hybridize with the target oligonucleotide (having a complementary sequence) and a target-cloning chain having the same nucleic acid sequence as the target oligonucleotide, and can therefore be advantageously used for the detection and / or amplification of the target oligonucleotide or target substance.

[0028] In one specific example, if the target substance is a substance other than a nucleic acid molecule, the nanoparticles may further include a binding agent for the target substance in addition to the partially double-stranded oligonucleotide described above. The binding agent for the target substance may be one or more selected from the group consisting of antibodies, aptamers, small molecule chemicals, etc., to which the target substance can partially bind (e.g., specifically bind).

[0029] substrate particles In this specification, the term “substrate particle” means all forms of particles having a surface capable of immobilizing a target substance (e.g., oligonucleotides, antibodies, peptides, polymer compounds, etc.).

[0030] As an example, a substrate particle is provided in which one or more (e.g., two or more) target-capturing strands (or target-capturing oligonucleotides) having a sequence that is hybridizable or complementary to a portion of the target oligonucleotide are immobilized (attached, adhered) to the surface. The target-capturing strands may be immobilized (attached) directly to the surface of the substrate particle or via a linker (second linker).

[0031] As described later, since the number of nanoparticles bound to the substrate particle is equal to the number of target-cloning chains contained in the nanoparticle multiplied by the number of NCR cycles, the number of target-capture chains fixed (attached) to the surface of each substrate particle must be large enough to cover the number of nanoparticles. For example, the number of target-capture chains contained in each substrate particle may be 10 or more, 50 or more, or 10 2 More than 10 pieces 3 More than 10 pieces 5 More than 10 pieces 6 More than 10 pieces 7 One or more, or 10 8or more (there is no particular limitation on the upper limit, which can be the maximum number of oligonucleotides that can adhere to the surface of the substrate particle, and can be appropriately determined based on the surface area of the substrate particle; for example, 10 12 , 10 11 , 10 10 or 10 9 , although it is not limited thereto), but it is not limited thereto.

[0032] The substrate particles may be made of a solid material. In order to facilitate collection / purification of the complex formed with the nanoparticles, the substrate particles may be one or more selected from the group consisting of magnetic particles and beads (e.g., magnetic beads), but are not limited thereto. In one example, the substrate particles may be made of a material selected from metals such as gold (e.g., gold nanoparticles), silver (e.g., silver nanoparticles), platinum, copper, alloys (e.g., containing two or more selected from the group consisting of gold, silver, platinum, copper, etc.), silica, hydrogel, carbon nanotubes, quantum dots, but are not limited thereto. In addition, the size (average diameter) of the substrate particles must be sufficiently larger than that of the nanoparticles, and can be 2 times or more, 5 times or more, 10 times or more, 20 times or more, 30 times or more, 40 times or more, or 50 times or more the size (average diameter) of the nanoparticles; for example, it can be 0.1~100μm, 0.1~50μm, 0.1~20μm, 0.1~10μm, 0.1~8μm, 0.1~5μm, 0.1~3μm, 0.5~100μm, 0.5~50μm, 0.5~20μm, 0.5~10μm, 0.5~8μm, 0.5~5μm, 0.5~3μm, 1~100μm, 1~50μm, 1~20μm, 1~10μm, 1~8μm, 1~5μm, or 1~3μm, but is not limited thereto. There is no particular limitation on the structure and / or morphology of the substrate particles; for example, they can have a solid structure, a core-shell structure, a hollow structure, and / or have a circular, elliptical, tubular, polygonal structure, etc., but are not limited thereto.

[0033] In one specific example, if the target substance is a substance other than a nucleic acid molecule, the substrate particles may further include a target-capture chain in addition to the target-capture chain described above. The target-capture substance may be one or more selected from the group consisting of antibodies, aptamers, small molecule chemicals, etc., to which the target substance can partially bind (e.g., specifically bind). In this case, the target-capture substance contained in the substrate particles and the target-binding substance contained in the nanoparticles may bind to different sites on the target substance. In other words, the binding sites of the capture substance and the binding substance on the target substance may not overlap.

[0034] In this application, the linker (first linker) that can be used to fix (attach) the template chain (target-cloning site) to the nanoparticle surface and the linker (second linker) that can be used to fix (attach) the target-capture chain to the substrate particle can be appropriately selected from all materials that can attach oligonucleotides to the surface of solid particles, and may be one or more selected from the group consisting of, for example, chemical functional groups (e.g., thiol groups (SH), amine groups (NH2, etc.), binding proteins (e.g., biotin, etc.)), but is not limited thereto, and may be appropriately selected depending on the surface material of the attached particle. The linker has ends (e.g., 3' end in the case of the template chain, 5' end in the case of the target-capture chain) that bind the template chain and the target-capture chain to the nanoparticle and substrate particle surfaces, respectively. The template chain and / or target-capture chain may be bound to the 'end' and / or the surface of the nanoparticles and / or substrate particles. The template chain and / or target-capture chain may further include spacers at the ends (e.g., the 3' end for the template chain and the 5' end for the target-capture chain) that are bound to the nanoparticles and / or substrate particle surfaces, respectively, to maintain a predetermined distance from the surface of the nanoparticles and / or substrate particles (if the chain includes a linker, the spacers may be between the template chain and the first linker (first spacer) and / or between the target-capture chain and the second linker (second spacer)). The spacers may be appropriately selected from all materials having a length / volume that can provide the predetermined distance, for example, oligonucleotides (e.g., Poly A (e.g., A 2~20The spacer may be one or more selected from the group consisting of poly A, polymers (e.g., polyethylene glycol (PEG)), alkyl groups having 1-6 carbon atoms, etc., but is not limited thereto. For example, the spacer may be poly A, PEG, or a combination thereof (first spacer), or poly A, alkyl groups having 1-6 carbon atoms, or a combination thereof (second spacer), but is not limited thereto.

[0035] Detection and / or amplification of target substances (nucleic acid molecules) Other examples include compositions (or applications), arrays, kits, and / or apparatus for amplification, detection, quantification, confirmation, and / or visualization of target substances comprising one or more substrate particles and one or more nanoparticles as described above. The compositions, arrays, kits, and / or apparatus may include the substrate particles and / or nanoparticles in a form dispersed and / or suspended in a suitable medium (e.g., a liquid medium).

[0036] Specifically, the compositions, arrays, kits, and / or apparatus may include: (a) One or more (e.g., two or more) substrate particles on which one or more (e.g., two or more) target-capture strands (or target-capture oligonucleotides) having a sequence that is hybridizable or complementary to a portion of the target oligonucleotide are immobilized (attached, glued) to the surface, and (b) One or more (e.g., two or more) nanoparticles, where each nanoparticle has one or more (e.g., two or more) partially double-stranded oligonucleotides on its surface. In this case, the nanoparticles and partially double-stranded oligonucleotides are as described above.

[0037] In one specific example, compositions, arrays, kits, and / or devices for amplification, detection, quantification, confirmation, and / or visualization of the target substance may include the following: (a) Substrate particles on which one or more (e.g., two or more or more) target-trapping chains having a sequence that can hybridize with or is complementary to a portion (first portion) of the target oligonucleotide are immobilized (attached, adhered) to the surface, and (b) One or more (e.g., two or more) nanoparticles, each nanoparticle containing one or more (e.g., two or more) partially double-stranded oligonucleotides on its surface, each partially double-stranded oligonucleotide containing: (1) The first chain (template chain) including the following: (i) A target-cloning site that can hybridize with the target oligonucleotide (complementary site), and (ii) A target-binding site that can hybridize with a portion (second portion) of the target oligonucleotide, wherein the target-cloning site and the target-binding site are linked to each other (for example, the 3' end of the target-cloning site may be linked to the 5' end of the target-binding site, or the 5' end of the target-cloning site may be linked to the 3' end of the target-binding site); and (2) A second strand (target-cloning strand) containing the same sequence as the target oligonucleotide (i.e., containing a sequence complementary to (hybridizable with) the target-cloning site of the template strand), At this time, the target-cloning strand hybridizes with the target-cloning site of the template strand to form a double-stranded structure (i.e., the double-stranded portion (site) of a partially double-stranded oligonucleotide), and the target-binding site of the template strand is exposed in a single-stranded form.

