KR20210095312A - Bio probe for detecting or determining wound and method for detecting or determining wound using the same - Google Patents

Abstract

The present invention relates to a bio probe which detects extracellular matrix metalloproteinase (MMP)-2 or MMP-9 in order to detect or determine a cut, and a composition for detecting or determining a cut, comprising the same. The bio probe of the present invention performs rapid diagnosis and easily determines a disease stage in cut diagnosis and management requiring fields, and can be used as a point of care (POC) solution.

Description

Bio probe for detecting or determining wound and method for detecting or determining wound using the same}

The present invention relates to a bioprobe for detecting extracellular matrix metal-containing protease (MMP) for detecting or determining a wound, and a composition for detecting or determining a wound comprising the same.

Due to the development of science and medical technology, human life in modern society is gradually enriched, and the average lifespan is increasing. However, the number of trauma patients whose tissues and organs are damaged or lose their functions due to safety accidents due to the aging of the population and increase in leisure activities according to these changes is also increasing. In particular, the most common tissue damage in daily life is a skin defect (chronic skin wound).

Pressure sores are one of the representative skin diseases, and can be defined as ulcers caused by tissue necrosis caused by impaired blood circulation due to continuous and repeated pressure applied mainly to the protrusions of bones. one of the diseases. Bedsores can also be caused by damage caused by diseases such as diabetic arterial occlusion, arteriosclerotic arterial occlusion, burns, etc. Accordingly, the increase in hospitalization period and medical expenses aggravates economic and psychological pain for patients, and thus their importance is being highlighted as an important social health problem.

As for the treatment methods for pressure sores, non-surgical conservative wound healing, skin transplantation, and, in the extreme, surgical treatment such as amputation, are applied depending on the cause and severity of the disease. In many cases, surgery is not possible for pressure sores, and even after surgery, a cure process through continuous observation and follow-up treatment is required. judgment is essential. However, until now, there is no indicator of objective selection for evaluating the degree of wounding in the patient's lesion area and determining the treatment method. When treatment is required, the need for a point-of-care (POC) solution is emerging.

Among various POC solutions, chemical biosensors are inexpensive and intuitive, enabling accurate measurements in a short time, so that not only surgeons but also people without surgical knowledge can use them easily. Moreover, the chemical POC biosensor has advantages in that it has high visibility because it is advantageous for remotely transmitting or databaseizing measurement data, and has a high probability of success when developing a small mobile sensor platform.

It is an object of the present invention to provide a bioprobe for detecting or determining a wound comprising a target peptide of extracellular matrix metalloprotease-2 (MMP-2) or extracellular matrix metalloprotease-9 (MMP-9).

Another object of the present invention is to provide a composition for detecting or determining a wound comprising the bioprobe, and a kit, biochip or biosensor comprising the same.

Another object of the present invention is to provide a method of providing information about a wound by detecting or determining a wound by using the bioprobe.

In order to achieve the above object, the present invention provides a bioprobe for detecting or determining a wound comprising a target peptide of MMP-2 or MMP-9.

In one aspect of the present invention, the bioprobe includes a target (substrate) peptide for MMP-2 or MMP-9 protease and a label bound to the peptide, wherein the label is capable of detecting the bioprobe of the present invention. Molecules include, but are not limited to, luminescent molecules, fluorochromes, fluorescent quenchers, lipids, colored molecules, radioactive isotopes, scintillators, biotin, avidin, streptavidin, protein A, protein G, antibody or its It may include any one or more selected from the group consisting of fragments, polyhistidine, Ni2+, Flag tags, myc tags, heavy metals, and enzymes.

In another aspect of the present invention, the bioprobe, a target (substrate) peptide for MMP-2 or MMP-9 protease; A complex of a fluorescent receptor and a donor, which are fluorescent substances bound to the target peptide, and exhibits fluorescence resonance energy transfer (FRET) upon cleavage of the target peptide by the protase.

More specifically, the fluorescent material may be a fluorescent protein, and it is sufficient as long as it is commonly used in the technical field of the present invention and is not limited thereto, but a green fluorescent protein (GFP), a modified green fluorescent protein (MGFP), Enhanced Green Fluorescent Protein (EGFP), Red Fluorescent Protein (RFP), Enhanced Red Fluorescent Protein (ERFP), Blue Fluorescent Protein (BFP), Enhanced Blue Fluorescent Protein (EBFP), Yellow Fluorescent Protein (YFP), Enhanced It may be selected from the group consisting of yellow fluorescent protein (EYFP), indigo fluorescent protein (CFP), and enhanced indigo blue fluorescent protein (ECFP).

