KR20200109188A - Aptamer functionalized vertical biosensor and use thereof - Google Patents

Abstract

The present invention relates to a biosensor capable of testing growth of microorganisms in blood and antibiotic sensitivity thereof in real time without prior culture, and to a use thereof.

Description

Aptamer functionalized vertical biosensor and its use {APTAMER FUNCTIONALIZED VERTICAL BIOSENSOR AND USE THEREOF}

The present invention relates to a biosensor capable of testing the growth of microorganisms in blood and susceptibility to antibiotics in real time without prior culture, and to a use thereof.

A test that looks for antibiotics that can inhibit the growth of bacteria is called an antibiotic sensitivity test. The antibiotic susceptibility test is a direct and important test that allows you to choose the type of antibiotic to use for your bacteria. In addition, when prescribing an appropriate antibiotic for a patient, it enables customized prescription in consideration of the prescription method, frequency, cost, and side effects. If the susceptibility result is used, it is possible to increase the treatment cost and reduce the disappointment of the guardian that may occur when empirical antibiotics are prescribed, and provide an opportunity to acquire bacterial resistance, reduce complications, and shorten the patient's recovery period.

The most common antibiotic susceptibility tests are the disk diffusion technique and the broth dilution technique. However, these methods require a process of culturing the bacteria for several days, then identifying the bacteria, and measuring turbidity. This process takes a long time and requires a lot of labor.

On the other hand, sepsis is a disease that occurs when blood is infected with bacteria, causing an inflammatory reaction in body tissues, causing death within a short time if severe. The causative agent of sepsis is E. coli, pneumococcal, Staphylococcus aureus, purulent streptococcus, Pseudomonas aeruginosa, etc. Bacteria penetrate through wounds and infiltrate into the blood, ingestion of food can cause infection, and inflammatory substances can be generated from the source of infection, causing sepsis. It is particularly prone to those with weak immunity or newborns.

The existing sepsis test method uses collected blood to pre-culture for more than 24 hours and then checks the presence of bacteria. Because of the pre-culture, it takes more than 24 hours to test for sepsis. If it is determined as sepsis, an antibiotic susceptibility test is performed. This is performed by measuring optical properties such as turbidity of the sample 10 to 24 hours after the pre-cultured sample is treated with antibiotics, so in general, the antibiotic sensitivity test is performed for 34 to 48 hours. It takes a long time.

Since the antibiotic sensitivity test time is long, in most cases, antibiotics are first prescribed based on clinical experience, and then the antibiotics with high sensitivity are changed when the antibiotic sensitivity test is performed. Therefore, antibiotic resistance may be a problem, and misuse of antibiotics may be caused, and thus, there is a need to develop a technology capable of faster testing.

Korean Patent Registration No. 10-1776698

It is to provide a biosensor comprising an aptamer that specifically binds to the microorganism of the present invention.

Another object of the present invention is to provide a kit for detecting microorganisms including the biosensor.

Another object of the present invention is to provide a method for detecting microorganisms using the biosensor.

Another object of the present invention is to provide a method of providing information necessary for diagnosing microbial infection using the biosensor.

Another object of the present invention is to provide a method for measuring antibiotic sensitivity of microorganisms using the biosensor.

One aspect of the present invention for achieving the above object is a substrate including glass; An electrode layer formed on the substrate and including interdigitated first and second electrodes; And an aptamer that is fixed on the substrate and specifically binds to microorganisms.

In the present invention, "biosensor" refers to a device, a device, etc. that investigates the properties of a substance by using the function of a living organism, and the shape thereof is not limited. Specifically, an electrode is formed on a substrate, and a change in capacitance or conductance generated when a target material is bonded between the electrodes may be measured to detect the presence of a material, bonding/separation. In particular, it may be a device or device capable of measuring or detecting the presence of microorganisms, growth of microorganisms, and susceptibility to antibiotics, and further, a device that provides information necessary for diagnosing the presence of microorganisms through the measurement or detection, or It can be a device.

