WO2018084607A1 - Biothiol detecting composition comprising redox regulation protein - Google Patents

Biothiol detecting composition comprising redox regulation protein Download PDF

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WO2018084607A1
WO2018084607A1 PCT/KR2017/012344 KR2017012344W WO2018084607A1 WO 2018084607 A1 WO2018084607 A1 WO 2018084607A1 KR 2017012344 W KR2017012344 W KR 2017012344W WO 2018084607 A1 WO2018084607 A1 WO 2018084607A1
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Prior art keywords
biothiol
dna
ohrr
protein
composition
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PCT/KR2017/012344
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French (fr)
Korean (ko)
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김영필
이진오
이진원
양윤모
김태욱
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한양대학교 산학협력단
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Priority claimed from KR1020170144833A external-priority patent/KR102451039B1/en
Application filed by 한양대학교 산학협력단 filed Critical 한양대학교 산학협력단
Priority to US16/347,321 priority Critical patent/US20190331690A1/en
Priority to CN201780082078.4A priority patent/CN110168367A/en
Publication of WO2018084607A1 publication Critical patent/WO2018084607A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a biothiol detection composition
  • a biothiol detection composition comprising a redox control protein, a biothiol detection method using the composition, and a biosensor / kit for biothiol detection.
  • Biothiol is a low molecular weight (LMW) thiol, unlike the thiols such as Cysteine, which are present in proteins, to regulate oxidative stress resistance and physiological activity in vivo in all living things, from bacteria to humans. Perform important functions. These biothiols are very sensitive to redox reactions, so they switch to functional groups such as SOH, SO 2 H, SNO, and SS, and act as a control switch of biomolecule activity. It is also widely detected in major body fluids such as tears.
  • LMW low molecular weight
  • Biothiols present in body fluids include cysteine (Cys), homocysteine (Hcy), glutathione (GSH), N-acetylcysteine (NAC), cysteamine (CA), ⁇ -Glutamylcysteine ( ⁇ -GluCys), Cysteinylglycine (CysGly), N-acetylcysteine (N-AC), Coenzyme A (CoA), Coenzyme B ( Coenzyme B, CoB), Coenzyme M (CoM), bacillithiol (BacT), mycothiol (MyT), ergothioneine (ERT), trypanothione (TrT) Etc.
  • cysteine Cys
  • Hcy homocysteine
  • GSH glutathione
  • NAC N-acetylcysteine
  • CA cysteamine
  • ⁇ -GluCys Cysteinylglycine
  • N-AC N-acet
  • Non-Patent Documents 1 and 2 changes in the concentrations of Cys, Hcy, and GSH in vivo are closely related to various kinds of diseases and reported to exist in different concentration ranges [Non-Patent Documents 1 and 2].
  • the total concentrations of Hcy, Cys, and GSH (total amounts oxidized and reduced) in the plasma are present at concentrations of 6-20 ⁇ M, 150-350 ⁇ M and 4-10 ⁇ M, respectively, but high oxidative conditions in plasma Hcy and Cys are present in almost oxidized form, so the free form is extremely small, less than about 0.2 ⁇ M for Hcy, and less than about 10 ⁇ M for Cys.
  • biothiol measurement method antibody-based immunoassays, which are currently sold in kit form, are widely used. However, all of the used antibodies do not directly recognize free biothiols, but instead oxidize the biothiols to bind proteins. It recognizes the form (such as serum albumin for blood). Thus, there is a limitation in that dynamic changes in free biothiol and total biothiol cannot be detected.
  • the measurement of free biothiol is a chemical sensor based on chemical bonding. Fluorescence induced by binding to biothiol by using variants of fluorescent dyes such as rhodamine, fluorescein, BODIPY, cyanine, flavone, and coumarin It is based on the change measurement [nonpatent literature 8]. Although these methods react rapidly with free biothiol, most of the materials that induce such fluorescence changes are synthetic compounds based on benzene ring variants and functional groups, which have very low solubility, are vulnerable to pH changes, and are measured using the -SH group of free thiol.
