Colorimetric biosensor, method for producing the same, and antibiotic susceptibility testing method using the same

Abstract

The present invention relates to a colorimetric biosensor, a method for producing the same, and a method for testing antibiotic susceptibility using the same. More specifically, the present invention can produce a colorimetric biosensor including a porous hydrogel structure including polydiacetylene and a hydrogel polymer (alginate, PEG-DA, etc.); and a microbial nutrient source, which can be used to produce a colorimetric biosensor for detecting microorganisms that can perform real-time measurement and exhibit excellent sensitivity, or a method for testing antibiotic susceptibility of microorganisms.

Description

[Technical Field]

[0001] The present invention relates to a colorimetric biosensor, a method for producing the same, and a method for testing antibiotic susceptibility using the same. [Background technology]

[0002] A colorimetric biosensor is a material or device that utilizes the colorimetric or fluorescent properties of nanomaterials to detect signals at the cellular or in vivo level, thereby providing easier and more useful information in data analysis.

[0003] Polydiacetylene (PDA) is an advantageous sensor material for such biosensors because it can be easily synthesized in aqueous solution, and because most biologically important target substances, such as DNA, proteins, carbohydrates, and ions, are hydrophilic. Furthermore, polydiacetylene is known to undergo a color transition to blue upon UV stimulation and to red upon external physical, chemical, or biological stimulation, without the need for additional catalysts or initiators.

[0004] Meanwhile, antibiotic susceptibility testing, a test used to select antibiotics that can inhibit the growth of microorganisms, is a direct and important test that allows for the selection of antibiotics to be used against microorganisms. Furthermore, when prescribing appropriate antibiotics for patients, it allows for customized prescriptions, taking into account prescribing method and frequency, cost, and side effects. Using susceptibility results can reduce the increased treatment costs and disappointment of caregivers that can occur when prescribing antibiotics empirically, and it also provides an opportunity to reduce bacterial resistance and complications, as well as shorten patient recovery times.

[0005] The most common antibiotic susceptibility tests are the disk diffusion technique and the broth dilution technique, but these methods require the bacterial culture to be cultured for several days, followed by the turbidity measurement, which is time-consuming and labor-intensive.

[0006] Therefore, there is a need to develop a method that allows real-time measurement, can reduce time and labor, and overcomes the problems of conventional antibiotic susceptibility testing methods.

[0007] Therefore, the inventors of the present invention prepared a colorimetric biosensor containing a porous hydrogel structure including polydiacetylene and a hydrogel polymer (alginate, PEG-DA, etc.) and a nutrient source for microorganisms, and realized that this can be used as a colorimetric biosensor for detecting microorganisms that can perform real-time measurements and exhibit excellent sensitivity, or as a method for testing the antibiotic susceptibility of microorganisms, thereby completing the present invention. Summary of the Invention The problem the task is trying to solve

[0008] The present invention was discovered in consideration of the above-mentioned problems, and the object of the present invention is to prepare a porous hydrogel structure comprising polydiacetylene and a hydrogel polymer (alginate, PEG-DA, etc.); and a colorimetric biosensor comprising a microbial nutrient source, and to apply the same to a method for testing the antibiotic susceptibility of microorganisms, which is capable of real-time measurement and exhibits excellent sensitivity.

[0009] The problems to be solved by the present invention are not limited to the problem(s) described above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0010] To achieve the above-mentioned object, the present invention provides a colorimetric biosensor comprising a porous hydrogel structure made of a hydrogel polymer and a polydiacetylene; and a microbial nutrient source.

[0011] The microbial nutrient source can be encapsulated within the porous hydrogel structure.

[0012] The microbial nutrient source may be formed in the shape of a shell surrounding the surface of the porous hydrogel structure.

[0013] The hydrogel polymer may be one or more selected from the group consisting of alginate, agarose, chitosan, poly(ethylene glycol) diacrylate (PEG-DA), hydroxyethylene methacrylate (HEMA), polyvinyl alcohol (PVA), and polyacrylamide (PAM).