The biosensor of the present invention includes an electrode layer, and specifically, may include an interdigitated microelectrode. The interdigitated microelectrode may have a structure in which bars arranged in a line form one pole, and two electrodes (a first electrode and a second electrode) face each other in a meshed state. The two electrodes serve as classical impedance measuring electrodes.

In the sensor using the interdigitate microelectrode, the distance between the first electrode and the second electrode may be 0.1 to 1000 μm, 1 to 500 μm, or 10 to 100 μm. By adjusting the separation distance between the first electrode and the second electrode within the above range, it is possible to accurately measure biomolecules at the micro level. In addition, the height of each electrode may be in the range of 50 to 5,000 nm, and the width of each electrode may be in the range of 1 to 500 μm or 10 to 100 μm. Each of the electrodes may independently be any one selected from the group consisting of gold, platinum, carbon electrodes, conductive polymers, and indium tin oxide (ITO), for example gold.

The biosensor may detect or measure all microorganisms and/or biomolecules thereof, and specifically, the microorganism may be Escherichia coli , Staphylococcus aureus , or Pseudomonas aeruginosa .

In the present invention, "aptamer" refers to a small single-stranded oligo nucleic acid capable of specifically recognizing a target substance with high or specific affinity.

Since the type of microorganism that specifically binds to a specific microorganism is different according to the type of the aptamer, the bound microorganism can be detected according to the type of aptamer that specifically binds to a specific microorganism. For example, when an aptamer specifically binding to E. coli is fixed on the substrate and a sample containing E. coli is processed on the sensor, if the sample contains E. coli, binding of the aptamer and E. coli and bound E. coli Since the change in capacitance and conductance will be detected by the electrode by the proliferation of the sample, it is possible to quickly detect that E. coli is contained in the sample, and to detect microorganisms in the sample through the target-specific binding characteristics of the aptamer. I can. Specifically, the aptamer may include one or more sequences selected from SEQ ID NO: 1 to SEQ ID NO: 3.

Further, specifically, the spacing between the first electrode and the second electrode may be 10 to 100 μm, but is not limited thereto, and the spacing between the first electrode and the second electrode may be appropriately changed as necessary.

In addition, the biosensor of the present invention may further include a wireless transmission unit for transmitting the result of measuring the capacitance and/or conductance, and this transmission method includes both data transmission by wire using a wire such as USB or a wireless method using a Bluetooth method. It can be carried out by a method that can be used conventionally including.

In addition, the biosensor of the present invention is connected to an LCR meter (LCR meter) capable of measuring capacitance and conductance to monitor changes in the concentration of microorganisms in real time, and at this time, such a microorganism sensor has a frequency of 0.1 to 100 kHz. 1 to 100 mV AC voltage can be supplied.

Further, specifically, the aptamer may be fixed between the first electrode and the second electrode of the electrode layer, but is not limited thereto, and the fixing position of the aptamer may be appropriately changed as needed.

In addition, specifically, the biosensor may further include a storage unit capable of receiving a sample to be detected therein. The storage unit may be made of at least one material selected from the group consisting of glass, polypropylene, polyethylene tetraphthalate, and polycarbonate, but is not limited thereto and may be applied by changing to an appropriate material as necessary.

In addition, the accommodating portion may have an upper portion of which is blocked or opened, and specifically, may be formed in a direction perpendicular to the substrate. For example, a commercially used well may be made of a plastic material. The storage unit may have a capacity of 10 μl to 10 ml. The microorganism can be treated with the culture medium.

In the present invention, "sample" refers to any substance in which microorganisms are present, or are estimated or predicted to exist. The sample may be natural or synthetic, and may be obtained through any means known to a person skilled in the art. Accordingly, the sample is a microbial culture medium (media), blood, tissue, cells, whole blood, plasma, serum, saliva, sputum, lymph, cerebrospinal fluid, intercellular fluid or urine, as well as samples of in vitro cell culture components, for example For example, it may include cellular components, cell culture medium, recombinant cells, and the like. It may also be collected from liquid, soil, air, food, or waste.

The microorganism culture medium (media) refers to a liquid or solid material used for proliferation, preservation, etc. of microorganisms, and may be used by transforming into a form appropriately required according to the type of microorganism.

