Multiple analyte detection methods

Abstract

The present invention provides a method for detecting multiple analytes that can effectively improve detection sensitivity, comprising the following steps: providing micron particles. The micron particles are coupled with at least one first ligand and include a body and a plurality of first protrusions formed on the surface of the body. Next, the micron particles are mixed with a plurality of analytes to form a first complex. Thereafter, the first complex is mixed with a plurality of second ligands with a plurality of labels, so that the plurality of second ligands bind to the plurality of analytes in the first complex and form a second complex. Finally, the plurality of labels of the plurality of second ligands in the second complex are detected.

Description

Technical Field

[0001] The present invention relates to a method for detecting an analyte, and in particular to a method for detecting multiple analytes. Background Art

[0002] Ligand binding assay is a detection method that relies on the affinity binding of ligand molecules to analytes, among which enzyme-linked immunosorbent assay (ELISA) is the most widely used detection method. Traditional ELISA uses the specific binding characteristics of antibodies and antigens to carry out detection in conjunction with enzyme reactions, and the technology has been quite mature. However, the disadvantage of ELISA is that the detection time is long and one reaction can only detect one analyte.

[0003] At present, the products used in multiplex protein immunoassays on the market are based on the traditional sandwich method, which first uses spherical microparticles with different colors to couple with antibodies, then allows the spherical microparticles coupled with antibodies to bind to the target antigen, and then uses detection antibodies with specific fluorescent markers to bind to the antigen to be analyzed, and then uses flow cytometry to analyze, thereby achieving the purpose of multiplex protein detection. However, the above-mentioned multiplex protein immunoassay still has the following disadvantages: it is necessary to use microparticles with multiple fluorescent colors, and it is necessary to set the fluorescence spectrum for microparticles with different fluorescent colors before detection, and it is necessary to distinguish particles with different markers for signal analysis during operation. Overall, the detection time is lengthy and the operation is complicated, which is easy to cause errors.

[0004] Therefore, it is urgent to develop a detection method that can analyze multiple analytes simultaneously and has convenient operation and high detection sensitivity. Summary of the invention

[0005] The present invention is directed to a method for detecting multiple analytes, which can have the effect of improving detection sensitivity.

[0006] According to an embodiment of the present invention, a method for detecting multiple analytes includes the following steps. A micron particle is provided. The micron particle is coupled with at least one first ligand and includes a body and a plurality of first protrusions formed on the surface of the body. Next, the micron particle is mixed with a sample including a plurality of analytes to form a first complex. Thereafter, the first complex is mixed with a plurality of second ligands with a plurality of first labels so that the plurality of second ligands bind to the plurality of analytes in the first complex and form a second complex. Finally, the plurality of first labels in the second complex are detected.

[0007] In the detection method according to an embodiment of the present invention, the above-mentioned micron particles are multi-protrusion microparticles. The body of the multi-protrusion microparticles includes a copolymer core, a polymer layer and a silicon base layer from the inside to the outside. A plurality of second protrusions are formed on the surface of the copolymer core. The average height of the plurality of second protrusions is 100 nanometers to 5000 nanometers.

[0008] In the detection method according to an embodiment of the present invention, the above-mentioned micron particles are multi-protrusion magnetic particles. The body of the multi-protrusion magnetic particles includes a copolymer core, a polymer layer, a magnetic material layer and a silicon-based layer from the inside to the outside. A plurality of second protrusions are formed on the surface of the copolymer core. The average height of the plurality of second protrusions is 100 nanometers to 5000 nanometers.

[0009] In the detection method according to the embodiment of the present invention, the ratio of the average height of the plurality of first protrusions to the average particle diameter of the body is 0.005 to 0.25.

[0010] In the detection method according to the embodiment of the present invention, the ratio of the average volume of the plurality of first protrusions to the average volume of the body is 1×10 -7 Up to 2x10 -2 .

[0011] In the detection method according to the embodiment of the present invention, the total volume of the plurality of first protrusions accounts for 10% of the average volume of the micron particles. -1 Up to 6x10 -1 .

[0012] In the detection method according to the embodiment of the present invention, the average number of the plurality of first protrusions is 5 to 500.

[0013] In the detection method according to the embodiment of the present invention, the average particle size of the above-mentioned micron particles is 1 micron to 20 microns.

[0014] In the detection method according to the embodiment of the present invention, the above-mentioned micron particles are non-spherical.

[0015] In the detection method according to an embodiment of the present invention, the coupling of the at least one first ligand to the micronized particles comprises non-covalent bonding, covalent bonding, avidin-biotin interaction, electrostatic adsorption, hydrophobic adsorption or a combination thereof.

[0016] In the detection method according to an embodiment of the present invention, the above-mentioned multiple analytes are located on the surface of the sample. The above-mentioned method of forming the first complex includes: allowing at least one first ligand to recognize and directly bind to the target located on the surface of the sample.

[0017] In the detection method according to an embodiment of the present invention, the at least one first ligand comprises a first specific antibody. The first specific antibody comprises an anti-human exosome surface antigen antibody, an anti-human blood cell surface antigen antibody, an anti-human immune cell surface antigen antibody, an anti-human tumor cell surface antigen antibody, or a combination thereof.

[0018] In the detection method according to an embodiment of the present invention, the above-mentioned sample includes human exosomes, human blood cells, human immune cells, human tumor cells or a combination thereof. The multiple analytes include surface antigens of human exosomes, surface antigens of human blood cells, surface antigens of human immune cells, surface antigens of human tumor cells or a combination thereof.

[0019] In the detection method according to an embodiment of the present invention, the plurality of second ligands include a plurality of second specific antibodies, including anti-human exosome surface antigen antibodies, anti-human blood cell surface antigen antibodies, anti-human immune cell surface antigen antibodies, anti-human tumor cell surface antigen antibodies, or a combination thereof.

[0020] In the detection method according to an embodiment of the present invention, the at least one first ligand includes multiple first ligands. The manner of forming the first complex includes: allowing the multiple first ligands to recognize and directly bind to multiple analytes.

[0021] In the detection method according to the embodiment of the present invention, the plurality of first ligands mentioned above include a plurality of nucleic acid probes, which include a plurality of primers or aptamers.

[0022] In the detection method according to the embodiment of the present invention, the aforementioned multiple analytes include multiple nucleic acid sequences with multiple second labels.

[0023] In the detection method according to an embodiment of the present invention, the plurality of second labels include biotin, a plurality of antigen regions or a combination thereof.

[0024] In the detection method according to an embodiment of the present invention, the plurality of second ligands mentioned above include specific proteins, which include anti-biotin antibody, avidin, streptavidin, neutravidin, a third specific antibody or a combination thereof.

[0025] In the detection method according to the embodiment of the present invention, the plurality of first labels mentioned above include a plurality of fluorescent labels or a plurality of luminescent labels.

[0026] In the detection method according to an embodiment of the present invention, the plurality of fluorescent labels include FITC, Alexa, PE, BV, PerCP, APC, Pacific Blue or a combination thereof. The plurality of luminescent labels include luciferase.

[0027] According to an embodiment of the present invention, a method for detecting multiple analytes includes the following steps: providing micron particles. The micron particles are coupled with at least a plurality of ligands and include a body and a plurality of protrusions formed on the surface of the body. Next, the micron particles are mixed with a sample including a plurality of analytes to form a complex, wherein the plurality of analytes carry a plurality of labels. Finally, the plurality of labels in the complex are detected.

[0028] In the detection method according to an embodiment of the present invention, the aforementioned multiple ligands include multiple nucleic acid probes. The aforementioned multiple labels include multiple fluorescent labels or multiple cold light labels or a combination thereof. The aforementioned multiple analytes include multiple nucleic acid sequences.

[0029] Based on the above, the micron particle of the present invention can increase the surface area of ​​the micron particle (i.e., the sum of the surface area of ​​the body and the surface area of ​​the first protrusions) by configuring a plurality of irregularly shaped first protrusions on the surface of the body. In this way, the micron particle can be coupled with more first ligands to identify and bind more targets, thereby enhancing the detection signal to improve the sensitivity of the detection.

[0030] In order to make the above features and advantages of the present invention more clearly understood, embodiments are given below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a flow chart of a method for detecting multiple analytes according to an embodiment of the present invention;

[0032] Figure 2 Schematic diagram of a process for detecting multiple analytes according to an embodiment of the present invention;

[0033] Figure 3A is a schematic cross-sectional view of a multi-protuberance particle according to an embodiment of the present invention;

[0034] Figure 3B is a cross-sectional schematic diagram of a multi-protuberance magnetic particle according to an embodiment of the present invention;

[0035] Figure 4 is a schematic flow chart of a method for detecting multiple analytes according to another embodiment of the present invention;

[0036] FIG. 5A to FIG. 5F To analyze the specifications of spherical micron particles or multi-protuberance microparticles using a scanning electron microscope and a particle size analyzer respectively;

[0037] 6A to 6F Comparative Examples 1 to 3 and Examples 1, 3 to 4 coupled with Anti-Human CD63 were used to detect the expression of CD9 in the exosomes of SKBr3;

[0038] FIG. 6G to FIG. 6L Comparative Examples 1 to 3 and Examples 1, 3 to 4 coupled with Anti-Human CD9 were used to detect the expression of CD9 in the exosomes of SKBr3;

[0039] FIG. 7A to FIG. 7C Example 1 and Example 2 coupled with Anti-Human CD9 were used to detect the expression of CD9 in the exosomes of SKBr3 in different solutions;

[0040] FIG. 8A to FIG. 8C Example 1 and Example 2 coupled with Anti-Human CD9 were used to detect the expression of CD81 in the exosomes of SKBr3 in different solutions;

[0041] 9A to 9D Example 2 coupled with Anti-Human CD9 is used to simultaneously detect the expression levels of any three of CD9, CD29, CD63 and CD81 in the exosomes of SKBr3;

[0042] FIG. 10A to FIG. 10D Example 2 coupled with Anti-Human Her2 is used to simultaneously detect the expression levels of any three of CD9, CD29, CD63 and CD81 in the exosomes of SKBr3;

[0043] FIG. 11A to FIG. 11D Example 2 is to use different first specific antibodies coupled to simultaneously detect the expression levels of CD9 and CD63 in exosomes in clinical specimens;

[0044] FIG. 12A to FIG. 12B The method is to use multi-spike magnetic microparticles coupled with AIV probes to detect AB nucleic acid to be analyzed labeled with biotin or AIV nucleic acid to be analyzed labeled with biotin.