  • Non-Patent Document 1 Persichilli, S., Gervasoni, J., Castagnola, M., Zuppi, C. & Zappacosta, B. A Reversed-Phase HPLC Fluorimetric Method for Simultaneous Determination of Homocysteine-Related Thiols in Different Body Fluids. Labmedicine 42, 657-662 (2011)
  • Non-Patent Document 2 Fiskerstrand, T., Refsum, H., Kvalheim, G. & Ueland, P. M. Homocysteine and Other Thiols in Plasma and Urine-Automated-Determination and Sample Stability. Clin Chem 39, 263-271 (1993))
  • Non-Patent Document 3 Seshadri, S. et al. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. New Engl J Med 346, 476-483, (2002)
  • Non-Patent Document 4 Refsum, H., Ueland, P. M., Nygard, O. & Vollset, S. E. Homocysteine and cardiovascular disease. Annu Rev Med 49, 31-62 (1998)
  • Non-Patent Document 5 Korean Patent Document 5
  • Non-Patent Document 6 El-Khairy, L., Ueland, P. M., Refsum, H., Graham, I. M. & Vollset, S. E. in Circulation Vol. 103 2544-2549 (2001).
  • Non-Patent Document 7 Andersson, A., Lindgren, A., Arnadottir, M., Prytz, H. & Hultberg, B. Thiols as a measure of plasma redox status in healthy subjects and in patients with renal or liver failure. Clin Chem 45, 1084-1086 (1999).
  • Non-Patent Document 8 Jung, H. S., Chen, X. Q., Kim, J. S. & Yoon, J. Recent progress in luminescent and colorimetric chemosensors for detection of thiols. Chem Soc Rev 42, 6019-6031, doi: Doi 10.1039 / C3cs60024f (2013).
  • the present inventors have made a research effort to solve the problem of the conventional detection method of the biothiol, it is possible to detect a low molecular weight biothiol using a redox-regulating protein, free biothiol and total Simultaneous measurement of (Total) biothiol was possible, and the present invention was completed by developing a composition for detecting biothiol which is excellent in terms of rapidity and storage stability as well as improved sensitivity by detecting even a small amount.
  • an object of the present invention is to provide a composition for detecting biothiol, which comprises a redox-regulating protein.
  • Another object of the present invention is to provide a method for detecting biothiol using the composition.
  • the present invention has another object to provide a biosensor using the composition.
  • the present invention has another object to provide a biochip using the composition.
  • the present invention provides a composition for detecting biothiol comprising a redox-regulating protein.
  • the present invention provides a method for detecting biothiol using the composition.
  • the present invention provides a biosensor using the composition.
  • the present invention provides a biochip using the composition.
  • the composition for detecting biothiol according to the present invention can rapidly measure the biothiol in free form in the body fluid.
  • the relative content ratio and change of total and free biothiol in body fluids can be detected in real time, making it possible to use biothiol as a major indicator of disease, thereby enabling prediction and warning of various diseases.
  • various redox stress changes associated with major diseases can be explained by the amount of change in biothiol, which can provide important technical, economic and social values for the identification and diagnosis of disease pathogenesis in the future.
  • 1 is a schematic diagram showing the principle that OhrR and dsDNA are dissociated by biothiol.
  • 3 is a result of confirming the binding of the OhrR protein and the target DNA using the electrophoresis method.
  • FIG. 9 is a schematic diagram showing a biothiol detection process according to an embodiment of the present invention.
  • FIG. 10 shows fluorescence measurement results according to the biothiol detection method of FIG. 9.
  • FIG. 11 is a chemiluminescence measurement result according to the biothiol detection method of FIG. 9.
  • 12 is a schematic diagram showing signal amplification of DNA.
  • Figure 13a shows the chemiluminescence measurement results for confirming the DNA signal amplification of Figure 12.
  • Figure 13b shows the results of electrophoresis to confirm the signal amplification of the DNA of Figure 12.
  • FIG. 14 is a schematic diagram of a biochip configuration for measuring biothiol using binding between OhrR and DNA.
  • FIG. 15 is a result of detecting biothiol using the biochip of FIG. 14.
  • FIG. 16 is a schematic diagram of the biosensor in strip form of FIG. 9.
  • a biothiol detection composition comprising a redox-regulating protein and a biothiol detection method using the same are included.
  • a biothiol detecting composition in addition to the redox control protein, further comprises a DNA bound to the redox control protein and a biothiol detection method using the composition thereof.