[0014] The microbial nutrient source may be one or more selected from the group consisting of Luria-Berani (LB) broth or LB agar medium, Tryptic Soy Broth (TSB) medium, Lactobacilli MRS medium, R2A medium (MB-R2230), Mueller-Hinton (MH) medium, and a medium containing casein hydrolysate (casamino acid).

[0015] The colorimetric biosensor can detect microorganisms by a change in color.

[0016] The microorganism may be one or more selected from the group consisting of isolated cells of bacteria, fungi, algae, protozoans, and metazoans.

[0017] The present invention also provides a method for testing antibiotic susceptibility, comprising culturing a microorganism in the presence of a colorimetric biosensor according to the present invention and an antibiotic.

[0018] If there is no colorimetric change in the colorimetric biosensor, it can be determined that the microorganism is susceptible to the antibiotic, and if there is a colorimetric change in the colorimetric biosensor, it can be determined that the microorganism is resistant to the antibiotic.

[0019] The present invention also provides an antibiotic susceptibility testing kit comprising the colorimetric biosensor of the present invention and an antibiotic.

[0020] The present invention also provides a method for producing a colorimetric biosensor, comprising the steps of: (a) obtaining a hydrogel precursor solution containing a polydiacetylene solution, a hydrogel polymer, and a microbial nutrient source; (b) adding the hydrogel precursor solution dropwise to a calcium chloride solution to obtain a hydrogel structure; and (c) irradiating the hydrogel structure with ultraviolet light having a wavelength of 200 to 300 nm.

[0021] The concentration of the hydrogel polymer in the hydrogel precursor solution may be 1.0 to 15.0% (w / v).

[0022] The concentration of the polydiacetylene solution may be 0.5 to 5 mM.

[0023] The concentration of the calcium chloride solution may be 0.5 to 20.0% (w / v).

[0024] The colorimetric biosensor includes a porous hydrogel structure formed from the hydrogel polymer and polydiacetylene, and a microbial nutrient source, and the microbial nutrient source may be encapsulated within the porous hydrogel structure, or may be formed in the form of a shell surrounding the surface of the porous hydrogel structure. [Effects of the Invention]

[0025] According to the present invention, a porous hydrogel structure comprising a polydiacetylene and a hydrogel polymer; and a colorimetric biosensor comprising a microbial nutrient source can be prepared, and the biosensor can be used to apply to a method for testing the antibiotic susceptibility of microorganisms, which is capable of real-time measurement and exhibits excellent sensitivity.

[0026] The effects of the present invention are not limited to the effects described above, but should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description of the present invention or the claims. [Brief explanation of the drawings]

[0027] [Figure 1] 1 is an image showing a process of detecting microorganisms using a colorimetric biosensor according to an embodiment of the present invention [colorimetric biosensor: before incubation with bacteria, blue → after incubation with bacteria, red]. [Figure 2] 1 shows images illustrating the process of preparing colorimetric biosensors in (a) layered form and (b) encapsulated form according to one embodiment of the present invention [PDA-Alginate Beads: blue, LB: yellow, PDA-Alginate-LB Beads: blue]. [Figure 3] 1A and 1B are images showing colorimetric biosensors in (a) layered form and (b) encapsulated form, fabricated according to one embodiment of the present invention. [Figure 4] These are images of experimental results showing the difference in sensitivity when LB broth medium is located (a) outside and (b) inside an encapsulated colorimetric biosensor [T1: 0 h, T2: 6 h, T3: 12 h, T4; 18 h and T5; 24 h]. [Figure 5]1 shows images of antibiotic susceptibility experiments using a colorimetric biosensor in encapsulated form. DETAILED DESCRIPTION OF THE INVENTION

[0028] Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. The embodiments of the present invention can be modified in various forms, and the scope of the present invention should not be construed as being limited to the following embodiments. These embodiments are provided to more completely explain the present invention to those skilled in the art. Therefore, the shapes of elements in the drawings are exaggerated to emphasize a clearer description.

