Janus particles for detecting target material and method for detecting target material using same

Abstract

The present invention relates to Janus particles for detecting a target material and a method for detecting a target material using same and, more specifically, to Janus particles for detecting a target material and a method for detecting a target material using same, wherein the Janus particles vary in orientation depending on whether a mass label is bound thereto, and, by including a first surface and a second surface satisfying formula 1, are simple and economical and have high sensitivity.

Description

Janus particle for detecting target substance and method for detecting target substance using the same

[0001] The present invention relates to a Janus particle for detecting a target substance and a method for detecting a target substance using the same.

[0002] Various methods are used to detect target substances in biological specimens such as blood, serum, plasma, cells, and tissues.

[0003] A method for detecting a target substance can be performed using a capture reagent and a reporter reagent, for example, through the following process: incubating the sample with the capture reagent to bind the target substance to the capture reagent; adding a labeled reporter reagent to bind to the target substance; washing away excess reporter reagent; and measuring the amount of reporter reagent through label analysis.

[0004] Various substances can be used as capture and reporter reagents, and analysis using antibodies is called immunoassay. Immunoassays can take various forms. For example, there is the sandwich immunoassay, in which a capture antibody captures a target substance and a reporter antibody is used to generate a measurable signal; and the competitive immunoassay, in which a binder is attached to a solid phase and the target substance competitively binds to the binder among the reagent and solution that generate the measurable signal.

[0005] The detection limit of conventional immunoassays is approximately 0.1 picomolar to 1 nanomolar, depending on the method. Various methods have been developed to improve the detection limit, such as a method that measures individual binding events of a target substance and counts these binding events as "on" or "off" events when the measured value exceeds a local threshold. This method can eliminate a lot of background noise and, in particular, can significantly improve the detection limit through digital analysis.

[0006] Examples of digital analysis include the Quanterix Single Molecule Array (SIMOA) system and the Merck Millipore Single Molecule Counting (SMC) system.

[0007] The SIMOA system captures target substances from a solution using antibody-coated paramagnetic beads. The beads are then washed, and an enzyme-labeled reporter antibody is added. The beads are washed again and loaded into a microwell array capable of holding only one bead per well. If the enzyme is attached to a bead, the fluorescent substrate in the well is flipped. If the fluorescence exceeds a threshold, each well is counted as an "on" or "off" event.

[0008] In the case of the SMC system, magnetic beads coated with capture antibodies are used to capture target substances in sandwich assays. When the target substance binds to the beads, fluorescently labeled reporter antibodies also bind to the beads. The beads are pulled by magnets to wash away excess reporter antibodies. Then, an elution buffer is added to induce the dissociation of the sandwich complex, and the sample is transferred to a measuring vessel. The presence of fluorescent labels is confirmed using a confocal fluorescence microscope that sequentially irradiates small sample volumes. If the signal of each individual measurement exceeds a threshold, it is counted as an "on" event for that measurement.

[0009] SIMOA and SMC systems have demonstrated significantly improved performance compared to the detection limits of conventional immunoassays.

[0010] However, SIMOA and SMC systems require multiple cleaning and transfer steps, making them complex, cumbersome, and expensive.

[0011] Therefore, there is a need to develop a target substance detection method that is simpler, more economical, and highly sensitive.

[0012] The present invention relates to the Ministry of Science and ICT’s Individual Basic Research “Dipanoscopy-based Microfluidic Platform for Analysis of Mechanophenotype and Intracellular Skeletal Structure of Single Cells” (Reference Year: 2023, Unique Project No.: 1711194851, Sub-project No.: 00210336, Performing Institution: Kyungpook National University Industry-Academic Cooperation Foundation, Current Project Period: 2023-03-01~2024-02-29) and “Dipanoscopy-based Microfluidic Platform for Analysis of Mechanophenotype and Intracellular Skeletal Structure of Single Cells” (Reference Year: 2024, Unique Project No.: 2710000941, Sub-project No.: 00210336, Performing Institution: Kyungpook National University Industry-Academic Cooperation Foundation, Current Project Period: 2024-03-01~2025-02-28), and the Ministry of Science and ICT It was derived from research conducted as part of the "KNU-ETRI Future Digital Convergence System Scale-up Cooperation Platform" pilot project for establishing an industry-research cooperation platform (Project base year: 2024, Project unique number: 2710001688, Sub-project number: 00304695, Project implementing agency: Kyungpook National University Industry-Academic Cooperation Foundation, Project period for the current year: 2024-02-01~2024-12-31).

