Biosensor using PET substrate, its manufacturing method and method for testing antibiotic susceptibility of microorganisms using the same

Abstract

The present invention relates to a biosensor for testing the antibiotic susceptibility of microorganisms, the biosensor comprising a sensor part including a substrate comprising polyethylene terephthalate and an electrode layer including interdigitated first and second electrodes formed on one or both sides of the substrate, a method for producing the biosensor, and a method for testing the antibiotic susceptibility of microorganisms using the biosensor.

Description

[Technical Field]

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

[0002] Among the various diseases that occur in the human body, there are those caused by microbial infections, including urinary tract infections and sepsis. To treat these microbial infections, antibiotics are needed to inhibit the growth of microorganisms or kill them. However, up until recently, over 100 types of antibiotics have been developed, and their effectiveness varies depending on the type of microorganism and whether the microorganism is resistant to antibiotics. Therefore, for patients with infectious diseases, it is necessary to select the antibiotic that is most effective against the microorganism in question. The diagnostic test for this purpose is called antibiotic susceptibility testing.

[0003] Currently, the most commonly used antibiotic susceptibility testing method is the broth dilution method, which involves culturing bacteria in a culture medium treated with various antibiotics for at least one day, identifying the bacteria, and measuring the turbidity of the culture medium to determine antibiotic resistance and susceptibility. However, this process requires a lot of time and labor. Therefore, there is a need to develop a method that allows for real-time testing, reduces the time and labor required, and overcomes the problems of conventional antibiotic susceptibility testing methods. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Korean Patent Registration No. 10-1776698 (2017.09.04.) Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides a biosensor for testing antibiotic susceptibility of microorganisms, comprising a substrate comprising polyethylene terephthalate and an electrode layer formed on one or both sides of the substrate and including interdigitated first and second electrodes.

[0006] However, the technical problems that the present invention aims to solve are not limited to the problems mentioned 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]

[0007] The present invention provides a biosensor for testing antibiotic susceptibility of microorganisms, comprising a substrate comprising polyethylene terephthalate and an electrode layer formed on one or both sides of the substrate and including interdigitated first and second electrodes.

[0008] The electrodes may be made of silver or gold.

[0009] The electrodes may be formed through screen printing, PCB or photolithography methods.

[0010] The biosensor may be used to test the antibiotic susceptibility of microorganisms by measuring in real time the change in electrical properties that occurs as the microorganisms grow due to antibiotic treatment.

[0011] In one embodiment of the present invention, there is provided a method for manufacturing the biosensor, which includes the steps of preparing a substrate comprising polyethylene terephthalate and forming an electrode layer comprising interdigitated first and second electrodes on the substrate.

[0012] In another embodiment of the present invention, there is provided a method for testing antibiotic susceptibility of a microorganism, comprising the steps of treating a sample containing a microorganism with the biosensor, treating the biosensor with an antibiotic, and measuring a change in electrical properties after the antibiotic treatment. [Effects of the Invention]

[0013] The biosensor according to the present invention comprises a substrate made of polyethylene terephthalate and an electrode layer formed on one or both sides of the substrate, the electrode layer including interdigitated first and second electrodes. Compared to substrates made of other materials, the biosensor has the advantages of shorter detection time for changes in microbial growth and superior discrimination. Furthermore, the biosensor not only enables rapid antibiotic susceptibility testing of microorganisms, but also allows for rapid identification of the minimum antibiotic concentration required to limit resistance.