[0045] Description of Reference Numerals

[0046] 110: Micron particles

[0047] 110a: Multi-spike particles

[0048] 110b: Multi-spike magnetic particles

[0049] 113: Copolymer core

[0050] 114: Polymer layer

[0051] 115: Silicon base

[0052] 111: Ontology

[0053] 111a, 113a, S1: Surface

[0054] 112: First protrusion

[0055] 113b: Second protrusion

[0056] 120: First Ligand

[0057] 13a: Surface antigen

[0058] 130a, 130b, 130c: analyte

[0059] 131a, 131b, 131c: Sequence part

[0060] 132a, 132b, 132c: Second mark

[0061] 140: First Complex

[0062] 150a, 150b, 150c: Second ligand

[0063] 151a, 151b, 151c: First mark

[0064] 160: Second Complex

[0065] H1, H2: average height

[0066] S: Sample

[0067] T: Target

[0068] S10, S20, S30, S40: Steps

[0069] D, D1: average particle size

[0070] D2: Shortest particle size

[0071] D3: Longest particle size

[0072] V1, V2: average volume DETAILED DESCRIPTION

[0073] Reference will now be made in detail to exemplary embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Whenever possible, the same reference numerals are used in the drawings and the description to refer to the same or like parts.

[0074] Figure 1 FIG. 4 is a flow chart of a method for detecting multiple analytes according to an embodiment of the present invention. Figure 2FIG. 4 is a schematic diagram of a process for detecting multiple analytes according to an embodiment of the present invention. Figure 3A FIG. 4 is a schematic cross-sectional view of a multi-protrusion particle according to an embodiment of the present invention. Figure 3B FIG. 4 is a schematic cross-sectional view of a multi-spike magnetic particle according to an embodiment of the present invention.

[0075] Please also refer to Figure 1 , Figure 2 , Figure 3A as well as Figure 3B First, step S10 is performed to provide a micron particle 110. In the present embodiment, the micron particle 110 includes a body 111 and a plurality of first protrusions 112 formed on a surface 111a of the body 111, so the micron particle 110 is non-spherical. In terms of appearance, the plurality of first protrusions 112 are irregular in shape and may be evenly or unevenly distributed on the surface 111a of the body 111. In essence, the plurality of first protrusions 112 and the body 111 are integrally formed, and the plurality of first protrusions 112 and the body 111 are seamlessly connected. In the present embodiment, the plurality of first protrusions 112 have an average height H1 (i.e., the average vertical distance from the top of the plurality of first protrusions 112 to the surface 111a of the body 111).

[0076] For more details, please also refer to Figure 2 , Figure 3A and Figure 3B In this embodiment, the micron particle 110 may be a multi-spike particle 110a or a multi-spike magnetic particle 110b, wherein the multi-spike particle 110a is non-magnetic, while the multi-spike magnetic particle 110b is magnetic. Figure 3AAs shown, the body 111 of the multi-protrusion particle 110a includes a copolymer core 113, a polymer layer 114 and a silicon-based layer 115 from the inside out. A plurality of second protrusions 113b are formed on the surface 113a of the copolymer core 113. The plurality of second protrusions 113b are irregular in shape and can be evenly or unevenly distributed on the surface 113a of the copolymer core 113. In essence, the plurality of second protrusions 113b are integrally formed with the copolymer core 113, and the plurality of second protrusions 113b and the copolymer core 113 are seamlessly connected. In the present embodiment, the average height H2 of the plurality of second protrusions 113b (i.e., the average vertical distance from the top of the plurality of second protrusions 113b to the surface 113a of the copolymer core 113) may be 100 nm to 5000 nm, such as about 200 nm to 4500 nm, about 500 nm to 4000 nm, about 800 nm to 3500 nm, about 300 nm to 1000 nm, about 600 nm to 1800 nm, about 750 nm to 2500 nm, about 900 nm to 3000 nm, about 1000 nm to 3600 nm, about 1500 nm to 4800 nm, etc., but not limited thereto. In the present embodiment, the polymer layer 114 and the silicon-based layer 115 are substantially formed sequentially on the copolymer core 113 in a conformal manner. Therefore, the plurality of first protrusions 112 of the multi-protrusion particle 110 a may be disposed substantially corresponding to the plurality of second protrusions 113 b of the copolymer core 113 , but the present invention is not limited thereto.

[0077] like Figure 3BAs shown, the body 111 of the multi-protrusion magnetic particle 110b includes a copolymer core 113, a polymer layer 114, a magnetic material layer 116 and a silicon-based layer 115 from the inside out. Among them, a plurality of second protrusions 113b are formed on the surface 113a of the copolymer core 113. The plurality of second protrusions 113b are irregular in shape and can be evenly or unevenly distributed on the surface 113a of the copolymer core 113. In essence, the plurality of second protrusions 113b and the copolymer core 113 are integrally formed, and the plurality of second protrusions 113b and the copolymer core 113 are seamlessly connected. In the present embodiment, the average height H2 of the plurality of second protrusions may be 100 nm to 5000 nm, for example, about 200 nm to 4500 nm, about 500 nm to 4000 nm, about 800 nm to 3500 nm, about 300 nm to 1000 nm, about 600 nm to 1800 nm, about 750 nm to 2500 nm, about 900 nm to 3000 nm, about 1000 nm to 3600 nm, about 1500 nm to 4800 nm, etc., but not limited thereto. In the present embodiment, the polymer layer 114, the magnetic material layer 116, and the silicon-based layer 115 are substantially formed in sequence on the copolymer core 113 in a conformal manner. The plurality of first protrusions 112 of the multi-protruding magnetic particles 110b may be substantially arranged corresponding to the plurality of second protrusions 113b of the copolymer core 113, but not limited thereto.

[0078] In the present embodiment, the copolymer core 113 is, for example, a styrene / divinylbenzene copolymer, a methyl methacrylate / triethylene glycol dimethacrylate copolymer, a methyl methacrylate / ethylene glycol dimethacrylate copolymer, a styrene / triethylene glycol dimethacrylate copolymer, a styrene / ethylene glycol dimethacrylate copolymer, or a methyl methacrylate / divinylbenzene copolymer, but the invention is not limited thereto. The polymer layer 114 may be a surface of the copolymer core 113 modified by a functional group, and includes at least one functional group (such as a carboxyl group, an amino group, or a combination thereof, but the invention is not limited thereto). The material of the silicon-based layer 115 may include tetramethyl silicate (TMOS), tetraethoxysilane (TEOS), 3-aminopropyltriethoxysilane (APTES), 3-glycidoxypropyltrimethoxysilane (GOPTS), but the invention is not limited thereto. The material of the magnetic material layer 116 may include paramagnetic material, superparamagnetic material, ferromagnetic material, ferrite magnetic material or a combination thereof, but is not limited thereto.

[0079] Please refer to Figure 2 , Figure 3A as well as Figure 3B In this embodiment, the average height H1 of the first protrusion 112 may be 0.1 micrometers (μm) to 5 micrometers, such as about 0.2 micrometers to 4.5 micrometers, about 0.3 micrometers to 4 micrometers, about 0.4 micrometers to 3.5 micrometers, about 0.5 micrometers to 3 micrometers, about 0.6 micrometers to 2.5 micrometers, etc., but not limited thereto. The average volume V1 of the first protrusion 112 may be 0.00052 cubic micrometers (μm 3 ) to 65 cubic microns, such as about 0.001 cubic microns to 60 cubic microns, about 0.01 cubic microns to 50 cubic microns, about 0.1 cubic microns to 40 cubic microns, etc., but not limited thereto. The average particle size D1 of the body 111 can be 1 micron (μm) to 20 microns, such as about 2 microns to 15 microns, about 3 microns to 18 microns, about 4 microns to 10 microns, about 5 microns to 12 microns, etc., but not limited thereto. The average volume V2 of the body 111 can be 0.52 cubic microns (μm 3 ) to 4200 cubic microns, such as about 1 cubic micron to 4000 cubic microns, about 10 cubic microns to 3800 cubic microns, about 20 cubic microns to 3600 cubic microns, about 30 cubic microns to 3300 cubic microns, about 40 cubic microns to 2900 cubic microns, about 50 cubic microns to 2500 cubic microns, about 60 cubic microns to 2300 cubic microns, about 80 cubic microns to 2000 cubic microns, about 100 cubic microns to 1000 cubic microns, etc., but not limited thereto. The ratio of the average height H1 of the first protrusions 112 to the average particle size D1 of the body 111 can be 0.005 to 0.25, such as about 0.001 to 0.23, about 0.05 to 0.20, about 0.01 to 0.18, about 0.05 to 0.15, etc., but not limited thereto. The ratio of the average volume V1 of the first protrusion 112 to the average volume V2 of the body 111 may be 1×10 -7 Up to 2x10 -2 , for example 1x10 -6 Up to 1.5x10 -2 , 1x10 -5 Up to 1.2x10 -2 , 1x10 -5 Up to 1x10 -2 The average particle size D of the micron particles 110 (i.e., the average of the shortest particle size and the longest particle size of the micron particles 110, for example, the average of the shortest particle size D2 and the longest particle size D3 of the multi-protruding particles 110a or multi-protruding magnetic particles 110b) may be 1 micron to 20 microns, for example, about 2 microns to 18 microns, about 3 microns to 15 microns, about 5 microns to 10 microns, etc., but is not limited thereto.