In addition, the fluorescent material may be an organic fluorescent dye, which is sufficient as long as it is commonly used in the technical field of the present invention and is not limited thereto, but fluorescein, fluorescein chlorotriazinyl ), rhodamine green, rhodamine red, tetramethylrhodamine, fluorescein isothiocyanate (FITC, Fluorescein isothiocyanate), Oregon green, Alexa Fluoro (Alexa Fluor), FAM, VIC, JOE, ROX, HEX, NED, PET, LIZ, Texas Red, TET, TRITC, TAMRA, Cyanine-based dyes and thiadicarbocyanine It may be selected from the group consisting of.

In order to achieve another object of the present invention, the present invention provides a composition for detecting or determining a wound comprising the bioprobe, and a kit, biochip or biosensor comprising the same.

In order to achieve another object of the present invention, the present invention comprises the steps of directly contacting the bio-probe to the affected part of the patient or to the sample; A method of providing information about a wound by detecting or determining a wound, comprising measuring a fluorescence resonance energy transfer caused by cleavage of a target peptide by MMP-9 or MMP-2 contained in the affected area or sample to provide.

The bioprobe of the present invention uses fluorescence resonance energy transfer caused by cleavage of the target peptide contained in the bioprobe according to the presence and concentration of MMP-9 or MMP-2 contained in the lesion or exudate of a chronic skin wound patient. It has the advantage of being able to easily detect the disease or determine the stage of the disease.

1 is a diagram showing a method of manufacturing a hydrogel array to which the FRET peptide of the present invention is bound.
2 is a diagram showing the structure and mechanism of action of the peptide complex used as the bioprobe of the present invention.
3 shows the results of measuring the concentration of MMP-2 contained in the exudate sample collected from the wounded patient's affected area. In case of A., the exudate collected from the affected area was diluted with saline, and B. was covered with the affected area. This is the result of measuring the concentration of each MMP-2 on exudates directly collected from gauze.
4 shows the results of measuring the concentration of MMP-9 contained in the exudate sample collected from the wounded patient's affected area. In case of A., the exudate collected from the affected area was diluted with saline, and B. This is the result of measuring the concentration of each MMP-9 on exudates directly collected from gauze.
Figure 5 shows the results of measuring the concentrations of MMP-2 and MMP-9 from the frozen storage of exudate samples from wounded patients.
FIG. 6 shows the intensity of fluorescence as a result of treating the hydrogel biosensor with MMP-9 for each concentration.
7 is a result of quantifying the fluorescence intensity visually displayed from the hydrogel biosensor by a software-based method.
8 shows the results of treating the hydrogel biosensor of the present invention with MMP-9, and measuring the fluorescence intensity according to the treatment concentration and time.
9 shows the results of processing a patient's sample in the hydrogel biosensor of the present invention, and measuring the fluorescence intensity according to time for each disease stage.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a bioprobe for detecting or determining a wound comprising a target peptide of metalloprotease-2 or metalloprotease-9. The present inventors confirmed the fact that wounds can be detected or staged by the type and concentration of extracellular matrix metalloprotease (MMP) among various substances isolated from the exudate of the affected area of patients with chronic skin wounds (bedsores). The invention was completed.

The 'wound' of the present invention is an injury that occurs when the skin is torn, peeled off, or punctured by injury, and depending on the symptoms or cause, burns, lacerations, epidermal wounds, chronic skin wounds, ulcers, trauma , post-surgical, childbirth, chronic wound, pressure ulcer, diabetic ulcer, damage caused by dermatitis, corneal ulcer, corneal epithelial detachment, keratitis and dry eye damage may be included. The wound of the present invention may be one or more selected from the above diseases, and particularly includes localized tissue necrosis, such as pressure ulcers, pressure sores, and chronic skin wounds.

In the present invention, 'extracellular matrix metalloprotease (Matrix metalloprotease, MMP)' is a proteolytic enzyme that plays a central role in the decomposition of the extracellular matrix, and is particularly closely related to pathological progression related to cancer cell invasion, metastasis, inflammation, etc. is known to have (Son et al. ,) MMP has so far more than 20 species (MMP-1, MMP-2, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-9) , MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19, MMP-20, MMP-21, MMP -22 and MMP-23) are known, and the MMP detected by the bioprobe of the present invention is not limited thereto, but preferably a peptide having substrate specificity for MMP-2 or MMP-9 may be included in the probe. And, it can be used for the purpose of diagnosing chronic skin wounds (bedsores) by detecting the MMPs.