[0013] The present invention aims to provide a Janus particle for detecting a target substance that is economical as it does not require complex equipment and has a simple process, and has high sensitivity, and a method for detecting a target substance using the same.

[0014] 1. A Janus particle whose orientation changes depending on whether a mass label is attached, comprising an uncoated first surface and a second surface formed of a coating layer, wherein the first surface and the second surface satisfy the following mathematical formula 1:

[0015] [Mathematical Formula 1]

[0016] ρ 제1면 < ρ 제2면

[0017] (in the food, ρ 제1면 is the density of the first plane, and ρ 제2면 is the density of the second plane.)

[0018] 2. In the above 1, a Janus particle to which at least one antibody or a fragment thereof is bound.

[0019] 3. In the above 1, the second surface and mass label are a Janus particle satisfying the following mathematical formula 2:

[0020] [Mathematical Formula 2]

[0021] m 제2면 < m mass

[0022] (during the meal, m 제2면 is the mass of the second plane, and m mass is the mass of the mass label.)

[0023] 4. In 1 above, the Janus particle and mass label are a Janus particle satisfying the following mathematical formula 3:

[0024] [Mathematical Formula 3]

[0025]

[0026] (wherein, x is the number of mass markers bound to the Janus particle, a is the radius of the Janus particle (μm), t is the thickness of the second plane (nm), b is the radius of the mass marker (μm), and ρ cap The density of the second plane (g / cm³) 3 ) and, ρ mass is the density of the mass label (g / cm³) 3 )am.)

[0027] 5. In the above 1, the first surface and the second surface are Janus particles distinguished by color.

[0028] 6. In the above 1, the first surface is 1 to 3 g / cm² 3 It has a density of , and the second plane is 10 to 25 g / cm³ 3 A Janus particle having the density of

[0029] 7. A Janus particle according to 1 above, wherein the first surface is made of any one material selected from the group consisting of SiO2, Si, and polymethacrylate (PMMA).

[0030] 8. A Janus particle according to 1 above, wherein the second surface is composed of a metal layer selected from the group consisting of Pt, Au, and Ag.

[0031] 9. A method for detecting a target substance, comprising: a step of placing the Janus particle of 1 above into a medium to self-align the Janus particle such that the first surface faces upward and the second surface faces downward; a step of placing a sample into the medium to react the sample with the self-aligned Janus particle on the medium; and a step of selecting a Janus particle rotated such that the first surface faces downward and the second surface faces upward, wherein the target substance contained in the sample binds to the first surface.

[0032] 10. A method for detecting a target substance according to 9 above, wherein the second surface is distinguished from the first surface by a color sensor.

[0033] 11. A method for detecting a target substance according to 9, further comprising the step of quantifying the target substance by analyzing the color change of an image taken from an upward or downward direction.

[0034] 12. A method for detecting a target substance according to 9, further comprising the step of supplying gas onto a medium to promote a reaction between a sample and self-aligned particles.

[0035] 13. A method for detecting a target substance according to 9 above, wherein the medium is any one selected from the group consisting of water, PBS, HEPES buffer, TES buffer, and MOPS buffer.

[0036] 14. A method for detecting a target substance according to 9, wherein the particle is any one selected from the group consisting of spherical, ellipsoidal, and columnar particles.

[0037] 15. In the above 9, the first surface is 1 to 3 g / cm² 3 It has a density of , and the second plane is 10 to 25 g / cm³ 3A method for detecting a target substance having a density.

[0038] 16. A method for detecting a target substance according to 9 above, wherein the first surface is made of any one material selected from the group consisting of SiO2, Si, and polymethacrylate (PMMA).

[0039] 17. A method for detecting a target substance according to 9 above, wherein the second surface is composed of a metal layer selected from the group consisting of Pt, Au, and Ag.