[0080] Please refer to Figure 2 In this embodiment, the micron particle 110 is coupled with at least one first ligand 120 ( Figure 1 The figure shows a first ligand as an example, but the invention is not limited thereto). The first ligand 120 is coupled to the surface 111a of the body 111 of the micron particle 110 and the first protrusion 112. The first ligand 120 is coupled to the micron particle 110 in a manner including non-covalent bonding, covalent bonding, avidin-biotin interaction, electrostatic adsorption, hydrophobic adsorption, or a combination thereof. In the present embodiment, the first ligand 120 includes, for example, a specific antibody, but the invention is not limited thereto.

[0081] Next, please refer to Figure 1 and Figure 2 , step S20 is performed to mix the micron particles 110 with the plurality of analytes 130a, 130b, 130c to form a first complex 140. In this embodiment, the plurality of analytes 130a, 130b, 130c are located on the surface S1 of the sample S. The method of forming the first complex 140 includes: allowing at least one first ligand 120 to recognize and directly bind to the target T located on the surface S1 of the sample S.

[0082] In the present embodiment, the first ligand 120 includes a first specific antibody, which has the ability to recognize and directly bind to the target T. The first specific antibody includes anti-human exosome surface antigen antibodies (such as Anti-Human CD9, Anti-Human CD63, Anti-Human CD29, Anti-Human CD81, Anti-Human Her2 Anti-Human EGFR, Anti-Human EpCAM, etc.), anti-human blood cell surface antigen antibodies (such as Anti-Human CD45, Anti-Human CD235a, etc.), anti-human immune cell surface antigen antibodies (such as Anti-Human CD3, Anti-Human CD19, etc.), anti-human tumor cell surface antigen antibodies (such as Anti-Human CEA, Anti-Human PD-L1, Anti-Human Her2, etc.) or a combination thereof, but is not limited thereto.

[0083] In this embodiment, the sample S includes human exosomes, human blood cells, human immune cells, human tumor cells, or a combination thereof, but is not limited thereto. The analytes 130a, 130b, and 130c include surface antigens of human exosomes (e.g., CD9, CD63, CD81, Her2, EGFR, EpCAM, etc.), surface antigens of human blood cells (e.g., CD45, CD235a, etc.), surface antigens of human immune cells (e.g., CD3, CD19, etc.), surface antigens of human tumor cells (e.g., CEA, PD-L1, Her2, etc.), or a combination thereof, but are not limited thereto.

[0084] In the present embodiment, the target T may be at least one surface antigen among human exosome surface antigens, at least one surface antigen among human blood cell surface antigens, at least one surface antigen among human immune cell surface antigens, at least one surface antigen among human tumor cell surface antigens, or a combination thereof, but is not limited thereto. The target T may be the same as or different from any one of the multiple analytes 130a, 130b, 130c. For example, in one embodiment, the target T is, for example, the surface antigen CD9 of human exosomes, and the analytes are, for example, the surface antigens CD9, CD63, and CD81 of human exosomes. In another embodiment, the target T is, for example, the surface antigen Her2 of human exosomes, and the analytes are, for example, the surface antigens CD9, CD63, and CD81 of human exosomes, but is not limited thereto. It is worth noting that even if the target T and the analyte are the same surface antigen, the target T and the analyte 130a will not be the same surface antigen. For example, if Figure 2 As shown, the target T and the analyte 130a are the same surface antigen 13a, wherein the portion recognized and bound by the first ligand 120 is the target T, and the other portion not bound by the first ligand 120 can be used as the analyte 130a for subsequent detection.

[0085] Next, step S30 is performed to mix the first complex 140 with a plurality of second ligands 150a, 150b, 150c with a plurality of first labels 151a, 151b, 151c, so that the plurality of second ligands 150a, 150b, 150c bind to the plurality of analytes 130a, 130b, 130c in the first complex 140 and form a second complex 160. For the sake of clarity, Figure 2 Only one sample S is shown to be combined with one second ligand (150a or 150b or 150c). In fact, as long as the sample S surface has multiple (or multiple) analytes 130a, 130b, 130c, the sample S can be combined with multiple (or multiple) second ligands 150a, 150b, 150c at the same time.

[0086] In this embodiment, the plurality of second ligands 150a, 150b, 150c are, for example, a plurality of second specific antibodies. In this embodiment, the plurality of second specific antibodies include anti-human exosome surface antigen antibodies, anti-human blood cell surface antigen antibodies, anti-human immune cell surface antigen antibodies, anti-human tumor cell surface antigen antibodies, or a combination thereof. Among them, the plurality of second specific antibodies may be the same as or different from the first specific antibody. The plurality of first markers 151a, 151b, 151c include a plurality of different fluorescent markers or cold light markers, but are not limited thereto. Fluorescent labels may include FITC, Alexa (e.g., AF-488, AF-594, AF-647, AF-700, etc.), PE (e.g., PE, PE-Cyanine5, PE-Cyanine7, PE-Dazzle594, etc.), PerCP (e.g., PerCP, PerCP-Cyanine5.5, etc.), BV (Brilliant Violet, e.g., BV421, BV450, BV510, BV570, BV605, BV650, BV711, BV750, BV785, etc.), APC (e.g., APC, APC-Cyanine7, etc.), Pacific Blue, or a combination thereof, but not limited thereto. Luminescent labels include luciferase, but not limited thereto. A person skilled in the art may select the first ligand, the first label, and the second ligand according to actual needs (e.g., the source of the sample or the amount of the analyte), and the present invention is not limited thereto.

[0087] Finally, step S40 is performed to detect the multiple first labels 151a, 151b, 151c of the multiple second ligands 150a, 150b, 150c in the second complex 160. The detection result can represent the relative content (or expression amount) of the analytes 130a, 130b, 130c on the surface S1 of the sample S. In this embodiment, for example, flow cytometry is used for detection, but it is not limited to this. So far, the detection method of multiple analytes in this embodiment has been substantially completed.

[0088] In this embodiment, compared to the general spherical micron-particles, the micron-particles 110 of this embodiment can increase the surface area of ​​the micron-particles 110 (i.e., the sum of the surface area of ​​the body and the surface area of ​​the first protrusions) by disposing a plurality of irregularly shaped first protrusions on the surface of the body. Thus, the micron-particles 110 of this embodiment can couple more first ligands 120 to identify and bind more target objects T, thereby enhancing the detection signal to improve the sensitivity of the detection.

[0089] In addition, since the surface of the magnetic material layer 116 of the multi-spike magnetic particle 110b of the present embodiment is a rough surface (or has small protrusions), the surface of the multi-spike magnetic particle 110b (i.e., the surface 111a of the body 111 and the surface of the first protrusion 112) is also a rough surface. Then, compared to the multi-spike magnetic particle 110a, since the surface of the multi-spike magnetic particle 110b is a rough surface, the multi-spike magnetic particle 110b can have a larger surface area, thereby allowing the multi-spike magnetic particle 110b to couple more first ligands 120, identify and bind more target objects T, and detect more analytes 130a, 130b, 130c, thereby enhancing the detection signal and improving the sensitivity of the detection. In addition, since the multi-spike magnetic particle 110b is magnetic, the detection time can be shortened, and the efficiency and convenience of the detection can be improved.

[0090] Other embodiments will be listed below for illustration. It must be noted that the following embodiments use the component numbers and some contents of the previous embodiments, wherein the same numbers are used to represent the same or similar components, and the description of the same technical contents is omitted. The description of the omitted parts can be referred to the previous embodiments, and the following embodiments will not be repeated.

[0091] Figure 4 FIG. 1 is a flow chart of a method for detecting multiple analytes according to another embodiment of the present invention. Figure 2 and Figure 4 , the method of this embodiment is Figure 2 The method is similar to that in the above method, but the main difference between the two is that at least one first ligand 120 includes multiple first ligands 120a, 120b, 120c, and the multiple first ligands 120a, 120b, 120c are, for example, multiple nucleic acid probes (for example, multiple primers or aptamers). The multiple analytes 130a, 130b, 130c include multiple nucleic acid sequences with multiple second labels 132a, 132b, 132c (for example, biotin, antigen regions, or a combination thereof, but not limited thereto). The multiple second ligands 150a, 150b, 150c include specific proteins (for example, anti-biotin antibodies, avidin, streptavidin, neutravidin, third specific antibodies, or a combination thereof, but not limited thereto).