Also, specifically, the biosensor may have a vertical shape.

In one embodiment of the present invention, a biosensor incorporating an aptamer that specifically binds a microorganism was used to measure changes in capacitance and conductux to measure bacterial growth and antibiotic sensitivity in real time. It was confirmed that the presence, growth and antibiotic sensitivity of microorganisms can be immediately confirmed in a blood sample by measuring an electrical signal in real time without the process of separating and extracting microorganisms using and culturing.

Another aspect of the present invention relates to a kit for detecting microorganisms including the biosensor. Specifically, the microorganism may be Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa.

The detection kit may have a solution, a lyophilized powder, a frozen solution, or a strip form, and each form may be formulated by a method conventional in the art. It may also include instructions for using the kit.

Another aspect of the present invention relates to a sepsis diagnostic kit comprising the biosensor. In one embodiment of the present invention, it was confirmed that the infection of Staphylococcus aureus and Pseudomonas aeruginosa, which are the causative agents of sepsis, can be detected by measuring changes in capacitance and conductance in microbial culture media and blood samples (FIGS. 6 and 6 7), it is possible to diagnose sepsis by using a kit including the biosensor of the present invention.

The diagnostic kit may have a solution, a lyophilized powder, a frozen solution, or a strip form, and each form may be formulated by a conventional method in the art. It may also include instructions for using the kit.

Another aspect of the present invention is a) contacting a target sample and the biosensor; And b) measuring a change in capacitance or conductance. Specifically, the microorganism may be Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa.

In addition, specifically, the sample may be a microorganism culture medium (media), blood, tissue, cells, whole blood, plasma, serum, saliva, sputum, lymph fluid, cerebrospinal fluid, intercellular fluid, or urine, but is not limited thereto. It may also be collected from liquid, soil, air, food, or waste.

The method for detecting microorganisms may further include determining that a microorganism is present when the sample is a microorganism culture medium and the capacitance or conductance increases.

In addition, when the sample is blood, the method for detecting microorganisms may further include determining that a microorganism is present when the capacitance or conductance decreases.

In one embodiment of the present invention, changes in capacitance and conductance in the microbial culture medium and blood samples were measured, and it was confirmed that capacitance and conductance increased when E. coli, Staphylococcus aureus, or Pseudomonas aeruginosa were present in the microbial culture medium ( Fig. 6). In addition, when E. coli, Staphylococcus aureus, or Pseudomonas aeruginosa were present in the blood, it was confirmed that the capacitance and conductance decreased (FIG. 7).

In particular, it was confirmed that all concentrations from 10 ³ CFU/ml to 10 CFU/ml can be detected, and it was confirmed that the biosensor of the present invention can detect microorganisms with high sensitivity without prior culture.

In another aspect of the present invention, a) contacting a subject patient sample with the biosensor; And b) measuring a change in capacitance or conductance of the biosensor. It relates to a method of providing information necessary for diagnosing microbial infection, including.

Specifically, the microorganism may be Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa, and more specifically, may provide information necessary for diagnosing sepsis.

In addition, specifically, the sample may be a microorganism culture medium (media), blood, tissue, cells, whole blood, plasma, serum, saliva, sputum, lymph fluid, cerebrospinal fluid, intercellular fluid, or urine, but is not limited thereto. It may also be collected from liquid, soil, air, food, or waste.

The method of providing information necessary for diagnosing microbial infection may further include determining that the sample is a microbial culture medium, determining that the sample is infected with the microbe if the capacitance or conductance increases.

In addition, the method of providing information necessary for diagnosing microbial infection may further include determining that the sample is infected with the microbe if the sample is blood and the capacitance or conductance decreases.

Another aspect of the present invention is a) contacting the biosensor with a subject patient sample; And b) measuring a change in capacitance or conductance of the biosensor. It provides a method for diagnosing sepsis, including.

In one embodiment of the present invention, it was confirmed that the infection of Staphylococcus aureus and Pseudomonas aeruginosa, which are the causative agents of sepsis, can be detected by measuring changes in capacitance and conductance in microbial culture media and blood samples (FIGS. 6 and 6 7), it is possible to diagnose sepsis by identifying the strain using this.

