Colorimetric detection of target materials based on hydrogel particles

Abstract

The present invention relates to colorimetric detection of target materials based on hydrogel particles. The colorimetric detection of target materials based on hydrogel particles according to an embodiment of the present invention comprises the following steps: reacting a sample containing a target material with a hydrogel particle loaded with a probe that specifically binds to the target material; accumulating and amplifying an insoluble colorimetric material in the hydrogel particle to mark the target material bound to the probe. Here, the hydrogel particle is made of a polymer network, and the insoluble colorimetric material is fixed in the polymer network by binding to the polymer network loaded probe.

Description

Technical Field

[0001] The present invention relates to a colorimetric detection method of a target analyte based on hydrogel particles, and more particularly to a method for detecting a target analyte by introducing a colorimetric reaction into hydrogel particles. Background Art

[0002] Encoded hydrogel particles have attracted wide attention in the fields of diagnostic medicine, drug screening, and molecular biochemistry, which require high detection performance for target biomolecules because they can perform multiple detections of target biomolecules with high sensitivity and specificity. The target analytes bound to the encoded hydrogel particles can be labeled with fluorescent materials. However, the use of expensive systems such as light sources, microscopes, and cameras is essential for fluorescence analysis, which makes it difficult to use fluorescent materials in places where there is usually no fluorescence analysis system. When exposed to light during the fluorescent labeling process or during the cleaning process after fluorescent labeling, fluorescent materials may lose their fluorescent properties due to fluorescence bleaching. In addition, non-specific binding of fluorescent materials to supports such as substrates or membranes may produce false positive signals, thereby affecting the reliability of quantitative analysis. Fluorescent materials can be used for qualitative analysis and quantitative analysis. Quantitative analysis using fluorescent materials also requires the same expensive system for quantitative analysis to determine the binding of target analytes. In addition, quantitative analysis should be performed in a limited space such as a darkroom to prevent fluorescence bleaching. Due to these problems, fluorescence analysis should only be performed in centralized laboratories and is difficult to expand to point-of-care testing (POCT) where qualitative analysis is the majority.

[0003] Therefore, it is necessary to develop new technologies capable of analyzing biomolecules with high sensitivity comparable to conventional fluorescence measurement without using expensive systems or having spatial limitations. Summary of the invention

[0004] Problems to be solved by the present invention

[0005] The present invention is directed to solving the problems of the prior art, and one aspect of the present invention provides a colorimetric detection method for a target analyte based on hydrogel particles, wherein a probe is loaded into the hydrogel particles, the target analyte specifically binds to the probe, and an insoluble colorimetric material is accumulated and amplified in the hydrogel particles.

[0006] Solutions to the problem

[0007] The method for colorimetric detection of target analytes based on hydrogel particles according to an embodiment of the present invention comprises (a) reacting a sample containing the target analyte with hydrogel particles loaded with probes that specifically bind to the target analyte and (b) accumulating and amplifying an insoluble colorimetric material in the hydrogel particles to label the target analyte bound to the probes, wherein the hydrogel particles form a polymer network, the probes bind to and are loaded into the polymer network, and the insoluble colorimetric material is immobilized on the polymer network.

[0008] The hydrogel particles can be geometrically shaped and coded to identify the probe.

[0009] The probes can be loaded onto the hydrogel particles during or after synthesis.

[0010] Each probe loaded after synthesis of the hydrogel particles may include a capture moiety that specifically binds to the corresponding target analyte and a functional group that is linked to the capture moiety and to an unreacted end in the form of a carbon-carbon double bond that is attached to the polymer network.

[0011] The probe may be a chemical compound, oligonucleotide, oligosaccharide, protein, antibody, peptide or aptamer that specifically binds to the target analyte.

[0012] The functional group may be selected from sulfhydryl (-SH) and amine (-NH 2 ).

[0013] Step (b) may include conjugating an enzyme to the target analyte bound to the probe and adding a substrate that reacts with the enzyme to generate an insoluble colorimetric material.

[0014] Conjugation of the enzyme may include adding a secondary binding material that specifically binds to the target analyte and adding an enzyme for binding to the secondary binding material.

[0015] The secondary binding material can be a compound, oligonucleotide, oligosaccharide, protein, antibody, peptide or aptamer that specifically binds to the target analyte.

[0016] The enzyme may be selected from the group consisting of alkaline phosphatase (ALP), β-galactosidase, peroxidase, luciferase, cytochrome P450, and combinations thereof.