[0014] Therefore, the use of the biosensor according to the present invention can dramatically reduce the time required for antibiotic susceptibility testing of microorganisms to within 4 hours due to its significantly superior sensitivity, and can be very effectively applied to treating microbial infections that require rapid diagnosis and treatment. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram illustrating a method for testing antibiotic susceptibility of microorganisms using a PET-based biosensor according to an embodiment of the present invention. [Figure 2a] 2a and 2b are schematic diagrams illustrating a biosensor using a PET substrate and an Ag electrode according to an embodiment of the present invention. [Figure 2b] 2a and 2b are schematic diagrams illustrating a biosensor using a PET substrate and an Ag electrode according to an embodiment of the present invention. [Figure 3a] 3a and 3b are schematic diagrams illustrating a biosensor using a PET substrate and an Au electrode according to an embodiment of the present invention. [Figure 3b]3a and 3b are schematic diagrams illustrating a biosensor using a PET substrate and an Au electrode according to an embodiment of the present invention. [Figure 4a] FIG. 4a is a graph showing the capacitance change of a microorganism measured using a biosensor with a PET substrate and an Ag electrode according to an embodiment of the present invention, and FIG. 4b is a graph showing the results of an antibiotic susceptibility test of a microorganism using a biosensor with a PET substrate and an Ag electrode according to an embodiment of the present invention. [Figure 4b] FIG. 4a is a graph showing the capacitance change of a microorganism measured using a biosensor with a PET substrate and an Ag electrode according to an embodiment of the present invention, and FIG. 4b is a graph showing the results of an antibiotic susceptibility test of a microorganism using a biosensor with a PET substrate and an Ag electrode according to an embodiment of the present invention. [Figure 5a] 5a and 5b are graphs showing the capacitance change of microorganisms measured using a biosensor with a PET substrate and Au electrodes according to one embodiment of the present invention, and FIG. 5c is a graph showing the results of an antibiotic susceptibility test of microorganisms using a biosensor with a PET substrate and Au electrodes according to one embodiment of the present invention. [Figure 5b] 5a and 5b are graphs showing the capacitance change of microorganisms measured using a biosensor with a PET substrate and Au electrodes according to one embodiment of the present invention, and FIG. 5c is a graph showing the results of an antibiotic susceptibility test of microorganisms using a biosensor with a PET substrate and Au electrodes according to one embodiment of the present invention. [Figure 5c] 5a and 5b are graphs showing the capacitance change of microorganisms measured using a biosensor with a PET substrate and Au electrodes according to one embodiment of the present invention, and FIG. 5c is a graph showing the results of an antibiotic susceptibility test of microorganisms using a biosensor with a PET substrate and Au electrodes according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present inventors have been researching a biosensor for testing the antibiotic susceptibility of microorganisms by measuring in real time the changes in electrical properties that occur as microorganisms grow due to antibiotic treatment. They have found that polyethylene terephthalate is an optimal substrate material and has significantly improved sensitivity, leading to the completion of the present invention.

[0017] The present invention will be described in detail below.

[0018] However, since the present invention can be modified in various ways and can have various forms, the specific examples and explanations described below are intended to aid in understanding the present invention and are not intended to limit the present invention to the specific disclosed forms. The scope of the present invention should be understood to include all modifications, equivalents, and alternatives that fall within the spirit and technical scope of the present invention.

[0019] As used herein, "antibiotic susceptibility," also known as antibiotic sensitivity, refers to the extent to which a microorganism is affected by a specific antibiotic, such as by inhibiting growth, inhibiting cell division, or killing cells. In conventional disk diffusion antibiotic susceptibility testing, a circular clear zone of a certain diameter is formed around the placement of an antibiotic disk, and a microorganism is considered to be susceptible to an antibiotic when the diameter is above a certain level. Antibiotic susceptibility test results can be classified as susceptible, intermediate resistant, and resistant (resistant), for example. To treat an infection caused by a microorganism, an antibiotic to which the microorganism is susceptible must be used. A microorganism interpreted as susceptible can be treated by prescribing the antibiotic in the recommended dose for the strain and site of infection. A microorganism interpreted as intermediate resistant can be treated by prescribing the antibiotic in the recommended dose for the strain and site of infection. A microorganism interpreted as intermediate resistant can be treated by prescribing the antibiotic in the recommended dose for the strain and site of infection, which may reduce the therapeutic effect, since the minimum inhibitory concentration of the antibiotic for the target microorganism is similar to the maximum concentration of the drug that can be prescribed. On the other hand, in the case of microorganisms that are interpreted as exhibiting the above-mentioned resistance, it is indicated that they cannot be treated with the maximum concentration of the drug that can be prescribed.

[0020] Biosensor for testing antibiotic susceptibility of microorganisms and method for manufacturing the same The present invention provides a biosensor for testing antibiotic susceptibility of microorganisms, comprising a substrate containing (or made of) polyethylene terephthalate, and an electrode layer formed on one or both sides of the substrate and including interdigitated first and second electrodes.