According to a conventional sepsis diagnosis method, blood is collected, pre-cultured in a measurement medium for several days, and then components in the blood are analyzed. In this process, it takes about 1 to 3 days to diagnose sepsis and consumes a lot of labor. However, when the biosensor of the present invention is used, the electrochemical signal of capacitance and conductance in the blood can be measured and the result can be checked in real time without a long-term pre-cultivation, so that the sepsis diagnosis time can be shortened.

Another aspect of the present invention is a) contacting a target sample and the biosensor; b) treating the desired antibiotic against the microorganism; And c) measuring a change in capacitance or conductance, it relates to a method for measuring antibiotic susceptibility of microorganisms.

Specifically, the microorganism may be Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa.

In addition, specifically, the sample may be a microorganism culture medium (media), blood, tissue, cells, whole blood, plasma, serum, saliva, sputum, lymph fluid, cerebrospinal fluid, intercellular fluid, or urine, but is not limited thereto. It may also be collected from liquid, soil, air, food, or waste.

In the present invention, "antibiotic susceptibility" is also referred to as antibiotic sensitivity, and refers to a microorganism (strain, etc.) affected by a specific antibiotic such as inhibition of growth. In the case of antibiotic treatment, it is said to have sensitivity when microorganisms cannot grow around the area treated with antibiotics, and infectious diseases caused by microorganisms can be treated using sensitive antibiotics.

The antibiotic sensitivity is determined by measuring the capacitance and conductance values before treatment with the target antibiotic, and the capacitance and conductance values changed after treatment with the antibiotic, and according to the change value, it is possible to check in real time whether the microorganisms are killed according to the sensitivity in a short time, and the capacitance and conductance change Sensitivity can be measured more precisely through For example, since the sensitivity according to the antibiotic concentration can also be quickly measured, the minimum amount can be used in the antibiotic concentration and dosage administered for treatment, thereby preventing misuse of antibiotics, and thereby reducing side effects. have.

The antibiotic may be gentamicin, but is not limited thereto, and any one capable of measuring sensitivity by the biosensor of the present invention may be included.

Specifically, when the sample is a microbial culture medium, it may further include the step of determining that sensitivity appears at the antibiotic treatment concentration if the increase in capacitance or conductance does not appear after the antibiotic treatment.

In particular, when the sample is blood, the step of determining that the sensitivity appears at the concentration of the antibiotic treatment if the capacitance or conductance does not decrease after the antibiotic treatment may be further included.

In one embodiment of the present invention, changes in capacitance and conductance in the microbial culture medium and blood samples were measured, and when antibiotics were treated in the microbial culture medium in which E. coli, Staphylococcus aureus or Pseudomonas aeruginosa were present, the capacitance and conductance increased. It was confirmed that it did not appear (FIG. 8). In addition, it was confirmed that the decrease in capacitance and conductance did not appear when antibiotics were treated in blood in which E. coli, Staphylococcus aureus or Pseudomonas aeruginosa were present (FIG. 9).

In particular, it was confirmed that the sensitivity can be measured up to a low concentration of 0.1 μg/ml of the antibiotic concentration, and it was confirmed that the biosensor of the present invention can measure the antibiotic sensitivity with high sensitivity without prior culture.

Accordingly, the biosensor of the present invention can improve the sensitivity of the sensor in the blood without prior culture by using an aptamer that specifically binds to the microorganism, and can directly measure the antibiotic sensitivity and the lowest concentration of the microorganism. As a result, there is an effect of simultaneously identifying microorganisms and measuring antibiotic sensitivity.

In addition, since it is possible to detect even minute changes in capacitance and conductance changes, there is an advantage in that it is possible to more accurately present the dosage and/or concentration of antibiotics for diagnosing and treating sepsis.

The aptamer-functionalized vertical biosensor of the present invention can shorten the identification time of microorganisms, and the antibiotic susceptibility test time, which took several days in the past, can be shortened to within 24 hours.