[0038] The target-capture chain must include a portion (first portion) of the target oligonucleotide and a site capable of hybridizing with it, and one end (e.g., the 5' end or 3' end) may be fixed (attached) to the substrate particle surface directly or via a linker (second linker).

[0039] In the target oligonucleotide, the portion on the substrate particle to which the target-capture chain is hybridized (first portion) and the portion on the nanoparticle to which the target-binding site of the first chain is hybridized (second portion) may be designed so as not to overlap with each other. For example, if the target-capture chain is hybridized with a portion of the 5' end of the target oligonucleotide (first portion), the target-binding site may be hybridized with a portion of the 3' end of the target oligonucleotide (second portion). In another example, if the target-capture chain is hybridized with a portion of the 3' end of the target oligonucleotide (first portion), the target-binding site may be hybridized with a portion of the 5' end of the target oligonucleotide (second portion).

[0040] In the compositions, arrays, kits, and / or apparatus provided in this application, when a target oligonucleotide (single-stranded) or a sample containing the same comes into contact with substrate particles, a portion of the target oligonucleotide (first portion) hybridizes with one or more target-capture chains on the substrate particles to generate one or more second portions of the target oligonucleotide that are exposed in a single-stranded state without hybridization. One or more of these exposed second portions bind to target-binding sites in the template chain on the nanoparticles, causing one or more nanoparticles to bind to the substrate particles and form a substrate particle-nanoparticle sandwich complex. Subsequently, through a dehybridization process, the target-cloning chain is dehybridized from the target-cloning site of the template chain and released to each of the nanoparticles. Since the released target-cloning chain has the same nucleic acid sequence as the target oligonucleotide, it can hybridize again with the target-capture chain on the substrate particles at the first portion, allowing the next cycle to be repeated. Through this process, as the cycle is repeated, the number of nanoparticles bound to the surface of the substrate particles increases geometrically and accumulates (concentrates). Theoretically, using one substrate particle as a reference, a number of nanoparticles equal to the number of target-cloning chains contained in the nanoparticle multiplied by the number of cycles will bind to and accumulate (concentrate) on the substrate particle [(number of target-cloning chains per nanoparticle)]. n[where n is the number of cycles]. This shows an amplification effect of the target oligonucleotide and / or a concentration effect of nanoparticles to a detectable level, for example, a level detectable to the naked eye (i.e., the presence or absence and / or concentration of the target oligonucleotide is easily detected by a color change of the solution due to the accumulated (concentrated) nanoparticles).

[0041] Other examples provide methods for amplifying, detecting, quantifying, confirming, and / or visualizing target substances using the substrate particles and nanoparticles. These methods may include: (A) Adding (or applying to or contacting) a target oligonucleotide or sample to a substrate particle containing multiple target-trapping chains on its surface, thereby hybridizing a portion (first portion) of the target oligonucleotide with the target-trapping chains; (B) A step of adding (or applying or contacting) a plurality of nanoparticles to the mixture obtained in step (A) above to form a complex between the substrate particles and the nanoparticles, wherein each nanoparticle contains a plurality of partially double-stranded oligonucleotides on its surface, and each partially double-stranded oligonucleotide contains the following: (1) The first chain (template chain) including the following: (i) A target oligonucleotide and a (complementary) target-cloning site that can hybridize with the target oligonucleotide, and (ii) A target-binding site that can hybridize with a portion (second portion) of the target oligonucleotide; and (2) A second strand (target-cloning strand) containing the same sequence as the target oligonucleotide, A complex is formed between the substrate particle and the nanoparticle by hybridization between the target-capture chain and the other portion (second portion) of the target oligonucleotide partially hybridized with the target-binding site on the substrate particle surface; and (C) A step of dehybridizing the partially double-stranded oligonucleotide and / or disassembling the complex to release a target-cloning chain having the same nucleic acid sequence as the target oligonucleotide.

[0042] In step (A) above, when a target oligonucleotide or sample is added to substrate particles having multiple target-capture chains on their surface, the target-capture chains can hybridize to a portion (first portion) of the target oligonucleotide, and can capture the target oligonucleotide present in the sample (if present in the sample).

[0043] The multiple nanoparticles used in step (B) each contain multiple partially double-stranded oligonucleotides on their surface, and each partially double-stranded oligonucleotide may include: (1) The first chain (template chain) including the following: (i) A target-cloning site that can hybridize with the target oligonucleotide (complementary site), and (ii) A target-binding site that can hybridize with a portion (second portion) of the target oligonucleotide, wherein the target-cloning site and the target-binding site are linked to each other (for example, the 3' end of the target-cloning site may be linked to the 5' end of the target-binding site, or the 5' end of the target-cloning site may be linked to the 3' end of the target-binding site); and (2) A second strand (target-cloning strand) containing the same sequence as the target oligonucleotide (i.e., containing a sequence complementary to (hybridizable with) the target-cloning site of the first strand).

[0044] At this time, the target-cloning chain hybridizes with the target-cloning site of the template chain to form a double-stranded structure (i.e., a double-stranded portion (site) of a partially double-stranded oligonucleotide), and the target-binding site of the template chain is exposed in a single-stranded form, allowing it to hybridize between the target-binding site and the other portion (second portion) of the target oligonucleotide that is partially hybridized (by the first portion) with the target-capture chain on the substrate particle, thereby forming a sandwich complex assembly between the substrate particle and the nanoparticle. In other words, the first portion of the target oligonucleotide is bound (hybridized) with the target-binding site of the template chain on the nanoparticle, and the second portion (not overlapping with the first portion) is bound (hybridized) with the target-capture chain on the substrate particle.

[0045] In step (C) above, the dehybridization of the partially double-stranded oligonucleotide and / or the disassembling of the sandwich complex assembly (which may be by dehybridization, for example) can be carried out by conventional dehybridization means. For example, the dehybridization can be carried out by one or more selected from the group consisting of increasing the pH (e.g., basic conditions), increasing the temperature (e.g., increasing to a range such as 50-100°C, 52-100°C, 55-100°C, 50-90°C, 52-90°C, 55-90°C, etc.) and decreasing the salt concentration, compared to the hybridization reaction. In one specific example (the example of this application), the salt solution was replaced with distilled water and the temperature was lowered, but the method is not limited thereto. As a result, the target oligonucleotide hybridized with the target-capture chain and target-binding site, and / or the target-cloning chain hybridized with the target-cloning site (both having identical nucleic acid sequences), are dehybridized and released in a single-stranded form. In addition, during the dehybridization process, the binding (hybridization) between the target oligonucleotide due to dehybridization and the target-binding site on the template chain on the nanoparticle and the target-capture chain on the substrate particle is also dissociated, and the nanoparticle and substrate particle that formed a complex through the target oligonucleotide are separated (released). The target-cloning chain and nanoparticle thus released can be used in subsequent cycles.

[0046] The above method may further include a step of separating, collecting, and / or concentrating the composite after step (B) (B-1), where step (C) can be performed on the separated, collected, and / or concentrated composite. The separation, collection, and / or concentration of the composite can be carried out by methods such as applying a magnetic field (e.g., using a magnet; if the substrate particles are magnetic particles) and / or centrifugation, but is not limited thereto, and can be carried out by conventional methods appropriately selected in consideration of the material, properties, etc., of the particles used.