[0017] The substrate can be selected from bromochloroindole phosphate (BCIP) / nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, p-nitrophenyl phosphate (PNPP), enhanced chemifluorescence (ECF), 4-chloronaphthol, 3,3'-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), 3,3',5,5'-tetramethylbenzidine (TMB), 4-chloronaphthol, 3,3'-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), 6-chloro-3-indolyl-β-D-galactopyranoside (Red-gal) and combinations thereof.

[0018] The target analyte may include one or more than one material selected from the group consisting of DNA, RNA, protein, exosome, and virus.

[0019] Features and advantages of the present invention will become apparent from the following description with reference to the accompanying drawings.

[0020] Before the detailed description of the present invention, it should be understood that in view of the principle that the inventor can appropriately define the concepts of terms and words in order to describe his / her invention in the best way, the terms and words used in the specification and claims should not be interpreted as having common and dictionary meanings but as meanings and concepts corresponding to the technical spirit of the present invention.

[0021] Effects of the Invention

[0022] According to the present invention, insoluble colorimetric materials are accumulated and amplified in hydrogel particles, instead of using fluorescent materials to label target analytes (e.g., nucleic acids and proteins) bound to the inside of hydrogel particles, and results with detection sensitivity and specificity comparable to those achieved using fluorescent materials can be obtained even without using expensive analytical systems or separate spaces.

[0023] According to the present invention, the target analyte in the particles labeled with the colorimetric material can only be detected by the naked eye or bright field image, and the target analyte can be quantified only by a simple system including a USB microscope and a smart phone, without the need for an expensive system. In addition, the colorimetric material accumulates and amplifies in the hydrogel particles, achieving performance comparable to the detection sensitivity and specificity that cannot be achieved with existing colorimetric reactions. The accumulation and amplification of the colorimetric material makes it possible to quantitatively analyze the target analyte with high reliability even in a place that is not a limited space such as a darkroom, because fluorescence bleaching does not occur. Therefore, the present invention can be widely used in the field of point-of-care testing (POCT) outside centralized laboratories and the quantitative analysis of target analytes such as nucleic acids and proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1FIG. 1 is a diagram schematically illustrating a process of synthesizing hydrogel particles used in a method for colorimetric detection of a target analyte based on hydrogel particles according to an embodiment of the present invention.

[0025] Figure 2 Shown by Figure 1 Plan view of the process for synthesizing hydrogel particles of different shapes.

[0026] Figure 3 To illustrate the binding of the probe to Figure 1 Figure 3. Process of synthesizing the residual unreacted ends in the hydrogel particles.

[0027] Figure 4 and Figure 5 The diagram illustrates a process of making a colorimetric material insolubilized by an enzyme-substrate reaction of a labeled enzyme in hydrogel particles after detecting a target analyte using a colorimetric detection method of a target analyte based on hydrogel particles according to an embodiment of the present invention.

[0028] Figure 6 The figure shows the results of single detection of three pre-eclampsia-related proteins by the colorimetric detection method in Experimental Example 3.3.

[0029] Figure 7 Shown is a graph revealing the results of single detection of three pre-eclampsia-related proteins by the colorimetric detection method in Experimental Example 3.3.

[0030] Figure 8 Shown is a graph revealing the results of multiplex detection of three pre-eclampsia-related proteins by a colorimetric detection method in Experimental Example 3.4.

[0031] Fig. 9 The figure shows the results of multiplex detection of three pre-eclampsia-related proteins by the colorimetric detection method in Experimental Example 3.4.

[0032] Fig.10 The results of the detection of proteins added to plasma by the colorimetric detection method in Experimental Example 3.5 were compared with the results of ELISA.

[0033] Fig.11 A diagram schematically illustrates the process of applying the colorimetric detection method to plasma extracted from real pre-eclampsia patients and analyzing it using a USB microscope and a smartphone in Experimental Example 3.5.

[0034] Fig.12 The results of multiplex detection of two proteins in plasma samples extracted from real pre-eclampsia patients and healthy subjects by a colorimetric detection method in Experimental Example 3.5 are shown.

[0035] Fig.13 The results of single detection of nucleic acid by the colorimetric detection method in Experimental Example 3.6 are shown.