  • redox-regulating protein refers to all proteins whose activity is regulated by the oxidation and reduction of proteins, typically OhrR present in some bacteria such as B. subtilis . (organic hydroperoxide regulator), PerR (Peroxide regulator), and OxyR (Oxygen regulator) present in some bacteria, including E. coli .
  • protein engineering may be used to modify redox regulatory protein active site amino acids, or to screen for orthologue proteins present in other types of organisms, including redox regulatory proteins that react more selectively with specific biothiols. can do.
  • the redox regulatory protein may include a variant capable of controlling binding affinity between biothiol and DNA or a protein in which a labeled protein is conjugated.
  • the conjugate form may include a fluorescent protein-OhrR, a light emitting protein-OhrR, a FLAG-OhrR, His6-OhrR, GSH-OhrR, Biotin-OhrR, and the like.
  • biothiol refers to a low molecular weight thiol having a molecular weight of 10 Da to 1,000 Da or less (preferably 10 Da to 500 Da), specifically cysteine (Cys), homocysteine (homocysteine, Hcy), glutathione (GSH), N-acetylcysteine (NAC), cysteamine (CA), ⁇ -glutamylcysteine ( ⁇ -GluCys), cysteinylglycine ( cysteinylglycine, CysGly), N-acetylcysteine (N-AC), Coenzyme A (CoA), Coenzyme B (Coenzyme B, CoB), Coenzyme M (Coenzyme M, CoM), bacillithiol , BacT), mycothiol (MyT), ergothioneine (ERT) and trypanothion (trypanothione, TrT) may be one or more selected from the group, but
  • biothiol can be used for various diseases related to cardiovascular disease, neurorodegenerative disease, cancer, cancer, kidney dysfunction, diabetes, diabetes mellitus, or bacterial and viral infections.
  • a marker it is an indicator that can detect abnormal biological reactions early.
  • the present invention also includes a biothiol detection composition comprising a redox control protein and a DNA bound to the redox control protein and a biothiol detection method using the composition.
  • biothiol can be detected using the principle of binding / dissociation of redox regulatory protein and DNA bound to redox regulatory protein.
  • the DNA may be, for example, one represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 used in the following Examples, and may be any compound that binds to a redox regulatory protein.
  • FIG. 1 The detection principle of such a biothiol is shown in FIG. 1 as an embodiment.
  • OhrR a representative example of the redox control protein, is an organic perperoxide (ROOH) sensor in bacteria.
  • OhrR is a homodimer and has one cysteine residue per monomer. In the state where the cysteine residue is reduced (-SH), OhrR maintains the form of binding to DNA (complex of OhrR and DNA), and OhrR is rapidly oxidized in the presence of organic peroxide (-SOH).
  • the oxidation is OhrR while maintaining engagement with the DNA, the environment in which the present bio-thiol to quickly react with the bio-thiol is released from the DNA (typically bio-thiol
  • Dissociation rates vary depending on the concentration and type of biothiol.
  • OhrR forms sulfenamide (-SN-) at a relatively slow rate by ROOH and slowly dissociates from DNA (t 1/2 ⁇ 10 min).
  • DNA bound to the redox regulatory protein may be coupled to a fluorescence factor or DNA-based enzyme (DNAzyme) to enable fluorescence, chemiluminescence, and absorption detection, or amplify DNA sequences to improve reaction sensitivity.
  • DNAzyme DNA-based enzyme
  • the specific kind of the fluorescent factor is not particularly limited, and examples thereof include rhodaman and its derivatives, fluorescein and its derivatives, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives, and erythrosine. At least one selected from the group consisting of derivatives thereof, eosin and derivatives thereof, and 4-acetamido-4'-isothiocyanatostilben-2,2'disulfonic acid. More specifically illustrating the fluorescent material that can be used in the present invention is as follows.
  • Rhodamine and its derivatives include 6-carboxy-X-rhodamine (ROX), 6-carboxyrodamine (R6G), lysamine rhodamine B sulfonyl chloride, rhodamine (Rhodamine B, rhodamine 123, Rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivatives (Texas Red) of sulforhodamine 101, N, N, N ', N'-tetramethyl-6-carboxyrodamine (TAMRA), tetramethyl rhodamine, tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid, terbium chelate derivatives, Alexa derivatives, Alexa-350, Alexa-488, Alexa-547 and Alexa-647, etc. May be mentioned;
  • Pyrene and its derivatives include pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4 (Cibacron® Brilliant Red 3B-A) and the like;
  • Erythrosine and its derivatives include erythrosin B, erythrosin isothiocyanate and ethidium;
  • Eosin and Eosin isothiocyanate etc. are mentioned as Eosin and its derivatives ;
  • the DNA-based enzyme is a peroxidase-mimicking DNA enzyme having peroxidase characteristics among various DNA-based enzymes (Wang Li et al. Insight into G-quadruplex-hemin DNAzyme / RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity.