In addition, since it is possible to quickly determine whether an infection is present with detection of microorganisms, and to quickly determine the type of susceptible antibiotic and the minimum antibiotic concentration required for administration, it can be effectively applied to customized treatment using antibiotics.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a schematic diagram showing a vertical biosensor functionalized with an aptamer prepared according to a preparation example of the present invention.
Figure 2 is a result showing the difference in the performance of the biosensor according to the presence or absence of aptamers, the blue line is blood not treated with bacteria, the red line is blood treated with bacteria on the sensor not functionalized with aptamer, and black line is aptamer This is the result of real-time measurement of changes in the capacitance of blood treated with bacteria in a functionalized sensor while bacteria are growing.
3 is a fluorescence image of a biosensor according to the presence or absence of an aptamer, showing green fluorescence when the aptamer is successfully functionalized.
4 is a fluorescence image of bacteria according to the presence or absence of an aptamer, which is a photograph of E. coli stained after measurement, and green fluorescence indicates bacteria.
5 is a photograph of a state in which blood is tested using a biosensor manufactured according to an embodiment of the present invention.
6 shows the growth of bacteria on the media measured using a biosensor, and shows real-time capacitance and conductance changes according to the type and concentration of bacteria on the media.
7 shows the growth of bacteria in blood measured using a biosensor, and shows real-time changes in capacitance and conductance according to type and concentration of bacteria in the blood.
FIG. 8 shows the antibiotic susceptibility of each bacteria on a media measured using a biosensor, and shows changes in capacitance and conductance measured in real time while varying the concentration of antibiotics for each bacteria on the media.
9 shows the sensitivity of antibiotics for each bacteria in the blood measured using a biosensor, and shows changes in capacitance and conductance measured in real time while varying the concentration of antibiotics for each bacteria in the blood.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited by the following examples.

Preparation Example 1. Preparation of experiment

Gentamicin antibiotics were purchased from Sigma Aldrich (US), and were prepared at concentrations of 0.001, 0.01, 0.1, 1 and 10 μg/ml. Gentamicin was dissolved in distilled water and used. The strains used in the experiments of E. coli (E. coli, ATCC 25922), Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853), a Staphylococcus aureus (S. aureus, ATCC 29213) was used as received from the pre-sale Medicine, Yonsei University Department of Microbiology, Difco Nutrient Broth media was used.

Sheep blood was purchased and used by Synergy Innovation (Seongnam, Korea). The aptamer was purchased and used from Xenotech (Daejeon, Korea).

Preparation Example 2. Fabrication of aptamer functionalized biosensor

In order to manufacture a biosensor functionalized with aptamers for detecting bacteria, a sensor with electrodes formed on a glass substrate (Comparative Example 1) and a sensor with electrodes formed on a glass substrate and functionalized with aptamers (Example 1) were fabricated, respectively. .

As shown in FIG. 1, the electrode was formed on a glass substrate using a photolithography process, and specifically, one rod arranged in a row forms a pole, and the other electrodes (the first electrode and the second electrode) are formed. A microelectrode of an interdigitated structure having a pair structure in which the electrode) faces each other with different poles is patterned so that the width of the electrode is 50 μm and the separation distance is 30 μm. (Cr) and 50 nm-thick gold (Au) were deposited to prepare an electrode.

In addition, an aptamer (Genotech, Daejeon, Korea) that induces specific binding of bacteria was treated on the substrate. The aptamer is an aptamer that specifically binds to bacteria, and the base sequence of the aptamer is shown in Table 1 below.