[0036] Fig.14 The results of multiplex detection of nucleic acids by the colorimetric detection method in Experimental Example 3.6 are shown. DETAILED DESCRIPTION

[0037] The objects, specific advantages, and novel features of the present invention will become more apparent from the following detailed description and preferred embodiments, examples of which are illustrated in the accompanying drawings.A detailed description of well-known technologies is avoided in order to avoid obscuring the subject matter of the present invention.

[0038] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0039] Figure 1 FIG. 1 is a diagram schematically illustrating a process of synthesizing hydrogel particles used in a method for colorimetric detection of a target analyte based on hydrogel particles according to an embodiment of the present invention. Figure 2 Shown by Figure 1 Plan view of the process for synthesizing hydrogel particles of different shapes. Figure 3 To illustrate the binding of the probe to Figure 1 Figure 3. Process of synthesizing the residual unreacted ends in the hydrogel particles. Figure 4 and Figure 5 The diagram illustrates a process of making a colorimetric material insolubilized by an enzyme-substrate reaction of a labeled enzyme in hydrogel particles after detecting a target analyte using a colorimetric detection method of a target analyte based on hydrogel particles according to an embodiment of the present invention.

[0040] like Figures 1 to 5 As shown, the colorimetric detection method of the target analyte based on the hydrogel particles according to the embodiment of the present invention includes: reacting a sample containing the target analyte with the hydrogel particles loaded with the probe that specifically binds to the target analyte; accumulating and amplifying the insoluble colorimetric material in the hydrogel particles to mark the target analyte bound to the probe. The hydrogel particles form a polymer network, the probe binds and is loaded into the polymer network, and the insoluble colorimetric material is fixed on the polymer network.

[0041] The method of the present invention detects the target analyte using a colorimetric reaction introduced into the hydrogel particles. Conventional fluorescence assays using fluorescent materials to detect target analytes require the use of expensive systems such as light sources, microscopes, and cameras for fluorescence analysis. When exposed to light, the fluorescent material may be fluorescently bleached and may non-specifically bind to the support to produce false positive signals, thereby affecting the reliability of quantitative analysis. Quantitative analysis using fluorescent materials also requires expensive systems and should also be performed in limited spaces such as darkrooms to prevent fluorescence bleaching. Due to these problems, fluorescence analysis should only be performed in centralized laboratories and is difficult to expand to point-of-care testing (POCT) where qualitative analysis is the majority. The present invention provides a solution to the problems of conventional fluorescence assays.

[0042] Specifically, the method of the present invention includes reacting a sample with hydrogel particles loaded with probes and accumulating and amplifying an insoluble colorimetric material.

[0043] First, the sample is allowed to react with the hydrogel particles loaded with probes. The hydrogel particles loaded with probes form a polymer network and have a plurality of pores extending from the outside to the inside. The probes are bound and loaded into the polymer network.

[0044] In the probe-loaded hydrogel particles, the probe binds to the target analyte. This reaction is similar to the reaction in solution, can be carried out in three dimensions, and can detect biomolecules with high specificity and sensitivity over a wide dynamic range. The probe specifically binds to the target analyte. Specifically, when a sample containing the target analyte is mixed with the probe-loaded hydrogel particles, the target analyte penetrates into the hydrogel particles through the pores of the hydrogel particles and specifically binds to the probe loaded in the hydrogel particles.

[0045] Reference Figure 2 , hydrogel particles loaded with probes can be encoded. Here, the hydrogel particles can be geometrically shaped and encoded to identify the probes. The hydrogel particles can be loaded with different probes. In this case, the hydrogel particles have different shapes as codes for identifying the probes. Therefore, the hydrogel particles and the loaded probes can be distinguished according to the code assigned to the particles, thereby enabling simultaneous detection of multiple target analytes. Figure 2 In the study, the hydrogel particles were encoded with up to eight rectangular gear-shaped protrusions. Alternatively, the hydrogel particles could be encoded with different geometric shapes.

[0046] Encoded probe-loaded hydrogel particles can be synthesized by flow lithography. Figure 1, UV light is irradiated onto a flowing precursor fluid to synthesize particles by flow photolithography. The UV light passing through the photomask causes polymerization of the precursor to synthesize particles having the same shape as the photomask. The fluid forms a laminar flow in the microchannel and can be configured in multiple parallel flows without mixing, thereby enabling the synthesis of multifunctional asymmetric particles after UV irradiation.