The "bioprobe" of the present invention includes a target (substrate) peptide for MMP-2 or MMP-9 protease, a fluorescent donor and a fluorescent acceptor complex bound to the target peptide, and the MMP- When the target peptide is cleaved by 2 or MMP-9, it may exhibit fluorescence resonance energy transfer (FRET).

'Fluorescence Resonance Energy Transfer (FRET)' is a non-radiative energy transfer phenomenon that occurs between two fluorescent materials in different emission wavelength bands. ) is a phenomenon in which the excitation level energy of the fluorescence acceptor is transferred to the fluorescence acceptor, and emission from the fluorescence acceptor is observed, or the fluorescence decrease of the fluorescence donor is observed.

In the present invention, the 'fluorescence acceptor' constitutes a probe in a fused form with the target peptide on the principle of FRET, and is a substance that transmits energy absorbed from the outside, and the 'fluorescence donor' is an externally absorbed probe. As a material that transmits energy, it has a characteristic of absorbing light from a fluorescent material or a light source.

In the case of the fluorescent acceptor / donor, but not limited to the type, green fluorescent protein (GFP), modified green fluorescent protein (modified green fluorescent protein), enhanced green fluorescent protein (EGFP), red Fluorescent Protein (RFP), Enhanced Red Fluorescent Protein (ERFP), Blue Fluorescent Protein (BFP), Enhanced Blue Fluorescent Protein (EBFP), Yellow Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (EYFP), Indigo Fluorescent Protein (CFP), and a fluorescent protein such as enhanced indigo blue fluorescent protein (ECFP) can be selected and used. In addition, fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, fluorescein isothiocyanate Acid (FITC, Fluorescein isothiocyanate), Oregon green, Alexa Fluor, FAM, VIC, JOE, ROX, HEX, NED, PET, LIZ, Texas Red, TET, TRITC, TAMRA, a cyanine-based dye, and a fluorescent dye such as thiadicarbocyanine can be selected and used. In addition, Dabcyl (4-(dimethylaminoazo)benzene-4-carboxylic acid) may be used as a widely used acceptor. In an embodiment of the present invention, FITC and Dabcyl were used as the fluorescent donor/acceptor FRET pair.

That is, in the present invention, when the two substances (Dabcyl / FITC) maintain a certain distance, the energy transfer phenomenon (FRET) from the donor (FITC) to the acceptor (Dabcyl) occurs. Although it maintains a constant state (energy is transferred from the FITC part to Dabcyl), when the probe (Protease) reacts and the light of a specific wavelength is irradiated, cleavage between the peptides occurs and the distance between the two substances is increased. As the distance increases, the FRET phenomenon does not occur, so the phenomenon in which fluorescence occurs at the excited side was used.

In addition, the bioprobe of the present invention may further include a 'quencher' capable of quenching the fluorescence of the fluorescent molecule. As the 'quencher', a quenching dye capable of absorbing light may be used, and as long as it can cover the entire fluorescence spectrum or fluorescence of the highest absorption wavelength of the fluorescent dye, it may be used without limitation.

The "target peptide" of the present invention is a peptide recognized and cleaved by MMP-2 or MMP-9 of the present invention, and may include any base or amino acid sequence capable of being recognized and cleaved by MMP-2 or MMP-9. In an embodiment of the present invention, a bioprobe including a peptide consisting of the amino acid sequence of SEQ ID NO: 1 (Gly-Pro-Leu-Gly-Met-Trp-Ser-Arg-Lys-Cys-) was used.

In one embodiment of the present invention, the 'bio probe' is a target (substrate) peptide for MMP-2 or MMP-9 protease, and a fluorescent donor/acceptor complex bound to the target peptide is micro-patterned free male of the hydrogel. It binds to the mid group (MAL), and the complex may exist in a form conjugated to the surface of the hydrogel.

The hydrogel is a cross-linked network-type high molecular polymer composed of one or more monomers, and is not limited to the type thereof, and includes polyethylene glycol (PEG), polyacrylamide, polyacrylic acid and The same synthetic polymer or natural polymer such as hyaluronic acid, chitosan, or dextran can be selected and used. In an embodiment of the present invention, a PEG hydrogel was selected and used, which is a known biocompatible scaffold that reduces non-specific binding of proteins and cells, and is widely used as a basic material for biosensors and tissue engineering.

