KR102154990B1 - Composition for bio-signal measurement and stimulation containing biocompatible conductive protein gel - Google Patents

Abstract

The present invention relates to a composition for measuring a living body signal or a composition for stimulating a living body including a biocompatible conductive protein gel containing a complex of a biocompatible protein and a conductive nanomaterial, and a use thereof, and biocompatibility that has emerged as a limitation of a conventional metal-based electrode. By solving the problem of hypermechanical strength and providing a non-degradable material, it is possible to measure and observe a minute electric signal with high sensitivity for a long period of time, providing excellent use for diagnosis and treatment of diseases.

Description

A composition for measuring and stimulating a biological signal including a biocompatible conductive protein gel {COMPOSITION FOR BIO-SIGNAL MEASUREMENT AND STIMULATION CONTAINING BIOCOMPATIBLE CONDUCTIVE PROTEIN GEL}

The present invention is a composition for measuring or stimulating a living body comprising a biocompatible conductive protein gel containing a complex of a biocompatible protein and a conductive nanomaterial, its use, a biosignal measurement and biostimulation system using the same, and a degenerative brain disease using the same It is about how to provide information to diagnose.

Bioelectrical signals, including brain neural activation signals and neural signals, contain a lot of information on physiodynamic activities, so observing and measuring them plays a role in understanding the phenomena occurring in the body and further discovering and treating diseases. very important. In particular, neurological disorders such as epilepsy can cause chronic damage to the central nervous system by causing abnormal electrical signals of cranial nerve cells, so not only measuring changes in cranial nerve currents that occur instantaneously, but also monitoring and analyzing for a long time to determine the progression of the disease It is necessary to do. However, the strength of a living body signal is very small, so there is a limitation in measuring a minute current with high sensitivity outside the living body. Therefore, the signal is measured after implantation in the body using an implantable neural electrode. Existing electrodes are mainly made of metal with excellent conductivity. However, since metal-based electrodes have relatively high mechanical strength compared to surrounding tissues, it is easy to cause wounds due to mechanical disharmony at the implantation site, and when implanted in the body for a long period of time, it causes a foreign body reaction, thus limiting the detection of nerve signals due to inflammation. In particular, in the case of the brain, which has the lowest mechanical strength among organs in the body, the above problems due to metal electrodes may become more prominent, and thus it is necessary to develop a biocompatible and flexible material that can replace metal. In order to solve these problems, research to amplify microcurrents by developing electrodes using polymer polymers and mesh-type electrodes having similar flexibility to biological tissues has been conducted, but research on developing new conductive materials in terms of materials is insufficient. to be.

As a solution to this problem, biocompatible hydrogels have excellent strengths in terms of biomaterials, and have advantages such as high moisture content and viscoelasticity control, so they are being studied in various fields such as drug delivery systems, biosensors, and tissue engineering. In particular, hydrogels are in the spotlight as implantable materials because they have the same flexibility as living tissues. Biocompatible hydrogels have been studied a lot based on natural proteins such as collagen and elastin, which have secured biosafety, but natural proteins have the disadvantage that it is difficult to separate and purify proteins, and chemical treatment for application due to the complexity of the structure is difficult. have. As the knowledge of the protein structure and protein self-assembly principle is expanding in recent years, self-assembly peptides through protein design and manipulation are in the spotlight as hydrogel components. Peptides made through design are easy to understand physical properties through the establishment of a predetermined tertiary structure, and can be used as useful materials by giving functionality that does not exist in existing natural proteins, so research is actively being conducted worldwide. However, biomaterials formed through existing self-assembled peptides have problems in biostability and persistence due to in vivo peptide-degrading enzymes. In addition, since it does not have electrical properties, there is a limitation in application, and biocompatibility cannot be guaranteed because a conductive material must be bonded through a chemical process.

Korean Patent Publication No. 10-2012-006138

Accordingly, the present inventors have completed the present invention by discovering that a biocompatible conductive protein gel that does not have a rejection reaction in a living body and is not degraded can be used for measuring a biological signal and stimulating a living body.

Accordingly, an object of the present invention is a composition for measuring or stimulating a bio-signal comprising a biocompatible conductive protein gel containing a complex of a biocompatible protein and a conductive nanomaterial, its use, a bio-signal measurement and bio-stimulation system using the same, and It is to provide a method of providing information for diagnosing degenerative brain diseases used.

In order to achieve the above object, a first aspect of the present invention provides a composition for measuring a biological signal or stimulating a living body comprising a biocompatible conductive protein gel containing a complex of a biocompatible protein and a conductive nanomaterial.

A second aspect of the present invention provides a medical implant comprising the composition for measuring a living body signal or stimulating a living body according to the present invention.