  • Nucleic Acids Research 44 (15); 7373-7384 (2016)] and RNA-sequencing DNA enzymes (RNA-cleaving DNA enzymes) [Meng Liu, Dingran Chang, and Yingfu Li. Discovery and Biosensing Applications of Diverse RNA-Cleaving DNAzymes, Accounts of Chemical Research, 50; 2273-2283 (2017)] may be, but is not limited to having one or more sequences selected from the group consisting of.
  • the signal amplification method binds a short DNA sequence to one end of the redox regulatory protein binding DNA (a single strand DNA template capable of DNA amplification) and additionally reacts Hairpin1 (HP1) and Hairpin 2 (HP2). DNA sequences are amplified without the use of PCR. DNA sequence and length, and the type of hairpin is not limited to those described in the following Examples (Table 1).
  • a tag including a biotin group, an alkyne group, an azide group, a thiol group, an amine group, etc. is attached to the 5 'end or 3' end of the DNA sequence to separate or bind DNA only on beads, nanoparticles, and chip surfaces. Can be.
  • biothiol detection means measuring biothiol using redox control.
  • the biothiol measurement may include gel electrophoresis, fluorescence anisotropy, matrix-assisted laser desorption ionization-time-of-flight mass spectrometer, surface plasmon resonance (SPR) , At least one selected from the group consisting of interferometry and bead measurement.
  • SPR surface plasmon resonance
  • the binding of redox regulator protein and biothiol can be quantitatively analyzed according to the type of biothiol, and the amount of free biothiol or total biothiol can be analyzed.
  • a biothiol can be detected by fluorescence or chemical luminescence by connecting a fluorescent label or a DNA-based enzyme to a DNA binding to a redox regulatory protein.
  • the redox regulatory protein may be combined with a FLAG tag, His6 tag, GSH tag, biotin tag, and the like, and the bound protein may be affinity beads (FLAG affinity beads, NTA-beads, glutathione beads, avidin). Series of beads, etc.) to detect biothiol.
  • composition for detecting biothiol according to the present invention can detect free biothiol and total biothiol in free form or detect free biothiol and total biothiol at the same time.
  • the composition when detecting the total biothiol, further comprises a reducing agent. Since OhrR binds only to the reduced free biothiol, in order to detect total biothiol in the sample, the total biothiol can be detected by OhrR by rapidly reducing the oxidized biothiol in the sample using a reducing agent.
  • the reducing agent is one or more selected from the group consisting of dithiothreitol (DTT), 2-mercaptoethanol (2-mercaptoenthanol), and TCEP (tris (2-carboxyethyl) phosphine), but is not limited thereto.
  • the present invention also includes a biothiol detection biochip comprising the composition.
  • the biochip is reacted with a redox regulatory protein on a plate onto which the DNA is immobilized to form a complex with a DNA capable of binding to a redox regulatory protein, as shown in FIG. 14.
  • the redox regulatory protein bound to the affinity tag is used to measure the detection signal by reducing the redox regulatory protein from the DNA attached to the surface of the biochip in the presence of biothiol. .
  • the present invention also includes a biosensor for detecting a biothiol comprising the composition.
  • a biosensor for detecting a biothiol in a strip form is included.
  • a sample introduction part including a fixation site capable of binding to a complex of a redox control protein and a DNA bound to the protein, the sample introduction part being configured to introduce a sample and the complex into a mixture;
  • a measurement unit configured to move the dissociated DNA to measure biothiol
  • biosensor for detecting biothiol comprising a.
  • the fixation site may be an antibody (eg FLAG tag-OhrR, Anti-FLAG antibody, anti-His6 antibody, etc.) or a receptor (eg His6 tag-OhrR, NTA or Biotin-OhrR, Strepavidin (including avidin, NeutrAvidin), etc.). It may be combined.