In one embodiment of the present invention, in the bioprobe, before the target peptide is cleaved, the acceptor/donor maintains a constant distance from each other while maintaining a state in which the acceptor FITC transfers energy to the donor Dabcyl. However, in the presence of MMP-2 or MMP-9, when light of a specific wavelength is irradiated, the target peptide is cleaved accordingly, and as the distance between FITC and Dabcyl increases, the FRET phenomenon does not occur anymore, Fluorescence is generated from the excited side.

More specifically, the target peptide is '-Gly-Pro-Leu-Gly-Met-Trp-Ser-Arg-Lys-Cys-', and the bioprobe including it is 'Dabcyl-Gly-Pro-Leu'. -Gly-Met-Trp-Ser-Arg-Lys(FITC)-Cys-'. Among the peptide sequences, 'Cys-' is linked to the free MAL group of the hydrogel by a thiol covalent bond. That is, when exposed to a specific wavelength in the Dabcyl-Gly-Pro-Leu-Gly-Met-Trp-Ser-Arg-Lys(FITC)-Cys-MAL-Group-hydrogel structure, the peptide bond of -Gly-Met- As it breaks, fluorescence is generated. In this study, Dabcyl-Gly-Pro-Leu-Gly- acts as an acceptor (Acceptor), and -Met-Trp-Ser-Arg-Lys(FITC)-Cys acts as a donor. In an embodiment of the present invention, the excitation wavelength used for measuring the fluorescence intensity of the bio-probe was 469±35 nm, and the emission wavelength was 525±39 nm, indicating green fluorescence upon light emission. The measurement was performed using a lens capable of measuring a GFP (Green Fluorescent Protein) wavelength, and the type is not limited as long as it can measure an increase in fluorescence intensity.

When MMP-2 or MMP-9 is added to the probe and irradiated with light of 469±35 nm, the peptide bond between '-Gly-Met-' is broken and the target peptide is 'Dabcyl-Gly-Pro-' Leu-Gly-' and 'Gly-Met-Trp-Ser-Arg-Lys(FITC)-Cys-'. At this time, the fluorescence was increased at the excited FITC side, and the emission wavelength was 525±39 nm, showing green fluorescence when emitted. It was confirmed that the intensity of fluorescence increases as the concentration of MMP-2 or MMP-9 increases.

As another aspect of the present invention, the present invention provides a composition for detecting or determining a wound comprising the bio-probe as an active ingredient.

The composition may be manufactured in the form of a biochip, a kit, a biosensor, etc. and used to detect a wound or determine the stage of a disease.

As another aspect of the present invention, the present invention provides a method comprising: contacting the bio-probe to a patient's lesion or sample; A method of providing information about a wound by detecting or determining a wound, comprising measuring a fluorescence resonance energy transfer caused by cleavage of a target peptide by MMP-9 or MMP-2 contained in the affected area or sample to provide.

The 'sample' of the present invention refers to a material to be analyzed that contains or is estimated to contain MMP-2 or MMP-9 to be analyzed, and may be a biological sample. The biological sample may include, but is not limited to, tissue, cells, whole blood, serum, plasma, tissue autopsy sample (skin, lymph node, spinal cord, brain, etc.), cell culture supernatant, ruptured eukaryotic cell or bacterial expression system, etc. there is. In particular, since the present invention is used for diagnosing a wounded patient and determining the degree of disease, the sample may be an exudate (blood, lymph, etc.) derived from the wounded patient.

Specifically, the method for providing information on the wound is based on analyzing the activity of MMP-2 or MMP-9 in a sample derived from the patient's lesion, and fluorescence intensity of a fluorescent protein or organic fluorescent dye. It is carried out by measuring with an analyzer or the like, and the fluorescence analyzer may be selected and used as long as it is used in the art.

Hereinafter, examples will be given to describe the present specification in detail. However, the embodiments according to the present specification may be modified in various other forms, and the scope of the present specification is not to be construed as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present specification to those of ordinary skill in the art.