[0047] For example, steps (A) to (C) can be repeated two or more times (steps (A) to (C) constitute one cycle). In this case, the target-cloning chain having the same sequence as the target oligonucleotide, released in single-strand form in step (C), is used again as the target oligonucleotide in step (A), and the nanoparticles released in step (C) are used as NCR probes to carry out the next cycle. In this case, one target oligonucleotide is amplified by the number of target-cloning chains contained in one nanoparticle, and in the next cycle, nanoparticles equal to the number of target-cloning chains are bound to one substrate particle to carry out the step. Therefore, one target oligonucleotide is amplified by the number of target-cloning chains contained in one nanoparticle multiplied by the number of cycles. In other words, the number of target oligonucleotides after the cycle is carried out is the initial number multiplied by m n It can be twice the initial number (where m is the number of target-cloning strands per nanoparticle (174 is exemplified in the example), and n is the number of cycles). The amplification level of a normal PCR is twice the initial number. n Compared to a multiple of the original, the amplification level of the NCR technology provided herein is significantly higher.

[0048] For example, the number of target-cloning chains (m) per nanoparticle may be 2 or more, 10 or more, 20 or more, 50 or more, 70 or more, 100 or more, 120 or more, 150 or more, or 170 or more (there is no special limit on the upper limit, and it may be the maximum number of oligonucleotides that can adhere to the surface of the nanoparticle (which can be appropriately determined based on the surface area of ​​the nanoparticle), for example, 10,000, 5,000, 2,500, 1,000, 750, 500, 250, or 200, but is not limited to these). For example, the number of target-cloning chains (m) per nanoparticle may be, but is not limited to, 2 to 1,000, 2 to 500, 50 to 1,000, 50 to 500, 100 to 1,000, or 100 to 500. For example, the number of cycles (n) may be, but is not limited to, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 2 to 20, 2 to 15, 2 to 10, 2 to 5, 3 to 20, 3 to 15, 3 to 10, or ~3 to 5.

[0049] In one specific example, when the target substance is a bioactive substance other than a nucleic acid molecule, a composition, array, kit, apparatus and / or method for amplification, detection, quantification, confirmation and / or visualization of the target substance, The substrate particles in (a) further include, in addition to the target-trapping chain described above, a target substance trapping material, The nanoparticles in (b) may further include, in addition to the aforementioned partially double-stranded oligonucleotide, a binding agent for the target substance.

[0050] The binding substance for the target substance may be one or more selected from the group consisting of antibodies, aptamers, small molecule chemicals, etc., to which the target substance can partially bind (e.g., specifically bind). In this case, the target substance capture substance contained in the substrate particles and the target substance binding substance contained in the nanoparticles may bind to different sites on the target substance. In other words, the binding sites of the capture substance and the binding substance on the target substance may not overlap.

[0051] When the target substance is a bioactive substance other than a nucleic acid molecule, when the target substance or sample is brought into contact with substrate particles on which a target substance capture agent is immobilized, a portion of the target substance binds to the capture agent. When nanoparticles containing the target substance binding agent are processed, the binding agent binds to the target substance at a site different from the binding site with the capture agent, forming a complex of the substrate particle and the nanoparticle. Once the complex is formed, the partially double-stranded oligonucleotide on the nanoparticle is dehybridized, releasing the target-cloning chain. Since the released target-cloning chain has the same nucleic acid sequence as the target oligonucleotide, a portion of it hybridizes with the target-capture chain on the surface of the substrate particle, and the subsequent process is the same as (A) to (C) described above, so that the bioactive substance other than a nucleic acid molecule can be amplified and / or detected by the target-cloning chain. In other words, the presence and / or level of the bioactive substance in the sample can be measured through the amplified target-cloning chain and / or accumulated (enriched) nanoparticles as described above.

[0052] In the compositions, arrays, kits, apparatus, and / or methods provided in this application for amplification, detection, quantification, confirmation, and / or visualization of target nucleic acid molecules or target substances, nanoparticles can be used in a sufficiently larger quantity (number) than substrate particles so that the nanoparticles are concentrated on the surface of the substrate particles. Theoretically, the minimum number of nanoparticles per substrate particle can be appropriately selected from 2 to the number of target-trapping chains per substrate particle, but is not limited thereto. For example, the amounts of nanoparticles and substrate particles used can be appropriately adjusted depending on the detection conditions, the characteristics of the target sequence, etc. (For example, if the target sequence has a nucleic acid sequence with strong hybridization (binding) strength (e.g., a "GC-rich" sequence), the amount of nanoparticles used can be relatively small, and if it has a sequence with a weak degree of hybridization (e.g., an "AT-rich" sequence), the amount of nanoparticles used can be relatively large, but is not limited thereto).

[0053] Compositions, arrays, kits, apparatus, and / or methods provided in this application for amplification, detection, quantification, confirmation, and / or visualization of target nucleic acid molecules or target substances may be enzyme-free or polymerase-free.

[0054] Furthermore, the methods provided in this application for amplification, detection, quantification, confirmation, and / or visualization of target nucleic acid molecules or target substances may be performed in liquid form.

[0055] For example, in addition to or as an alternative to detection / quantification / visualization by the nanoparticles themselves by using visible nanoparticles, the target-cloning (target-amplification) chain can be labeled for detection / quantification / visualization. For instance, the target-cloning (target-amplification) chain can be labeled with a conventional labeling agent. There are no particular restrictions on the labeling agents that can be used; any labeling agent that can be used for labeling oligonucleotides can be selected and used, and may, but is not limited to, fluorescent substances (e.g., fluorescent dyes and / or fluorescent proteins).

[0056] The NCR technology provided herein can be applied to the multiplexing and / or amplification of two or more different target substances by using two or more nanoparticles for each different target substance.

[0057] More specifically, the compositions, arrays, kits, apparatus, and / or methods provided herein for the amplification, detection, quantification, confirmation, and / or visualization of target substances provide for the simultaneous amplification, detection, quantification, confirmation, and / or visualization of two or more target substances (target nucleic acid molecules) by using two or more nanoparticles containing a template chain and a target-cloning chain for each of the two or more target substances (target nucleic acid molecules) and / or two or more substrate particles containing a target-capture chain for each of the two or more target substances (target nucleic acid molecules). In the multiple detection / amplification, if one or more of the two or more target substances are substances other than nucleic acid molecules, the nanoparticles may further contain binding substances for the target substances other than nucleic acid molecules.

[0058] In the aforementioned multiplex detection / amplification, by labeling the target-cloning chain differently for each target substance, two or more target substances can be amplified, detected, quantified, confirmed, and / or visualized simultaneously.

[0059] The compositions, arrays, kits, apparatus, and / or methods provided herein can achieve considerably high detection sensitivity for target substances. For example, the detection sensitivity of the compositions, arrays, kits, apparatus, and / or methods provided herein for target substances is 10 based on the amount of target substance (e.g., DNA (e.g., gDNA) or RNA) in the sample. -7 ng or more, 10 -6 ng or more, 10 -5 ng or more, 10 -4 ng or more, or 10 -3 It can be greater than ng (i.e., the lower limit of the amount of the target substance in the sample that can be detected (detection limit) is 10 -7 ng, 10 -6 ng, 10 -5 ng, 10 -4 ng or 10 -3(This can be ng). This exhibits significantly higher detection sensitivity than conventional PCR using polymerase (e.g., at least 10 times, at least 100 times, at least 1,000 times, or at least 10,000 times) (see Figure 12b). In one specific example, using an NCR probe loaded with 174 target-cloning strands per nanoparticle, after three NCR cycles, the dynamic range of the target substance (DNA), based on solution color change or UV-Vis absorbance change (e.g., measured at wavelengths of 400-650 nm), appeared to be around 100 zM to 100 pM. Furthermore, the Limit of Detection (LoD) observed in the integrated UV-Vis spectrum was found to be approximately 2 times, 10 times, 50 times, 100 times, 500 times, 1,000 times, 5,000 times, or 10,000 times higher for NCR compared to PCR (see Figure 12c).