A third aspect of the present invention provides a medical electrode comprising the composition for measuring a living body signal or stimulating a living body according to the present invention.

A fourth aspect of the present invention is an electrical stimulation generator; An electrode electrically connected to the electrical stimulation generator; And it provides a biological signal measurement and biological stimulation system comprising a medical implant according to the present invention connected to the electrode.

A fifth aspect of the present invention is an electrical stimulation generator; And a medical electrode according to the present invention that is electrically connected to the electrical stimulation generator.

A sixth aspect of the present invention is the step of inserting a medical implant or a medical electrode according to the present invention into brain tissue; Monitoring brain nerve signals by applying electrical stimulation; And it provides a method of providing information for diagnosing degenerative brain disease comprising the step of comparing the monitored brain nerve signal with a normal brain nerve signal.

A seventh aspect of the present invention provides a skull substitute comprising the composition for measuring or stimulating a living body according to the present invention.

The present invention relates to a novel bio-signal measurement and biostimulation use of a biocompatible conductive protein gel, solving the problems of biocompatibility and mechanical strength, which have emerged as limitations of conventional metal-based electrodes, and providing a non-degradable material It enables long-term measurement and observation of signals with high sensitivity. In particular, the biocompatible conductive protein gel of the present invention can measure minute signals of the brain or nerves for diagnosis of diseases such as degenerative brain diseases and neurological diseases, so that diseases can be accurately and quickly diagnosed. In addition, by controlling the concentration and conductivity of the biocompatible conductive protein gel, it is possible to measure various biological signals, so that it can be applied to diagnosis of various diseases.

In addition, the biocompatible conductive protein gel of the present invention can be applied to treatment methods such as deep brain stimulation because electrical stimulation can be applied to living tissues.

1A shows maldi-tope data of a fluorescently labeled β-peptide according to an embodiment of the present invention.
1B shows the absorbance (black) and spectral (red) spectra of the fluorescently labeled β-peptide according to an embodiment of the present invention.
1C is an electron microscope image taken after injecting a conductive protein gel containing a fluorescently-labeled β-peptide according to an embodiment of the present invention into the brain of a mouse.
Figure 2a shows the results of measuring the storage elasticity (G') and loss elasticity (G") according to the concentration of the conductive protein gel in the strain mode according to an embodiment of the present invention.
2B shows the results of measuring storage elasticity (G') and loss elasticity (G") according to the concentration of the conductive protein gel in a frequency sweep mode according to an embodiment of the present invention.
2C is a photograph of a protein gel prepared by increasing the concentration of the β-peptide/carbon nanotube complex to 0, 0.004, 0.020, and 0.040% unit volume weight according to an embodiment of the present invention.
2D is a graph showing the storage elasticity (G') according to the concentration of the β-peptide/carbon nanotube complex in the frequency sweep mode according to an embodiment of the present invention.
3A shows the result of measuring the conductivity of the protein gel according to the concentration of the β-peptide/carbon nanotube complex in an embodiment of the present invention.
3B shows the results of measuring the impedance of the protein gel according to the concentration of the β-peptide/carbon nanotube complex in an embodiment of the present invention.
4A shows the results of in vitro biocompatible cell experiments of laminin, protein gel, and conductive protein gel according to an embodiment of the present invention.
4B shows absorbance results showing cell proliferation of laminin, protein gels, and conductive protein gels over time in an embodiment of the present invention.
4C shows the results of an in vivo biocompatibility experiment of a conductive protein gel according to an embodiment of the present invention. Green fluorescence: microglia, red fluorescence: Astrosite.
Figure 5a schematically shows the epilepsy mouse model experiment according to an embodiment of the present invention.
Figure 5b shows the EEG signal measurement results measured in the epilepsy mouse model experiment according to an embodiment of the present invention. EEG 1 (blue), EEG 2 (red).
Figure 6a shows a glass pipet for injection containing a conductive protein gel in an embodiment of the present invention.
6B shows chronic recording data obtained by measuring a brain tissue injected with a protein gel and an EEG signal in an epilepsy mouse model experiment according to an embodiment of the present invention.

Hereinafter, embodiments and embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art can easily implement the present invention.

However, the present invention may be implemented in various different forms, and is not limited to the embodiments and examples described herein. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present invention, and similar reference numerals are assigned to similar parts throughout the specification.

Throughout the specification of the present invention, when a member is said to be located "on" another member, this includes not only the case where a member is in contact with the other member, but also the case where another member exists between the two members.

Throughout the specification of the present invention, when a certain part "includes" a certain constituent element, it means that other constituent elements may be further included rather than excluding other constituent elements unless otherwise stated. The terms "about", "substantially", etc. of the degree used throughout the specification of the present invention are used at or close to the numerical value when manufacturing and material tolerances specific to the stated meaning are presented, and the present invention To aid in understanding of, accurate or absolute figures are used to prevent unreasonable use of the stated disclosure by unscrupulous infringers. As used throughout the specification of the present invention, the term "step (to)" or "step of" does not mean "step for".