Example 1. Sample Collection

In order to obtain exudates (human derivatives) from chronic skin wounds (bedsores), the research is conducted after approval (IRB No.: B-1805-468-305) by the Bioethics Review Committee of Seoul National University Bundang Hospital. did. For the protection of the subject, the exudate (sample) was collected from the patient's affected area after the written instructions and consent form for consent to the provision of human materials were prepared. In order to collect more exudates, exudates were collected from the lesion site in patients with pressure ulcers in their 20s and 80s in stage 3 of the National Pressure Ulcer Advisory Pane (NPUAP). The age and gender of the collected patients are shown in Table 1 below.

The target NPUAP stage 3 patient contains an ulcer that has caused a loss of wide and deep skin thickness and is expanding to the subcutaneous tissue layer. After washing the wound with 10mL saline, the exudate is collected, and the patient The exudate absorbed in the gauze that covered the wound area was collected, and the expression level of MMP-2 or MMP-9 was measured from the two samples.

Example 2. Preparation of bioprobes

2-1. Hydrogel microwell fabrication

The bioprobe of the present invention was prepared using a polyethylene glycol (PEG) hydrogel. The functional hydrogel composition and its micropatterning method were prepared according to the method described in the known literature (son et al, Anal Chem. 2013 December 17; 85(24): 11893-11901). Hydrogel rings (inside diameter 100 μm, outside diameter 200 μm) were prepared by UV-initiated polymerization of PEG-DA and Acryl-PEG-Mal. Dehydrate 20% (v/v) PEG-DA (MW 700), 40% (v/v) PEG (MW 200), 10% (v/v) 20 mM Acryl-PEG-Mal and 1% Irgacure 2959 Monomer solutions for functional hydrogels were prepared by dissolving them in ionized water. The monomer solution was loaded onto the silane-treated glass and then covered with a cover glass (24*30*0.13 mm) to create a uniform monomer solution layer. Slides were irradiated through a photomask for 1.5 s by a 365 nm UV light source ( ^{80 mW/cm 2} ; OmniCure Series 1000, Lumen Dynamics Group, Lumen Dynamics Group, Luman Dynamics Group, Lusion Dynamics Group, Ontario, Canada), followed by deionized water. After washing with nitrogen gas, it was dried.

2-2 Conjugation of target peptide and hydrogel

As a target peptide sequence having substrate specificity for MMP-2 or MMP-9, a peptide of the Gly-Pro-Leu-Gly-Met-Trp-Ser-Arg-Lys-Cys (GPLGMWSRKC) sequence (GL Biochem, Shanghai, China) ) to FITC and DABCYL as a FRET acceptor/donor pair to prepare a peptide fusion (Dabcyl-Gly-Pro-Leu-Gly-Met-Trp-Ser-Arg-Lys (FITC)-Cys-). The peptide was incubated with the hydrogel prepared in 2-1. The free MAL group (Maleimide group) on the hydrogel and the Cystein group of the peptide were bonded to each other by a thiol covalent bond to prepare a hydrogel microarray in which the peptide fusion was fixed on the surface of the hydrogel.

The manufacturing process is shown in FIGS. 1 and 2 .

Example 3. Confirmation of the expression level of MMP through ELISA

3-1. Confirmation of expression level of MMP-2

The expression level of MMP-2 contained in the sample collected from Example 1 was confirmed using the target peptide prepared in Example 2 using the ELISA method.

에서 밤새 인큐베이션하여 면역 플레이트 상에 고정시켰다. 1차 항체를 코팅시킨 플레이트를 PBS, 0.1 % Tween 20 (PBST)으로 1회 세척하고, 200㎕의 블로킹 용액 (PBS, 2 % BSA, 0.02 % NaN₃)을 2시간 동안 실온에서 처리하였다. 삼출물을 코팅된 플레이트에 첨가하고 실온에서 2시간 동안 인큐베이션 하였다. PBST로 플레이트를 3회 세척한 후, 0.1 % BSA로 희석된 MMP 이차 항체를 첨가하고 플레이트를 1시간 30분 동안 인큐베이션 하였다. 이어서, 플레이트를 PBST로 3회 세척하고 PBS에 희석된 스트렙타비딘 퍼옥시다제 (0.3μg/ml) 100 ul를 처리하였다. PBST로 3회 세척 후, 효소 반응 발색을 위해 100 μl의 TMB 용액을 첨가 후 100 μl의 1M H₂SO₄를 첨가하여 중단시켰다. 흡광도는 450nm에서의 마이크로 플레이트 리더를 사용하여 검출하였다.First, MMP primary antibody dissolved in PBS solution was