[0060] The NCR technologies (compositions, arrays, kits, devices, and / or methods) provided herein are applicable to quantitative diagnostic platforms. These compositions, arrays, kits, devices, and / or methods can be applied to the amplification, detection, and quantification of a variety of target substances, replacing or extending existing conventional PCR technologies, and may also be applied to the diagnosis of diseases and / or symptoms associated with such target substances. Furthermore, these compositions, arrays, kits, devices, and / or methods may also be applied to rapid diagnostic technologies such as Lateral Flow Assay (LFA), and may, for example, be applied to rapid diagnostic kits such as strip sensors.

[0061] As mentioned above, this application develops a nanoparticle chain reaction (NCR) technique that modifies PCR, which involves repeating enzyme-based target-amplifying thermal cycles, to detect target substances using visible nanoparticles (e.g., colorimetric detection). The application will be described more specifically below, with reference to the drawings, using the example where the target substance is DNA and the nanoparticles are gold nanoparticles (AuNPs):

[0062] The target DNA is amplified through numerous hybridized target-cloning DNAs released by nanoparticles (DNA-AuNPs) to which DNA identical to the target DNA (target-cloning DNA) and complementary sequences partially form a double helix. This amplification can be achieved by forming and separating a target DNA sandwich complex using DNA-modulated (binding) magnetic particles and releasing the target cloning DNA strands along with the target DNA strands from the nanoparticles through a desalting process. The NCR techniques provided herein (number of target-cloning DNAs per nanoparticle) n The target DNA can be increased geometrically by n, where n is the number of cycles. Furthermore, the NCR technique dramatically increases the number of nanoparticles in the solution, making the distinct color change of the solution induced by plasmon nanoparticles visible to the naked eye. The NCR cycle can be repeated until the number of accumulated DNA-nanoparticles reaches a sufficiently high level for visual colorimetric detection or quantification based on a UV-Vis spectrophotometer.

[0063] More specifically, after applying a magnetic field to separate the sandwich complex, the target-cloning DNA loading AuNP (NCR probe) is released from the complex by dehybridization. The target-cloning DNA sequence is exactly identical to the target site sequence of the target DNA and is preloaded onto the NCR probe surface before the assay. In this process, numerous target-cloning DNA strands are released along with the target sequence. The released target-cloning strands are used in subsequent cycles to form a sandwich complex between the NCR probe and magnetic particles (magnetic probe), inducing a high level of opportunity for the formation of the NCR probe-magnetic particle complex (Figure 1a). Once the NCR probe, which has been loaded with target-cloning strands, dehybridizes the target-cloning strands, the NCR probe becomes an unloaded NCR probe without the target-cloning strands (see Figure 3). Figure 3 schematically illustrates the release of the target-cloning chain from a loaded NCR probe. The loaded NCR probe contains pre-hybridized target-cloning chain (green) and target binding sites of the template chain (red). When the loaded NCR probe is dehybridized, the pre-hybridized target-cloning chain is released, and the loaded NCR probe becomes an unloaded NCR probe, which is then used for the next cycle of target amplification.

[0064] As the NCR cycle repeats, NCR probes accumulate to high levels, leading to an increased concentration of plasmon AuNPs. This causes the solution to turn red in proportion to the concentration, and the total number of target chains and captured NCR probes in the reaction solution increases geometrically (Figure 1b). Scanning electron microscope (SEM) images of the NCR probe-magnetic particle sandwich complex confirm that as the NCR cycle repeats, the number of NCR probes captured on the magnetic particle surface increases, and the red color intensity of the solution increases (Figures 1c, 2d, and 4).

[0065] To experimentally prove the above, the anthrax lethal factor DNA sequence was detected using the NCR technology provided in this application. Given the increasing importance of field detection techniques that can rapidly and sensitively detect infectious pathogens without special equipment, anthrax-related genes were tested as a representative example of such infectious pathogens.

[0066] As illustrated in Example 1.3, an NCR probe can be produced by attaching (bonding) a thiolated template chain to gold nanoparticles (AuNPs), and then adding an excess amount of target-cloning chain so that the target-cloning chain is partially hybridized to the template chain.

[0067] In this case, since the target-binding site in the template chain is complementary to a portion of the target-cloning chain, undesirable hybridization between the target-cloning chain and the target-binding site may occur. This can generate an inactive NCR probe that cannot perform its intended function, severely reducing the selectivity and sensitivity of the NCR assay. Therefore, by adding target complements (toehold sequences) to induce toehold-mediated displacement, the target-cloning chain that has undesirably hybridized to the target-binding site of the template chain can be removed, completely exposing the target-binding site of the template chain and opening it up for binding to the target sequence, thereby forming an active NCR probe (Figure 1d). The gel electrophoresis results of the inactive NCR probe and the NCR probe (with toehold sequence added) are shown in Figure 2a. As shown in Figure 2a, it can be confirmed that the addition of the toehold sequence caused the intended toehold-mediated displacement that exposed the target-binding site. Furthermore, the dynamic light scattering (DVR) measurements of the inactive and active NCR probes are shown in Figure 8. As can be seen in Figure 8, the inactive NCR probe had a larger diameter than the active NCR probe, with the unloaded NCR probe showing the largest diameter. The zeta potential measurements are shown in Figure 9. The zeta potential results in Figure 9 show the surface negative charge state due to DNA fused to the NCR probe surface, and Figure 10 shows that AuNP, template-modulated (fused) AuNP, inactive NCR probe, and active NCR probe generate diverse UV-Vis spectra. In particular, an absorption peak due to DNA was observed at 260 nm, and an absorption peak due to gold nanoparticles was observed at 532 nm. Importantly, after two NCR cycles, the improved selectivity of the active NCR probe was demonstrated by comparing the inactive and active NCR probes (Figures 2b, 8).The active NCR probe showed red color only at target sequences, while showing little detectable color at non-target and single-base mismatch sequences. Conversely, the inactive NCR probe showed strong red color at target, non-target, and single-base mismatch sequences, demonstrating very low selectivity for target sequences.

[0068] The increased number of captured NCR probes corresponds to the amplified number of target nucleic acids due to NCR cycle repetitions. Importantly, the AuNP-induced red intensity of the NCR assay product increases with increasing NCR cycle count (Figure 2c). The macroscopic detection limit of the NCR assay was confirmed to be low, approximately 1 pM, 1 fM, and 100 zM after 1, 2, and 3 NCR cycles, respectively. Overall, the red intensity of the NCR assay product gradually increased with increasing target DNA concentration. For comparison, 10 pM non-target and single-nucleotide mismatch sequence detection was performed via the NCR assay, and in both cases, no detectable solution color change was observed. UV-Vis measurements were also performed to more accurately quantify the amount of target DNA by integrating the spectral changes based on AuNPs. To include a wide range of AuNP-induced spectral changes, the amount of AuNPs was determined by integrating the UV-Vis spectral region at wavelengths of 400–650 nm (Figure 2d). Importantly, a more pronounced increase in detection signal intensity was observed as the target concentration increased with increasing NCR cycle recovery, resulting in a very wide dynamic range from 100 zM to 100 pM target concentrations after three NCR cycles.