Throughout the specification of the present invention, the term "combination of these" included in the expression of the Makushi format refers to one or more mixtures or combinations selected from the group consisting of components described in the expression of the Makushi format, the It means to include one or more selected from the group consisting of components.

The present invention provides a composition for measuring a living body signal or stimulating a living body comprising a biocompatible conductive protein gel containing a complex of a biocompatible protein and a conductive nanomaterial.

In one embodiment of the present invention, the complex of the biocompatible protein and the conductive nanomaterial is in a concentration of 0.001 to 10% w/v in the biocompatible conductive protein gel, for example, 0.001 to 1% w/v, 0.001 to It may be included in a concentration of 0.5% w/v or 0.004 to 0.04% w/v.

In one embodiment of the present invention, the biocompatible protein may include an amino acid sequence consisting of β-amino acids. The biocompatible protein may include an amino acid sequence represented by SEQ ID NO: 1, for example, may include a sequence of (KVKEVFFVKEVFFVKEVY) n (1 ≤ n ≤ 3) by repetition of the sequence of SEQ ID NO: 1. . Lysine (K) may be added to the N-terminus or C-terminus of the amino acid sequence.

In one embodiment of the present invention, since the biocompatible protein is not degraded by an enzyme, for example, a proteolytic enzyme, the biocompatible conductive protein gel can be stably maintained in vivo. The enzyme includes, for example, trypsin, chymotrypsin, subtilisin, and the like.

In one embodiment of the present invention, the conductive nanomaterial is a carbon nanotube (CNT), fullerene (C60), a conductive polymer nanowire, a conductive polymer nanotube, a conductive polymer nanoparticle, a metal nanowire, It may be selected from the group consisting of metal nanoparticles and combinations thereof, and may be, for example, carbon nanotubes.

In one embodiment of the present invention, the complex of the biocompatible protein and the conductive nanomaterial is prepared by dissolving the biocompatible protein in a buffer solution to prepare a protein solution; Preparing a conductive nanomaterial aqueous solution by dispersing the conductive nanomaterial in an aqueous glycerol solution; Forming a complex by mixing the protein solution and the conductive nanomaterial aqueous solution; Concentrating the complex; And adding a biocompatible protein subjected to fibrosis to the concentrated complex, followed by gelation.

The step of forming the complex may be formed by self-assembling proteins in nanomaterials. When the complex is formed, the protein solution and the conductive nanomaterial aqueous solution may be mixed in a mass ratio of 1:1 to 10:1, and the protein may have a concentration of 1 mg/mL or more. Various biological signals can be measured by adjusting the ratio of the complex of the biocompatible protein and the conductive nanomaterial according to the biological signal to be measured.

In addition, the present invention provides a medical implant and a medical electrode comprising a biocompatible conductive protein gel containing a complex of a biocompatible protein and a conductive nanomaterial.

In one embodiment of the present invention, the medical implant and the medical electrode may be implanted in a living tissue, for example, brain tissue, cerebral blood vessels, cartilage, spinal cord, muscle, skin, and the like.

The medical implant can be implanted into a living body and then connected to an electrode to receive electrical stimulation, so that a living body signal can be measured and an electrical stimulation can be applied to the living body. Accordingly, the medical implant can be used for diagnosis and treatment of degenerative brain diseases, neurological diseases, pain, arthritis, and the like.

In the medical electrode, a biocompatible conductive protein gel containing a complex of a biocompatible protein and a conductive nanomaterial according to the present invention may be fixed on the surface of the electrode. The medical electrode can receive electrical stimulation after being implanted in a living tissue, so that a living body signal can be measured, and an electrical stimulation can be applied to the living body. Accordingly, the medical implant can be used for diagnosis and treatment of degenerative brain diseases, neurological diseases, pain, arthritis, and the like.

In addition, the present invention provides a bio-signal measurement and bio-stimulation system including a medical implant or medical electrode according to the present invention.

In one embodiment of the present invention, the bio-signal measurement and bio-stimulation system comprises: an electrical stimulation generator; An electrode electrically connected to the electrical stimulation generator; And a medical implant according to the present invention connected to the electrode, or an electrical stimulation generator; It may be configured to include a medical electrode according to the present invention electrically connected to the electrical stimulation generator. The bio-signal measurement and bio-stimulation system may further include a Fourier transducer that converts the measured bio-signal, an information analyzer that processes the converted signal, and a display that displays processed information.