  • an antibody eg FLAG tag-OhrR, Anti-FLAG antibody, anti-His6 antibody, etc.
  • a receptor eg His6 tag-OhrR, NTA or Biotin-OhrR, Strepavidin (including avidin, NeutrAvidin), etc.
  • the biosensor for detecting a biothiol in the form of a strip is, for example, as shown in FIG. 16.
  • the oxidized / reduced amount of low molecular weight biothiol present in the blood can be measured relatively.
  • the sample introduction part is a fixed site capable of binding to the OhrR-dsDNA complex, and immobilizes an antibody (e.g., an anti-FLAG antibody or an anti-His6 antibody) or an affinity receptor (e.g., an avidin family, NTA, etc.) Plasma)
  • OhrR-dsDNA is mixed and dropped in the sample introduction, the mixed solution flows to the right by the chromatographic principle to pass through the fixed site, the OhrR-dsDNA is bound to the fixed site without moving anymore.
  • dsDNA bound to OhrR is rapidly dissociated in the presence of biothiol in the sample, and only dsDNA is moved to the right side, and it is detected by chemiluminescence by reacting with the substrate by DNAzyme in dsDNA at the right measuring part of the sensor.
  • biothiol using the redox control protein according to the present invention has the following differentiation and superiority compared to the conventional biothiol detection method.
  • the sensitivity is significantly improved and the sample volume to be measured can be reduced to a minimum. Since monomers of OhrR are 1: 1 (molar ratio) with biothiol, reaction sensitivity can be improved by measuring proteins (eg, OhrR) of macromolecules that react with biothiol (eg, OhrR and OhrR + bio). Mass spectrometry of thiols).
  • proteins eg, OhrR
  • the signal and amplification factors are introduced into the DNA site that binds to the protein and the DNA signal is controlled to be controlled by the binding or dissociation process with the protein, compared with the conventional methods (chromatography, immunoassay, chemical sensor-based analysis) High sensitivity can be obtained.
  • the OhrR protein it is possible to measure not only the whole biothiol but also the free form of the biothiol using only 1-2 ⁇ L of blood, thereby reducing the amount of sample to be analyzed.
  • OhrR a redox control protein
  • a redox control protein can be expressed in large quantities and can be stored for a long time because it is not easily degraded at room temperature in a relatively small size (17 kD).
  • oxidizing conditions eg oxygen and hydrogen peroxide
  • -9 M or less is so high that it is not easily dissociated under normal conditions, so the background signal can be kept very low when using protein-DNA binding.
  • OhrR a redox regulator protein, reacts only with low molecular weight biothiol in the presence of organic oxides and does not react with thiol groups present in macromolecular protein, so that only low molecular weight biothiol can be detected.
  • free biothiol and total biothiol can be measured simultaneously.
  • Redox proteins can be used for rapid detection.
  • the reaction time can be significantly reduced.
  • the second reaction rate between the Cys residues and the peroxide is from about 10 4 - 10 5 M -1 Cys residues present in or as a common protein s -1 glass Bio-thiol Tens of thousands or hundreds of thousands of times faster than the peroxide reaction rate (about 1 M -1 s -1 ), and the secondary reaction rate of biothiol and redox regulatory protein was about 10 3 M -1 s -1 . -2 -10 1 M -1 s -1 ) thousands of times faster.
  • the redox regulatory protein which senses the biothiol, is immediately dissociated from the DNA (50% dissociation time, t 1/2 ⁇ 0.5 min), thereby reacting with the sample within a few minutes to rapidly induce a resultant signal.
  • Fluorescence anisotropy was used to measure real-time DNA binding activity of OhrR.
  • Buffer 20 mM Tris (pH 8.0) 150 mM NaCl, 5% Glycerol (vol / vol)
  • Measurement time every 10s measurement
  • Measurement conditions ex 492 nm; slit width 15 nm, em 520 nm; slit width 20 nm, integration time 1 s
  • OhrR sequence Direct cloning in B. subtilis strains. MENKFDHMKLENQLCFLLYASSREMTKQYKPLLDKLNITYPQYLALLLLWEHETLTVKKM GEQLYLDSGTLTPMLKRMEQQGLITRKRSEEDERSVLISLTEDGALLKEKAVDIPGTILGLSKQSGEDLKQLKSALYTLL ETLHQKN (SEQ ID NO: 3)
  • OhrR and fluorescence (6FAM, 6-carboxyfluorescein) labeled OhrR binding DNA was dissolved in 3 mL of buffer using a LS55 luminescence spectrometer (PerkinElmer).