[0069] Subsequently, the fluorescence signals of the Cy3-labeled template strand and the Cy5-labeled target-cloning strand were measured to quantify the effect of increasing NCR cycles on the amount of target DNA. An NCR probe was formed using the fluorophore-modulated (labeled) strand, and the AuNP was finally lysed with KCN to completely release the DNA strand for fluorescence-based quantification of the oligonucleotide. Figure 2e shows that as the number of NCR cycles increased up to the 5th cycle, the number of target-cloning strands increased more rapidly over a very wide dynamic range. Nanoparticles provide a high surface-to-volume ratio, allowing a large number of nucleic acid molecules to be fused to the surface of the AuNP. In this example, the average number of template strands was 430 per AuNP, and the number of pre-hybridized target-cloning strands was 174 per AuNP (Figures 9, 10). Therefore, a single NCR probe can release up to 174 target-cloning strands per NCR cycle. In principle, PCR targets a sequence of 2 n On the other hand, the NCR provided in this application is theoretically 174 to the power of n (174), which is the number of target-cloning chains bound to a single nanoparticle. n The target strand can be amplified by a factor of 1 / 2 (where n is the number of cycles). However, it should be noted that accumulated unloaded NCR probes may interfere with target-mediated hybridization between the loaded NCR probe and the DNA-bound magnetic particles. The analytical results in this embodiment showed that the unloaded NCR probe was preferred over the loaded NCR probe for target binding (Figure 11). Therefore, the amplification efficiency of the NCR cycles is not currently maximized in the assay and further optimization is needed. Nevertheless, the NCR assay enabled macroscopic detection of zeptomolar target concentrations after only three NCR cycles. [Effects of the Invention]

[0070] This invention provides a detection and / or diagnostic technique that utilizes a novel nanoparticle chain reaction (NCR) technology to stably amplify, detect, and visualize even minute amounts of target substances. Because the NCR technology does not use enzymes, it eliminates the need for additional equipment and achieves extremely high detection sensitivity, making it applicable as an in-situ detection / diagnostic method. Compared to existing PCR, it can detect longer DNA (e.g., genomic DNA) with very high sensitivity, thereby improving the detection limit. Furthermore, the NCR provided herein contributes to improving the detection limit of lateral flow assays (rapid kits) for in-situ detection, and can therefore be usefully applied to lateral flow assays (rapid kits). [Brief explanation of the drawing]

[0071] [Figure 1a] Figure 1 illustrates the principle of nanoparticle chain reaction, and Figure 1a shows one cycle of the nanoparticle chain reaction; [Figure 1b] Figure 1b schematically shows a repeating NCR assay cycle; [Figure 1c] Figure 1c is an SEM image of a sandwich composite of NCR probes and magnetic substrate particles starting at a target analyte concentration of 10 pM (Scale bar, 200 nm; inset shows the color appearance as the number of unloaded NCR probes increases). [Figure 1d] Figure 1d schematically shows the preparation process for the synthesis of NCR probes via the toehold-mediated strand displacement reaction; [Figure 1e] Figure 1e is a schematic diagram of an NCR probe containing the target-binding site (red) on the target-cloning chain (green) and template chain (red). [Figure 2a]Figure 2 shows the results of a colorimetric NCR assay, and Figure 2a shows the results of gel electrophoresis performed to illustrate the desired NCR probe preparation process, demonstrating that different DNA lengths are obtained at each stage of NCR probe preparation. [Figure 2b] Figure 2b is a graph showing NCR results (UV-Vis spectra) for various concentrations of analytes (target substances). It shows that non-target sequences (N) and single-base mismatch sequences (S) are not detected, while target substances are detected with high sensitivity (above 100 zM), and that the detection capability is proportional to the number of cycles. [Figure 2c] Figure 2c shows the NCR assay results detectable with the naked eye. [Figure 2d] Figure 2d shows the NCR assay results detectable with the naked eye. [Figure 3] Figure 3 schematically shows the release of the target-cloning chain from the loaded NCR probe. [Figure 4] Figure 4 shows an SEM image of the sandwich complex during the NCR assay, which was performed at a target sequence concentration of 10 pM. The image shows that the formation of the sandwich complex increases with increasing NCR cycles (Scale bar: 1 μm). [Figure 5] Figure 5 is a graph showing the UV-Vis absorbance region measurement results after two NCR assay cycles. [Figure 6] Figure 6 is a graph showing the results of quantifying the target-cloning strand on the NCR probe. [Figure 7] Figure 7 is a graph showing the results of quantifying the template chain on the NCR probe. [Figure 8] Figure 8 is a graph showing the dynamic light scattering (DLS) measurement results of the NCR probe. [Figure 9] Figure 9 shows the zeta potential measurement results of the NCR probe. [Figure 10] Figure 10 is a graph showing the UV-Vis spectral measurement results of the NCR probe. [Figure 11] Figure 11 is a graph showing the degree of target chain capture by NCR probes during loading (including a partially double-stranded oligonucleotide hybridized with the target-cloning chain) and unloading (including only the template chain). [Figure 12a] Figure 12 shows the results of a colorimetric NCR assay compared to PCR, and Figure 12a is a schematic diagram comparing the gDNA detection processes between NCR and PCR. [Figure 12b] Figure 12b shows the results of detecting gDNA extracted from bacteria by NCR or PCR. The NCR assay shows that the DNA is detectable with the naked eye (detection limit: 10⁻⁷ ng), while PCR shows that it can be confirmed through gel electrophoresis (detection limit: 10⁻³ ng). [Figure 12c] Figure 12c is a graph comparing the NCR and PCR analysis results (UV-Vis spectrum). [Modes for carrying out the invention]

[0072] The present invention will be described in more detail below based on examples. However, the following examples are merely illustrative of the content of the present invention, and the scope of the rights of the present invention is not limited by the following examples.

[0073] Example 1 1.1. Material preparation 50-nm citrate-stabilized gold nanoparticles were purchased from BBI Solutions (Cardiff, UK). Dynabeads® M-270 carboxylic acid was purchased from Thermo Fisher Scientific (Waltham, MA, USA). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received without further purification. Oligonucleotides used in the following examples were obtained from Integrated DNA Technologies, Inc. Distilled water was obtained from Millipore system (>18.0 MΩ, Milli-Q) and used in all experiments. A mixture of Perfect Hyb® Plus Hybridization buffer (Sigma-Aldrich) and 1x PBS buffer in a 1:3 ratio was used as the hybridization buffer for the NCR assay.

[0074] To experimentally demonstrate the effectiveness of this application, the target oligonucleotide (hereinafter also referred to as the target chain or target sequence) selected was the DNA base sequence of the anthrax lethal factor (GAG GGA TTA TTG TTA AAT ATT GAT AAG GAT; Sequence ID No. 1), and subsequent tests were conducted. In the detection of infectious pathogens, the importance of field detection technology that can detect rapidly and sensitively without special equipment is increasing, so anthrax-related genes, which are representative examples of infectious pathogens, were selected as the target for detection.

[0075] 1.2. Preparation of substrate particles (magnetic probes) Carboxylic acid-functionalized magnetic particles (2.8 μm in diameter) were thoroughly vortexed for 10 minutes before use. 200 μl of the magnetic particles were transferred to a new tube. The magnetic particles were washed twice with 25 mM 2-[N-morpholino]ethanesulfonic acid (MES) buffer (pH 4.8) and resuspended in 20 μl of the same buffer. 8 μl of N-ethyl-N'-[3-dimethylaminopropyl]carbodiimide hydrochloride (100 mg / ml) dissolved in 100 mM MES (pH 4.8) and 5'-aminated oligonucleotide (target-capture chain) (10 nmol; 5'-NH2-(CH2)3-A) were added. 10 -ATC CTT ATC AAT ATT-3':NH2-(CH2)3-A 10 -[SEQ ID NO: 3]) mixture was added to the washed magnetic particle solution. After incubation at room temperature for 4 hours with vortexing, the magnetic particles were washed three times and dispersed in hybridization buffer. The magnetic particles obtained above were used as substrate particles for target substance detection in the following examples.