[0029] Conventionally, the most commonly used antibiotic susceptibility tests were the disk diffusion technique and the broth dilution technique. However, these methods required the bacteria to be cultured for several days, followed by the identification of the bacteria and the measurement of turbidity, which had the drawback of being time-consuming and labor-intensive.

[0030] Therefore, there is a need to develop a method that allows real-time measurement, reduces time and labor, and overcomes the problems of conventional antibiotic susceptibility testing methods.

[0031] In order to solve the above-mentioned problems, the present invention aims to prepare a porous hydrogel structure comprising a polydiacetylene and a hydrogel polymer; and a colorimetric biosensor comprising a microbial nutrient source, and to apply the same to a method for testing the antibiotic susceptibility of microorganisms, which is capable of real-time measurement and exhibits excellent sensitivity.

[0032] Colorimetric Biosensors The present invention provides a colorimetric biosensor comprising a porous hydrogel structure made of a hydrogel polymer and polydiacetylene (PDA); and a microbial nutrient source.

[0033] According to one embodiment of the present invention, the colorimetric biosensor can detect microorganisms through a color change. Specifically, when the colorimetric biosensor is co-cultured with microorganisms, the microbial nutrients contained within the sensor diffuse to the outside, and the microorganisms utilize these nutrients as a growth source, resulting in proliferation. Biomolecules, which are metabolic products produced by the proliferation of the microorganisms, penetrate into the hydrogel through the pores of the porous hydrogel structure and stimulate the PDA, thereby inducing a color change in the colorimetric biosensor. Referring to FIG. 1, it can be seen that the color of the colorimetric biosensor is blue before incubation with bacteria, but changes to red after the microbial incubation progresses due to the color change mechanism described above.

[0034] More specifically, microorganisms, particularly bacteria, have the ability to recognize growth sources in their surrounding environment and grow. That is, bacteria can gather and grow in the direction of a growth source. Utilizing this characteristic, the present invention incorporates a nutrient source that can act as a growth source and PDA into the hydrogel so that bacteria can grow in close proximity to the PDA sensor material.

[0035] In addition, PDA has the characteristic that various stimuli (temperature, pH, electrical / physical stimuli, etc.) on its surface cause internal structural changes, which result in a color change from blue to red. Therefore, it is possible that some of the metabolic products produced by bacteria interact with the PDA surface and are stimulated to cause a color change.

[0036] This color change can be visually confirmed and can also be analyzed numerically using an image processing device, making it possible to determine the presence and growth rate of microorganisms.

[0037] More specifically, the colorimetric biosensor can measure the presence and growth rate of microorganisms through a color transition from blue to red. The colorimetric biosensor can exhibit a blue color and, as described above, can exhibit a color transition to red due to biomolecules produced by the microorganisms during the culture process with the microorganisms.

[0038] According to one embodiment of the present invention, the hydrogel polymer may be at least one selected from the group consisting of, but not limited to, alginate, agarose, chitosan, poly(ethylene glycol) diacrylate (PEG-DA), hydroxyethylene methacrylate (HEMA), polyvinyl alcohol (PVA), and polyacrylamide (PAM). Specifically, the hydrogel polymer is alginate, which can exhibit superior color change sensitivity.

[0039] According to one embodiment of the present invention, the porous hydrogel structure may be, but is not limited to, a spherical shape, specifically, a bead shape.

[0040] According to one embodiment of the present invention, the microbial nutrient source may be formed in a shell shape surrounding the surface of the porous hydrogel structure (see FIG. 3a), which allows the microorganisms to more easily access the microbial nutrient source, thereby improving sensitivity to microorganisms.

[0041] According to one embodiment of the present invention, the microbial nutrient source may be encapsulated within the porous hydrogel structure (see FIG. 3b), which surprisingly demonstrated improved sensitivity to microorganisms compared to when the microbial nutrient source is encapsulated on the surface of the structure.