[0040] 18. A method for detecting a target substance according to 9 above, wherein the target substance is any one selected from the group consisting of nucleotides, nucleic acids, amino acids, peptides, lipids, carbohydrates, and proteins.

[0041] The Janus particles of the present invention enable simple detection of target substances, thus offering advantages in terms of time and cost.

[0042] The Janus particles of the present invention are economical because they can be used to detect target substances without complex equipment such as optical devices.

[0043] The Janus particles of the present invention can be used to detect various types of substances.

[0044] The Janus particles of the present invention can detect target substances with high sensitivity.

[0045] The Janus particles of the present invention can detect target substances contained in biological samples with high performance.

[0046] The target substance detection method of the present invention is advantageous in terms of time and cost because the process is simple.

[0047] The present invention is economical because it can detect target substances without complex equipment such as optical devices.

[0048] The present invention can be used to detect various substances.

[0049] The present invention can detect target substances with high sensitivity.

[0050] The present invention can detect target substances contained in biological samples with high performance.

[0051] FIGS. 1a, 1b and FIG. 2 are schematic diagrams of Janus particles for detecting target substances according to one embodiment.

[0052] FIG. 3 schematically illustrates the process of manufacturing Janus particles according to one embodiment.

[0053] Figure 4 is an image of a Janus particle produced according to Example 1 of the present invention.

[0054] FIG. 5 is a schematic diagram showing the principle of Example 3 of the present invention.

[0055] Figure 6 shows the observation results of Example 3 of the present invention.

[0056] Figure 7 shows the results of confirming the reaction between the Janus particle and the mass label for detecting the target substance.

[0057] FIG. 8 is a schematic diagram showing the process of Example 5 of the present invention.

[0058] Figures 9a and 9b are images of the experimental results of Examples 5-1 and 5-2, respectively.

[0059] FIG. 10 is a schematic diagram illustrating the digital analysis method of Example 6 of the present invention.

[0060] FIGS. 11a and FIGS. 11b illustrate the experimental process according to Example 6 of the present invention.

[0061] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the claims. Throughout the specification, the same reference numerals refer to the same components.

[0062]

[0063] The present invention relates to Janus particles for detecting target substances.

[0064] The present invention relates to a Janus particle whose orientation changes depending on whether a mass label is attached, comprising a first surface that is not coated and a second surface that is composed of a coating layer.

[0065] The Janus particle of the present invention satisfies the following mathematical formula 1:

[0066] [Mathematical Formula 1] ρ 제1면 < ρ 제2면 (in the food, ρ 제1면 is the density of the first plane, and ρ 제2면 is the density of the second plane.).

[0067] In the present invention, the Janus particles may be spherical, ellipsoidal, or columnar, and preferably spherical.

[0068] In one embodiment, the Janus particle may include a capture reagent capable of binding to a target substance on its first surface, and the type of capture reagent may be selected according to the type of target substance to be detected.

[0069] In one embodiment, the capture reagent may be a nucleic acid, carbohydrate, antigen, peptide, protein, or antibody, and preferably may be an antibody or a fragment thereof.

[0070] In the present invention, the first and second surfaces of the Janus particle can be distinguished by color.

[0071] In the present invention, a mass label can be attached to the first surface of the Janus particle.

[0072] In the present invention, the mass label is a type of reporter reagent and consists of a substance heavier than the mass of the second face of the Janus particle.

[0073] In one embodiment, the mass label can be bound to a capture reagent bound to the first surface of the Janus particle through a target substance.

[0074] In one embodiment, the second face and mass label of the Janus particle may satisfy Equation 2: [Equation 2] m 제2면 < m mass (during the meal, m 제2면 is the mass of the second plane, and m mass is the mass of the mass label.)

[0075] In the present invention, since the density of the second face of the Janus particle is greater than the density of the first face and the mass of the mass label is greater than the mass of the second face of the Janus particle, the orientation of the Janus particle may vary depending on whether the mass label is bound.