[0092] Specifically, please refer to Figure 4First, the micronized particles 110 coupled with a plurality of first ligands 120a, 120b, 120c are mixed with a plurality of analytes 130a, 130b, 130c, so that the plurality of first ligands 120a, 120b, 120c recognize and directly bind to the corresponding (or sequence-complementary) sequence portions 131a, 131b, 131c of the plurality of analytes 130a, 130b, 130c, and form a first complex 140. Next, the first complex 140 is mixed with a plurality of second ligands 150a, 150b, 150c with a plurality of first labels 151a, 151b, 151c, so that the plurality of second ligands 150a, 150b, 150c bind to the second labels 132a, 132b, 132c of the plurality of analytes 130a, 130b, 130c in the first complex 140, and form a second complex 160. Finally, the plurality of first labels 151a, 151b, 151c of the plurality of second ligands 150a, 150b, 150c in the second complex 160 are detected.

[0093] In addition, in some other embodiments, the second labels 132a, 132b, 132c may also be the first labels 151a, 151b, 151c. Thus, when the first ligands 120a, 120b, 120c are combined with the sequence parts 131a, 131b, 131c of the analytes 130a, 130b, 130c, the formed first complex 140 is provided with a plurality of fluorescent labels or a plurality of luminescent labels, and thus can be directly detected without adding a second ligand to form a second complex. Thus, the detection method of multiple analytes of this embodiment has been substantially completed.

[0094] The following will be combined with drawings and embodiments to illustrate the technical means adopted by the present invention to achieve the purpose. However, the following embodiments and the accompanying drawings are only auxiliary explanations and are not used to limit the present invention.

[0095] [Example]

[0096] Example 1: Specification Analysis of Multi-Spike Microparticles

[0097] In this embodiment, the specifications of general spherical micron particles or multi-protrusion microparticles are analyzed using a scanning electron microscope (SEM) and a particle size analyzer (Multisizer). The results are as follows: FIG. 5A to FIG. 5F As shown, Figure 5A , Figure 5C and Figure 5E To analyze the results of spherical microparticles, Figure 5B , Figure 5D and Figure 5E This is the result of analyzing multi-protruding particles.

[0098] Depend on FIG. 5A to FIG. 5F It can be seen that Figure 5A The average particle size of the spherical micron particles is about 2.421 microns. Figure 5B The average particle size of the multi-protuberant particles is about 3.280 microns. Figure 5C The average particle size of the spherical micron particles is about 4.541 microns. Figure 5D The average particle size of the multi-protuberant particles is about 4.153 microns. Figure 5E The average particle size of the spherical micronized particles is about 7.568 microns and Fig. 5F The average particle size of the multi-protuberant microparticles is about 7.189 microns.

[0099] Next, Figure 5B , Figure 5D and Fig. 5F The specifications of the multi-protuberant particles are further analyzed. Figure 5B In the embodiment, the average volume of the multi-protrusion particles is about 13.86 cubic microns, the average particle size of the body is about 2.5 microns, the average volume of the body is about 6.14 cubic microns, the average height of the first protrusion is about 1.2 microns, the average volume of the first protrusion is about 0.679 cubic microns, and the total volume of the first protrusion is 7.72 cubic microns. Figure 5B In the multi-protrusive particles, the ratio of the average height of the first protrusions to the average particle size of the body is about 0.48, the ratio of the average volume of the first protrusions to the average volume of the body is about 0.111, each multi-protrusive particle has about 11 first protrusions, and the total volume of the first protrusions is 55.7% of the volume of the multi-protrusive particle.

[0100] exist Figure 5D The average volume of the multi-protrusion particles is about 49.09 cubic microns, the average particle size of the body is about 4.5 microns, the average volume of the body is about 35.78 cubic microns, the average height of the first protrusion is about 0.8 microns, the average volume of the first protrusion is about 0.268 cubic microns, and the total volume of the first protrusion is 13.31 cubic microns. That is, Figure 5D In the multi-protrusive particles, the ratio of the average height of the first protrusions to the average particle size of the body is about 0.18, the ratio of the average volume of the first protrusions to the average volume of the body is about 0.007, each multi-protrusive particle has about 49 first protrusions, and the total volume of the first protrusions is 27.1% of the volume of the multi-protrusive particle.

[0101] And in Fig. 5FIn the embodiment, the average volume of the multi-protrusion particles is about 201 cubic microns, the average particle size of the body is about 7.5 microns, the average volume of the body is about 166 cubic microns, the average height of the first protrusion is about 1 micron, the average volume of the first protrusion is about 0.392 cubic microns, and the total volume of the first protrusion is 35 cubic microns. That is to say, in Fig. 5F In the multi-protrusive particles, the ratio of the average height of the first protrusions to the average particle size of the body is about 0.133, the ratio of the average volume of the first protrusions to the average volume of the body is about 0.0024, each multi-protrusive particle has about 89 first protrusions, and the total volume of the first protrusions is 17.4% of the volume of the multi-protrusive particle.

[0102] In particular, the ratio of the average height (or average volume) of the first protrusions of the multi-protrusion particle to the average particle diameter (or average volume) of the body may be related to the surface morphology (ie, overall appearance) of the multi-protrusion particle. Figure 5B In the multi-protrusion particle, the ratio of the average height of the first protrusion to the average particle diameter of the body is greater than 0.25, and the ratio of the average volume of the first protrusion to the average volume of the body is greater than 2x10 -2 ,therefore, Figure 5B The surface morphology of the multi-protrusion particle is irregular, that is, it is difficult to clearly identify the positions of the main body and the first protrusion. Figure 5D Multi-spike particles and Fig. 5F In the multi-protrusion particles, the ratio of the average height of the first protrusions to the average particle diameter of the body is between 0.005 and 0.25, and the ratio of the average volume of the first protrusions to the average volume of the body is between 1x10 -7 Up to 2x10 -2 Between, therefore, Figure 5D Multi-spike particles and Fig. 5F The multi-protrusion particles can all identify the positions of the main body and the first protrusion.

[0103] Example 2: Test of the grafting amount of specific antibodies

[0104] In this embodiment, the first specific antibody (Anti-Human CD9, Anti-Human CD63 or Anti-Human Her2) is first coupled to general spherical micron particles and the micron particles of this embodiment (multi-protuberance microparticles or multi-protuberance magnetic microparticles) respectively. Then, the spherical micron particles coupled with the first specific antibody, the multi-protuberance microparticles coupled with the first specific antibody, and the multi-protuberance magnetic microparticles coupled with the first specific antibody are tested and compared for the grafting amount of the specific antibody. The types and particle sizes of the micron particles used in this embodiment are shown in Table 1.

[0105] Table 1: Types and sizes of micron particles

[0106] Types of microparticles Micron particle size (μm) Example 1 Polyparticles 8.5 Example 2 Multi-particle magnetic particles 8.5 Example 3 Polyparticles 2.5 Example 4 Polyparticles 4.5 Comparative Example 1 Spherical micron particles 8.5 Comparative Example 2 Spherical micron particles 2.5 Comparative Example 3 Spherical micron particles 4.5

[0107] In this embodiment, the steps of coupling Anti-Human CD9 (or Anti-Human CD63, or Anti-Human Her2) to spherical microparticles, multi-spike microparticles, and multi-spike magnetic microparticles are as follows: (1) 1×10 6The particles with amino groups modified on the surface (i.e., the spherical micronized particles of Comparative Examples 1 to 3; the multi-protruding microparticles of Examples 1 and Examples 3 to 4; the multi-protruding magnetic microparticles of Example 2) were washed three times with 200 μL of MES buffer solution. Then, 20 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), 20 mg of NHS (N-Hydroxysulfosuccinimide sodium salt) and 4 mg of PAA (15 kDa poly acrylic acid) were dissolved in 400 μL of MES buffer solution and mixed with the washed particles. Then, the mixture was mixed and reacted for 30 minutes at room temperature using a vortex at a speed of 1000 rpm. Then, the particles were collected, wherein the spherical micronized particles and the multi-protruding microparticles were collected by centrifugation at a speed of 10,000 rpm for 3 minutes; the multi-protruding magnetic microparticles were collected by a magnet and stayed on the magnet for at least 1 minute. After removing the reaction solution, the particles were washed three times with 200 μL of MES buffer solution, and 20 μL of Anti-Human CD9 (or Anti-Human CD63, or Anti-Human Her2) in a pH 3 Citric Acid-PBS solution (0.625 M Citric Acid dissolved in PBS solution, pH 3.0) was added and reacted overnight at 4° C. to couple Anti-Human CD9 (or Anti-Human CD63, or Anti-Human Her2) to the surface of the particles. Then, 200 μL of bovine serum albumin solution (10 mg / mL BSA dissolved in MES solution) was added and reacted overnight at 4° C. to cover the remaining surface of the particles that was not coupled with Anti-Human CD9 (or Anti-Human CD63, or Anti-Human Her2). (2) Collect the particles after the reaction is completed. The spherical micron particles and multi-spike microparticles are collected by centrifugation: 10,000 rpm, 3 minutes; the multi-spike magnetic microparticles are collected by magnet, and stay for at least 1 minute each time. Remove the reaction solution, wash the particles three times with PBS solution (0.1% BSA and 0.01% Sodium azide dissolved in PBS), and finally disperse the particles coupled with Anti-Human CD9 (or Anti-Human CD63, or Anti-Human Her2) in 100 μL PBS solution. (3) Use a cell automatic counter to calculate the concentration of the particles, and the concentration is about 3 to 8×10 6 / mL of particles with Anti-Human CD9 (or Anti-Human CD63, or Anti-Human Her2) coupled on the surface.