In one embodiment of the present invention, the medical implant and the medical electrode may be implanted in a nervous system, for example, a body part that generates electrical signals throughout the body. Specifically, the medical implants and medical electrodes may be implanted into biological tissues such as brain tissue, cerebral blood vessels, cartilage, spinal cord, muscle, skin, etc., and by measuring a biological signal by transmitting electrical stimulation to the biological tissue after transplantation The onset or progression of the disease can be checked, and the disease can be treated by applying electrical stimulation to the living tissue.

In one embodiment of the present invention, the electrical stimulation may have a microvoltage, for example, a voltage of about 1 mV to about 10 V and a frequency of about 1 to about 200 hz. For example, the electrical stimulation applied to the mouse may be about 1 mV to about 5 mV, and the electrical stimulation applied to the human may be about 0.5 V to about 5 V, but is not limited thereto.

In one embodiment of the present invention, the disease may include degenerative brain disease, neurological disease, pain, arthritis, spinal cord disease, neuronal disease, muscle disease, and the like. The degenerative brain disease may include, for example, Alzheimer's disease, Parkinson's disease, stroke, dementia, Huntington's disease, Pick's disease, and Creutzfeld-Jakob's disease. The neurological diseases may include, for example, epilepsy, seizures, seizure-related disorders, rapid brain injury, chronic brain damage, chronic headache, migraine, movement disorders, and the like.

In one embodiment of the present invention, the biological signal measurement and biological stimulation system may be used for deep brain stimulation. Deep brain stimulation is an electrical therapy for treating epilepsy, pain, Parkinson's disease, etc. A medical device called a cerebral coordinator is implanted into the brain to measure signals from the brain and send electrical stimulation to a specific part of the brain to send chronic pain, Parkinson's disease. , Tremors, and dystonia are known as effective treatment methods. The medical implant or the medical electrode according to the present invention may be implanted into the brain using a cerebral regulator to stimulate deep brain, and thus can be effectively applied to the treatment of diseases.

In addition, the present invention includes the steps of inserting a medical implant or medical electrode according to the present invention into brain tissue; Monitoring brain nerve signals by applying electrical stimulation; And it provides a method of providing information for diagnosing degenerative brain disease comprising the step of comparing the monitored brain nerve signal with a normal brain nerve signal.

In one embodiment of the present invention, the degenerative brain disease may include Alzheimer's disease, Parkinson's disease, stroke, dementia, Huntington's disease, Pick's disease, Creutzfeld-Jakob's disease, and the like.

In addition, the present invention provides a cranial substitute comprising a composition for measuring or stimulating a biological signal comprising a biocompatible conductive protein gel containing a complex of a biocompatible protein and a conductive nanomaterial.

In one embodiment of the present invention, the skull substitute may be implanted in the skull and then subjected to electrical stimulation through it, so that the onset or progression of a disease is confirmed by measuring a biological signal, and an electrical stimulation is applied to the biological tissue Can cure.

Advantages and features of the present invention, and a method of achieving them will become apparent with reference to embodiments described below in detail. Hereinafter, the present invention will be described in detail by examples. However, these examples are intended to specifically illustrate the present invention, and the scope of the present invention is not limited to these examples.

[ Example ]

Manufacturing example 1. Preparation of biocompatible conductive protein gel

Biocompatible β- Peptide Produce

The β-peptide was synthesized using a microwave synthesizer (Discover SPS, 200-240V/50/60Hz) of CEM. Using the solid-phase peptide synthesis method, the C-terminal β-amino acid activated with HBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) is converted into an inactive solid H-rink amide resin ( PCAS 0.53mmol substitution) was synthesized by Fmoc (fluorenylmethyloxycarbonyl carbamate) method.

First, the resin was expanded in N,N-dimethylformamide (DMF) for 30 minutes and then deprotected 3 times at 80° C. for 2 minutes using 20% piperidine (DMF solution). After that, when the β-amino acid was coupled to the solid support, the ratio of amino acid: HBTU: DIEA (N,N-Diisopropylethylamine): resin was 3:2.5:4:1. After mixing β-amino acid, HBTU, and DIEA well, the reaction in the resin was carried out three times at 70°C for 2 minutes.

After each deprotection and coupling step, it was washed three times, alternately with DMF and methylene chloride (DCM). After 6 residues from the C-terminus were synthesized, the deprotection and coupling reaction time was increased to 4 minutes, and after 12 residues were synthesized, the reaction time of the deprotection was increased to 5 minutes while increasing to 8 minutes. After all β-amino acid residues were synthesized, the Fmoc protecting group of the amino acid at the N-terminus was removed by the same deprotection reaction as in the previous step. Finally, samples that may remain in ethanol were removed and dried under vacuum.