[0042] The microbial nutrient source may be one or more selected from the group consisting of Luria-Berani broth (LB) or LB agar medium, tryptic soy broth (TSB) medium, Lactobacilli MRS medium, R2A medium (MB-R2230), Mueller-Hinton (MH) medium, and a medium containing casein hydrolysate (casamino acid). Specifically, the microbial nutrient source may be LB medium. More specifically, the microbial nutrient source may be, but is not limited to, an LB broth medium composed of tryptone, yeast extract, and NaCl, or an LB agar medium containing a mixture of tryptone, yeast extract, and NaCl with agar. The microbial nutrient source is not limited to the above-mentioned types, and any microbial nutrient source may be used as long as it can be used as a growth source for the target microorganisms to be detected.

[0043] The microorganism may be one or more species selected from the group consisting of isolated cells of bacteria, fungi, algae, protozoans, and metazoans, but is not limited thereto. Specifically, the microorganism may be a bacterium. More specifically, the microorganism may be a bacterium of the genus Escherichia or Pseudomonas, which are typical Gram-negative bacteria, or a bacterium of the genus Staphylococcus or Enterococcus, which are typical Gram-positive bacteria.

[0044] According to one embodiment of the present invention, the culture of the present invention can be performed in sterilized water. Since the medium components required for culturing microorganisms are contained in the nutrient source of the present invention and are present inside or outside the porous hydrogel structure, a separate medium for culturing microorganisms is not required, and the culture can be performed in sterilized water, but is not limited thereto.

[0045] The culture is carried out to obtain the proliferation of the microorganism to be detected. The techniques and conditions for culturing microorganisms are well known to those skilled in the art, who are particularly familiar with the nutrient medium, optimal growth temperature (e.g., 37°C for many mammalian pathogenic bacteria), and necessary conditions for the growth of the microorganism according to the present invention for each microorganism. The culture time will vary for each microorganism depending on the growth rate and generation time, i.e., the time required for a microorganism to divide into two progeny microorganisms. Generally, the culture time is less than 72, 48, or 24 hours. Furthermore, the culture will be discontinued as soon as the desired information, i.e., detection, quantification, or antibiotic susceptibility, is obtained.

[0046] Applications of colorimetric biosensors The present invention provides a method for antibiotic susceptibility testing comprising culturing a microorganism in the presence of a colorimetric biosensor according to the present invention and an antibiotic.

[0047] The antibiotic susceptibility, also called antibiotic sensitivity, refers to the effect of a specific antibiotic on the growth of the microorganism (e.g., bacterial strain), and according to conventional antibiotic susceptibility testing methods, a microorganism is considered to be susceptible if it cannot grow around the area where the antibiotic is applied. The results of antibiotic susceptibility testing are, for example, classified as susceptible, intermediate susceptible (intermediate resistant), and resistant (resistant). Antibiotics that are susceptible must be used to treat infections caused by microorganisms, and infections caused by strains that are susceptible can be treated with the recommended dose of antibacterial agent for that species and the site of infection. Intermediate susceptibility means that the minimum inhibitory concentration of the antibacterial agent for the test strain is similar to the blood or tissue concentration, and therefore the therapeutic effect is lower than that for susceptible strains. This means that treatment is effective when the infection occurs in a site where the antibacterial agent is concentrated, such as urine, or when the maximum amount of the drug that can be administered is administered. Resistance means that the agent cannot be treated with the blood concentration when administered at the normal dose.

[0048] According to one embodiment of the present invention, if there is no colorimetric change in the colorimetric biosensor, it can be determined that the microorganism is susceptible to the antibiotic, and if there is a colorimetric change in the colorimetric biosensor, it can be determined that the microorganism is resistant to the antibiotic.

[0049] Specifically, as described above, when the colorimetric biosensor is cultured with microorganisms, the microbial nutrients contained within the sensor migrate to the outside, and the microorganisms utilize these nutrients as a growth source, resulting in proliferation. Biomolecules, which are metabolic products produced by the proliferation of such microorganisms, penetrate into the hydrogel through the pores of the porous hydrogel structure, stimulating the PDA and inducing a color change in the colorimetric biosensor.