  • Anisotropy Asnis
  • Anisotropy increases, and when it dissociates with DNA, Anisotropy decreases.
  • three representative biothiols, Cys (Cysteine), Hcy (Homocysteine), and GSH (Glutathione) were treated at various concentrations (0, 1, 2, 4, 8, 16, 32, 64 ⁇ M).
  • CHP cumene hydroperoxide
  • one of the representative organic peroxides at 300 sec.
  • Figure 2 confirms that the higher the biothiol concentration, the faster the DNA dissociation rate of OhrR. 2 shows anisotropy value when OhrR dissociates with DNA depending on the concentration of biothiol. The time it takes to decrease from OhrR to DNA Time to dissociate).
  • Fluorescent probe (FAM) -coupled double strand DNA (200 nM) [SEQ ID NOs: 1 and 2] and OhrR were mixed at the concentrations shown in FIG. 3 and reacted at room temperature for 30 minutes, followed by polyacrylamide gel (7%).
  • electrophoresis 25 mA, 30 min
  • the dsDNA band was measured with a fluorescence measuring instrument (Model KIF-300, Korea Lab Tech, Korea). It can be seen that the fluorescence band of dsDNA shifted upward from the concentration of OhrR of about 1.6 ⁇ M (about 8 times the concentration of DNA).
  • PAGE electrophoresis is not a result of measuring protein-DNA binding in real time, and a large amount of protein is required for OhrR-to-DNA binding (hence the actual binding constant It is not possible to measure with this method.) It can be used as a method to easily check the coupling without a specific device.
  • Example 3 without fluorescent label DNA and OhrR The bond between proteins In biothiol The process of being dissociated by At photorefractive index Measured experiment
  • a device capable of measuring biolayer interferometry (Blitz, Fortebio, USA) was used to measure the degree of binding between DNA and protein on the optical fiber surface.
  • binding buffer, washing buffer TBS (Tris 20 mM, NaCl 150 mM)
  • biotin-coupled double strand DNA [SEQ ID NOS: 1 and 2] was dissolved in the TBS buffer at the above concentration and flowed for 120 seconds on the streptavidin-coated optical fiber, and then washed with a buffer and washed with OhrR protein TBS buffer. At 120 ° C., the solution was further flowed for 120 seconds to associate with double strand DNA bound to an optical sensor. Even after the association was completed, dissociation of the DNA-OhrR complex was confirmed by additionally flowing only the buffer for 120 seconds.
  • Biolayer interferometry measurements inevitably involve changes in the refractive index of the optical sensor when the biomaterial (DNA or OhrR) is bound to the optical sensor surface (refractive index is positively correlated with the concentration of the biomaterial). It applies the principle of converting the change value into thickness.
  • OhrR was strongly bound to dsDNA to maintain a binding thickness value of about 4.3 nm (minus the value of the dissociation equilibrium minus dsDNA binding equilibrium), and similarly, after mixing only one of CHP or biothiol with OhrR protein Even when spilled into DNA, it can be seen that OhrR binds to DNA effectively.
  • This method can be used as a method to effectively compare the reactivity to various types and concentrations of biothiol without separately labeling dsDNA or OhrR with phosphors on the optical sensor surface.
  • MALDI-TOF MS (Maltitop Mass Spectrometer, Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometer) was used to confirm the quantitative analysis of the binding of OhrR to biothiol.
  • Biothiol (Cys, Hcy, GSH) was added to 1 mL of buffer (20 mM Tris (pH 8.0) 100 mM NaCl, 5% Glycerol) in 300 nM OhrR (0, 1, 2, 4, 8, 16). , 32, 64 ⁇ M). After reacting 3 ⁇ M CHP for 2 minutes to each, 110 ⁇ L of 100% TCA was used to stop the oxidation reaction and precipitated OhrR. In order to alkylate the precipitated OhrR, 50 mM Iodoacetamide was treated to block the reduced cysteine residue of OhrR.