Separation from the solid resin was carried out by reacting the resin synthesized at 95:2.5:2.5 with Clivis solution TFA (Trifluoroacetic acid):TIS (Triisopropylsilane):distilled water for 2 hours, and then the resin was filtered with an asbestos filter. The Clivis solution in the filtered solution was evaporated under nitrogen gas, and when a precipitate was formed, it was precipitated with diethyl ether stored cold. The substance precipitated here is a β-peptide. After drying the β-peptide precipitate in a vacuum state, it was dissolved in distilled water and acetonitrile (ACN) and lyophilized.

The lyophilized β-peptide was subjected to maldi-tope mass spectrometry to confirm the mass 2516 of the biocompatible protein β-VhexS of the present invention.

The β-peptide synthesized by the solid-phase peptide method requires purification using high-performance liquid chromatography to increase the purity, so it was purified using a Waters prep 150 LC system and an XBridge BEH300 Prep C4 5um column.

Biocompatible β- Peptide Gel manufacturing

In order to prepare a physical gel of β-protein, the lyophilized β-protein was stored at room temperature for about 30 minutes to adjust the β-protein temperature to room temperature (15°C to 25°C). A protein gel was prepared by dissolving the protein adjusted to room temperature in a buffer solution and ultrapure water according to the concentration and pH conditions and storing for about 3 hours. In other words, in order to prepare a protein gel having different physical properties, gels according to the concentration of the protein (1.25, 2.5, 5, 10, 20, 30, 50 mg/mL) were prepared, and also proteins with different chemical properties. To prepare a gel, a protein gel was prepared according to the pH of the solvent dissolving the protein (pH 4.5, 6.0, 7.5, 9.0, 10.5).

β- Peptide / CNT Composite manufacturing

After taking 20mg of single-walled carbon nanotube (CNT) in a 50mL Falcon tube, it was dissolved in 1 wt% 20mL glycerol aqueous solution. For SWNT dispersion, the tip of the tip sonicator (max power 125W) was placed at 1/3 of the prepared solution, and ultrasonic waves were applied for 20 minutes at 2 seconds/1 second on/off with 40% power. At this time, the Falcon tube containing the solution was sufficiently immersed in ice. As a result of the ultrasound, the SWNT glycerol solution exhibited even black light. Transfer 3 mL of this solution to a 15 mL Falcon tube and centrifuge at 7,000 rpm for 10 minutes to settle the CNTs.

After removing all the supernatant with a Pasteur pipette, 3 mL of distilled water was added to the settled SWNT and redispersed. At this time, if the dispersion was not good, ultrasonic waves were applied. After centrifuging the redispersed solution, the same process was repeated twice.

5.5 mg of β-peptide was taken and dissolved in 5 mL 20 mM Tris, 100 mM NaCl buffer, and the peptide solution was transferred to a Falcon tube with SWNT from which glycerol was removed. The falcon tube containing this solution was sufficiently immersed in ice, and ultrasonic waves were applied for 30 minutes at 60% power and 2 seconds/1 second on/off conditions using a tip sonicator. As a result, the resulting black solution was dispersed by surrounding the CNT with β-peptide. The dark supernatant obtained by centrifuging for 10 minutes at 7,000 rpm to selectively obtain only CNTs dispersed by self-assembly of the peptides in this solution is the β-peptide/CNT complex of the present invention.

conductivity Peptide Gel manufacturing

The synthesized β-peptide/SWNT was concentrated 10 times using a centrifugal filter device (Microcon-30kDa, MRCF0R030).

In this preparation example, each 1 mL of β-peptide/SWNT solution was concentrated to 100 μL. Concentrated β-peptide/CNT solution and 100uL of a 20mg/mL β-peptide solution whose viscosity had already increased due to fibrosis in a pH 7.4 Tris-HCl buffer solution were mixed and stored at room temperature for one day. In order to adjust the final concentration to 20 mg/mL, a conductive peptide gel was completed through a 2-fold reconcentration process using a centrifugal filter device.

Example 1. Fluorescent β- Peptide - Cy3 Manufacturing and absorbance analysis

When the β-peptide gel synthesized as in Preparation Example 1 was injected into the body, in order to confirm its location by fluorescence, β-peptide and Cyanine 3 carboxylic acids (Cy3, Lumiprobe) were conjugated to show fluorescence. For effective synthesis, a microwave synthesizer of CEM (Discover SPS, 200-240V/50/60Hz) was used. First, after synthesis of the last β-amino acid of β-peptide synthesized in solid resin, the Fmoc protecting group was 8 at 80℃ using 80% N,N-dimethylformamide (DMF) and 20% piperidine solution. It was deprotected 5 times for a minute. After that, Cy3:HBTU(2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate):DIEA(N,N-Diisopropylethylamine):resin is 1.5:2.5:4:1 After mixing well, the reaction was carried out in a resin at 73° C., 11 minutes, and 30W. After that, magnetic stirring was performed at room temperature for 1 hour. During the synthesis process, the resin color changed from yellow to reddish pink. After that, it was washed three times with DMF and methylene chloride (dichloromethane, DCM) alternately.