[0047] The precursor fluid may include a photocurable monomer having a carbon-carbon double bond (C=C) as a functional group and a photosensitive initiator. The photocurable monomer having a carbon-carbon double bond as a functional group may be, for example, methacrylate, maleimide, vinyl sulfone, acrylate or acrylamide. The precursor fluid may also include a pore former such as polyethylene glycol. Deionized water (DI water) may be used as a solvent for dispersing the pore former. In the hydrogel particles synthesized using the precursor fluid, the photocurable monomer molecules are polymerized to form a polymer network structure. During the synthesis of the particles, the highly reactive and biochemically unstable carbon-carbon double bonds in the monomer molecules are cross-linked to form a network. The carbon-carbon double bonds are converted into single bonds with very low reactivity after cross-linking. However, not all functional groups of the monomer molecules present in the precursor fluid are converted during the flow lithography process, and some of the carbon-carbon double bonds remain unreacted. Some unreacted carbon-carbon double bonds are bonded to the network. That is, after the reaction, some functional groups in the monomer molecules are cross-linked in the network, but some functional groups in the monomer molecules remain unreacted. The unreacted functional groups connected to the network in the form of carbon-carbon double bonds are defined as unreacted terminals.

[0048] Since the unreacted ends are not removed even by washing after particle synthesis, they are present in hydrogel particles synthesized by flow lithography, e.g. Figure 3 The probe binds to the unreacted end of the polymer network.

[0049] Each probe may include a capture portion that specifically binds to the target analyte and a functional group that is connected to the capture portion and bound to the unreacted end. The functional group of the probe that is bound to the unreacted end in the form of a carbon-carbon double bond may be selected from a sulfhydryl group (-SH) and an amine group (-NH 2 ). The thiol-ene click reaction is used to react between a carbon-carbon double bond and a thiol group. The thiol-ene click reaction is very rapid, with a yield of almost 100%, and does not produce side reactions or byproducts. The aza-Michael addition reaction can be used to react between a carbon-carbon double bond and an amine group.

[0050] These reactions can be free radical reactions, catalytic reactions or spontaneous reactions. The capture portion of the probe can be cross-linked with the inside of the hydrogel particles. According to the free radical reaction, the hydrogel particles are dispersed in a medium such as water or a solvent, a capture portion (probe) containing a functional group that can react with the unreacted end by a free radical reaction is added and dispersed, a photosensitive initiator or a thermal initiator is added, and light (UV) or thermal energy is applied within a predetermined time period to induce a covalent bond between the carbon-carbon double bond and the functional group. As a result, the probe is loaded into the hydrogel particles and the unreacted end is removed. Catalytic reaction refers to a catalyst-mediated reaction. The catalytic reaction uses an organic catalyst instead of the initiator used in the free radical reaction to induce a covalent bond between the carbon-carbon double bond and the functional group. As a result, the probe is loaded and the unreacted end is removed. Spontaneous reaction is suitable for loading probes that are sensitive to free radicals or catalysts. According to the spontaneous reaction, the probe is cross-linked with the hydrogel particles by electrons moving in the solution. The electrons in the buffer or polar solvent act as nucleophiles to form covalent bonds between the functional group and the carbon-carbon double bond.

[0051] However, the synthesis of encoded hydrogel particles is not necessarily limited to flow lithography. Different processes including replica molding can be used to synthesize encoded hydrogel particles. Replica molding is a process for synthesizing particles by loading a fluid into a micro mold engraved with an intaglio micro pattern and irradiating ultraviolet light onto the fluid. The shape of the particle is the same as the intaglio pattern engraved on the micro mold.

[0052] The probe for detecting the target analyte can be loaded not only after the hydrogel particles are synthesized, but also during the synthesis of the hydrogel particles. The probe (capture moiety) is a material substance that can specifically bind to the target analyte. For example, the probe (capture moiety) can be a compound, oligonucleotide, oligosaccharide, protein, antibody, peptide or aptamer that specifically binds to the target analyte. In particular, the antibody as the probe (capture moiety) specifically binds to the target analyte through an antigen-antibody reaction. The target analytes are not particularly limited as long as they specifically bind to the probe (capture moiety). The target analyte can include one or more than one material selected from DNA, RNA, protein, exosome, and virus.