[0076] In one embodiment, the Janus particle and the mass label may satisfy Equation 3: [Equation 3] (wherein, x is the number of mass markers bound to the Janus particle, a is the radius of the Janus particle (μm), t is the thickness of the second plane (nm), b is the radius of the mass marker (μm), and ρ cap The density of the second plane (g / cm³) 3 ) and, ρ mass is the density of the mass label (g / cm³) 3 )am.)

[0077] In the present invention, the thickness of the second surface refers to the thickness of the coating layer.

[0078] In the present invention, a represents the radius of the Janus particle (μm), t represents the thickness of the second face of the Janus particle (nm), and b represents the radius of the mass label (μm) (Fig. 1a). For example, if the radius of the Janus particle is 4 μm, the thickness of the second face of the Janus particle is 30 nm, and the radius of the mass label is 1.5 μm, then 4 can be substituted for a in Equation 3, 30 for t, and 1.5 for b.

[0079] In the present invention, the density is g / cm³ 3 It has units. For example, ρ cap and ρ mass silver g / cm 3 It has units.

[0080] In one embodiment, the density of the first plane is 0.1 to 10 g / cm³ 3 It can be, for example, 0.1 to 9 g / cm³ 3 , 0.1 to 8 g / cm² 3 , 0.1 to 7 g / cm² 3 , 0.1 to 6 g / cm² 3 , 0.1 to 5 g / cm² 3 , 0.1 to 4 g / cm² 3 , 0.1 to 3 g / cm² 3 , 0.1 to 2 g / cm³ 3 , 0.1 to 1 g / cm³ 3 , 0.5 to 10 g / cm³ 3 , 0.5 to 9 g / cm² 3 , 0.5 to 8 g / cm² 3 , 0.5 to 7 g / cm² 3 , 0.5 to 6 g / cm² 3 , 0.5 to 5 g / cm² 3 , 0.5 to 4 g / cm² 3 , 0.5 to 3 g / cm² 3 , 0.5 to 2 g / cm²3 , 0.5 to 1 g / cm³ 3 , 1 to 10 g / cm² 3 , 1 to 9 g / cm² 3 , 1 to 8 g / cm² 3 , 1 to 7 g / cm² 3 , 1 to 6 g / cm² 3 , 1 to 5 g / cm² 3 , 1 to 4 g / cm² 3 , 1 to 3 g / cm² 3 , 1 to 2 g / cm² 3 or 2 to 3 g / cm³ 3 It could be.

[0081] In one embodiment, the density of the second plane is 3 to 30 g / cm³ 3 It can be, for example, 3 to 29 g / cm³ 3 , 3 to 28 g / cm² 3 , 3 to 27 g / cm² 3 , 3 to 26 g / cm² 3 , 3 to 25 g / cm² 3 , 3 to 24 g / cm² 3 , 3 to 23 g / cm² 3 , 3 to 22 g / cm² 3 , 5 to 30 g / cm² 3 , 5 to 29 g / cm² 3 , 5 to 28 g / cm² 3 , 5 to 27 g / cm² 3 , 5 to 26 g / cm² 3 , 5 to 25 g / cm² 3 , 5 to 24 g / cm² 3 , 5 to 23 g / cm² 3 , 5 to 22 g / cm² 3 , 10 to 30 g / cm² 3 , 10 to 29 g / cm² 3 , 10 to 28 g / cm² 3 , 10 to 27 g / cm² 3 , 10 to 26 g / cm² 3, 10 to 25 g / cm² 3 , 10 to 24 g / cm² 3 , 10 to 23 g / cm³ 3 , 10 to 22 g / cm² 3 , 15 to 30 g / cm² 3 , 15 to 29 g / cm³ 3 , 15 to 28 g / cm² 3 , 15 to 27 g / cm² 3 , 15 to 26 g / cm² 3 , 15 to 25 g / cm² 3 , 15 to 24 g / cm² 3 , 15 to 23 g / cm³ 3 , 15 to 22 g / cm² 3 , 20 to 30 g / cm² 3 , 20 to 29 g / cm² 3 , 20 to 28 g / cm² 3 , 20 to 27 g / cm² 3 , 20 to 26 g / cm² 3 , 20 to 25 g / cm² 3 , 20 to 24 g / cm² 3 , 20 to 23 g / cm² 3 , 20 to 22 g / cm² 3 or 21 to 22 g / cm² 3 It could be.