[0021] The present invention also provides a method for manufacturing the biosensor, comprising the steps of: providing a substrate comprising polyethylene terephthalate; and forming an electrode layer comprising interdigitated first and second electrodes on the substrate.

[0022] The biosensor according to the present invention may be used to test the antibiotic susceptibility of microorganisms, specifically, to test the antibiotic susceptibility of microorganisms by measuring in real time the change in electrical properties that occurs as the microorganisms grow due to antibiotic treatment. In this case, the electrical properties may include capacitance, impedance, resistance, reactance, etc. Therefore, when measuring changes in electrical properties, there is an advantage that sensitivity can be increased even with a lower concentration and a smaller amount of sample compared to when measuring changes in optical properties.

[0023] First, the biosensor according to the present invention includes a substrate containing polyethylene terephthalate. Polyethylene terephthalate is a non-conductive polymer, which has the advantages of shorter detection time for changes in microbial growth and superior discrimination power compared to substrates made of other materials such as epoxy, polyimide, and glass. Furthermore, the biosensor not only enables rapid testing of microbial antibiotic susceptibility, but also allows for rapid identification of the minimum antibiotic concentration required to limit resistance.

[0024] Next, the biosensor according to the present invention includes an electrode layer including interdigitated first and second electrodes, which is formed on one or both sides of the substrate. The electrode layer may have a structure in which aligned rods form one pole, and two electrodes (the first and second electrodes) face each other in an interdigitated manner. The two electrodes function in the same way as classical impedance measurement electrodes. The distance between the first and second electrodes may be 0.1 μm to 1000 μm, 1 μm to 500 μm, or 10 μm to 300 μm. By adjusting the distance between the first and second electrodes within this range, precise measurement of even micro-level biomolecules is possible. The height of the first electrode and the second electrode may be in the range of 50 nm to 5,000 nm, and the width of the first electrode and the second electrode may be in the range of 0.1 μm to 1,000 μm, 1 μm to 500 μm, or 10 μm to 300 μm. Specifically, the electrodes may be made of silver or gold.

[0025] The electrodes may be formed by screen printing, PCB, or photolithography. Specifically, if the electrodes are made of silver, they may be formed by screen printing. If the electrodes are made of gold, they may be formed by PCB. The screen printing or PCB method has the advantage of being about 1 / 100 cheaper in manufacturing cost than the photolithography method.

[0026] The biosensor according to the present invention may further include a storage unit. The storage unit may store an electrode layer, an antibiotic, and a microorganism therein. The storage unit may be formed perpendicular to the substrate. Specifically, when the biosensor is used to measure the antibiotic susceptibility of a microorganism, the microorganism or the antibiotic may be treated inside the storage unit. The storage unit may have an open top.

[0027] The storage section may be made of one or more materials selected from the group consisting of glass, polypropylene (PP), polyethylene terephthalate (PET), and polycarbonate (PC). A commercially available example is a plastic well. The storage section may have a volume of 10 μl to 1 ml. The microorganisms may be treated together with a culture medium, and in this sense, the storage section may contain the culture medium for the microorganisms.

[0028] The culture medium, also called a culture medium or a culture solution, refers to a liquid or solid material used for the growth, preservation, etc. of microorganisms, and its specific composition may vary depending on the type of microorganism. A liquid form is preferred, and it may be blood or contain blood.

[0029] The microorganism may be a bacterium, and the bacterium is preferably, but not limited to, a gram-positive bacterium, a gram-negative bacterium, or an antibiotic-resistant strain thereof. Specifically, the gram-positive bacterium is Bacillus subtilis. The Gram-negative bacterium may be any one or more selected from the group consisting of Staphylococcus aureus, Enterococcus faecalis, and Staphylococcus epidermidis, and the Gram-negative bacterium may be any one or more selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella typhimurium.

[0030] The antibiotics are not limited to any particular type, and include any antibiotics whose sensitivity can be measured by the biosensor according to the present invention. Specifically, the antibiotics include ampicillin, tetracycline, gentamicin, erythromycin, vancomycin, linezolid, methicillin, oxacillin, cefotaxime, rifampicin, amikacin, kanamycin, Tobramycin, Neomycin, Ertapenem, Doripenem, Imipenem / Cilastatin, Meropenem, Ceftazidime, Cefapime, Ceftaroline, Ceftobiprole, Aztreonam, Piperacillin, Polymyxin B B), colistin, ciprofloxacin, levofloxacin, moxifloxacin, gatifloxacin, tigecycline, combinations thereof and derivatives thereof, and is preferably, but not limited to, ampicillin or tetracycline.