[0076] 1.3. Fabrication of NCR probe Disulfide-modulated template chain (5'-TAA CAA TAA TCC CTC ATC CTT ATC AAT ATT TAA CAA TAA TCC CTC (A) 10 -3';[Sequence ID 2]-(A) 10 The 3'-thiolated template chain (5'-[SEQ ID NO: 2]-PEG6-A) was reduced using 100 mM dithiothreitol (DTT) dissolved in 100 mM phosphate buffer (pH 8) via incubation at room temperature for 2 hours. 10 A (SH-3') sample was prepared. After reduction, excess DTT was removed using a NAP-5 column (Fisher Scientific). The newly reduced thiolated template chain was prepared in 10 steps. 3The mixture was added to a distilled aqueous solution containing 0.1% (v / v) SDS (Sodium Dodecyl Sulfate) and 5.17 moles of gold nanoparticles (AuNPs) with an average diameter of 50 nm. The resulting mixture was incubated at room temperature for 2 hours, and then adjusted to a final phosphate concentration of 100 mM (pH 7.4). Six preparative portions of 2 M NaCl containing 0.01% SDS were added at 30-minute intervals to achieve a final salt concentration of 0.3 M. The mixture was heated at 80°C for 10 minutes after each salt addition. The final mixture was gradually cooled at room temperature and incubated overnight to obtain a suspension of gold nanoparticles with numerous template chains bonded to the surface. The suspension was washed by centrifugation (8000 rpm, 10 minutes), and the resulting particle solution was redispersed in distilled water. Template-bound gold nanoparticles were incubated at 55°C for 10 minutes in 300 mM NaCl buffer with an excess of target-cloning strand (5'-GAG GGA TTA TTG TTA AAT ATT GAT AAG GAT-3'; SEQ ID NO: 1), and then cooled at room temperature for 1 hour. The excess target-cloning strand was removed by centrifugation (11000 rpm, 2 min). An excess of toehold DNA (SEQ ID NO: 4) in 40 mM MgCl2 buffer containing 0.1% (v / v) SDS was added to the same tube and incubated at 43°C for 30 minutes, then cooled at room temperature for 3 hours to obtain an NCR probe in which the template strand was bound to the surface and the target-cloning strand was partially hybridized to the template strand. After sequential hybridization, the obtained NCR probe was washed four times by centrifugation (11000 rpm, 2 min) and redispersed in hybridization buffer.

[0077] The sequences of oligonucleotides used in Examples 1.2 and 1.3 are summarized in Table 1 below:

[0078] [Table 1]

[0079] 1.4. NCR Assay Before amplification, PerfectHyb (trademark) Plus Hybridization Buffer (Sigma-Aldrich) was prepared by mixing it with 1x PBS buffer in a 1:3 ratio. For hybridization, 100 μl of the target sequence (GAG GGA TTA TTG TTA AAT ATT GAT AAG GAT; SEQ ID NO: 1) and 10 μl (5 mg / ml) of magnetic substrate particles (Example 1.2) were placed in a tube and mixed, then incubated for 10 minutes. 15 μl (1 OD) of NCR probe (Example 1.3) (5.17 x 10) was added to the same tube. -2 After adding pmole / ml (775 amole), the mixture was incubated for 20 minutes. The magnetic substrate particles were washed three times with hybridization buffer through a magnetic separator, then dispersed in 30 µl of distilled water and incubated at 80°C for 2 minutes to dehybridize. For the next cycle of the assay, the salt concentration was increased by adding hybridization buffer, and the mixture was incubated for 10 minutes. NCR probe 15 μl (1 OD) (5.17 x 10 -2 The assay product was subjected to a higher red color intensity by repeating the process of adding pmole / ml (775 amole), incubating for 20 minutes, washing the nucleic acid, and releasing it.

[0080] 1.5. UV-Vis Measurement of NCR Assay Products NCR assay products were immediately separated via a magnetic separator and cultured at 80°C for nucleic acid release before UV-Vis measurement. The absorbance spectra of each released NCR assay product were measured using an ultraviolet-visible spectrometer (S-3100, SCINCO). Red UV-Vis intensity measured from a gold nanoparticle probe (NCR probe) was measured at 532 nm.

[0081] 1.6. Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis was performed to verify the process of preparing active NCR probes, including the toehold-mediated displacement reaction. A 15% (w / v) polyacrylamide gel was prepared using 30% (w / v) acrylamide / Bis solution, deionized water, 5X TBE (Tris-Borate-EDTA) buffer, ammonium persulfate, and TEMED (tetramethylethylenediamine). Gold nanoparticles bound to DNA (Example 1.3) were dissolved by treatment with 0.1 M KCN ​​solution. The gold-dissolved DNA sample containing a loading dye was added to each well of a 1.5 mm thick gel. A 20 bp DNA ladder was loaded into another well to confirm the DNA molecular weight on the gel. Electrophoresis was performed on the gel by applying an electric field (130 V) for 70 minutes using a power supply. After electrophoresis, the gel was stained with SYBR Green I (Lonza) for 10 minutes, and the gel was imaged using a gel imager with UV excitation at 306 nm.

[0082] 1.7. Scanning electron microscopy (SEM) The sandwich complex of magnetic substrate particles and NCR probe in the presence of the target sequence (SEQ ID NO: 1) in each cycle was observed using a field emission scanning electron microscope (FE-SEM; SUPRA 55VP, Carl Zeiss, Germany). The structural particles of the sandwich complex were loaded onto a copper grid, coated with platinum (EM ACE200, Leica, Austria), and imaged. FE-SEM imaging was performed at NICEM (National Instrumentation Center for Environmental Management; Seoul National University, Seoul, South Korea).

[0083] 1.8. Quantification of oligonucleotides AuNP-based PCR probes were prepared using fluorescent dye-modified oligonucleotides for the quantification of target oligonucleotides. Fluorescence signals were measured using a fluorometer (F200 Pro, TECAN). The template and target-cloning strands were labeled at their 5' ends with Cy3 dye (template strand) and Cy5 dye (target-cloning strand), respectively. After salting and NCR assay, gold nanoparticles were dissolved in 10 mM KCN. Fluorescence intensity was measured using the remaining fluorescent-labeled oligonucleotides. Calibration curves of known dye sample concentrations were used for quantification.

[0084] 1.9. Analysis of particle properties through dynamic light scattering (DLS) and zeta potential measurement. To analyze the size and surface charge characteristics of the NCR probe and magnetic substrate particles, the NCR probe and / or magnetic substrate particles were dispersed in a distilled aqueous solution. Then, the particle size and charge were measured using a dynamic light scattering photometer (Malvern Instrument, Malvern), and the average value after three measurements was used.

[0085] Data availability The data supporting the plots in this paper and other findings of this study are available from the corresponding author upon reasonable request.

[0086] Example 2. Colorimetric NCR assay The magnetic probes and NCR probes prepared in Examples 1.2 and 1.3, respectively, were treated with a 10 pM concentration of target oligonucleotides (GAG GGA TTA TTG TTA AAT ATT GAT AAG GAT; SEQ ID NO: 1). The sandwich complexes formed between the NCR probes and magnetic probes in each cycle were observed using a scanning electron microscope (SEM) (see Example 1.7), and the color change of the reaction solution was observed visually. The resulting SEM images and the color change of the solution are shown in Figure 1c (Scale bar, 200 nm; inset shows the color appearance as the number of unloaded NCR probes increases) and Figure 4 (Scale bar: 1 μm). As these results show, it can be confirmed that as the NCR cycle is repeated, the number of NCR probes trapped on the magnetic probe surface increases, and the red color intensity of the solution increases.

[0087] Furthermore, as in Example 1.3, gel electrophoresis was performed with an NCR probe prepared by adding the toehold sequence (SEQ ID NO: 4) to confirm whether undesirable binding occurred between the target-binding site on the template strand and the target-cloning strand. This was done by referring to Example 1.6. For comparison, the same test was performed on an NCR probe that had not been treated with toehold DNA. The gel electrophoresis results obtained are shown in Figure 2a. As shown in Figure 2a, when the toehold sequence is added as in Example 1.3, suitable binding occurs between the target-cloning site on the template strand and the target-cloning strand without binding between the target-binding site on the template strand and the target-cloning strand, resulting in an activated NCR probe with the target-binding site exposed on a single strand. Conversely, when the toehold sequence is not added, binding occurs between the target-binding site on the template strand and the target-cloning strand, resulting in an inactive NCR probe with the target-binding site not exposed. These results indicate that the addition of the toehold sequence resulted in toehold-mediated displacement, which was intended to expose the target-binding site.

[0088] Example 3. NCR assay (UV-Vis spectrum) The NCR assay results, depending on the target oligonucleotide concentration or the presence or absence of the toehold sequence, were confirmed by UV-Vis spectroscopy (see Examples 1.4 and 1.5).