[0108] Next, the particles with Anti-Human CD9 (or Anti-Human CD63, or Anti-Human Her2) coupled to the surface were tested for the amount of specific antibody grafted. The steps are as follows: (1) Add 5uL Anti-mouseIgG-FITC to 50μL (1250 particles) of particles with Anti-Human CD9 (or Anti-Human CD63, or Anti-Human Her2) coupled to the surface, and then add PBS solution to a total reaction volume of 200μL. Mix and react for 30 minutes at room temperature using a test tube oscillator at a speed of 1500rpm. After the reaction is completed, the above particles are collected, among which the spherical microparticles and multi-spike microparticles are collected by centrifugation at a speed of 10000rpm for 3 minutes; the multi-spike magnetic microparticles are collected by a magnet and stay on the magnet for at least 1 minute. (2) Remove the reaction solution and wash twice with 200μL PBS solution. (3) After washing, 100 μL of PBS solution was added, and the obtained particles were analyzed for fluorescence signals by flow cytometry to measure the grafting amount of Anti-Human CD9 (or Anti-Human CD63, or Anti-Human Her2) (expressed as the average fluorescence intensity, that is, the stronger the average fluorescence intensity, the greater the grafting amount); (4) Statistical analysis was performed, and the test results were expressed as multiples of fluorescence intensity, among which Table 2 is a comparison of the grafting amount of spherical micron particles (Comparative Example 1), multi-spike microparticles (Example 1) and multi-spike magnetic microparticles (Example 2) with the same particle size (8.5 μm). Table 3 is a comparison of the grafting amount of spherical micron particles (Comparative Examples 1 to 3) and multi-spike microparticles (Examples 1, 3 to 4) with different particle sizes (2.5, 4.5, 8.5 μm). Table 4 compares the grafting amounts of spherical microparticles (Comparative Examples 1 to 3) and multi-protruding microparticles (Examples 1, 3 to 4) having the same particle size (2.5, 4.5, and 8.5 microns).

[0109] Table 2: Comparison of the grafting amount of the first specific antibody (Anti-Human CD9, Anti-Human CD63 or Anti-Human Her2) in Comparative Example 1, Example 1 and Example 2

[0110]

[0111]

[0112] From the results in Table 2, it can be seen that when the first specific antibody is Anti-Human CD9, the grafting amount of Example 1 is about 1.71 times that of Comparative Example 1. When the first specific antibody is Anti-Human CD63, the grafting amount of Example 1 is about 4.55 times that of Comparative Example 1. In other words, regardless of whether the first specific antibody is Anti-Human CD9 or Anti-Human CD63, the grafting amount of Example 1 is greater than that of Comparative Example 1. Therefore, compared with general spherical microparticles, the multi-protuberance microparticles of this embodiment can significantly couple more first specific antibodies.

[0113] In addition, when the first specific antibody is Anti-Human CD9, the grafting amount of Example 2 is about 2.24 times that of Example 1. When the first specific antibody is Anti-Human Her2, the grafting amount of Example 2 is about 3.81 times that of Example 1. In other words, regardless of whether the first specific antibody is Anti-Human CD9 or Anti-Human Her2, the grafting amount of Example 2 is greater than that of Example 1. Therefore, compared with the multi-spike microparticles of this embodiment, the multi-spike magnetic microparticles of this embodiment can significantly couple more first specific antibodies.

[0114] Table 3: Comparison of the grafting amount of the first specific antibody (Anti-Human CD9 or Anti-Human CD63) in Comparative Examples 1 to 3 and Examples 1, 3 to 4

[0115]

[0116] Table 4: Comparison of the grafting amount of the first specific antibody (Anti-Human CD9 or Anti-Human CD63) in Comparative Examples 1 to 3 and Examples 1, 3 to 4

[0117]

[0118]

[0119] From the results in Table 3, it can be seen that, regardless of whether it is spherical micron particles or multi-protruding microparticles, the larger the particle size, the greater the surface area that can be coupled with the first specific antibody, thereby increasing the grafting amount of the first specific antibody. From the results in Table 4, it can be seen that compared to spherical micron particles (Comparative Example 3 or Comparative Example 1), multi-protruding microparticles with similar particle sizes (Example 4 or Example 1) can significantly couple more first specific antibodies. Compared to spherical micron particles with a particle size of approximately 2.5 microns (Comparative Example 2), multi-protruding microparticles with a particle size of approximately 2.5 microns (Example 3) may have an uneven surface morphology (please refer to Figure 5B), resulting in lower detected fluorescence signals.

[0120] Example 3: Detection of surface antigens of exosomes from breast cancer cell line SKBr3

[0121] In this embodiment, spherical microparticles (Comparative Example 1, Comparative Example 2, Comparative Example 3) coupled with a first specific antibody (Anti-Human CD9 or Anti-HumanCD63), multi-spike microparticles (Example 1, Example 3, Example 4), multi-spike magnetic microparticles (Example 2) and a fluorescently labeled second specific antibody (Anti-Human CD9-Alexa488 or Anti-Human CD81-APC) are used to detect the expression of exosome surface antigens located on the surface of exosomes. Take Comparative Example 1 as an example for illustration: Specifically, first, Comparative Example 1 coupled with Anti-Human CD9 (or Anti-Human CD63) is mixed with exosomes carrying a variety of exosome surface antigens, so that Anti-Human CD9 (or Anti-Human CD63) can recognize and directly bind to CD9 (or CD63) on the surface of the exosome to form a first complex; then, the first complex is mixed with Anti-Human CD9-Alexa488 (or Anti-Human CD81-APC) so that Anti-Human CD9-Alexa488 (or Anti-Human CD81-APC) binds to CD9 (or CD81) on the surface of the exosome in the first complex to form a second complex; then, the second complex is analyzed by fluorescence signal using a flow cytometer to detect the expression of exosome surface antigens. Wherein, the above-mentioned exosomes are exosomes purified from breast cancer cell line (SKBr3). The above-mentioned spherical micron particles include spherical micron particles with a particle size of about 2.5 microns (Comparative Example 2), spherical micron particles with a particle size of about 4.5 microns (Comparative Example 3) and spherical micron particles with a particle size of about 8.5 microns (Comparative Example 1). The above-mentioned multi-protruding microparticles include multi-protruding microparticles with a particle size of about 2.5 microns (Example 3), multi-protruding microparticles with a particle size of about 4.5 microns (Example 4) and multi-protruding microparticles with a particle size of about 8.5 microns (Example 1). The above-mentioned multi-protruding magnetic microparticles include multi-protruding magnetic microparticles with a particle size of about 8.5 microns (Example 2).

[0122] In this embodiment, the exosomes of Comparative Examples 1-3, Examples 1, 3-4 or Example 2 coupled with Anti-Human CD9 (or Anti-Human CD63) are mixed with SKBr3 so that Anti-Human CD9 (or Anti-Human CD63) can recognize and directly bind to CD9 (or CD63) on the surface of the exosomes to form the first complex. The steps are as follows: 50 μL (1250 particles) of Comparative Examples 1-3, Examples 1, 3-4 or Example 2 react with 20-50 μL of SKBr3 exosomes, then add PBS solution to a total reaction volume of 200 μL, mix and react at room temperature for 90 minutes. After the reaction is completed, the first complex containing Comparative Examples 1-3 or the first complex of Examples 1, 3-4 is collected by centrifugation at a speed of 10000 rpm for 3 minutes; the first complex containing Example 2 is collected by a magnet and left on the magnet for at least 1 minute. The reaction solution is removed and the first complex is washed twice with 200 μL of PBS solution.

[0123] In this embodiment, the first complex is mixed with Anti-Human CD9-Alexa488 (or Anti-Human CD81-APC) to bind Anti-Human CD9-Alexa488 (or Anti-Human CD81-APC) to CD9 (or CD81) on the surface of the exosomes in the first complex and form a second complex. The steps are as follows: 100 μL of Anti-Human CD9-Alexa488 (or Anti-Human CD81-APC) is added to the first complex, and then mixed and reacted for 30 minutes at room temperature on a test tube shaker at a speed of 1500 rpm to form a second complex. After the reaction is completed, the second complex containing Comparative Examples 1 to 3 or the second complex containing Examples 1, 3 to 4 is collected by centrifugation at a speed of 10000 rpm for 3 minutes; the second complex containing Example 2 is collected by a magnet and left on the magnet for at least 1 minute. The reaction solution is removed, and the second complex is washed twice with 200 μL of PBS solution.

[0124] In this embodiment, the steps of performing fluorescence signal analysis on the second complex to detect the expression of exosome surface antigens are as follows: 100 μL of PBS solution is added to the washed second complex (i.e., the second complex containing Comparative Example 1, the second complex containing Example 1, or the second complex containing Example 2), and a flow cytometer is used to perform fluorescence signal analysis to detect the expression of exosome surface antigens (i.e., average fluorescence intensity). The results are shown in FIG. FIG. 6A to FIG. 6L , FIG. 7A to FIG. 7C as well as FIG. 8A to FIG. 8C shown.