[0082] Preferably, the first surface is 1 to 3 g / cm² 3 It has a density of 10 to 25 g / cm³, and the second surface has a density of 10 to 25 g / cm³ 3 It can have a density of.

[0083] In one embodiment, the first surface of the Janus particle may be made of any one material selected from the group consisting of SiO2, Si, and polymethacrylate (PMMA).

[0084] In one embodiment, Janus particles can be produced by depositing or coating one side of a particle of a specific material with another material.

[0085] In one embodiment, Janus particles can be produced by depositing or coating one side of a particle of a specific material with metal.

[0086] In one embodiment, the second surface may be composed of a metal layer selected from the group consisting of Pt, Au, and Ag.

[0087] The Janus particle according to the present invention can be used for detecting a target substance, and the target substance may be a nucleotide, nucleic acid, amino acid, peptide, protein, lipid, carbohydrate, drug, steroid, or hormone.

[0088] In one embodiment, the target substance may be any one selected from the group consisting of nucleotides, nucleic acids, amino acids, peptides, lipids, carbohydrates, and proteins.

[0089] For example, the first surface of the Janus particle is made of SiO2 and the second surface is deposited with Pt, and an antibody may be bound to the first surface (Fig. 1b). In this case, the Janus particle can bind to a target substance (antibody) that can bind complementarily using the antibody bound to the first surface, and thus can be used to detect the target substance.

[0090]

[0091] The present invention also relates to a method for detecting a target substance using Janus particles for detecting the target substance.

[0092] The present invention relates to a method for detecting a target substance, comprising the steps of: placing the Janus particle into a medium to self-align the Janus particle such that the first surface faces upward and the second surface faces downward; placing a sample into the medium to react the sample with the self-aligned Janus particle on the medium; and selecting a Janus particle that has been rotated such that the first surface faces downward and the second surface faces upward, wherein the target substance contained in the sample binds to the first surface.

[0093] The target substance detection method of the present invention can be performed using Janus particles having two sides of different densities.

[0094] In the present invention, since the Janus particles are composed of two faces having different densities, they can self-align in a certain orientation in a fluid, such as a medium.

[0095] In the present invention, the Janus particles may be spherical, ellipsoidal, or columnar, but the shape of the Janus particles is not limited as long as the Janus particles can self-align due to the difference in density between the two faces constituting the Janus particles in the medium.

[0096] In one embodiment, the Janus particle may be spherical.

[0097] In the present invention, the density of the first surface of the Janus particle may be smaller than the density of the second surface.

[0098] In one embodiment, the Janus particle may include a capture reagent capable of binding to a target substance on its first surface, and the type of capture reagent may be selected according to the type of target substance to be detected.

[0099] In one embodiment, the capture reagent may be a nucleic acid, carbohydrate, antigen, peptide, protein, or antibody, and preferably may be an antibody.

[0100] In the present invention, the first and second surfaces of the particle may have different colors.

[0101] In one embodiment, the first surface and the second surface of the particle can be distinguished by a color sensor.

[0102] In the present invention, since the first and second surfaces of the particles have different densities and colors, the particles self-align in a certain orientation in the medium, and the orientation of the self-aligned particles can be distinguished by color.

[0103] For example, the first surface is 1 to 3 g / cm² 3It has a density of 10 to 25 g / cm³, and the second surface has a density of 10 to 25 g / cm³ 3 It can have a density of.

[0104] The target substance detection method of the present invention may further include a step of quantifying the target substance by analyzing the color change of an image taken from the upward or downward direction.

[0105] In the present invention, the medium is not limited to any type as long as it is a liquid that does not react with particles.

[0106] In the present invention, the medium may be selected by a person skilled in the art as appropriate depending on the type of particle used in the target substance detection method.

[0107] In one embodiment, the medium may be water or a buffer solution. In one embodiment, the medium may be any one selected from the group consisting of PBS (phosphate buffered saline), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid) buffer, and MOPS (3-(N-morpholino)propanesulfonic acid) buffer.