It was incubated overnight and fixed on an immunoplate. The plate coated with the primary antibody was washed once with PBS, 0.1 % Tween 20 (PBST), and 200 μl of a blocking solution (PBS, 2 % BSA, 0.02 % NaN ₃ ) was treated at room temperature for 2 hours. The exudate was added to the coated plate and incubated at room temperature for 2 hours. After washing the plate 3 times with PBST, an MMP secondary antibody diluted with 0.1% BSA was added and the plate was incubated for 1 hour and 30 minutes. Then, the plate was washed 3 times with PBST and treated with 100 ul of streptavidin peroxidase (0.3 μg/ml) diluted in PBS. After washing 3 times with PBST, 100 μl of TMB solution was added for enzymatic color development, and then 100 μl of 1M H ₂ SO ₄ was added to stop it. Absorbance was detected using a microplate reader at 450 nm.

As a result, as shown in FIG. 3A , when the exudate derived from the affected area was diluted with saline and measured, MMP_2 was detected at concentrations of 0.63, 0.84, 0.40, and 3.80 ng/mL, respectively, depending on the severity of the wound. In addition, as shown in FIG. 3B , in the case of a sample directly taken from the gauze in which the exudate was absorbed, concentrations of 1.90, 4.89, 1.25 and 22.19 ng / mL were measured, respectively. Although there was a difference in concentration in both the case of direct measurement and the case of dilution measurement, it was confirmed that there was the same effect in determining the degree of disease.

3-2. Confirmation of expression level of MMP-9

In the same manner as in 3-1, the expression level of MMP-9 contained in the sample collected from Example 1 was confirmed using the target peptide prepared in Example 2 using the ELISA method.

As a result, as shown in FIG. 4A, when the exudate derived from the affected area was diluted with saline and measured, concentrations of 0.63, 2.75, 19.13, and 32.82 ng/mL were detected, respectively, depending on the degree of wound deepening, as shown in FIG. 4B. , in the case of samples taken directly from the gauze in which the exudate was absorbed, the concentrations were 7.55, 7.84, 278.22 and 2987.30 ng/mL, respectively.

From the above results, it was confirmed that the expression level of MMP-9 was remarkably proportional to the stage and severity of the wound, and in particular, it was confirmed that the expression level of MMP-2 was increased by 1 to 100 times than that of MMP-2.

3-3. Confirmation of expression levels of MMP-2 and MMP-9 from frozen samples

After cryopreserving each of the collected samples, the concentrations of MMP-2 and MMP-9 before and after cryopreservation were measured in the same manner as in 3-1 and 3-2. As a result, as shown in FIG. 5 , it was confirmed that the expression levels of MMP-2 and MMP-9 were preserved even after freezing storage in both diluted and undiluted samples of the exudate of the patient.

Example 4. Confirmation of expression level of MMP-9 through fluorescence-based micropatterning hydrogel biosensor

Using the hydrogel biosensor prepared in Example 2, the change in fluorescence intensity according to the concentration of MMP-9 was measured.

In the prepared hydrogel biosensor, MMP-9 was dissolved in working buffer (50 mM Tris-HCl, 1 mM CaCl2, and 0.05% Triton X-100; pH 7.5) to obtain 0, 0.16, 0.31, 1.25, 2.5, Each was treated at a concentration of 5 μg/ml for 20 minutes, The intensity of fluorescence was measured using a fluorescence microscope (Lionheart FX fluorescence microscope (BioTek Instruments, Inc., Winooski, VT, USA). When measured, the excitation wavelength is 469±35 nm and the emission wavelength is 525±39 nm. Green fluorescence is indicated For the image analysis for the measurement, the fluorescence of each hydrogel was measured over time using ZEN software (Carl Zeiss Microscopy, Jena, Germany).

As a result, as shown in FIGS. 6 and 7 , it was confirmed that, according to the concentration of MMP-9, the fluorescence intensity of the biosensor increased in proportion to the concentration.

In addition, in order to observe the intensity of fluorescence according to the treatment time, after treating the biosensor with MMP-9, the fluorescence intensity was measured for 60 minutes at 10-minute intervals. As a result, as shown in FIG. 8, when the hydrogel biosensor of the present invention was incubated with MMP-9, it was confirmed that the fluorescence intensity increased and then saturated at a certain time (20 minutes).