[0053] Then, the insoluble colorimetric material is accumulated and amplified. The insoluble colorimetric material labels the target analyte bound to the probe to determine the binding of the target analyte. The insoluble colorimetric material is locally aggregated in the hydrophilic environment and is fixed in the polymer network of the probe-loaded hydrogel particles. As a result, the insoluble colorimetric material does not flow out of the particles but is accumulated and amplified in the hydrogel particles. Figure 4As shown, the insoluble colorimetric material labeling the target analyte in the particles can only be detected by the naked eye or bright field images. Therefore, the target analyte can be quantified by a simple system including a USB microscope and a smartphone without the need for an expensive system. The accumulation and amplification of the colorimetric material in the hydrogel particles can achieve performance comparable to the detection sensitivity and specificity of fluorescence assays, and the target analyte can be quantified with high reliability even in a place other than a limited space such as a darkroom because photobleaching does not occur.

[0054] In one embodiment where the insoluble colorimetric material is accumulated and amplified in the hydrogel particles, an enzyme is conjugated to a target analyte bound to the probe, and a substrate for the enzyme reaction is added to produce the insoluble colorimetric material. The enzyme may be selected from alkaline phosphatase (ALP), β-galactosidase, peroxidase, luciferase, cytochrome P450, and combinations thereof. The substrate can be selected from bromochloroindole phosphate (BCIP) / nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, p-nitrophenyl phosphate (PNPP), enhanced chemifluorescence (ECF), 4-chloronaphthol, 3,3'-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), 3,3',5,5'-tetramethylbenzidine (TMB), 4-chloronaphthol, 3,3'-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), 6-chloro-3-indolyl-β-D-pyranogalactoside (Red-gal), and combinations thereof.

[0055] For example, alkaline phosphatase (ALP) and 5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium (BCIP / NBT) can be used as enzyme and substrate, respectively. In this case, the target analyte captured in the probe-loaded hydrogel particles is labeled with the enzyme ALP and the BCIP / NBT substrate solution is added to the hydrogel particles, resulting in the accumulation and amplification of the insoluble colorimetric material through the enzyme-substrate reaction in the hydrogel particles. As a result, the hydrogel particles appear dark purple (see Figure 5 ). The target analyte can be enzyme-labeled by a secondary binding material that specifically binds to the target analyte bound to the probe. The secondary binding material can be a compound, oligonucleotide, oligosaccharide, protein, antibody, peptide or aptamer that specifically binds to the target analyte. The secondary binding material and the enzyme can be added in sequence to induce binding between the secondary binding material and the enzyme. Alternatively, a secondary binding material polymerized with the enzyme can be added. In one embodiment, the secondary binding material can be a secondary antibody that specifically binds to the target analyte through an antigen-antibody reaction. In this embodiment, the biotinylated secondary antibody specifically binds to the target analyte captured by the probe, streptavidin-polymerized ALP (streptavidin-ALP) is added to bind streptavidin to biotin, and BCIP / NBT is added, resulting in the accumulation and amplification of insoluble colorimetric materials in the hydrogel particles.

[0056] In general, according to the present invention, when the hydrogel particles loaded with probes are mixed with a sample and left for a predetermined time for a detection reaction, only the material specific to the probe loaded into the particles is bound to the probe. After the detection reaction is completed, it is necessary to identify the binding between the probe and the target analyte to determine whether the target analyte is bound to the hydrogel particles. According to conventional fluorescence assays, the binding sites are labeled with fluorescent materials and the presence and intensity of fluorescence are analyzed using a fluorescence analyzer including a light source, a microscope, and a camera. In contrast, according to the present invention, the accumulation and amplification of insoluble colorimetric materials in the hydrogel particles can achieve performance comparable to the detection sensitivity and specificity of fluorescence assays while avoiding the problems of fluorescence assays.

[0057] Mode for Carrying Out the Invention

[0058] The present invention will be explained more specifically with reference to the following experimental examples.

[0059] 1. Experimental example

[0060] 1.1. Fabrication of microfluidic chip

[0061] The microfluidic chip for synthesizing hydrogel particles was designed using AutoCAD (Autodesk, California, USA) and printed on a photomask (Han & All technology, South Korea). SU-8 25 is a negative photoresist, coated on a silicon wafer with a thickness of 52 μm, and then the SU-8 master mold was constructed by photolithography. A mixture of PDMS (Corning, USA) and a curing agent with a weight ratio of 10:1 was poured into the SU-8 master mold and fixed at 70°C for 8 hours. The cured PDMS was separated from the SU-8 master mold and cut into slices. Thereafter, a 1.0 mm and 10.0 mm biopsy punch was used to drill the inlet and outlet of the channel engraved on the slice. A mixture of PDMS and a curing agent with a weight ratio of 10:1 was poured onto a glass slide and fixed at 70°C for 25 minutes. The prepared slices were attached to the surface of the glass slide and cured overnight at 70°C to make a microfluidic chip.