  • the mass signal value of OhrR-Cys was reduced in arteriosclerosis model mouse blood compared to normal mouse blood, and the mass signal value of OhrR-Cys was higher in 45 year old male smoker blood than in 25 year old non-smoker blood. It can be seen that the decrease. In other words, given that OhrR reacts with free biothiol in a fast time, it can be observed that the concentration of free biothiol (particularly free cysteine) present in the blood is relatively decreased in the mouse atherosclerosis model and in the smoker model. Can be. As such, it was confirmed that the amount of free biothiol can be effectively analyzed by mass spectrometry of OhrR even with a small amount of blood of various disease patients.
  • DTT results in the reduction of the disulfide bonds of all kinds of (oxidized) biothiols to form a free form.
  • CHP accelerates the dissociation rate of the OhrR DNA from the oxidized OhrR again with the rapidly reduced free form of biothiol, thereby measuring the total amount of biothiol (DTT).
  • cyclization is performed by internal binding between thiol molecules of DTT and thus does not participate in the reduction reaction of additional biothiol and does not react with OhrR (see left figure of FIG. 7).
  • Plasma volume 2 ⁇ L (purchased from Norma Mouse serum, Jackson ImmunoResearch)
  • Plasma collected from the mice was reacted with-/ + 1 mM DTT at RT (23-25 ° C.) for 1 hour. Thereafter, 2 ⁇ L of Plasma was reacted with 25 ⁇ M of OhrR and 50 ⁇ M of CHP for 10 minutes (total reaction at 2.5 ⁇ L). After the reaction, 1 mL of 10% TCA was treated to precipitate proteins containing OhrR. In order to prevent further oxidation of the reduced cysteine of precipitated OhrR, the alkylating agent Iodoacetamide was treated at a concentration of 50 mM to react with the reduced cysteine.
  • the reduced biothiol reacts rapidly with OhrR in the presence of CHP (within about 5 minutes) even in the presence of about 1 mM of DTT. Afterwards, biothiol can be dissociated into OhrR by DTT. It can be seen that. From this, it is preferable to measure OhrR and CHP within about 5 minutes after treatment to detect the amount of total biothiol after treatment with DTT. These conditions were analyzed by MALDI-TOF MS, and it was confirmed that the amount of free cysteine can be measured when DTT is not treated in mouse plasma and the amount of total cysteine when DTT is treated. (Right figure of FIG. 7).
  • Free cysteine and total cysteine were detected after addition of cysteine concentrations in mouse blood using MALDI-TOF MS and OhrR. That is, it was confirmed through MALDI-TOF MS analysis that the amount of biothiol analyzed by OhrR is increased when the oxidized biothiol is reduced by treating DTT with mouse serum.
  • Plasma volume 2 ⁇ L (from Norma Mouse serum, Jackson ImmunoResearch)
  • Example 9 phosphor labeling on dsDNA
  • Example 10 ssDNAzyme on dsDNA
  • the label is an example in which this experimental procedure is commonly applied and only the measuring method is different.
  • M2 FLAG affinity bead (sigma, A2220) was added to the tube by the number of samples, and then FLAG tagged OhrR (2 ⁇ M) was bound for 1 hour so as to have a total volume of 200 ⁇ L (OhrR bound to His6 tag). If so, you can use NTA-Bead).
  • OhrR binding dsDNA 100 nM, SEQ ID NO: 1 and 2) to which phosphor or DNAzyme was bound was bound to OhrR bound beads for 30 minutes.
  • FAM was combined with OhrR-binding dsDNA as a fluorescence factor and 200 ⁇ L of the final supernatant was transferred to a 96 well plate, and fluorescence was measured using a multi plate reader (Variokan, Thermo Scientific). Fluorescence measurements were made after obtaining fluorescence signal values at 525 nm emission wavelengths obtained from 480 nm excitation wavelengths, and then comparing the fluorescence values of each well with the wells containing no buffers but only buffers as reference values.
  • biothiol amount can be detected effectively.
  • TBS Tris 20 mM, NaCl 150 mM
  • DNAzyme (5'-GG GTT GGG CGG GAT GGG-3 '[SEQ ID NO: 6], synthesized by IDT)
  • NeutrAvidin ® is put into a suspension 20 ⁇ L of the coated beads (Thermo Scientific Inc., USA) was shaking 30 min at room temperature.
  • TBS Tris Buffered Saline, 20 mM Tris, NaCl 150 mM, pH 7.4
  • 180 ⁇ L of TBS buffer and 20 ⁇ L of 1 ⁇ M Hemin (Calbiochem, USA) (in TBS) solution were mixed to induce the activity of DNA enzymes.