[0050] In this case, if an antibiotic is present around the biosensor, the growth of microorganisms that are sensitive to the antibiotic will cease, and they will no longer produce metabolic biomolecules, making it impossible to observe the colorimetric change in the colorimetric biosensor.On the other hand, the growth of microorganisms that are resistant to the antibiotic will continue, and they will produce metabolic biomolecules, making it possible to observe the colorimetric change in the colorimetric biosensor.

[0051] As described above, the colorimetric biosensor according to the present invention can provide a method for testing antibiotic susceptibility of microorganisms, which is capable of real-time measurement and exhibits excellent sensitivity.

[0052] According to one embodiment of the present invention, the antibiotic may be one or more selected from the group consisting of penicillin antibiotics, glycopeptide antibiotics, quinolone antibiotics, cephalosporin antibiotics, tetracycline antibiotics, sulfonamide antibiotics, polyether antibiotics, and peptide antibiotics, but is not limited thereto. Specifically, the antibiotic may be a penicillin antibiotic or a glycopeptide antibiotic.

[0053] The present invention also provides an antibiotic susceptibility testing kit comprising the colorimetric biosensor of the present invention and an antibiotic. Specifically, the colorimetric biosensor can be provided as a conventional kit for determining the susceptibility or resistance of a target substance (e.g., a microorganism) to a specific antibiotic.

[0054] As a specific example, the antibiotic susceptibility testing kit may comprise a well plate containing the colorimetric biosensor or the antibiotic, or a well plate containing both the colorimetric biosensor and the antibiotic. In this case, the antibiotic may be contained in different concentrations in each well of the kit. For example, media containing different concentrations of the antibiotic to be tested, such as 0.25, 0.5, 1, 2, 4, 8, 16, or 32 μg / mL, may be placed in each well of the kit. The amount of media varies depending on the volume of each well of the multi-well plate, but typically, an average of 200 μL may be placed in each well.

[0055] Each well of the kit may further contain sterile water. As described above, the medium components necessary for culturing microorganisms are contained in the nutrient source according to the present invention and are present inside or outside the porous hydrogel structure, so that a separate medium for culturing microorganisms is not required and the culture can be performed using sterile water, but the culture method is not limited thereto.

[0056] Method for preparing a colorimetric biosensor The present invention provides a method for preparing a colorimetric biosensor, comprising the steps of: (a) obtaining a hydrogel precursor solution containing a polydiacetylene (PDA) solution, a hydrogel polymer, and a microbial nutrient source; (b) adding the hydrogel precursor solution dropwise to a calcium chloride solution to obtain a hydrogel structure; and (c) irradiating the hydrogel structure with ultraviolet light having a wavelength of 200 to 300 nm.

[0057] According to one embodiment of the present invention, step (a) is a step of preparing a hydrogel precursor solution for forming a hydrogel structure, which can be performed by mixing a PDA solution, a hydrogel polymer, and a microbial nutrient source.

[0058] The concentration of the hydrogel polymer in the hydrogel precursor solution may be 1.0-15.0% (w / v), 5.0-13.0% (w / v), or 8.0-10.0% (w / v). When the concentration of the hydrogel polymer is 1.0% (w / v) or higher, excellent gel formation is achieved. When the concentration is 15.0% (w / v) or lower, excellent shape retention of the spherical porous hydrogel structure and easy handling due to appropriate viscosity are achieved. Surprisingly, the critical effect can be maintained even when the mixing ratio of the PDA solution and the microbial nutrient source is varied, as long as the hydrogel polymer concentration range is maintained.

[0059] The concentration of the PDA solution may be 0.5 to 5 mM, 0.5 to 4 mM, or 1.0 to 3 mM. The concentration of the PDA solution can affect the process of obtaining the hydrogel structure in step (b), and within the above concentration range of the PDA solution, the formation of the hydrogel structure is facilitated.