[0062] 1.2. Stop-Flow Lithography (SFL) Setup

[0063] The hydrogel particles were synthesized by stopped-flow lithography (see Figure 1). For SFL, a UV and pressure control system was built using a custom circuit board and LabView (National Instruments, Texas, USA). The microfluidic chip was placed on an inverted microscope (Zeiss, Germany) and the precursor was injected into the microfluidic chip through a pipette tip. Here, the precursor was injected through air, and the pressure of the air was controlled by a pressure regulator. A photomask engraved with different patterns was fixed to the microscope field stop and an LED lamp was used as the source of the cured precursor. The intensity of the UV was maintained at 2200 mW cm -2 .

[0064] 1.3. Preparation of antibody-functionalized hydrogel particles

[0065] The precursor injected into the microfluidic chip consists of 20% (volume / volume) polyethylene glycol diacrylate (SigmaAldrich, USA, PEG700DA), 40% (volume / volume) polyethylene glycol 600 (Sigma Aldrich, PEG600) as a pore former, 35% (volume / volume) deionized water, and 5% (volume / volume) Darocur1173 (Sigma Aldrich) as a photoinitiator. The precursor injected into the microfluidic chip synthesizes hydrogel particles through a continuous cycle of flow (400ms), stop (200ms), UV contact (65ms), and duration (335ms). Different photomasks are used to encode proteomes. The synthesized particles are rinsed 3 times in 1xPBST (phosphate buffer containing 0.005% Tween 20). Next, 12 μL of recombinant capture antibodies (12 μg / μL of P1GF, 6 μg / μL of sFIT-1, 6 μg / μL of sEng) were reacted with 16.5 μL of particles (~75 per μL) and stirred with 1.5 μL of different functional PEG linkers (Thiol-PEG 2000-NHS) at 1500 rpm, 25° C. The resulting antibody-conjugated hydrogel particles were stored in 1× PBST at 4° C.

[0066] 1.4. Protein detection by colorimetric reaction

[0067] Protein detection reactions were performed in 80 μL volumes. In each detection reaction, 40 μL (~50 μL) of hydrogel particles loaded with antibodies in 1x PBST were mixed with 2x target proteins in FBS. The reaction was allowed to proceed for 2 hours at 1500 rpm and 25°C. After the reaction was complete, the reaction mixture was rinsed 3 times with 1x PBST and secondary antibodies (15 ng / μL of P1GF, 125 ng / μL of sFIT-1, 12.5 ng / μL of sEng) were added. The reaction was again performed for 1 hour at 1500 rpm and 25°C.

[0068] After rinsing 3 times with 1x PBST, the hydrogel particles were enzymatically labeled with streptavidin-AP in 1% BSA. Finally, 50 μL of BCIP / NBT solution was mixed with the particles in a microtube, and then the reaction was reacted at 25°C for 7 minutes. After the reaction was completed, the reaction mixture was rinsed 3 times with DI water + Tween 20 solution. Bright field images were obtained by connecting a laptop (Insancommerce, Korea), a 3D printer, and a USB microscope with a domestic light source system.

[0069] 2. Comparative Example

[0070] ELISA

[0071] 100 μL capture antibody (4 μg / μL P1GF, 2 μg / μL sFIt-1, 2 μg / μL sEng) was applied to a 96-well microplate and rinsed 3 times with 1x PBST. The wells were closed with 1% BSA. The dilutions of the target protein at different concentrations in FBS were injected into the wells and the reaction was allowed to proceed at room temperature for 2 hours. After rinsing the wells, 100 μL detection antibody (60 ng / μL P1GF, 500 ng / μL sFIt-1, 50 ng / μL sEng) was added to the wells. The reaction was carried out at room temperature for 2 hours. After rinsing 3 times, streptavidin-HRP was added to the well plate. The reaction was carried out at room temperature for 20 minutes. The well plate was rinsed 3 times, 100 μL substrate solution was added, and then the reaction was carried out for 20 minutes. After injecting 50 μL stop solution, the optical density was measured using an enzyme reader.