Sequence number bacteria Aptamer sequence One Coli 5′-GCA ATG GTA CGG TAC TTC CCC ATG AGT GTT GTG AAA TGT TGG GAC ACT AGG TGG CAT AGA GCC GCA AAA GTG CAC GCT ACT TTG CTA A-3′ 2 Staphylococcus aureus 5′-GCA ATG GTA CGG TAC TTC CTC GGC ACG TTC TCA GTA GCG CTC GCT GGT CAT CCC ACA GCT ACG TCA AAA GTG CAC GCT ACT TTG CTA A-3′ 3 Pseudomonas aeruginosa 5'-CCC CCG TTG CTT TCG CTT TTC CTT TCG CTT TTG TTC GTT TCG TCC CTG CTT CCT TTC TTG- (CH 2) 3 '-SH-3'

First, piranha (sulfuric acid (H ₂ SO ₄ ): hydrogen peroxide (H ₂ O ₂ ) = 3: 1 v/v) is treated on a glass substrate to generate a hydroxy group (-OH), and 3-aminopropyltriethoxysilane (3-aminopropyltriethoxysilane, APTES) A 10% ethanol solution was treated and reacted with a hydroxy group to generate an amino group (-NH ₂ ). Thereafter, a 0.1M ethanol solution of succinic anhydride was treated to generate a carboxyl group (-COOH). Then, N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)/en-hydroxy-succinimide (N- Hydroxy-succinimide, NHS) was added to a 1M solution of 2-(ene-morpholino)ethanesulfonic acid (MES) in a ratio of 2:1 v/v. After activating the carboxyl group, the aptamer was fixed on the electrode by reacting the amino group on the surface of the aptamer.

Experimental Example 1. Checking the ability to measure the capacitance change of the sensor according to the presence or absence of an aptamer

Using the sensor manufactured according to Preparation Example 2, the ability to measure the change in capacitance of the sensor was confirmed. Specifically, E. coli (10 ³ CFU/ml) was treated for each of a sensor having an electrode formed on a glass substrate (Comparative Example 1) and a sensor having an electrode formed on a glass substrate and functionalized with an aptamer (Example 1), thereby changing the capacitance. Was measured.

As shown in Figure 2, it was measured that the capacitance changes as the bacteria in the blood bind/proliferate, and the aptamer functionalized glass of Example 1 compared to the glass substrate sensor (red) of Comparative Example 1. In the substrate sensor (black), it was found that the capacitance decreases rapidly from 6 hours later.

In particular, in the case of Example 1, the capacitance change was measured within a short time, which indicates that the measurement of the capacitance change with remarkably high sensitivity is possible in the case of a biosensor having a structure in which an aptamer is bonded to a glass substrate.

In addition, as shown in FIG. 3, green fluorescence was observed in the electrode in which the aptamer was successfully functionalized, but green fluorescence was not observed in the electrode in which the aptamer was successfully functionalized, and as shown in FIG. 4, the aptamer was successfully functionalized. It was confirmed that more bacteria were bound to the aptamer on the electrode, so that more green fluorescence was observed.

Experimental Example 2. Real-time measurement of growth by bacterial concentration

Using the biosensor prepared according to Preparation Example 2, the growth of bacteria by concentration was confirmed in real time. Specifically, as shown in FIG. 5, a biosensor electrode functionalized with aptamers in a vertical form is fixed to a 2 ml vial, and media (control, blood) and Escherichia coli that do not contain bacteria, Staphylococcus aureus and Pseudomonas aeruginosa (10, 10 ² and 10 ³ CFU/ml, respectively) were treated on the sensor, respectively, and then the agglomeration of bacteria was prevented in an incubator at 37°C without prior culture. For 18 hours, while shaking (shaking), changes in the capacitance and conductance in the blood were measured.

2-1. Real-time bacterial concentration measurement in media

Real-time bacterial concentration in the media was measured using this method. As a result, as shown in FIG. 6, when the media containing no bacteria was treated (control), there was no significant change in capacitance and conductance (green), and when functionalized with a bacteria-specific aptamer, the capacitance and The change in conductance was significant. In addition, it was confirmed that bacteria can be successfully detected in all concentration ranges from a high concentration (10 ³ CFU/ml) to a low concentration (10 CFU/ml).

2-2. Real-time measurement of bacteria concentration in blood

Real-time bacterial concentration in blood was measured using the above method. As a result, as shown in FIG. 7, it was confirmed that capacitance and conductance decreased when bacteria were included in the blood, and thus it was possible to distinguish between a case without bacteria and a case including bacteria in the blood. In particular, it was confirmed that bacteria can be successfully detected in all concentration ranges from a high concentration (10 ³ CFU/ml) to a low concentration (10 CFU/ml) in the blood as in the media. This indicates that the biosensor of the present invention can successfully detect bacteria in various concentration ranges, and in particular, bacteria can be identified within 24 hours, which is a fairly short time without prior culture.