[0060] The concentration of the calcium chloride solution may be 0.5 to 20.0% (w / v), 0.5 to 10.0% (w / v), 0.5 to 5.0% (w / v), or 0.5 to 1.5% (w / v), which has the effect of further improving the sensitivity of the resulting colorimetric biosensor.

[0061] According to one embodiment of the present invention, step (b) is a step of obtaining a gelled hydrogel structure by reacting the hydrogel precursor solution with the calcium chloride, and a process of stirring the calcium chloride solution using a magnetic bar may be performed simultaneously to prevent the dropped droplets from forming clumps.

[0062] According to an embodiment of the present invention, after step (b), the gelled hydrogel structure may be further washed with distilled water.

[0063] According to one embodiment of the present invention, step (c) may be a step of obtaining a blue-colored colorimetric biosensor by irradiating the hydrogel structure with ultraviolet light having a wavelength of 200-300 nm, 230-280 nm, 240-270 nm, or 250-260 nm. When irradiated with ultraviolet light in these wavelength ranges, a color transition to blue can be easily achieved.

[0064] The colorimetric biosensor includes a porous hydrogel structure made of the hydrogel polymer and polydiacetylene, and the microbial nutrient source. The microbial nutrient source may be encapsulated inside the porous hydrogel structure, or may be formed in the shape of a shell surrounding the surface of the porous hydrogel structure, but the shape is not limited thereto.

[0065] The matters mentioned in relation to the colorimetric biosensor of the present invention, its uses and its manufacturing method are equally applicable unless they contradict each other.

[0066] The above description has been given by way of an embodiment to illustrate the technical concept of the present invention, and various modifications and variations may be made by those skilled in the art without departing from the essential characteristics of the present invention. Therefore, the embodiments described herein are for illustrative purposes only and do not limit the technical concept of the present invention. The scope of the present invention should be interpreted by the scope of the claims, and all technical concepts within the scope equivalent thereto should be interpreted as being within the scope of the present invention. The present invention will be described in more detail below with reference to examples.

[0067] <Example> Example 1. Preparation of an encapsulated colorimetric biosensor A hydrogel precursor solution was prepared by mixing 3 mM PDA (polydiacetylene) solution, alginate (9% (w / v) of the hydrogel precursor solution), and LB broth. The hydrogel precursor solution was dripped into a beaker filled with 1% (w / v) calcium chloride (CaCl) solution through a syringe needle. The calcium chloride solution was continuously stirred with a magnetic bar to prevent the droplets from clumping, allowing the hydrogel precursor solution to react with the calcium chloride and form a gel. After gelation was complete, the hydrogel construct was removed from the calcium chloride solution, washed with distilled water, and then irradiated with 254 nm UV light. The PDA inside the hydrogel construct turned blue, forming an encapsulated colorimetric biosensor (see Figures 2b and 3b).

[0068] Example 2. Preparation of a layered colorimetric biosensor A hydrogel precursor solution was prepared by mixing 3 mM PDA (polydiacetylene) solution and alginate (9% (w / v) of the hydrogel precursor solution). The hydrogel precursor solution was then added dropwise through a syringe needle to a beaker filled with 1% (w / v) calcium chloride (CaCl2) solution. The calcium chloride solution was continuously stirred with a magnetic bar to prevent the droplets from clumping, causing the hydrogel precursor solution to react with the calcium chloride and gelate. The hydrogel constructs that had completed gelation were removed from the calcium chloride solution and washed with distilled water. They were then irradiated with UV light at a wavelength of 254 nm, which transformed the color of the PDA inside the hydrogel constructs into blue. The hydrogel constructs that had completed gelation were then pipetted into hydrophobic liquid LB agar medium, covering them with a thin film. The coated LB agar medium solidified rapidly at low temperatures, ultimately producing a colorimetric biosensor with a layered bacterial growth source (see Figures 2a and 3a).

[0069] Experimental example 1. Sensitivity comparison experiment When LB broth medium was placed on the outside and inside of an encapsulated colorimetric biosensor (a) and (b), a comparison experiment of the sensor sensitivity was carried out and is shown in FIG.