[0108] In the present invention, water is used to refer collectively to distilled water, such as primary distilled water, secondary distilled water, and tertiary distilled water.

[0109] In the present invention, the specimen may be a biological specimen, such as blood, serum, plasma, cells, or tissue.

[0110] The target substance detection method of the present invention may further include the step of supplying a gas onto a medium to promote the reaction between the sample and the self-aligned particles. The supplied gas can cause at least some of the particles present in the medium to react more actively with the mass label by propelling them.

[0111] The target substance detection method of the present invention may further include the step of pre-reacting a sample with a mass label.

[0112] In the present invention, the mass label is a type of reporter reagent and consists of a substance capable of binding to a target substance and a substance heavier than the mass of the second face of the Janus particle (Fig. 2).

[0113] In one embodiment, the type of substance capable of binding to the target substance included in the reporter reagent may be selected according to the type of target substance to be detected, and may be, for example, nucleic acid, carbohydrate, antigen, peptide, protein, or antibody.

[0114] In the step of pre-reacting the sample of the present invention with a mass label, if a target substance is present in the sample, the mass label can be bound to the target substance.

[0115] In the present invention, since the mass label is heavier than the second surface of the particle, when a sample that has been pre-reacted with the mass label is reacted with a self-aligned particle in a medium, the target substance contained in the sample binds to the first surface and can rotate so that the first surface faces downward and the second surface faces upward.

[0116] In the present invention, the target substance may be a nucleotide, nucleic acid, amino acid, peptide, protein, lipid, carbohydrate, drug, steroid, or hormone.

[0117] In one embodiment, the target substance may be any one selected from the group consisting of nucleotides, nucleic acids, amino acids, peptides, lipids, carbohydrates, and proteins.

[0118] The above description of the method for detecting a target substance using Janus particles for detecting a target substance can be applied in the same way as the description of the Janus particles for detecting a target substance described above, to the extent that it does not contradict each other.

[0119]

[0120] Hereinafter, the present invention will be described in detail with reference to examples to specifically explain the invention. However, the following examples are provided merely to facilitate a better understanding of the invention, and the content of the invention is not limited by the following examples.

[0121]

[0122] Examples

[0123]

[0124] The Janus particles of the present invention were fabricated through experiments designed as follows, and additional experiments were performed to apply them to a target substance detection method:

[0125] 1) Setting the target substance as streptavidin and accordingly fabricating Janus particles using streptavidin silica particles; 2) Fabricating a mass label by attaching biotin, which has binding affinity to the target substance; 3) Verifying whether the particle reaction can be accelerated to improve the target substance detection performance; 4) Verifying the reaction between the Janus particles and the mass label; 5) Verifying the alignment of Janus particles based on whether the target substance is bound; 6) Quantitative analysis of the target substance using the orientation and color change of Janus particles based on whether the target substance is bound.

[0126]

[0127] Example 1. Fabrication of Janus particles for target substance detection

[0128] 20 μL of 7.88 μm streptavidin silica particles (SPHERO™ Catalog no. SVSIP-60-5; concentration: 73,645 particles / μL) were placed in an e-tube, and 20 μL of ethanol was added to adjust the total volume of the solution to 40 μL. The solution was vortexed for 30 seconds. A silicon wafer was sliced ​​into pieces measuring 0.5 cm × 0.5 cm, and the surface was treated with O2 plasma to make it hydrophilic. Then, 10 μL of the previously prepared solution was dropped onto the surface of the silicon wafer slices to perform drop casting, thereby arranging the particles into a monolayer. The silicon wafer was placed in a sputtering apparatus, and Pt sputtering was performed under the conditions of Table 1 to deposit a 30 nm thick layer of Pt on one side of the particles (Fig. 3).

[0129] Vacuum 10 Pa, Output Current 19 mA, Time 60 seconds

[0130] After Pt sputtering was completed, a silicon wafer piece containing Janus particles and 400 μL of PBS were placed in an e-tube to recover the particles, and sonication was performed for 10 minutes. The particles were precipitated by centrifugation at 4000 rpm for 30 seconds, and the supernatant was removed to obtain Janus particles with Pt deposited on one side. The results of observing and photographing the Janus particles under a microscope are shown in Figure 4.