Experimental Example 3. Measurement of sensitivity according to the type of antibiotic

After measuring the growth of bacteria using a vertical biosensor functionalized with an aptamer specific for bacteria as in Experimental Example 2 on the substrate prepared according to Preparation Example 2, the sensitivity to antibiotics was measured.

As a control, media and blood (blood: media = 1: 4) not treated with antibiotics and bacteria were used, and gentamicin at concentrations of 0, 0.001, 0.01, 0.1, 1 and 10 μg/ml After the treatment, the capacitance and conductance changes were measured for 18 hours while shaking in an incubator at 37°C.

3-1. Antibiotic susceptibility measurement in media

Antibiotic sensitivity in the media was measured using the above method. As a result, as shown in FIG. 8, when bacteria were cultured in the sensor and treated with gentamicin antibiotics, differences in capacitance and conductance changes were observed according to the concentration of the antibiotic.

In the case of antibiotics with a concentration higher than MIC according to the antibiotic minimum inhibitory concentration (MIC) for each bacteria, the changes in capacitance and conductance were similar to those of the media without antibiotics and bacteria treatment. This is because the growth of bacteria is inhibited by antibiotics, and the sensor of the present invention is developed with antibiotics of 0.1 μg/ml or more for E. coli, 1 μg/ml or more for Staphylococcus aureus, and 0.1 μg/ml or more for Pseudomonas aeruginosa. It was confirmed that what is suppressed can be detected.

In addition, when antibiotics at a lower concentration than the MIC were treated, the growth of bacteria was inhibited, but after a certain period of time, normal bacterial growth patterns were exhibited.

3-2. Measurement of sensitivity to antibiotics in the blood

Antibiotic sensitivity in blood was measured using the above method. As a result, as shown in FIG. 9, when antibiotics with a concentration higher than MIC were treated in the blood, changes in capacitance and conductance were similar to those of blood without antibiotics and bacteria. Conversely, when antibiotics at a lower concentration than MIC were treated, a general pattern of bacterial growth was observed after a certain period of time.

In particular, in the blood, the sensor of the present invention can detect inhibition of growth in antibiotics of 0.1 μg/ml or more for E. coli, 1 μg/ml or more for Staphylococcus aureus, and 1 μg/ml or more for Pseudomonas aeruginosa. Confirmed.

The above results indicate that using the biosensor functionalized with aptamer of the present invention, it is possible to detect low concentration of bacteria without a long cultivation, as well as sensitively detect sensitivity to antibiotics. This suggests that it can be used for confirming the dosage of antibiotics by sensitively detecting the sensitivity of antibiotics.

In particular, the biosensor of the present invention with high sensitivity and high sensitivity while performing the detection step immediately without the step of isolating and culturing the bacteria to be detected in the blood allows monitoring and screening for infection and treatment of the strain in real time. I did it.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and the concept of equivalents thereof should be construed as being included in the scope of the present invention.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> APTAMER FUNCTIONALIZED VERTICAL BIOSENSOR AND USE THEREOF <130> 18PP31204 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> aptamer of E.coli <400> 1 gcaatggtac ggtacttccc catgagtgtt gtgaaatgtt gggacactag gtggcataga 60 gccgcaaaag tgcacgctac tttgctaa 88 <210> 2 <211> 88 <212> DNA <213> Artificial Sequence <220> <223> aptamer of S. aureus <400> 2 gcaatggtac ggtacttcct cggcacgttc tcagtagcgc tcgctggtca tcccacagct 60 acgtcaaaag tgcacgctac tttgctaa 88 <210> 3 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> aptamer of P. aeruginosa <400> 3 cccccgttgc tttcgctttt cctttcgctt ttgttcgttt cgtccctgct tcctttcttg 60 60

Claims (27)