[0070] Specifically, the encapsulated colorimetric biosensors prepared in Example 1 were prepared with or without LB broth medium. The wells containing the biosensors without LB medium were filled with 10 CFU / mL E. coli and LB medium (Figure 4a). The wells containing the biosensors with LB medium were filled with 10 CFU / mL E. coli alone (Figure 4b). The LB broth medium was prepared at the same concentration to ensure consistent experimental conditions. The biosensors were incubated at 37°C to induce E. coli growth and the associated color change. Photographs of each well plate were taken every 0, 6, 12, 18, and 24 hours, and the color changes were compared.

[0071] Figure 4 shows that the color change of PDA occurs more quickly when LB broth is present inside the biosensor (Figure 4b) than when it is not (Figure 4a). Specifically, when LB broth is present inside the colorimetric biosensor, a color change (blue to red) occurs after 6 hours (T2). When LB broth is present outside the colorimetric biosensor, a color change (blue to red) occurs after 18 hours (T4). Therefore, we can conclude that E. coli recognizes the LB broth inside the biosensor, grows on the biosensor surface, and the biomolecules released as a result move more easily into the biosensor, resulting in a more rapid PDA color change.

[0072] Experimental Example 2: Antibiotic susceptibility test The encapsulated colorimetric biosensor prepared in Example 1 was subjected to an antibiotic susceptibility test, and the results are shown in FIG.

[0073] Specifically, encapsulated colorimetric biosensors were prepared according to Example 1 and placed in wells containing 2 and 4 μg / mL of ampicillin antibiotics (FIGS. 5B and 5C) and wells containing 0.25, 2, and 4 μg / mL of vancomycin antibiotics (FIGS. 5D, 5E, and 5F). Additionally, for antibiotic susceptibility testing, 10 CFU / mL of Methicillin-Resistant Staphylococcus Aureus (MRSA) bacteria, which are resistant to ampicillin but sensitive to vancomycin, were placed in each well. For comparative experiments, wells containing only the colorimetric biosensor and bacteria (FIG. 5A) and only the colorimetric biosensor (FIG. 5G) were also prepared. To induce the growth of MRSA and the accompanying color change of the biosensor, the wells were incubated at 37°C, and photographs of each well plate were taken every 0, 5, 6, 7, 9, 12, 13, and 15 hours, and the color changes were compared.

[0074] Referring to Figure 5, when the colorimetric biosensor was incubated with MRSA alone, a color change (blue to red) occurred after 7 hours (t = 7) (Figure 5A). In contrast, in the absence of both antibiotics and bacteria, no color change was observed even after 15 hours (t = 15) (Figure 5G). When 2 μg / mL ampicillin was added to the well containing the MRSA and colorimetric biosensor, a color change (blue to red) occurred after 9 hours (t = 9) (Figure 5B). When 4 μg / mL ampicillin was added, a color change (blue to red) occurred after 13 hours (t = 13) (Figure 5C). It can be concluded that MRSA, due to its resistance to ampicillin, caused a color change in the colorimetric biosensor when co-cultured. Next, when MRSA was co-cultured with 0.25 μg / mL vancomycin in a well containing a colorimetric biosensor, a color change (blue to red) occurred after 9 hours (t = 9) (Figure 5D). However, when 2 or 4 μg / mL vancomycin was co-cultured, no color change was observed (Figure 5E, Figure 5F). In the case of MRSA, when co-cultured with vancomycin, no color change occurred at 2 μg / mL, indicating that the MRSA strain is susceptible to vancomycin at a concentration of 2 μg / mL.

[0075] Therefore, it was confirmed that the colorimetric biosensor according to the present invention can be applied to a colorimetric biosensor for detecting microorganisms or a method for testing the antibiotic susceptibility of microorganisms, which is capable of real-time measurement and exhibits excellent sensitivity.