[0125] 6A to 6F Comparative Examples 1 to 3 and Examples 1, 3 to 4 coupled with Anti-Human CD63 were used to detect the expression of CD9 in the exosomes of SKBr3. Specifically, Fig. 6A , Figure 6C as well as Fig. 6E Spherical micronized particles with diameters of about 2.5 microns, 4.5 microns, and 8.5 microns (Comparative Example 2, Comparative Example 3, and Comparative Example 1) were used for testing. Figure 6B , Fig.6D as well as Fig. 6F The multi-protuberance particles (Example 3, Example 4 and Example 1) with particle sizes of about 2.5 microns, 4.5 microns and 8.5 microns were used for testing. Fig. 6A and Figure 6B It can be seen that compared with the spherical microparticles with a particle size of 2.5 μm (Comparative Example 2), the multi-protrusion microparticles with a particle size of 2.5 μm (Example 3) may have a lower detected fluorescence signal due to the uneven surface morphology. Figure 6C (or Fig. 6E ) showed that when the number of exosomes in Comparative Example 3 (or Comparative Example 1) (0, 1.05×10 7 , 1.05×10 8 , 1.05×10 9 ) increases, Comparative Example 3 (or Comparative Example 1) can bind to more exosomes, so that more CD9 and a higher average fluorescence intensity can be detected. Fig.6D (or Fig. 6F ) results show that when the number of exosomes in Example 4 (or Example 1) (0, 1.05×10 7 , 1.05×10 8 , 1.05×10 9 ) increases, Example 4 (or Example 1) can bind to more exosomes, so that more CD9 and higher mean fluorescence intensity can be detected. FIG. 6C to FIG. 6F The results show that, compared with Comparative Example 3 (or Comparative Example 1) containing spherical microparticles, Example 4 (or Example 1) containing multi-spike microparticles can bind to more exosomes, thereby detecting more CD9 and a higher average fluorescence intensity.

[0126] FIG. 6G to FIG. 6L Comparative Examples 1 to 3 and Examples 1, 3 to 4 coupled with Anti-Human CD9 were used to detect the expression of CD9 in the exosomes of SKBr3. Specifically, Figure 6G , Fig.6I as well as Figure 6KSpherical micronized particles with diameters of about 2.5 μm, 4.5 μm, and 8.5 μm (Comparative Example 2, Comparative Example 3, and Comparative Example 1) were used for testing. Figure 6H , Figure 6J as well as Figure 6L The multi-protuberance particles (Example 3, Example 4 and Example 1) with particle sizes of about 2.5 microns, 4.5 microns and 8.5 microns were tested respectively. Figure 6G and Figure 6H It can be seen that compared with the spherical microparticles with a particle size of 2.5 μm (Comparative Example 2), the multi-protrusion microparticles with a particle size of 2.5 μm (Example 3) may have a lower detected fluorescence signal due to the uneven surface morphology. Fig.6I (or Figure 6K ) showed that when the number of exosomes in Comparative Example 3 (or Comparative Example 1) (0, 1.05×10 7 , 1.05×10 8 , 1.05×10 9 ) increases, Comparative Example 3 (or Comparative Example 1) can bind to more exosomes, so that more CD9 and a higher average fluorescence intensity can be detected. Figure 6J (or Figure 6L ) results show that when the number of exosomes in Example 4 (or Example 1) (0, 1.05×10 7 , 1.05×10 8 , 1.05×10 9 ) increases, Example 4 (or Example 1) can bind to more exosomes, so that more CD9 and higher mean fluorescence intensity can be detected. FIG. 6I to FIG. 6L The results show that, compared with Comparative Example 3 (or Comparative Example 1) containing spherical microparticles, Example 4 (or Example 1) containing multi-spike microparticles can bind to more exosomes, thereby detecting more CD9 and a higher average fluorescence intensity.

[0127] It is worth noting that FIG. 6A to FIG. 6L In general, the larger the particle, the stronger the signal. The larger the particle size and the more spikes, the better the analysis signal. The flow cytometer is suitable for analyzing beads with larger and uniform particle size, which has a better analysis signal.

[0128] FIG. 7A to FIG. 7CIn order to detect the expression of CD9 in the exosomes of SKBr3 in different solutions, Example 1 and Example 2 coupled with Anti-Human CD9 were used respectively. Specifically, the exosomes of SKBr3 were first placed in DMEM culture medium (i.e., the supernatant of the culture medium of SKBr3 was collected), PBS solution (i.e., the exosomes of SKBr3 were purified and then placed in PBS solution) or plasma containing EDTA (i.e., the exosomes of SKBr3 were purified and then placed in plasma containing EDTA). Next, Example 1 and Example 2 coupled with Anti-Human CD9 were used to detect the expression of CD9 in the exosomes of SKBr3 in DMEM culture medium, and the results are as follows. Fig. 7A As shown; Example 1 and Example 2 coupled with Anti-Human CD9 were used to detect the expression of CD9 in the exosomes of SKBr3 in PBS solution, and the results are as follows Figure 7B As shown; Example 1 and Example 2 coupled with Anti-Human CD9 were used to detect the expression of CD9 in the exosomes of SKBr3 in plasma containing EDTA, and the results are as follows Figure 7C As shown. Among them, the above plasma comes from healthy people.

[0129] Depend on Fig. 7A The results show that, compared with Example 1 containing multi-spike microparticles, Example 2 containing multi-spike magnetic microparticles can bind to more exosomes in the DMEM culture medium, thereby detecting more CD9 and a higher mean fluorescence intensity. Figure 7B The results show that, compared with Example 1 containing multi-spike particles, Example 2 containing multi-spike magnetic particles can bind to more exosomes in the PBS solution, thereby detecting more CD9 and a higher average fluorescence intensity. Figure 7C The results show that, compared with Example 1 containing multi-spike particles, Example 2 containing multi-spike magnetic particles can bind to more exosomes in plasma containing EDTA, thereby detecting more CD9 and a higher mean fluorescence intensity. FIG. 7A to FIG. 7C The results show that Example 1 containing multi-spike microparticles or Example 2 containing multi-spike magnetic microparticles can detect exosomes in not only PBS solution, but also DMEM culture medium or more complex environments such as plasma containing EDTA.

[0130] FIG. 8A to FIG. 8CIn order to detect the expression of CD81 in the exosomes of SKBr3 in different solutions, Example 1 and Example 2 coupled with Anti-Human CD9 were used respectively. Specifically, the exosomes of SKBr3 were first placed in DMEM culture medium (i.e., the supernatant of the culture medium of SKBr3 was collected), PBS solution (i.e., the exosomes of SKBr3 were purified and then placed in PBS solution) or plasma containing EDTA (i.e., the exosomes of SKBr3 were purified and then placed in plasma containing EDTA). Next, Example 1 and Example 2 coupled with Anti-Human CD9 were used to detect the expression of CD81 in the exosomes of SKBr3 in DMEM culture medium, and the results are as follows. Fig. 8A As shown; Example 1 and Example 2 coupled with Anti-Human CD9 were used to detect the expression of CD81 in the exosomes of SKBr3 in PBS solution, and the results are as follows Figure 8B As shown; Example 1 and Example 2 coupled with Anti-Human CD9 were used to detect the expression of CD81 in the exosomes of SKBr3 in plasma containing EDTA, and the results are as follows Figure 8C As shown. Among them, the above plasma comes from healthy people.

[0131] Depend on Fig. 8A The results show that, compared with Example 1 containing multi-spike particles, Example 2 containing multi-spike magnetic particles can bind to more exosomes in the DMEM culture medium, thereby detecting more CD81 and a higher mean fluorescence intensity. Figure 8B The results show that, compared with Example 1 containing multi-spike particles, Example 2 containing multi-spike magnetic particles can bind to more exosomes in the PBS solution, thus more CD81 can be detected and the average fluorescence intensity is higher. Figure 8C The results show that, compared with Example 1 containing multi-spike particles, Example 2 containing multi-spike magnetic particles can bind to more exosomes in plasma containing EDTA, thereby detecting more CD81 and a higher mean fluorescence intensity. FIG. 8A to FIG. 8C The results show that Example 1 containing multi-spike microparticles or Example 2 containing multi-spike magnetic microparticles can detect exosomes in not only PBS solution, but also DMEM culture medium or more complex environments such as plasma containing EDTA.

[0132] Example 4: Detection of surface antigens of exosomes from breast cancer cell line SKBr3

[0133] 9A to 9DExample 2 coupled with Anti-Human CD9 is used to simultaneously detect the expression levels of any three of CD9, CD29, CD63 and CD81 in the exosomes of SKBr3. Specifically, this example uses similar experimental steps to Example 3 to simultaneously detect multiple surface antigens of the exosomes of the breast cancer cell line SKBr3, so it will not be repeated here. The difference between this example and Example 3 is that in this example, the exosomes of SKBr3 are first placed in plasma containing EDTA. Next, Example 2 coupled with Anti-Human CD9 and a fluorescently labeled second specific antibody (Anti-HumanCD9-Alexa488, Anti-Human CD63-PerCPCy5.5 and Anti-Human CD81-APC) are used to simultaneously detect the expression levels of CD9, CD63 and CD81 in the exosomes, and the results are as shown in the figure. Fig. 9A As shown; Example 2 coupled with Anti-Human CD9 and a second specific antibody (Anti-Human CD29-PE, Anti-Human CD63-PerCPCy5.5 and Anti-Human CD81-APC) labeled with fluorescent markers were used to simultaneously detect the expression of CD29, CD63 and CD81 in exosomes, and the results are shown Fig. 9B As shown; Example 2 coupled with Anti-Human CD9 and a second specific antibody (Anti-Human CD9-Alexa488, Anti-Human CD29-PE and Anti-Human CD63-PerCPCy5.5) with a fluorescent label were used to simultaneously detect the expression of CD9, CD29 and CD63 in exosomes, and the results are shown Fig. 9C As shown; Example 2 coupled with Anti-Human CD9 and multiple secondary specific antibodies (Anti-HumanCD9-Alexa488, Anti-Human CD29-PE and Anti-Human CD81-APC) with multiple fluorescent labels were used to simultaneously detect the expression of CD9, CD29 and CD81 in exosomes, and the results are shown Fig.9D As shown. Among them, the above plasma comes from healthy people. In addition, 9A to 9D In the figure, the expression level of exosome CD9 is represented by the symbol □, the expression level of exosome CD29 is represented by the symbol ╳, the expression level of exosome CD63 is represented by the symbol ○, and the expression level of exosome CD81 is represented by the symbol △.