[0072] 3. Analysis and evaluation

[0073] 3.1. Synthesis of Encoded Hydrogel Particles

[0074] Reference Figure 1 and Figure 2 , cylindrical encoded hydrogel particles with up to 8 gear-like protrusions were synthesized by stopped-flow lithography (SFL). After the encoded hydrogel particles were synthesized, the thiolated antibody was bound to the non-reactive end in the form of a carbon-carbon double bond of the hydrogel particles through a thiol-ene reaction. As a result, the antibody was loaded into the hydrogel particles (see Figure 3 ). Binding of antibodies during the synthesis of the particles may induce aggregation of the antibodies due to incompatibility with the photoinitiator. However, binding of antibodies in the protein stabilization solution after the particles are synthesized allows the antibodies to be loaded into the particles at a high density. Therefore, loading the antibodies into the synthesized hydrogel particles ensures a higher analytical sensitivity than binding of antibodies during the synthesis of the particles.

[0075] 3.2. Colorimetric reaction

[0076] In this experimental example, color development was induced by an enzyme-substrate reaction in the hydrogel particles. To this end, alkaline phosphatase (ALP) and 5-bromo-4-chloro-3-indolyl phosphate / nitroblue tetrazolium (BCIP / NBT) were used as enzyme and substrate, respectively. For the colorimetric reaction in the hydrogel particles, the antigen was specifically bound to the capture antibody and the biotinylated secondary antibody. The biotinylated secondary antibody was bound to streptavidin-ALP. Thereafter, the BCIP / NBT substrate solution was introduced. The BCIP / NBT substrate was converted into an insoluble colorimetric material by the enzyme-substrate reaction of the ALP enzyme in the hydrogel. The insoluble colorimetric material was accumulated and amplified. In this process, the hydrogel particles were dyed dark purple (see Figure 4 and Figure 5 ). The insoluble colorimetric material is locally aggregated in the hydrophilic environment and immobilized on the network of hydrogel particles.

[0077] 3.3. Single protein detection

[0078] The detectable range and minimum concentration of three preeclampsia-related proteins (P1GF, Flt-1, and endoglin) were studied. Figure 6 and Figure 7 shown. Figure 6 Shown are the results of a single assay for three preeclampsia-related proteins using a colorimetric detection method. Figure 7 Shown are images revealing the results of a single detection of three pre-eclampsia-related proteins by a colorimetric detection method.

[0079] The results showed that the detection ranges of P1GF, Flt-1, and endoglin were 41.3 pg / μL to 7500 pg / μL, 136.3 pg / μL to 30000 pg / μL, and 73.5 pg / μL to 15000 pg / μL, respectively, which exceeded the ELISA results obtained by measuring the absorbance using a spectrometer (P1GF was 31.2 pg / μL to 2000 pg / μL, Flt-1 was 125 pg / μL to 8000 pg / μL, and endoglin was 125 pg / μL to 8000 pg / μL).

[0080] 3.4. Multiplex protein detection

[0081] Multiplex detection of three proteins (P1GF, Flt-1 and endoglin) was performed. Figure 8 and Fig. 9 shown. Figure 8 Shown are images revealing the results of multiplexed detection of three pre-eclampsia-related proteins by a colorimetric detection method. Fig. 9 Shown are the results of multiplexed detection of three preeclampsia-related proteins using a colorimetric detection approach.

[0082] In both single and multiplex assays, three proteins (P1GF, Flt-1, and endoglin) showed no cross-reactivity in 8 cases according to the presence or absence of each protein. The recovery rates of P1GF were 92.6%, Flt-1 was 125.2%, and endoglin was 122.9%, all of which were within the generally acceptable range (70% to 130%).

[0083] 3.5. Detection of proteins in plasma samples

[0084] After adding different concentrations of PlGF to plasma samples of healthy subjects, PlGF was detected by ELISA which is generally used for colorimetric reaction and protein detection. Fig.10 The results of the colorimetric detection of proteins spiked into plasma were compared with the results of the ELISA.

[0085] Reference Fig.10 , the colorimetric reaction and ELISA showed a linear relationship, indicating that the colorimetric reaction can be applied to the detection of proteins in real plasma samples.

[0086] Fig.11 Figure 2 is a schematic diagram illustrating the application of the colorimetric detection method to plasma extracted from real preeclampsia patients and the process of analysis using a USB microscope and a smartphone. Based on the above results, plasma samples from real preeclampsia patients and healthy subjects were subjected to multiplex detection of P1GF and Flt-1. The results are shown in Figure 2. Fig.12 shown. Fig.12 Shown are the results of multiplexed detection of two proteins in plasma samples extracted from real pre-eclampsia patients and healthy subjects by colorimetric detection method.