[0131]

[0132] Example 2. Fabrication of a mass label for detecting a target substance

[0133] 20 μL of 2.38 μm silica particles (Batch no. SiO2-R-SC276-2; concentration: 1,589,886 particles / μL) were placed in an e-tube, and 20 μL of ethanol was added to wash the particles. To modify the surface of the silica particles with amine groups (-NH2), 200 μL of an APTES solution diluted to 5% by adding 1 mL of ethanol to 66 μL of 99% (3-Aminopropyl)triethoxysilane (APTES; CAS no. 919-30-2) was added to the e-tube, and the solution and particles were mixed by vortexing for 30 seconds. After reacting on a shaker for 30 minutes, the reaction was subsequently carried out in a 60°C oven for 30 minutes. Then, the particles were washed three times with ethanol to completely remove the APTES solution, and 200 μL of PBS was added to resuspend the particles. 10 μL of a 10 mM NHS-biotin solution, prepared by dissolving 1 mg of NHS-biotin (EZ-Link™; Catalog no. 20217) in 293 μL of N,N-Dimethylformamide (DMF; CAS no. 68-12-2), was added and reacted in a shaker for 30 minutes. Then, the particles were washed twice with PBS to completely remove any unreacted NHS-biotin solution remaining, and a biotin-bound mass label was obtained.

[0134]

[0135] Example 3. Confirmation of whether the particle can promote the reaction

[0136] 5 μL (73,645 particles / μL) of Janus particles prepared in Example 1 were dropped into a 30% hydrogen peroxide solution (CAS no. 7722-84-1) and observed. As soon as the particles were added to the hydrogen peroxide, oxygen bubbles began to form on the Pt surface of the particles as shown in Fig. 5, and as the bubbles burst, the Janus particles were observed to be propelled (Fig. 6). Through this, it was confirmed that the Janus particles in the solution gain propulsion, thereby improving reaction efficiency.

[0137]

[0138] Example 4. Confirmation of the reaction between Janus particles and mass labels

[0139] 100 μL of 1% BSA solution was added to 2 μL (1,589,886 particles / μL) of the biotin-bound mass label prepared in Example 2 and incubated for 30 minutes. Then, the 1% BSA solution was completely removed by washing three times with PBS. 5 μL (73,645 particles / μL) of Janus particles prepared in Example 1 and 200 μL of 0.05% Tween 20 buffer were added to 2 μL (1,589,886 particles / μL) of the 1% BSA-coated mass label and reacted for 30 minutes to obtain Janus particles bound to the mass label. The results of microscopic observation and imaging are shown in Figure 7.

[0140]

[0141] Example 5. Observation of Janus particle arrangement

[0142]

[0143] Example 5-1. Confirmation of the arrangement of Janus particles for target substance detection

[0144] 300 μL of triple-distilled water was added to the wells of a 96-well microplate (Corning® 96 Well EIA / RIA Assay Microplate; Catalog no. CLS3590). 20 μL of the Janus particles prepared in Example 1 were extracted, dropped into the wells, and mixed by pipetting. After waiting 10 minutes for the particles to settle completely to the bottom, the arrangement of the particles was observed under a microscope. It was confirmed that the black Pt side of the Janus particles faced downward and the white side faced upward. Images taken from the downward direction were obtained for digital analysis (Figs. 8 and 9a).

[0145]

[0146] Example 5-2. Confirmation of the arrangement of Janus particles combined with mass labels

[0147] 300 μL of triple-distilled water was added to the wells of a 96-microwell plate. 20 μL of the 200 μL of mass-labeled Janus particles obtained in Example 4 was extracted, dropped into the wells, and mixed by pipetting. After waiting 10 minutes for the particles to completely settle to the bottom, the arrangement of the particles was observed under a microscope, and images for digital analysis were obtained (Fig. 9b).

[0148]

[0149] Example 6. Digital analysis according to the arrangement of Janus particles

[0150] Using the Janus particle image obtained in Example 5-1, threshold values ​​required for image analysis were set in the Image J program. By entering the 'Analyze → Analyze Particles' menu, Size and Circularity values ​​were specified so that only black particles could be counted (Fig. 11a).