Separation from the solid resin was carried out by reacting the resin synthesized at 95:2.5:2.5 with Clivage solution TFA (Trifluoroacetic acid):TIS (Triisopropylsilane):distilled water through magnetic stirring for 2 hours, and then the resin was filtered with an asbestos filter. The solution in the filtered solution was evaporated under nitrogen gas, and when a precipitate formed, it was precipitated with diethyl ether stored cold. The precipitated β-peptide/cy3 was dried in a vacuum state, dissolved in distilled water, and lyophilized. The lyophilized β-peptide/Cy3 was confirmed to have a mass of 2956.3 through maldi-tope mass spectrometry (FIG. 1A).

The absorbance and spectral spectra were obtained and quantified through photoluminescence spectrum analysis of the synthesized β-peptide/Cy3 (Fig. 1b).

1C is a photograph of a protein gel containing β-peptide/Cy3 injected into the mouse cerebral cortex and observed with a two-photon electron microscope, and shows an example of the use of β-peptide/Cy3.

Example 2. β- Peptide Gel and conductivity β- Peptide Analysis of the physical properties of the gel

Measurement of a gap of 0.2 mm at 25°C using a 25 mm parallel plate of a dynamic rheometer (MCR 302, Anton Paar) to check the physical properties of the β-peptide gel and the conductive protein gel containing the β-peptide/CNT complex depending on the concentration The viscoelasticity of the protein gel was measured under conditions. For β-peptide gel, samples were prepared at concentrations of 30 and 50 mg/ml, respectively. First, strain was observed at an amplitude of 1 rad/s at each frequency from 0 to 10% in order to confirm the linear viscoelastic region where the physical properties did not change. As a result, it was confirmed that the protein gel having a concentration of 50 mg/ml had a linear limit of 1% and that the 30 mg/ml protein gel had a higher value (FIG. 2A). In this result, the difference between the storage elasticity (G') representing the elasticity of the sample and the loss elasticity (G") representing the properties of the viscous part of the 50 mg/ml protein gel is the difference between G'and G" of 30 mg/ml protein. It was found that it had a more solid property at a higher concentration than that. The frequency sweep was measured by varying the frequency from 0 to 10 rad/s at 1% strain. As a result, it was confirmed that both samples had a stable solid structure by confirming G'>G" in the low frequency range (FIG. 2b).

In addition, protein gel samples were prepared by increasing the concentration of the β-peptide/carbon nanotube complex to 0, 0.004, 0.020, and 0.040% unit volume weight in order to measure the change in physical properties according to the content of carbon nanotubes (Fig. 2c. ). As a result of measuring G'of each sample in the frequency sweep mode, it was confirmed that all samples had similar G'values, and that a small amount of carbon nanotubes did not change the physical properties of the protein gel (FIG. 2D).

Example 3: conductivity Peptide Control and analysis of gel electrical properties

In order to analyze the electrical properties of the peptide gel according to the content of the β-peptide/carbon nanotube complex, sample the concentration of the β-peptide/carbon nanotube complex at 0, 0.004, 0.01, 0.02, 0.03, 0.04% unit volume. Ready. Each sample was measured using IVIUM-n-Stat for impedance and conductivity analysis. For the conductivity measurement, the conductive peptide gel was injected into a polyethylene microtube having a diameter of 380 μm, which is a non-conductive material, and two copper wires having the same diameter were placed at both ends of the tube at a distance of 8 mm. The value obtained by changing the change in current from -1.0 V to 1.0 V was converted into conductivity through Equation 1. (R = average resistance, V = voltage change, = current change, = conductivity, = resistivity, L = distance between two copper electrodes, A = cross-sectional area)

(Equation 1)

3A shows the change in conductivity according to the content of the β-peptide/carbon nanotube complex. In the case of a protein gel not containing carbon nanotubes, the conductivity is 0 S/m, whereas the protein gel containing 0.004% of the complex is conductive. This increased to 48.5 mS/m. In the case of the 0.04% sample with the highest carbon nanotube content, the conductivity increased to 76 mS/m, but the increase in conductivity due to the increase in the concentration of the conductive nanomaterial increases the probability of connecting the path through which electricity can flow, and the resistance value decreases. Is predicted as a result of Theoretically, the rate of increase in conductivity should be proportional to the increase in the concentration of carbon nanotubes, but a percolation threshold in which the electrical conductivity rapidly increases with a small amount of 0.004% was observed, and then a saturation curve was obtained.