A substrate including glass;
An electrode layer formed on the substrate and including interdigitated first and second electrodes; And
Biosensor comprising; an aptamer fixed on the substrate and specifically binding to microorganisms. The method of claim 1,
The microorganism is Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa. The method of claim 1,
The aptamer comprises one or more sequences selected from SEQ ID NO: 1 to SEQ ID NO: 3, biosensor. The method of claim 1,
A biosensor having a spacing between the first electrode and the second electrode of 10 to 100 μm. The method of claim 1,
The aptamer is a biosensor fixed between the first electrode and the second electrode of the electrode layer. The method of claim 1,
The biosensor further comprises a storage unit capable of receiving a sample to be detected therein. The method of claim 6,
The storage unit is made of at least one material selected from the group consisting of glass, polypropylene, polyethylene tetraphthalate, and polycarbonate. The method of claim 6,
The sample is a microbial culture medium (media), blood, tissue, cells, whole blood, plasma, serum, saliva, sputum, lymph fluid, cerebrospinal fluid, intercellular fluid or urine. The biosensor according to any one of claims 1 to 8, wherein the biosensor has a vertical shape. A kit for detecting microorganisms, comprising the biosensor of any one of claims 1 to 8. The method of claim 10,
The microorganism is E. coli, Staphylococcus aureus or Pseudomonas aeruginosa, a kit for detecting microorganisms. A kit for diagnosing sepsis, comprising the biosensor of any one of claims 1 to 8. a) contacting the target sample with the biosensor of any one of claims 1 to 8; And
b) measuring a change in capacitance or conductance. The method of claim 13,
The microorganisms are Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa. The method of claim 13,
The sample is a microorganism culture medium (media), blood, tissue, cells, whole blood, plasma, serum, saliva, sputum, lymph fluid, cerebrospinal fluid, intercellular fluid or urine. The method of claim 13,
When the sample is a microorganism culture medium (media),
If the capacitance or conductance increases, the method further comprising determining that the microorganism is present. The method of claim 13,
If the sample is blood,
If the capacitance or conductance decreases, it further comprises determining that the microorganism is present. a) contacting the subject patient sample with the biosensor of any one of claims 1 to 8; And
b) measuring a change in capacitance or conductance of the biosensor; Containing, a method of providing information necessary for diagnosing a microbial infection. The method of claim 18,
The microorganism is Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa, a method of providing information necessary for diagnosing microbial infection. The method of claim 18,
The sample is a microorganism culture medium (media), blood, tissue, cells, whole blood, plasma, serum, saliva, sputum, lymph fluid, cerebrospinal fluid, intercellular fluid, or urine. A method of providing information necessary for diagnosing a microbial infection. The method of claim 18,
When the sample is a microorganism culture medium (media),
The method of providing information necessary for diagnosing microbial infection, further comprising the step of determining that the microbial infection is infected when the capacitance or conductance increases. The method of claim 18,
If the sample is blood,
If the capacitance or conductance decreases, the method of providing information necessary for diagnosing microbial infection further comprises the step of determining that the microbe is infected. a) contacting the target sample with the biosensor of any one of claims 1 to 8;
b) treating the desired antibiotic against the microorganism; And
c) measuring a change in capacitance or conductance, a method for measuring antibiotic susceptibility of microorganisms. The method of claim 23,
The microorganism is Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa, a method for measuring antibiotic susceptibility of microorganisms. The method of claim 23,
The sample is a microorganism culture medium (media), blood, tissue, cells, whole blood, plasma, serum, saliva, sputum, lymph fluid, cerebrospinal fluid, intercellular fluid, or urine. The method of claim 23,
When the sample is a microorganism culture medium (media),
If the increase in capacitance or conductance does not appear after the antibiotic treatment, the method of measuring antibiotic sensitivity of a microorganism further comprises the step of determining that sensitivity appears at the corresponding antibiotic treatment concentration. The method of claim 23,
If the sample is blood,
If the decrease in capacitance or conductance does not appear after the antibiotic treatment, the method of measuring antibiotic sensitivity of a microorganism further comprises the step of determining that sensitivity appears at the corresponding antibiotic treatment concentration.

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