[0089] To confirm target specificity, the same test was performed using a non-target sequence (N; SEQ ID NO: 6) and a single-base mismatch sequence (S; SEQ ID NO: 5) as comparative examples.

[0090] The results obtained above are shown in Figures 2b to 2d.

[0091] Figure 2b is a graph showing NCR results (UV-Vis spectra) for target sequences at various concentrations. It shows that non-target sequences (N) and single-base mismatch sequences (S) are not detected, while target sequences are detected with high sensitivity (detection limit: ≥100 zM), and that detection ability is proportional to the number of cycles. Figures 2c (1 cycle) and 2d (1-3 cycles) show the results of the NCR assay as observed visually. This also shows that the NCR assay does not detect non-target sequences and single-base mismatch sequences, but can detect target sequences with high sensitivity. These results demonstrate that the NCR assay provided in this application is target-specific and can amplify / detect / visualize target substances with high sensitivity. Furthermore, Figure 2c shows that the activated NCR probe with the target-binding site of the template chain exposed can clearly distinguish between N and S sequences and target sequences with the naked eye.

[0092] Furthermore, the UV-Vis absorbance region after two NCR assay cycles was measured and is shown in Figure 5. In Figure 5, the initial target sequence concentration was 10 pM, N and S represent the non-target sequence and single-base mismatch sequence, respectively, and the optical extinction value was measured at 532 nm (Error bars show the sd of the extinction values ​​from three independent experiments). As shown in Figure 5, the activated NCR probe with the target-binding site of the template chain exposed allows for clear visual differentiation of the N and S sequences from the target sequence.

[0093] Example 4. Quantification of oligonucleotides The oligonucleotides on the NCR probe prepared in Example 1.3 were quantified using the method of Example 1.8, and the results are shown in Figures 6 and 7.

[0094] Figure 6 shows the results of quantifying target-cloning strands on the NCR probe. The number of target-cloning strands on AuNP was calculated by measuring the fluorescence intensity. NCR probes prepared using target-cloning strands labeled with Cy5 dye were used in the test. Standard curves were prepared for known concentrations of Cy5 dye and serially diluted samples. The NCR probe (AuNP) was dissolved in 10 mM KCN ​​and used, and the blue triangles on the standard curve indicate the fluorescence intensity measured in the NCR probe solution.

[0095] Figure 7 shows the results of quantifying the template chain on the NCR probe. The number of template chains per AuNP was calculated by measuring the fluorescence intensity. NCR probes obtained by conjugating a template chain labeled with Cy3 dye to an AuNP were used in the test. Standard curves were prepared for diluted samples and known concentrations of Cy3 dye. The NCR probe (AuNP) was used dissolved in 10 mM KCN. The red triangles in the standard curve represent the fluorescence intensity measured between the template chain and the conjugated AuNP.

[0096] Example 5. Dynamic light scattering (DLS) measurement results Dynamic light scattering (DLS) measurements were performed on the NCR probe referring to Example 1.9, and the results are shown in Figures 8-11.

[0097] Figure 8 shows the results of dynamic light scattering (DLS) measurements of the NCR probe. DLS was used to measure the change in particle size during the NCR probe manufacturing process. Gold nanoparticles deformed (bound) to the template strand showed a Z-average size of 78.82 nm. Inactive NCR probes manufactured through target-cloning strand hybridization showed a Z-average size of 91.28 nm, indicating a longer DNA length than the template-bound gold nanoparticles. After removing excess target-cloning strand via Toehold sequencing, the active NCR probe showed a Z-average size of 69.51 nm, confirming a reduced DNA length. Although the base pairs of the template-deformed AuNPs and the NCR probes were identical, the active NCR probes contained double-stranded helical DNA, resulting in a smaller Z-average size.

[0098] Figure 9 shows the zeta potential measurement results of the NCR probe. Surface charge changes during the NCR probe fabrication process can be measured by zeta potential.

[0099] Figure 10 shows the UV-Vis spectral measurement results of the NCR probe. UV-Vis absorbance spectra were measured at various stages during NCR probe fabrication. The NCR probe shows that the UV-Vis data shifts to blue as the DNA strands are bonded to the surface of the gold nanoparticles.

[0100] Figure 11 is a graph showing the degree of target sequence capture by loading (containing a partially double-stranded oligonucleotide hybridized with the target-cloning strand) and unloading (containing only the template strand) NCR probes. The unloading NCR probe is generated during the NCR assay and interferes with the formation of the desired sandwich complex in the next cycle. To determine the effect of the unloaded NCR probe during the NCR assay, the kinetics of the unloaded and loaded NCR probes were measured through a single-cycle NCR assay. For comparison, both probes were tested identically against a mixture added to the same concentration as the target sequence, i.e., 10 pM. The mixtures of loaded and unloaded NCR probes were prepared in a 1:1 ratio. The unloading NCR probe was found to exhibit a better response than the loading NCR probe.

[0101] Example 6. Comparison of colorimetric NCR assay and PCR The genetic DNA (gDNA) of the bacterium Bacillus cereus was detected using the colorimetric NCR assay described in this application and existing PCR methods, and the results were compared.

[0102] Since the bacteria share SEQ ID NO: 1, which was used as a target in Examples 1.1 to 1.3, the NCR assay was performed using the magnetic probes and NCR probes prepared in Examples 1.1 to 1.3, in the manner of Example 1.4.

[0103] PCR was performed using the following primer sequence and conditions: F-primer:5'-CTGGCTCAGGATGAACGC-3'(Sequence ID 7) R-primer:5'-CGACTTCGGGTGTTACAA-3'(SEQ ID NO: 8)

[0104] PCR conditions: Repeat the following three stages 35 times: 1 minute at 95°C, 30 seconds at 53°C, and 1 minute 30 seconds at 72°C.