To measure the impedance of the conductive protein gel, each electrode was first prepared by immersing each electrode in 137 mM NaCl (Sodium Chloride), 2.7 mM KCl (Potassium Chloride), 11.9 mM phosphate buffer solution, and Ag/AgCl as a reference electrode and platinum as a counter electrode. Was used. The measurement range was measured with a voltage of 25 mVrms from 0.1 Hz to 10 kHz AC. 3B shows a change in the impedance value according to the content of the β-peptide/carbon nanotube composite frame, and it was confirmed that the impedance decreased as the carbon nanotube content increased. The impedance of the β-peptide gel is strongly influenced by the current frequency, which implies a capacity characteristic. However, in the presence of carbon nanotubes, the impedance was more resistant than the capacitor.

Example 4. Biocompatibility in vitro/in vivo analysis of conductive protein

Cell proliferation was observed through Cell Counting Kit-8 (CCK-8) analysis to confirm in vitro biocompatibility. The β-peptide gel and the protein gel containing the β-peptide/carbon nanotube complex were prepared at a concentration of 10 mg/ml each, and the β-peptide/carbon nanotube complex was mixed at a volume concentration of 0.02%. After coating the prepared protein gel and laminin for control contrast on a cell culture 96-well plate, neuroblastoma cells (SY-SH5Y) were seeded at 2.0 x 10 ⁴ per well. In order to reduce the error range and obtain an average value, the number of n per sample was designated as 5, and absorbance was measured according to the CCK-8 protocol after incubation at 37°C for 2, 18, 48, and 60 hours. 4A shows cell proliferation over time, and FIG. 4B shows absorbance showing the degree of cell proliferation. As a result, compared to the number of cells grown on laminin, it was confirmed that cells grown on the β-peptide gel and the protein gel containing the β-peptide/carbon nanotube complex sleep better, and through this, the conductive protein gel was not cytotoxic. I can confirm.

In vivo biocompatibility assays were tested in mouse brain tissue. First, a 50 mg/ml protein gel containing 0.02% β-peptide/carbon nanotube complex and 4 μM β-peptide/Cy3 was injected into a medical polyethylene tube with an inner diameter of 0.2 mm by injection. After inserting the tube into the cerebral cortex of an artificial-skull bone inserted mouse (Korean Patent No. 10-1597131), the activity of Microglia cells and Astrocyte cells was activated for 20 days. It was observed through a photon electron microscope (SPX-8, Leica Microsystems) (Fig. 4c). Through this, the biocompatibility was verified by seeing that the activity of the microglia cells and astrosite cells in the area where the protein gel was exposed was the same as that of the cells around the medical tube and did not show much activity.

Example 5. Measurement and analysis of epilepsy signals using conductive gel

In order to measure and analyze a biological signal in a mouse model using a biocompatible conductive gel, it was prepared as follows. First, the conductive protein gel was prepared to contain 0.02% of the β-peptide/carbon nanotube complex, and the impedance value was 3.67 MΩ.

Adult male C57BL mice (10-23 weeks, 25-30g) were used as experimental animals, and all individuals were reared in a dark/light cycle of 12 hours. The experimental animal was anesthetized in a 2-2.5% isoflurane induction chamber for about 5 minutes, and then moved to the stereotaxic frame to fix the head. During the operation, the anesthesia was maintained with 1.2-1.5% isoflurane, and the animal's body temperature was monitored to maintain 37°C using a DC temprature control system. Thereafter, the skin above the skull of the experimental animal was incised, hemostasis was performed, and a microscrew to be used as a baselined was inserted into the skull above the left olfactory bulb so as to touch the brain surface. In addition, microscrews were additionally inserted to strengthen the attachment of the metal frame to the skull above the cerebellum. Thereafter, the surface of the skull was covered with the exception of the EEG (eletroencephalogram) measurement and the drug injection space using a resin. After that, three burr holes (diameter: ~500 μm) were drilled in the range (bregma 1 to 2 mm, lateral 1 to 3 mm) corresponding to the somatosensory cortex area using a dental drill. The three holes were made to form a triangular shape so that each hole was located at the same distance of about 1mm. Of these, two holes were made for the electrode to measure EEG, and the other one was made to inject drugs to make the seizure model.

To make a mouse epilepsy model, a 4-AP (4-Aminopyridine) drug was injected into the cerebral cortex to create an acute epilepsy model. During EEG recording, anesthesia was maintained at a concentration of 1-1.2% isoflurane. A silver wire was used as the EEG measurement electrode, and it was put into the burr hole and attached to the brain surface. EEG data was measured at 40,000 Hz using Plexon Onmiplex recording systems.

In this example, the EEG measurement was performed in a total of 3 steps (Fig. 5A).

1) The spontaneous EEG signal was simultaneously measured at the first EEG electrode (EEG 1) and the second EEG electrode (EEG 2).