[0087] Therefore, the concentrations of P1GF and Flt-1 in plasma samples vary greatly, making it difficult to perform multiplex testing. The amount of Flt-1 and the ratio of Flt-1 / P1GF used to distinguish preeclampsia were significantly different between the patient and normal groups. However, there was no significant difference in the amount of PlGF, which was due to the small number of cases in the patient and normal groups (normal: 5 cases, patient: 5 cases).

[0088] 3.6. Nucleic acid testing

[0089] Nucleic acids are detected using a colorimetric reaction. Results are as follows Fig.13 and Fig.14 shown. Fig.13 Results are shown for a single detection of nucleic acids by a colorimetric detection method. Fig.14The results of multiplex detection of nucleic acid by colorimetric detection method are shown. The detectable LoD of nucleic acid detected by colorimetric reaction is 33.72 amol (see Fig.13 High specificity was also observed in cross-reactivity assays using two targets (see Fig.14 ).

[0090] Although the present invention has been described herein with reference to specific embodiments, these embodiments are not intended to limit the present invention but are provided for purposes of explanation. It will be apparent to those skilled in the art that modifications and improvements can be made without departing from the spirit and scope of the present invention.

[0091] Simple modifications and improvements of the present invention belong to the scope of the present invention, and the specific scope of the present invention will be clearly defined in the claims.

[0092] Industrial Applications

[0093] According to the present invention, the target analyte is specifically bound to the probe loaded into the hydrogel particles, and even without using an expensive analytical system or a separate space, the insoluble colorimetric material is accumulated and amplified in the hydrogel particles to obtain a result comparable to the detection sensitivity and specificity achieved by using a fluorescent material. Therefore, the present invention is considered to be industrially applicable.

Claims

1. A method for colorimetric detection of a target analyte based on hydrogel particles, the method comprising (a) reacting a sample containing the target analyte with a hydrogel particle loaded with a probe that specifically binds to the target analyte, (b) accumulating and amplifying an insoluble colorimetric material in the hydrogel particle to label the target analyte bound to the probe, wherein the hydrogel particle forms a polymer network, the probe binds to and is loaded into the polymer network, and the insoluble colorimetric material is immobilized on the polymer network; in, The samples contain different target analytes, Among them, the hydrogel particles can be loaded with different probes, so that different target analytes are bound to different hydrogel particles respectively. wherein each hydrogel particle has a code formed in a geometric shape for identifying a different probe, and wherein step (b) comprises conjugating an enzyme to a target analyte bound to the probe, and adding a substrate that reacts with the enzyme to produce an insoluble colorimetric material.

2. The method according to claim 1, wherein the probe is loaded during or after the synthesis of the hydrogel particles.

3. The method of claim 2, wherein each probe loaded after synthesis of the hydrogel particles comprises a capture moiety that specifically binds to the corresponding target analyte and a functional group that is connected to the capture moiety and to an unreacted end in the form of a carbon-carbon double bond connected to the polymer network.

4. The method of claim 1, wherein the probe is a compound, oligonucleotide, oligosaccharide, protein, antibody, peptide or aptamer that specifically binds to the target analyte.

5. The method according to claim 3, wherein the functional group is selected from the group consisting of thiol (-SH) and amine (-NH2).

6. The method of claim 1, wherein the conjugation of the enzyme comprises adding a secondary binding material that specifically binds to the target analyte and adding an enzyme for binding to the secondary binding material.

7. The method of claim 6, wherein the secondary binding material is a compound, oligonucleotide, oligosaccharide, protein, antibody, peptide or aptamer that specifically binds to the target analyte.

8. The method of claim 1, wherein the enzyme is selected from the group consisting of alkaline phosphatase (ALP), β-galactosidase, peroxidase, luciferase, cytochrome P450, and combinations thereof.

9. The method of claim 1, wherein the substrate is selected from the group consisting of bromochloroindole phosphate (BCIP) / nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, p-nitrophenyl phosphate (PNPP), enhanced chemifluorescence (ECF), 4-chloronaphthol, 3,3'-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), 3,3',5,5'-tetramethylbenzidine (TMB), 4-chloronaphthol, 3,3'-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), 6-chloro-3-indolyl-β-D-galactopyranoside (Red-gal), and combinations thereof.

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