[0031] The biosensor measuring device according to the present invention may further include a wireless transmitter for transmitting the measurement results of the change in electrical properties. Such transmission may be performed by any commonly available method, including wired data transmission using a data cable such as a USB, or wirelessly using a Bluetooth method.

[0032] The biosensor according to the present invention may be connected to a measuring instrument capable of measuring electrical properties to monitor changes in microbial concentration in real time, and in this case, an AC voltage of 1 mV to 1 V having a frequency of 10 Hz to 1 MHz may be supplied to the biosensor.

[0033] Antibiotic susceptibility testing method for microorganisms

[0034] The present invention provides a method for testing antibiotic susceptibility of a microorganism, comprising the steps of treating a sample containing a microorganism with the biosensor, treating the biosensor with an antibiotic, and measuring a change in electrical properties after the antibiotic treatment.

[0035] First, the method for testing antibiotic susceptibility of microorganisms according to the present invention includes treating a sample containing microorganisms with the biosensor.

[0036] The biosensor has been described above, so a duplicated description will be omitted.

[0037] The sample contains microorganisms, which have been described above, and therefore will not be described again. The sample may be in the form of a composition and may be collected from any of liquids, soil, air, food, waste, the intestines of animals and plants, tissues derived from animals and plants, blood, urine, tears, saliva, or sweat, etc., and the animals and plants include the human body. The sample may also be microorganisms extracted from a specimen of human blood and cultured and amplified in a blood culture medium or an isolation culture medium. The sample may also be a sample whose bacterial species cannot be identified using a mass spectrometer or other identification kits.

[0038] Next, the method for testing antibiotic susceptibility of a microorganism according to the present invention includes a step of treating the sample-treated biosensor with an antibiotic.

[0039] The antibiotics have been described above, so a repeated explanation will be omitted.

[0040] Next, the method for testing antibiotic susceptibility of a microorganism according to the present invention includes measuring changes in electrical properties after treatment with the antibiotic.

[0041] Specifically, the antibiotic susceptibility test measures changes in electrical properties before and after antibiotic treatment, and the changes can be used to quickly determine the antibiotic resistance and viability of microorganisms in real time, depending on whether they are resistant to antibiotics. The presence or absence of resistance can be more precisely determined through changes in electrical properties such as capacitance, impedance, resistance, and reactance. For example, the minimum inhibitory concentration (MIC) of a microorganism can also be measured, which can minimize the antibiotic concentration administered for treatment, select the optimal antibiotic depending on whether resistance is present, and prevent the misuse or overuse of antibiotics, thereby reducing side effects.

[0042] As described above, the biosensor according to the present invention is characterized by comprising a substrate comprising polyethylene terephthalate and an electrode layer including interdigitated first and second electrodes formed on one or both sides of the substrate, and has the advantages of shorter detection time for changes in microbial growth and superior discrimination power compared to substrates made of other materials. Furthermore, it not only enables rapid antibiotic susceptibility testing of microorganisms but also allows for rapid identification of the minimum antibiotic concentration required to limit resistance.

[0043] Therefore, the use of the biosensor according to the present invention can dramatically reduce the time required for testing the antibiotic susceptibility of microorganisms to within 4 hours due to its significantly superior sensitivity, and can be very effectively applied to treating microbial infections that require rapid diagnosis and treatment.

[0044] Preferred examples are presented below to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention, and are not intended to limit the scope of the present invention. [Example]

[0045] <Example>

[0046] Preparation Example 1: Experimental Preparation

[0047] The strain used in the experiment was Escherichia coli (ATCC25922), and the standard strain was purchased from ATCC in the United States. To cultivate the strain, Mueller-Hinton media (MH media), a culture medium recommended by the American standard CLSI and the European standard EUCAST, was purchased from DIFCO and used according to the recommended manufacturing method in the product instructions. After culturing in the media for 24 hours, the strain culture was adjusted to a McFarland OD of 0.5 and then diluted 1 / 100 with the culture medium (strain concentration: 5 × 10 5 ~10 6 cfu / mL) were applied to the sensor.