In addition, the hydrogel biosensor was incubated with the exudate sample of the patient collected in Example 1, and the intensity of fluorescence over time was measured in the same manner. As a result, as shown in FIG. 9 , when an exudate sample of an actual pressure ulcer patient was treated, the fluorescence intensity increased over time, and it was confirmed that the saturation state was reached. The time to reach the saturation state was different depending on the severity of the disease, but it was confirmed that most of the samples of bedsore patients increased the fluorescence intensity of the biosensor of the present invention. However, when the exudate of the actual patient is applied, it was confirmed that substances other than MMP-9 may affect the measurement results as the stage becomes longer.

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated by the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (17)

A bioprobe for detecting or determining a wound comprising a target peptide of metalloprotease-2 (MMP-2) or metalloprotease-9 (MMP-9).
The bio-probe for detecting or determining a wound according to claim 1, wherein the bio-probe further comprises a detectable label.
3. The method of claim 2, wherein the detectable label is a luminescent molecule, a chemiluminescent molecule, a fluorochrome, a fluorescent quencher, a lipid, a colored molecule, a radioactive isotope, a scintillator, biotin, avidin, streptavidin, protein A, protein G, antibody or fragment thereof, polyhistidine, Ni2+, Flag tag, myc tag, heavy metal, and a bioprobe for wound detection or determination, characterized in that at least one selected from the group consisting of enzymes.
The bioprobe for detecting or determining a wound according to claim 1, wherein the bioprobe comprises a complex of a donor and a fluorescent acceptor, which is a fluorescent substance bound to the target peptide.
[Claim 5] The bio-probe for detecting or judging a wound according to claim 4, wherein the fluorescent material is selected from a fluorescent protein and an organic fluorescent dye.
The method of claim 5, wherein the fluorescent protein is green fluorescent protein (GFP), modified green fluorescent protein (MGFP), enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), consisting of blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), indigo fluorescent protein (CFP), and enhanced indigo blue fluorescent protein (ECFP) A bioprobe for detecting or judging a wound, characterized in that at least one selected from the group.
According to claim 5, wherein the organic fluorescent dye is fluorescein (fluorescein), fluorescein chlorotriazinyl (fluorescein chlorotriazinyl), rhodamine green (rhodamine green), rhodamine red (rhodamine red), tetramethyl rhodamine ( tetramethylrhodamine), Fluorescein isothiocyanate (FITC, Fluorescein isothiocyanate), Oregon green, Alexa Fluor, FAM, VIC, JOE, ROX, HEX, NED, PET, LIZ, Texas Red (Texas Red), TET, TRITC, TAMRA, cyanine (Cyanine) dyes and thiadicarbocyanine (thiadicarbocyanine) for wound detection or determination bioprobe, characterized in that selected from the group consisting of.
According to claim 1, wherein the bio-probe exhibits fluorescence resonance energy transfer (FRET) upon cleavage of the target peptide by MMP-2 or MMP-9, the bio-probe for wound detection or determination.
The bioprobe for detecting or determining a wound according to claim 1, wherein the target peptide is GPLGMWSRKC.
The bio-probe for detecting or determining a wound according to claim 1, wherein the bio-probe is Dabcyl-GPLGMWSRK(FITC)C.
The method of claim 1, wherein the wound is burns, lacerations, epidermal wounds, ulcers, trauma, post-surgical, childbirth, chronic wounds, bedsores, pressure ulcers, diabetic ulcers, dermatitis ( dermatitis) damage, corneal ulcer, corneal epithelial detachment, keratitis, and any one or more selected from the group consisting of damage due to dry eye, a bioprobe for detecting or judging a wound.
A composition for detecting or determining a wound comprising the bio-probe according to any one of claims 1 to 11 as an active ingredient.
A biochip for detecting or determining a wound comprising the bioprobe according to any one of claims 1 to 11.
A bio-kit for detecting or determining a wound comprising the bio-probe according to any one of claims 1 to 11.
The biosensor for detecting or judging a wound according to any one of claims 1 to 11, wherein the bioprobe is bonded to a hydrogel.
The method of any one of claims 1 to 11, comprising: contacting the bioprobe according to any one of claims 1 to 11 to the affected part of the patient or treating the sample; and
A method of providing information related to detection or determination of a wound, comprising the step of analyzing MMP-2 or MMP-9 activity by the bio-probe.
The method of claim 16, wherein the analyzing the activity of MMP-2 or MMP-9 is performed by measuring the intensity of a fluorescent material included in the bio-probe. How to provide.

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