[0105] The comparative results of the colorimetric NCR assay and PCR obtained above are shown in Figures 12a-12c. Figure 12a is a schematic diagram comparing the gDNA detection processes between NCR and PCR. In the case of the NCR assay, no equipment is required for detection and it can be performed in a shorter time than PCR. Figure 12b shows the results of detecting gDNA extracted from bacteria by NCR or PCR. The NCR assay allows for macroscopic detection (detection limit: 10). -7 PCR can be confirmed through gel electrophoresis results (detection limit: 10 ng), indicating that the results can be confirmed through gel electrophoresis (detection limit: 10 -3 Figure 12c is a graph comparing NCR and PCR analysis results (UV-Vis spectrum), showing that the NCR assay is approximately 10% faster than the PCR assay. 4 It can be confirmed that it exhibits twice the sensitivity. The present invention includes, for example, the following embodiments: [Embodiment 1] (1) Template strand including the following: (i) A target-cloning site complementary to the target oligonucleotide, and (ii) A target-binding site complementary to a portion of the target oligonucleotide; and (2) A target-cloning strand containing the same sequence as the target oligonucleotide. Includes, The target-cloning site of the template chain and the target-cloning chain bind complementaryally to form a double helix, and the target-binding site is single-stranded. Partially double-stranded oligonucleotide. [Embodiment 2] The oligonucleotide according to Embodiment 1, wherein the template chain is 10-100 nt in length. [Embodiment 3] comprising a plurality of partially double-stranded oligonucleotides, Each partially double-stranded oligonucleotide is, (1) Template strand including the following: (i) A target-cloning site complementary to the target oligonucleotide, and (ii) A target-binding site complementary to a portion of the target oligonucleotide; and (2) A target-cloning strand containing the same sequence as the target oligonucleotide. Includes, The target-cloning site of the template chain and the target-cloning chain bind complementaryally to form a double helix, and the target-binding site is single-stranded. A nanoparticle in which one end of the aforementioned template chain is immobilized on the surface. [Embodiment 4] The nanoparticles according to Embodiment 3, wherein the nanoparticles are gold, silver, silica, platinum, copper, or two or more alloys selected from the group consisting of gold, silver, platinum, copper, etc. [Embodiment 5] (a) Substrate particles on which a portion (first portion) of the target oligonucleotide and multiple target-trapping chains complementary to it are immobilized on the surface, and (b) Nanoparticles containing multiple partially double-stranded oligonucleotides Includes, Each partially double-stranded oligonucleotide is, (1)(i) A target-cloning site complementary to the target oligonucleotide, and (ii) A template chain containing a target-binding site complementary to a portion (second portion) of the target oligonucleotide; and (2) Target-cloning chain containing the same sequence as the target oligonucleotide Includes, The target-cloning site of the template chain and the target-cloning chain bind complementaryally to form a double helix, and the target-binding site is single-stranded. One end of the template chain is immobilized on the surface of the nanoparticles. The first and second portions of the target oligonucleotide do not overlap. Compositions for amplification, detection, quantification, or visualization of target substances. [Embodiment 6] The composition according to Embodiment 5, wherein the substrate particles and nanoparticles are two or more types of different target oligonucleotides and are for use in amplification, detection, quantification, or visualization of two or more target substances. [Embodiment 7] A method for amplifying, detecting, quantifying, or visualizing a target substance, comprising the following steps: (A) Adding a target oligonucleotide or sample to substrate particles containing multiple target-trapping chains on their surface, thereby hybridizing a portion (first portion) of the target oligonucleotide with the target-trapping chains; (B) A step in which a plurality of nanoparticles are added to the mixture obtained in step (A) above to form a complex between substrate particles and nanoparticles, wherein each nanoparticle contains a plurality of partially double-stranded oligonucleotides on its surface, and each partially double-stranded oligonucleotide contains: (1) (i) a target-cloning site complementary to the target oligonucleotide, and (ii) A template chain containing a target-binding site complementary to a portion (second portion) of the target oligonucleotide; and (2) A target-cloning strand containing the same sequence as the target oligonucleotide, The target-cloning site of the template chain and the target-cloning chain bind complementaryally to form a double helix, and the target-binding site is single-stranded. A complex between the substrate particle and the nanoparticle is formed by hybridization between the target-binding site and the other portion (second portion) of the target oligonucleotide that is partially hybridized with the target-trapping chain on the surface of the substrate particle; and (C) A step of releasing a target-cloning chain having the same nucleic acid sequence as the target oligonucleotide by dehybridizing the partially double-stranded oligonucleotide, disassembling the complex, or both. [Embodiment 8] The method according to Embodiment 7, characterized in that the target-cloning chain released in step (C) is used as the target oligonucleotide in step (A), and steps (A) to (C) are repeated two or more times. [Embodiment 9] The method according to Embodiment 7, wherein the substrate particles and nanoparticles are two or more types for different target oligonucleotides, and the method is characterized by amplification, detection, quantification, or visualization of two or more target substances. [Embodiment 10] The method according to any one of Embodiments 7 to 9, characterized in that polymerase is not used. [Embodiment 11] A kit for amplification, detection, quantification, or visualization of a target substance, comprising the composition described in Embodiment 5 or 6.

Claims

1. A composition for amplification, detection, quantification, or visualization of a target oligonucleotide, (1) Template strand including the following: (i) A target-cloning site complementary to the target oligonucleotide, and (ii) A target-binding site complementary to a portion of the target oligonucleotide; and (2) A target-cloning strand containing the same sequence as the target oligonucleotide. It contains a partially double-stranded oligonucleotide, The target-cloning site of the template chain and the target-cloning chain bind complementaryally to form a double helix, and the target-binding site is single-stranded. A composition wherein the target oligonucleotide is an oligonucleotide that is amplified, detected, quantified, or visualized.

2. The composition according to claim 1, wherein the mold chain has a length of 10-100 nt.

3. A composition for amplification, detection, quantification, or visualization of a target oligonucleotide, The nanoparticles contain multiple partially double-stranded oligonucleotides, Each partially double-stranded oligonucleotide is, (1) Template strand including the following: (i) A target-cloning site complementary to the target oligonucleotide, and (ii) A target-binding site complementary to a portion of the target oligonucleotide; and (2) A target-cloning strand containing the same sequence as the target oligonucleotide. Includes, The target-cloning site of the template chain and the target-cloning chain bind complementaryally to form a double helix, and the target-binding site is single-stranded. One end of the template chain is immobilized on the surface of the nanoparticles, A composition wherein the target oligonucleotide is an oligonucleotide that is amplified, detected, quantified, or visualized.

4. The composition according to claim 3, wherein the nanoparticles are gold, silver, silica, platinum, copper, or two or more alloys selected from the group consisting of gold, silver, silica, platinum, and copper.

5. (a) A substrate particle on which a portion (first portion) of the target oligonucleotide and a plurality of target-trapping chains complementary to it are immobilized on its surface, and (b) Nanoparticles containing multiple partially double-stranded oligonucleotides on their surface Includes, Each partially double-stranded oligonucleotide is, (1) (i) A target-cloning site complementary to the target oligonucleotide, and (ii) A template chain containing a target-binding site complementary to a portion (second portion) of the target oligonucleotide; and (2) Target-cloning strand containing the same sequence as the target oligonucleotide Includes, The target-cloning site of the template chain and the target-cloning chain are complementaryly bound to form a double helix, the target-binding site is single-stranded, and the target-capture chain is single-stranded. One end of the template chain is immobilized on the surface of the nanoparticles. The first and second portions of the target oligonucleotide do not overlap. The target oligonucleotide is an oligonucleotide that is amplified, detected, quantified, or visualized. Compositions for amplification, detection, quantification, or visualization of target oligonucleotides.

6. The composition according to claim 5, wherein the substrate particles and nanoparticles are two or more types of different target oligonucleotides, and are used for amplification, detection, quantification, or visualization of two or more target oligonucleotides.

7. Methods for amplifying, detecting, quantifying, or visualizing target oligonucleotides, including the following steps: (A) A step of adding a target oligonucleotide or sample to substrate particles having a plurality of target-capture chains on its surface, thereby hybridizing a portion (first portion) of the target oligonucleotide with the target-capture chain, wherein the target-capture chain is complementary to the portion (first portion) of the target oligonucleotide and is single-stranded; (B) A step in which a plurality of nanoparticles are added to the mixture obtained in step (A) above to form a complex between the substrate particles and the nanoparticles, wherein each nanoparticle contains a plurality of partially double-stranded oligonucleotides on its surface, and each partially double-stranded oligonucleotide contains the following: (1) (i) A target-cloning site complementary to the target oligonucleotide, and (ii) A template chain containing a target-binding site complementary to a portion (second portion) of the target oligonucleotide; and (2) A target-cloning strand containing the same sequence as the target oligonucleotide, The target-cloning site of the template chain and the target-cloning chain bind complementaryally to form a double helix, and the target-binding site is single-stranded. A complex between the substrate particle and the nanoparticle is formed by hybridization between the target-binding site and the other portion (second portion) of the target oligonucleotide that is partially hybridized with the target-trapping chain on the surface of the substrate particle. The target oligonucleotide is an oligonucleotide that is amplified, detected, quantified, or visualized; and (C) A step of releasing a target-cloning chain having the same nucleic acid sequence as the target oligonucleotide by dehybriding the partially double-stranded oligonucleotide, dissociating the complex, or both.

8. The method according to claim 7, characterized in that steps (A) to (C) are repeated two or more times, using the target-cloning chain released in step (C) as the target oligonucleotide in step (A).

9. The method according to claim 7, characterized in that the substrate particles and nanoparticles are two or more types of different target oligonucleotides, and are used to amplify, detect, quantify, or visualize two or more target oligonucleotides.

10. The method according to any one of claims 7 to 9, characterized in that polymerase is not used.

11. A kit for amplification, detection, quantification, or visualization of a target oligonucleotide, comprising the composition according to claim 5 or 6.

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