[0048] The antibiotics used in the experiment were ampicillin and tetracycline, purchased from Sigma. Depending on the properties of each antibiotic, they were dissolved in deionized and autoclaved ultrapure water, 100% DMSO, or 100% ethanol (different solvents may be used for different antibiotics. For example, tetracycline may be dissolved in 100% ethanol and then diluted with autoclaved ultrapure water). The resulting solution was adjusted to a concentration of 8 mg / mL, filtered through a 22 μm filter, and then diluted with autoclaved ultrapure water to a concentration of 0.5–8 mg / mL before being applied to the sensor.

[0049] Example 1: Preparation of a biosensor (PET substrate-Ag electrode) for evaluating antibiotic susceptibility of microorganisms To fabricate biosensors for assessing the antibiotic susceptibility of microorganisms, we screen-printed a fine silver ink (E-Base SC-PE09F4) onto a 250 μm thick substrate of non-conductive polymer polyethylene terephthalate (PET) (SKC product, SW94M) to form interdigitated Ag electrodes (thickness: ~150 μm) (Figures 2a and 2b). For the 16-channel and 64-channel interdigitated sensors, the electrodes were ~150 μm high and 100 μm wide, with a 100 μm gap, on a 4.5 mm diameter sensor. A housing made of polypropylene was then attached to the Ag electrodes using waterproof double-sided tape or heat-sealing tape, and the sensors were sterilized using O3 before use. In addition, the Ag electrode and the pad for measuring an electric signal were allowed to be electrically connected through a via hole.

[0050] Comparative Example 1: Preparation of a biosensor (epoxy substrate-Ag electrode) for evaluating the antibiotic susceptibility of microorganisms The sensor was fabricated in the same manner as in Example 1, except that an epoxy substrate was used instead of the PET substrate.

[0051] Example 2: Preparation of a biosensor (PET substrate-Au electrode) for evaluating the antibiotic susceptibility of microorganisms To fabricate biosensors for assessing the antibiotic susceptibility of microorganisms, we fabricated sensors by printing gold electrodes (80.1 μm total thickness, 50 μm Cu, 30 μm Ni, 0.1 μm Au) on both sides of a 250 μm thick non-conductive polymer substrate (polyethylene terephthalate (PET) (SKC product, SW94M)) using a PCB fabrication process involving electroless and electroplating (see Figures 3a and 3b). For the 16-channel and 192-channel interdigitated gold sensors, the electrodes were ~80 μm high and 100 μm wide, with a 100 μm gap, on a 2.5 mm diameter sensor. A housing made of polypropylene was then attached to the gold electrodes using waterproof double-sided tape or heat-sealing tape, and the sensors were sterilized using O3 before use. In addition, the Au electrode and the pad for measuring an electric signal were allowed to be electrically connected through a via hole.

[0052] Comparative Example 2: Preparation of a biosensor (epoxy substrate-Au electrode) for evaluating the antibiotic susceptibility of microorganisms The sensor was fabricated in the same manner as in Example 2, except that an epoxy substrate was used instead of the PET substrate.

[0053] Comparative Example 3: Preparation of a biosensor (polyimide substrate-Au electrode) for evaluating antibiotic susceptibility of microorganisms A sensor was fabricated in the same manner as in Example 2, except that a polyimide substrate was used instead of a PET substrate.

[0054] Comparative Example 4: Preparation of a biosensor (glass substrate-Au electrode) for evaluating antibiotic susceptibility of microorganisms A sensor was fabricated in the same manner as in Example 2, except that a glass substrate was used instead of a PET substrate and a photolithography method was used instead of a PCB method.

[0055] Experimental Example 1: Measuring capacitance changes of microorganisms using a sensor

[0056] The ability to measure the capacitance change of microorganisms was confirmed using the sensors prepared in Examples 1 and 2 and Comparative Examples 1 to 4. Specifically, for each of the sensors prepared in Examples 1 and 2 and Comparative Examples 1 to 3, 10 Escherichia coli (ATCC25922) was placed in a 5 ~10 7 After treatment with 1000 cells / ml, the change in capacitance due to the growth of E. coli (ATCC25922) was measured while culturing it in MH media at 37°C. On the other hand, for the sensor prepared in Example 4, 1 to 10 E. coli (E. coli U556 or E. coli U433) or Staphylococcus aureus (S. aureus T82 or S. aureus P101) was cultured. 5 After treatment with cells / ml, the capacitance change due to the growth of Escherichia coli (E. coli U556 or E. coli U433) or Staphylococcus aureus (S. aureus T82 or S. aureus P101) was measured while culturing at 37°C.