[0134] Depend on 9A to 9DThe results show that even if the exosomes are located in a more complex environment of plasma containing EDTA, the detection method of this embodiment can still use Example 2 (i.e., multi-spike magnetic microparticles coupled with Anti-Human CD9) and any three fluorescently labeled second specific antibodies (i.e., any three of Anti-Human CD9-Alexa488, Anti-Human CD29-PE, Anti-Human CD63-PerCPCy5.5, and Anti-Human CD81-APC) to simultaneously detect the expression of any three exosome surface antigens (i.e., any three of CD9, CD29, CD63, and CD81) on the exosomes, so as to achieve the purpose of simultaneously detecting multiple analytes. In addition, although this embodiment takes the simultaneous detection of three analytes as an example, the present invention does not limit the number of analytes that can be detected simultaneously. That is to say, in some embodiments, two or more analytes can also be detected simultaneously.

[0135] FIG. 10A to FIG. 10D The expression of any three of CD9, CD29, CD63 and CD81 in the exosomes of SKBr3 was simultaneously detected using Example 2 coupled with Anti-Human Her2. Specifically, the exosomes of SKBr3 were first placed in plasma containing EDTA. Then, Example 2 coupled with Anti-Human Her2 and a second specific antibody with a fluorescent label (Anti-Human CD9-Alexa488, Anti-Human CD63-PerCPCy5.5 and Anti-HumanCD81-APC) were used to simultaneously detect the expression of CD9, CD63 and CD81 in the exosomes. The results are shown in FIG. Fig. 10A As shown; Example 2 coupled with Anti-Human CD9 and a second specific antibody (Anti-Human CD29-PE, Anti-Human CD63-PerCPCy5.5 and Anti-Human CD81-APC) labeled with fluorescent markers were used to simultaneously detect the expression of CD29, CD63 and CD81 in exosomes, and the results are shown Fig. 10B As shown; Example 2 coupled with Anti-Human CD9 and a second specific antibody (Anti-Human CD9-Alexa488, Anti-Human CD29-PE and Anti-Human CD63-PerCPCy5.5) with a fluorescent label were used to simultaneously detect the expression of CD9, CD29 and CD63 in exosomes, and the results are shown Fig. 10CAs shown; Example 2 coupled with Anti-Human CD9 and multiple secondary specific antibodies (Anti-Human CD9-Alexa488, Anti-Human CD29-PE and Anti-Human CD81-APC) with multiple fluorescent labels were used to simultaneously detect the expression of CD9, CD29 and CD81 in exosomes, and the results are shown Fig. 10D As shown. Among them, the above plasma comes from healthy people. In addition, FIG. 10A to FIG. 10D In the figure, the expression level of exosome CD9 is represented by the symbol □, the expression level of exosome CD29 is represented by the symbol ╳, the expression level of exosome CD63 is represented by the symbol ○, and the expression level of exosome CD81 is represented by the symbol △.

[0136] Depend on FIG. 10A to FIG. 10D The results show that even if the exosomes are located in a more complex environment of plasma containing EDTA, the detection method of this embodiment can still utilize Example 2 (i.e., multi-spike magnetic microparticles coupled with Anti-Human Her2) and any three fluorescently labeled second specific antibodies (i.e., any three of Anti-Human CD9-Alexa488, Anti-HumanCD29-PE, Anti-Human CD63-PerCPCy5.5, and Anti-Human CD81-APC) to simultaneously detect the expression of any three exosome surface antigens (i.e., any three of CD9, CD29, CD63, and CD81) on exosomes, so as to achieve the purpose of simultaneously detecting multiple analytes.

[0137] In addition, comparison Fig. 9A and Fig. 10A , Fig. 9B and Fig. 10B , Fig. 9C and Fig. 10C as well as Fig.9D and Fig. 10D It can be seen from the results that whether the multi-spike magnetic microparticles coupled with Anti-Human CD9 or Anti-Human Her2 are used to analyze the expression of exosome surface antigens (i.e., CD9, CD29, CD63, and CD81) of SKBr3, the detection results are highly consistent, indicating that the detection method of this embodiment has high accuracy and small error.

[0138] In addition, comprehensive 9A to 9D and FIG. 10A to FIG. 10DThe results show that whether the multi-spike magnetic microparticles coupled with Anti-Human CD9 or Anti-Human Her2 are used to bind to the exosomes of SKBr3, and any three fluorescently labeled second specific antibodies (i.e., any three of Anti-Human CD9-Alexa488, Anti-Human CD29-PE, Anti-Human CD63-PerCPCy5.5 and Anti-Human CD81-APC) are used to analyze the expression of exosome surface antigens (i.e., CD9, CD29, CD63 and CD81), the expression of CD9 is generally the highest, and the expression of CD81 is slightly higher than or similar to the expression of CD63 and CD29, indicating that even if there are more than two variation parameters in the detection method of this embodiment, the detection results obtained are still comparable.

[0139] Example 5: Detection of surface antigens of exosomes from clinical specimens

[0140] FIG. 11A to FIG. 11D Example 2, which is coupled with different first specific antibodies, is used to simultaneously detect the expression of CD9 and CD63 in the exosomes of the plasma of the clinical specimens. Among them, the first specific antibody is Anti-Human CD9, Anti-Human Her2, Anti-Human EGFR or Anti-Human EpCAM, and the clinical specimens are from the plasma of 8 benign tumors (Benign), 20 tubular breast cancers (Luminal), 8 Her2-positive breast cancers (Her2+) and 4 triple-negative breast cancers (TN). The second specific antibodies with fluorescent labels are Anti-Human CD9-Alexa488 and Anti-Human CD63-PE. Specifically, this embodiment uses similar experimental steps to Example 3 to simultaneously detect CD9 and CD63 in the exosomes of the clinical specimens, so it will not be repeated here. The difference between this embodiment and Example 3 is that in this embodiment, the exosomes in different clinical specimens are first placed in plasma containing EDTA. Next, Example 2 coupled with Anti-Human CD9 was used to simultaneously detect the expression of CD9 and CD63 in exosomes. The results are as follows: Fig.11A As shown; Example 2 coupled with Anti-Human Her2 was used to simultaneously detect the expression of CD9 and CD63 in exosomes, and the results are as follows Fig. 11B As shown; Example 2 coupled with Anti-Human EGFR was used to simultaneously detect the expression of CD9 and CD63 in exosomes, and the results are as follows Fig. 11CAs shown; Example 2 coupled with Anti-Human EpCAM was used to simultaneously detect the expression of CD9 and CD63 in exosomes, and the results are as follows Fig.11D shown.

[0141] Depend on FIG. 11A to FIG. 11D It can be seen from the results that for different clinical specimens, the detection method of this embodiment can still use Example 2 (i.e., multi-spike magnetic particles coupled with Anti-Human CD9, Anti-Human Her2, Anti-Human EGFR or Anti-HumanEpCAM) and two fluorescently labeled second specific antibodies (i.e., Anti-Human CD9-Alexa488 and Anti-Human CD63-PE) to simultaneously detect the expression levels of two exosome surface antigens (i.e., CD9 and CD63) on exosomes, so as to achieve the purpose of simultaneously detecting multiple analytes.

[0142] Example 6: Detection of nucleic acid

[0143] Probe coupling method

[0144] In this embodiment, a probe (Avian Influenza Virus primer, AIV probe) is first coupled to the multi-spike magnetic particles of this embodiment, and then a nucleic acid sequence labeled with biotin (AB nucleic acid to be analyzed or AIV nucleic acid to be analyzed) is added for analysis.

[0145] In this embodiment, the AIV probe has a nucleic acid sequence with sequence identification number: 1, the AB nucleic acid to be analyzed has a nucleic acid sequence with sequence identification number: 2, and the AIV nucleic acid to be analyzed has a nucleic acid sequence with sequence identification number: 3. The detailed sequences are shown in the table below.

[0146]

[0147] In this embodiment, the steps of coupling the AIV probe to the multi-spike magnetic particles are as follows: (1) 6The multi-spike magnetic particles modified with amino groups on their surfaces were washed three times with 200μL of MES buffer solution. Then, 20mg of EDC, 20mg of NHS and 4mg of PAA were dissolved in 400μL of MES buffer solution and mixed with the washed multi-spike magnetic particles. Then, the mixture was mixed and reacted for 30 minutes at room temperature using a test tube vortex at a speed of 1000rpm. Then, the multi-spike magnetic particles were collected with a magnet and kept on the magnet for at least 1 minute. The reaction solution was removed and the multi-spike magnetic particles were washed three times with 200μL of MES buffer solution, and then 20μL of AIV probe and 80μL of PBS solution were added, and the reaction was carried out at room temperature for 2 hours at a speed of 1000rpm. (2) The multi-spike magnetic particles that have completed the reaction were collected with a magnet and kept on the magnet for at least 1 minute. The reaction solution was removed and the multi-spike magnetic microparticles were washed three times with Tris solution (25 mM Tris, pH 7.4), each time for at least 1 minute. Finally, the multi-spike magnetic microparticles coupled with the AIV probe were dispersed in 100 μL Tris solution. (3) The concentration of the multi-spike magnetic microparticles was calculated using an automatic cell counter and the concentration was about 3 to 8 × 10 6 / mL of multi-spike magnetic microparticles with AIV probes coupled on their surface.