[0076] The above detailed description exemplifies the present invention. Furthermore, the above description illustrates and describes preferred embodiments of the present invention, and the present invention can be used in various other combinations, modifications, and environments. That is, changes and modifications are possible within the scope of the inventive concept disclosed herein, the scope of equivalents to the disclosed disclosure, and / or the scope of skill or knowledge in the art. The disclosed examples illustrate the best modes for embodying the technical ideas of the present invention, and various modifications are possible as required for specific fields of application and uses of the present invention. Therefore, the above detailed description of the invention is not intended to limit the present invention to the disclosed embodiments. Furthermore, the appended claims should be construed to include other embodiments.

Claims

1. A porous hydrogel structure comprising a hydrogel polymer and a polydiacetylene; and A colorimetric biosensor comprising a microbial nutrient source, the microbial nutrient source comprising one or more selected from the group consisting of Luria-Berani (LB) broth, LB agar medium, tryptic soy broth (TSB), Lactobacilli MRS medium, R2A medium (MB-R2230), Mueller-Hinton (MH) medium, and a casein hydrolysate (casamino acid)-containing medium.

2. The colorimetric biosensor of claim 1 , wherein the microbial nutrient source is encapsulated within the porous hydrogel structure.

3. The colorimetric biosensor according to claim 1 , wherein the microbial nutrient source is formed in the shape of a shell surrounding the surface of the porous hydrogel structure.

4. 2. The colorimetric biosensor of claim 1, wherein the hydrogel polymer comprises one or more selected from the group consisting of alginate, agarose, chitosan, poly(ethylene glycol) diacrylate (PEG-DA), hydroxyethylene methacrylate (HEMA), polyvinyl alcohol (PVA), and polyacrylamide (PAM).

5. The colorimetric biosensor according to claim 1 , wherein the colorimetric biosensor detects microorganisms through a color change.

6. 2. The colorimetric biosensor of claim 1, wherein the microorganism comprises one or more selected from the group consisting of isolated cells of bacteria, fungi, algae, protozoans, and metazoans.

7. A method for testing antibiotic susceptibility, comprising culturing a microorganism in the presence of the colorimetric biosensor according to any one of claims 1 to 6 and an antibiotic.

8. determining that the microorganism is susceptible to the antibiotic when there is no colorimetric change in the colorimetric biosensor; 8. The antibiotic susceptibility testing method according to claim 7, wherein the microorganism is determined to be resistant to the antibiotic when a colorimetric change is detected in the colorimetric biosensor.

9. A kit for testing antibiotic susceptibility, comprising the colorimetric biosensor according to any one of claims 1 to 6 and an antibiotic.

10. (a) obtaining a hydrogel precursor solution comprising a polydiacetylene solution, a hydrogel polymer, and a microbial nutrient source; (b) adding the hydrogel precursor solution dropwise to a calcium chloride solution to obtain a hydrogel structure; and (c) irradiating the hydrogel structure with ultraviolet light having a wavelength of 200 to 300 nm; The method for producing a colorimetric biosensor, wherein the microbial nutrient source comprises one or more selected from the group consisting of Luria-Berani (LB) broth, LB agar medium, tryptic soy broth (TSB), Lactobacilli MRS medium, R2A medium (MB-R2230), Mueller-Hinton (MH) medium, and a casein hydrolysate (casamino acid)-containing medium.

11. 11. The method for producing a colorimetric biosensor according to claim 10, wherein the concentration of the hydrogel polymer in the hydrogel precursor solution is 1.0 to 15.0% (w / v).

12. 11. The method for producing a colorimetric biosensor according to claim 10, wherein the concentration of the polydiacetylene solution is 0.5 to 5 mM.

13. 11. The method for producing a colorimetric biosensor according to claim 10, wherein the concentration of the calcium chloride solution is 0.5 to 20.0% (w / v).

14. The colorimetric biosensor includes a porous hydrogel structure composed of a hydrogel polymer and polydiacetylene; and the microbial nutrient source. The method for producing a colorimetric biosensor described in claim 10, wherein the microbial nutrient source is encapsulated inside the porous hydrogel structure or formed in the form of a shell surrounding the surface of the porous hydrogel structure.

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