[0057] As shown in Figure 4a, the sensor prepared in Example 1, which uses a PET substrate and Ag electrodes, showed a large change in capacitance within a short period of time, demonstrating its excellent discrimination ability and short detection time for changes in microbial growth. On the other hand, the sensor prepared in Comparative Example 1, which uses an epoxy substrate and Ag electrodes, showed only a slight change in capacitance.

[0058] As shown in Figures 5a and 5b, the sensor prepared in Example 2, which uses a PET substrate and Au electrodes, similarly measured a large change in capacitance within a short period of time, confirming its short detection time for changes in microbial growth and excellent discrimination ability. On the other hand, the sensors prepared in Comparative Examples 2 and 3, which use epoxy or polyimide substrates and Ag electrodes, both showed only a slight change in capacitance. Furthermore, the sensor prepared in Comparative Example 4, which uses a conventional glass substrate and Au electrodes, also showed only a slight change in capacitance.

[0059] Experimental example 2: Antibiotic susceptibility testing of microorganisms using sensors

[0060] The antibiotic susceptibility of microorganisms was measured using the sensors prepared in Examples 1 and 2. Specifically, for each of the sensors prepared in Examples 1 and 2, ampicillin was prepared at a concentration of 0.5 to 8 μg / ml, and tetracycline was prepared at a concentration of 0.5 to 8 μg / ml, and the sensor surface was treated with each of these, and then 10 Escherichia coli (ATCC25922) was immediately introduced. 5 cells / ml and monitored the capacitance change in real time. The antibiotic susceptibility of the microorganisms was measured using the sensor prepared in Example 1. As shown in Figure 4b, the sensor prepared in Example 1, which used a PET substrate-Ag electrode, showed a significant increase in capacitance change after treatment with ampicillin, whereas no change in capacitance was observed after treatment with tetracycline. This confirms that E. coli (ATCC25922) is resistant to ampicillin and sensitive to tetracycline.

[0061] As shown in Figure 5c, the sensor prepared in Example 2, which used a PET substrate-Au electrode, also showed a large increase in capacitance change after treatment with ampicillin, whereas no change in capacitance was observed after treatment with tetracycline. This confirms that E. coli (ATCC25922) is resistant to ampicillin and sensitive to tetracycline.

[0062] That is, when the sensors prepared in Examples 1 and 2 are used to treat ampicillin-resistant microorganisms and then ampicillin, a significant change in capacitance is observed within 1 to 2 hours. Therefore, the sensors prepared in Examples 1 and 2 enable rapid antibiotic susceptibility testing of microorganisms, and in the case of multichannel interdigitated sensors, they can simultaneously measure multiple antibiotics, providing rapid information on antibiotics to which the microorganism is highly susceptible. Another advantage is that the minimum antibiotic concentration required to limit resistance can be quickly determined.

[0063] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not restrictive.

Claims

1. Providing a substrate made of polyethylene terephthalate; and 1. A method for manufacturing a biosensor, comprising forming an electrode layer comprising interdigitated first and second electrodes on the substrate, The electrode is made of silver or gold, The electrodes are formed by a screen printing method or a PCB (Printed Circuit Board) method, the biosensor further comprises a housing; The method, wherein the spacing between the first electrode and the second electrode is 10 μm to 300 μm, the height of the first electrode and the second electrode is 50 nm to 5,000 nm, and the width of the first electrode and the second electrode is 10 μm to 300 μm.

2. The method described in claim 1, wherein the biosensor is for measuring the antibiotic sensitivity of a microorganism by measuring in real time changes in electrical properties that occur as the microorganism grows due to antibiotic treatment.

3. A step of processing a sample containing microorganisms so that the sample is contained inside a storage portion of a biosensor manufactured by the method described in claim 1; treating the sample-treated biosensor housing with an antibiotic; and A method for testing antibiotic susceptibility of a microorganism, comprising the step of measuring a change in electrical characteristics after treatment with the antibiotic.

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