2) A conductive protein gel was injected into the burr hole where EEG 1 was measured and a protein gel containing no CNT complex was injected into the burr hole to cover the tissue exposed around the electrode with the protein gel. Thereafter, EEG signals were simultaneously measured at both electrodes.

3) Signals induced by injecting 4-AP into the other hole were simultaneously measured at both electrodes.

As a result of the measurement, it was confirmed that the EEG signal induced by the drug was amplified by the electrode injected with the protein gel containing the β-peptide/carbon nanotube complex. As a result of this, it was confirmed that the conductive protein gel was capable of measuring vital signs with high sensitivity (FIG. 5B).

In addition, the EEG signal was measured after injecting a conductive protein gel into the brain of the epilepsy animal in a different way. The EEG signal was measured by connecting electrodes to the injection site and the control site on the 6th day after injecting the conductive protein gel into one part of the brain using the glass pipette of FIG. 6A. As can be seen in FIG. 6B, it was confirmed that the conductive protein gel was capable of measuring bio signals with high sensitivity.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in 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.

<110> Research & Business Foundation SUNGKYUNKWAN UNIVERSITY INSTITUTE FOR BASIC SCIENCE <120> COMPOSITION FOR BIO-SIGNAL MEASUREMENT AND STIMULATION CONTAINING BIOCOMPATIBLE CONDUCTIVE PROTEIN GEL <130> PN1909-434 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 1 Lys Val Lys Glu Val Phe Phe Val Lys Glu Val Phe Phe Val Lys Glu 1 5 10 15 Val Tyr

Claims (16)

A composition for measuring a biological signal of a brain tissue or stimulating a living body in order to diagnose epilepsy comprising a biocompatible conductive protein gel containing a complex of a biocompatible protein and a conductive nanomaterial,
The biocompatible protein contains the amino acid sequence represented by SEQ ID NO: 1,
The complex of the biocompatible protein and the conductive nanomaterial is contained in 0.02 (w/v)% of the total volume of the composition, and the impedance of the composition is 3.67 MΩ,
The biocompatible protein is not degraded by enzymes,
A composition for measuring vital signs of brain tissue or stimulating a living body. delete The method of claim 1,
A composition for measuring a biological signal of a brain tissue or stimulating a living body comprising the sequence of (KVKEVFFVKEVFFVKEVY)n (1≤n≤3) by repetition of the sequence of SEQ ID NO: 1. The method of claim 1,
The biocompatible protein is not degraded by enzymes, a composition for measuring a biological signal of a brain tissue or stimulating a living body. The method of claim 1,
The conductive nanomaterial is a carbon nanotube (CNT), fullerene (C60), a conductive polymer nanowire, a conductive polymer nanotube, a conductive polymer nanoparticle, a metal nanowire, a metal nanoparticle, and combinations thereof. A composition for measuring a biological signal of a brain tissue or stimulating a living body comprising one selected from the group consisting of. A medical implant comprising a composition for measuring or stimulating a living body of a brain tissue according to claim 1. The method of claim 6,
The medical implant is a medical implant that receives electrical stimulation by connecting electrodes after implantation in brain tissue. A medical electrode in which the composition for measuring a biological signal of a brain tissue according to claim 1 or stimulating a living body is fixed to the surface of the electrode. Electrical stimulation generator; An electrode electrically connected to the electrical stimulation generator; And the medical implant according to claim 6 connected to the electrode. A biological signal measurement and biological stimulation system of a brain tissue for diagnosing epilepsy. Electrical stimulation generator; And a medical electrode according to claim 9 that is electrically connected to the electrical stimulation generator. A biological signal measurement and biological stimulation system of a brain tissue for diagnosing epilepsy. The method of claim 9 or 10,
The medical implant or medical electrode is inserted into the brain tissue, the biological signal measurement and biological stimulation system of the brain tissue for diagnosing epilepsy. The method of claim 9 or 10,
The system is for deep brain stimulation, a biological signal measurement and biological stimulation system of brain tissue for diagnosing epilepsy. The method of claim 9 or 10,
The electrical stimulation is a bio-signal measurement and bio-stimulation system of a brain tissue for diagnosing epilepsy, which has a voltage of 1 mV to 10 V and a frequency of 1 to 200 hz. A skull substitute comprising a composition for measuring or stimulating a living body of a brain tissue according to claim 1. The method of claim 14,
A skull substitute that can give electrical stimulation through the skull substitute. Inserting a medical implant comprising the composition of claim 1 into the brain tissue of an individual other than a human;
Monitoring brain nerve signals by applying electrical stimulation; And
A method of measuring a biological signal of a brain tissue for diagnosing epilepsy, comprising the step of comparing the monitored brain nerve signal with a normal brain nerve signal.

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