[0148] Nucleic acid detection using multi-spike magnetic microparticles coupled with probes

[0149] In this embodiment, multi-spike magnetic microparticles coupled with AIV probes are used to detect AB nucleic acid to be analyzed or AIV nucleic acid to be analyzed with biotin, and the steps are as follows: (1) 30 μL of hybridization solution (TEGOHybridization solution) is mixed with 30 μL of different concentrations (0.05 μM, 0.1 μM, 0.5 μM, 15 μM, 10 μM) of AB nucleic acid to be analyzed or AIV nucleic acid to be analyzed with biotin. Then, 50 μL (1250 particles) of multi-spike magnetic microparticles with AIV probes coupled on the surface are added, and mixed and reacted at 60° C. with a test tube oscillator (vortex) at a speed of 1000 rpm for 30 minutes, wherein the AIV nucleic acid to be analyzed with biotin will be specifically paired with the AIV probe in the multi-spike magnetic microparticles coupled with the AIV probe to form a first complex. After the reaction is completed, the multi-spike magnetic microparticles are collected by a magnet. Next, after removing the reaction solution, wash twice with 200 μL PBS solution. (2) Add 100 μL of fluorescent anti-biotin antibody (Anti-Biotin PE) to the multi-spike magnetic particles, mix, and react at room temperature for 30 minutes, wherein the fluorescent anti-biotin antibody will bind to the biotin in the above-mentioned first complex to form a second complex. After the reaction is completed, collect the multi-spike magnetic particles with a magnet, remove the reaction solution, and wash twice with 200 μL PBS buffer solution (PBS solution, pH 7.4). (3) After washing, add 150 μL PBS buffer solution, and perform fluorescence signal analysis by flow cytometry. The results are as follows. Fig. 12A and Fig. 12B shown.

[0150] like Fig. 12A As shown, since the AB nucleic acid to be analyzed with biotin will not bind to the AIV probe in the multi-spike magnetic microparticles coupled with the AIV probe, the first complex will not be formed, and further the second complex will not be formed, so the measured average fluorescence intensity is close to 0.

[0151] like Fig. 12B As shown, the AIV nucleic acid to be analyzed with biotin will specifically pair with the AIV probe in the multi-spike magnetic microparticles coupled with the AIV probe to form a first complex, and then the biotin in the first complex will combine with the anti-biotin antibody with fluorescence to form a second complex, so the average fluorescence intensity can be measured. In addition, the value of the average fluorescence intensity will basically increase with the increase of the concentration of the AIV nucleic acid to be analyzed with biotin.

[0152] In summary, the micron particle of the present invention can increase the surface area of ​​the micron particle (i.e., the sum of the surface area of ​​the body and the surface area of ​​the first protrusions) by disposing a plurality of irregularly shaped first protrusions on the surface of the body. Thus, the micron particle of the present invention can couple more first ligands to identify and bind more target objects T, thereby enhancing the detection signal to improve the sensitivity of the detection.

[0153] In addition, compared to multi-spike particles, since the surface of the magnetic material layer of the multi-spike magnetic particles of the present invention is a rough surface (or has small protrusions), the surface of the multi-spike magnetic particles (i.e., the surface of the body and the surface of the first protrusion) is also a rough surface, so that the multi-spike magnetic particles can have more surface area, and thus the multi-spike magnetic particles can couple more first ligands, and identify and bind more targets to enhance the detection signal and improve the sensitivity of the detection. Furthermore, since the multi-spike magnetic particles are magnetic, the detection time can be shortened, and the efficiency and convenience of the detection can be improved.

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Sequence Listing <110> Industrial Technology Research Institute <120> Multiple analyte detection methods <130> 117290-CN-PA <150> US 63 / 130,857 <151> 2020-12-28 <150> US 63 / 194,188 <151> 2021-05-28 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 30 <212> DNA <213> Artificial sequence <220> <223> AIV probe <400> 1 gtctacgctg cagtcctcgc tcactgggca 30 <210> 2 <211> 33 <212> DNA <213> Artificial sequence <220> <223> AB Nucleic acid to be analyzed <400> 2 aaaaaaaaaa aaaatcctgg agctaagtcc gta 33 <210> 3 <211> 90 <212> DNA <213> Artificial sequence <220> <223> AIV nucleic acid to be analyzed <400> 3 caagaccaat cctgtcacct ctgactaagg ggattttagg gtttgtgttc acgctcaccg 60 tgccccagtga gcgaggactg cagcgtagac 90

Claims

1. A method for detecting multiple analytes for purposes other than disease diagnosis and treatment, characterized in that: include: A micron particle is provided, wherein the micron particle is coupled with at least one first ligand, and the micron particle comprises: The body; and a plurality of first protrusions formed on a surface of the body; Mixing the micronized particles with a sample including a plurality of analytes to form a first complex; mixing the first complex with a plurality of second ligands with a plurality of first labels so that the plurality of second ligands bind to the plurality of analytes in the first complex to form a second complex; and detecting the plurality of first labels in the second complex, The ratio of the average volume of the plurality of first protrusions to the average volume of the body is 1×10 -7 Up to 2x10 -2 The total volume of the plurality of first protrusions accounts for 10% of the average volume of the micron particles. -1 Up to 6x10 -1 , The micron particles are multi-protrusion particles, and the body of the multi-protrusion particles includes a copolymer core, a polymer layer and a silicon-based layer from the inside to the outside. A plurality of second protrusions are formed on the surface of the copolymer core.

2. The method according to claim 1, characterized in that The average height of the second protrusions is 100 nanometers to 5000 nanometers.

3. The method according to claim 1, characterized in that The micron particles are multi-protrusion magnetic particles, and the body of the multi-protrusion magnetic particles includes a copolymer core, a polymer layer, a magnetic material layer and a silicon-based layer from the inside out. A plurality of second protrusions are formed on the surface of the copolymer core, and the average height of the plurality of second protrusions is 100 nanometers to 5000 nanometers.

4. The method according to claim 1, characterized in that: A ratio of an average height of the plurality of first protrusions to an average particle diameter of the body is 0.005 to 0.

25.

5. The method according to claim 1, characterized in that: The average number of the plurality of first protrusions is 5 to 500, and the average particle size of the micron particles is 1 micron to 20 microns. The method according to claim 1 , wherein the micronized particles are non-spherical.

7. The method according to claim 1, characterized in that The plurality of analytes are located on the surface of the sample, and the method of forming the first complex includes: allowing the at least one first ligand to recognize and directly bind to a target located on the surface of the sample.

8. The method according to claim 7, characterized in that The at least one first ligand comprises a first specific antibody, and the first specific antibody comprises an anti-human exosome surface antigen antibody, an anti-human blood cell surface antigen antibody, an anti-human immune cell surface antigen antibody, an anti-human tumor cell surface antigen antibody, or a combination thereof.

9. The method according to claim 7, characterized in that: The sample includes human exosomes, human blood cells, human immune cells, human tumor cells, or a combination thereof, and the plurality of analytes include surface antigens of the human exosomes, surface antigens of the human blood cells, surface antigens of the human immune cells, surface antigens of the human tumor cells, or a combination thereof.

10. The method according to claim 7, characterized in that The plurality of second ligands include a plurality of second specific antibodies, and the plurality of second specific antibodies include anti-human exosome surface antigen antibodies, anti-human blood cell surface antigen antibodies, anti-human immune cell surface antigen antibodies, anti-human tumor cell surface antigen antibodies, or a combination thereof.

11. The method according to claim 1, characterized in that The at least one first ligand includes a plurality of first ligands, and the method of forming the first complex includes: allowing the plurality of first ligands to recognize and directly bind to the plurality of analytes.

12. The method according to claim 11, characterized in that The multiple first ligands include multiple nucleic acid probes, and the multiple nucleic acid probes include multiple primers or aptamers. The multiple analytes include multiple nucleic acid sequences with multiple second labels, and the multiple second labels include biotin, multiple antigen regions or a combination of the foregoing.

13. A method for detecting multiple analytes for purposes other than disease diagnosis and treatment, characterized in that: include: Provided are micron particles, wherein the micron particles are coupled with a plurality of ligands, and the micron particles include: The body; and a plurality of protrusions formed on a surface of the body; mixing the microparticles with a sample comprising a plurality of analytes to form a complex, wherein the plurality of analytes are labeled with a plurality of labels; and detecting the multiple labels in the complex, The ratio of the average volume of the plurality of first protrusions to the average volume of the body is 1×10 -7 Up to 2x10 -2 The total volume of the plurality of first protrusions accounts for 10% of the average volume of the micron particles. -1 Up to 6x10 -1 , The micron particles are multi-protrusion particles, and the body of the multi-protrusion particles includes a copolymer core, a polymer layer and a silicon-based layer from the inside to the outside. A plurality of second protrusions are formed on the surface of the copolymer core.

14. The method according to claim 13, characterized in that The plurality of ligands include a plurality of nucleic acid probes, the plurality of labels include a plurality of fluorescent labels, a plurality of luminescent labels or a combination thereof, and the plurality of analytes include a plurality of nucleic acid sequences.

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