[0151] In the image taken from the downward direction, since the particles flipped in combination with the mass label show a white side rather than a black side, the number of white particles was calculated by subtracting the number of particles counted as black from the total number of particles (Fig. 10). To obtain a calibration curve for quantitative analysis of the concentration of the target substance, the ratio of the number of white particles to the total number of particles was measured (Fig. 11b).

Claims

1. As a Janus particle whose orientation changes depending on whether a mass label is bound, It includes an uncoated first surface and a second surface composed of a coating layer, and The above-mentioned first surface and the above-mentioned second surface are Janus particles satisfying the following mathematical formula 1: [Mathematical Formula 1] r 제1면 < p 제2면 (in the food, ρ 제1면 is the density of the first plane, and ρ 제2면 is the density of the second plane.).

2. A Janus particle according to claim 1, wherein the first surface is to which at least one antibody or a fragment thereof is attached.

3. The Janus particle of Claim 1, wherein the second surface and the mass label satisfy the following Equation 2: [Mathematical Formula 2] m 제2면 < m mass (during the meal, m 제2면 is the mass of the second plane, and m mass is the mass of the mass label.).

4. The Janus particle of Claim 1, wherein the Janus particle and the mass label satisfy the following Equation 3: [Mathematical Formula 3] (wherein, x is the number of mass markers bound to the Janus particle, a is the radius of the Janus particle (μm), t is the thickness of the second plane (nm), b is the radius of the mass marker (μm), and ρ cap The density of the second plane (g / cm³) 3 ) and, ρ mass is the density of the mass label (g / cm³) 3 )am.).

5. The Janus particle of Claim 1, wherein the first surface and the second surface are distinguished by color.

6. In Claim 1, the first surface is 1 to 3 g / cm² 3 It has a density of 10 to 25 g / cm³, and the second surface has a density of 10 to 25 g / cm³ 3 A Janus particle having the density of 7. A Janus particle according to claim 1, wherein the first surface is made of any one material selected from the group consisting of SiO2, Si, and polymethacrylate (PMMA).

8. A Janus particle according to claim 1, wherein the second surface is composed of a metal layer selected from the group consisting of Pt, Au, and Ag.

9. A step of placing the Janus particles of Claim 1 into a medium and self-aligning the Janus particles such that the first surface faces upward and the second surface faces downward; A step of placing a sample into the medium and reacting the sample with the self-aligned Janus particles on the medium; and A method for detecting a target substance, comprising the step of selecting Janus particles in which the target substance contained in the sample binds to the first surface and is rotated so that the first surface faces downward and the second surface faces upward.

10. A method for detecting a target substance according to claim 9, wherein the second surface is distinguished from the first surface by a color sensor.

11. A method for detecting a target substance according to claim 9, further comprising the step of quantifying the target substance by analyzing the color change of an image taken from the upward or downward direction.

12. A method for detecting a target substance according to claim 9, further comprising the step of supplying a gas onto the medium to promote a reaction between the sample and the self-aligned particles.

13. A method for detecting a target substance according to claim 9, wherein the medium is any one selected from the group consisting of water, PBS, HEPES buffer, TES buffer, and MOPS buffer.

14. A method for detecting a target substance according to claim 9, wherein the particle is any one selected from the group consisting of spherical, ellipsoidal, and columnar bodies.

15. In claim 9, the first surface is 1 to 3 g / cm² 3 It has a density of 10 to 25 g / cm³, and the second surface has a density of 10 to 25 g / cm³ 3 A method for detecting a target substance having a density.

16. A method for detecting a target substance according to claim 9, wherein the first surface is made of any one material selected from the group consisting of SiO2, Si, and polymethacrylate (PMMA).

17. A method for detecting a target substance according to claim 9, wherein the second surface is composed of a metal layer selected from the group consisting of Pt, Au, and Ag.

18. A method for detecting a target substance according to claim 9, wherein the target substance is any one selected from the group consisting of nucleotides, nucleic acids, amino acids, peptides, lipids, carbohydrates, and proteins.

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