  • beads were precipitated in a total of 200 ⁇ L. It was left to stand. Then, 200 ⁇ L of the mixed solution containing beads was placed in a tube, mounted on a luminometer (Model Glo-Max 20/20, Promega, USA), and then ECL reaction solution (G-healthcare, USA) A (Luminol + H 2 O 2 Solution, 50 ⁇ L) and 100 ⁇ L of a mixture of B (enhancer, 50 ⁇ L) were added and the chemiluminescence intensity was measured immediately.
  • a luminometer Model Glo-Max 20/20, Promega, USA
  • OhrR-binding dsDNA sequence is linked to the ssDNAzyme (a DNAzyme having the property of Horseradish peroxidase) by the T9 linker [SEQ ID NO: 4], and the other strand is introduced with a biotin to introduce the DNA probe (SEQ ID NO: 5).
  • ssDNAzyme a DNAzyme having the property of Horseradish peroxidase
  • T9 linker [SEQ ID NO: 4]
  • the other strand is introduced with a biotin to introduce the DNA probe (SEQ ID NO: 5).
  • OhrR protein was used in the experiment after expression / purification in Escherichia coli by combining the flag peptide (DYKDDDDK, expressed in direct strain after making recombinant DNA).
  • the DNA probe portion is rapidly dissociated by adding CHP and cysteine in the sample.
  • the dissociated DNA probe is present in the supernatant after centrifugation.
  • the supernatant is reacted with the avidin-bound bead, washed, and then combined with dsDNA and hemin in the bead-containing solution, followed by luminol reaction. This can induce strong chemiluminescence.
  • Example 11 DNA Signal amplification method and confirmation
  • OhrR + F template forward DNA
  • OhrR_R complementary reverse DNA
  • Modified OhrR-F shows the addition of a short DNA sequence for amplification (Table 1).
  • hairpin 1 and hairpin 2 are added to the final reaction product, DNA sequences are amplified and can be detected by various methods (electrophoresis, fluorescence, luminescence) [FIG. 12].
  • All DNA oligos were used after production order through IDT. 20 ⁇ L of 100 ⁇ M Modified OhrR_F and OhrR-R were mixed, and a double strand sequence was prepared by binding to OhrR by heating at 95 ° C. for 10 minutes and then slowly cooling at room temperature. Using this double strand DNA as a template, 2 ⁇ L of 5 ⁇ M template, 2 ⁇ L of 20 ⁇ M hairpin 1 and 2 ⁇ L of 20 ⁇ M hairpin 2 were reacted at room temperature for 30 minutes at a total volume of 20 ⁇ L. Thereafter, 8 ⁇ L of each sample was amplified by agarose gel electrophoresis, and the length of the DNA band was visually confirmed by UV.
  • HP1 and HP2 are single stranded and have a band size of less than 50 bp.
  • no chain reaction occurs (no change in the band position on the DNA gel).
  • H1 is first combined with the template to release the hairpin structure of H1.
  • the opened H1 sequence is then combined with the H2 sequence to make H2 an open structure.
  • it is observed at a size larger than the DNA band size of each template DNA and H1, and the remaining amount of H1 still remains below because it is relatively larger than template DNA. can see.
  • a biochip method is measured by using His6-tag of OhrR. In the presence of biothiol, OhrR is separated from dsDNA attached to the surface of biochip.
  • the OabR-immobilized Hisprobe-HRP which targets the His-tag of OhrR, binds to dsDNA immobilized in the well, resulting in the highest absorption of TMB.
  • biothiol is present with CHP, OhrR is dissociated from dsDNA immobilized on the surface of the biochip and is removed by washing. Therefore, hisprobe-HRP is combined to reduce the absorption signal of TMB reacting with HRP.
  • the biothiol was observed to have a greater effect of reducing the absorption signal than L-cysteine and Homocysteine or GSH under the same concentration conditions [FIG. 15].
  • the strip sensor illustrated in FIG. 16 may be composed of three main parts (sample introduction part, reaction part, and measuring part), and cellulose, nitrocellulose and glass fiber as membranes for fabricating the strip sensor. -fiber) membrane and the like can be used.
  • the structure and characteristics of each site are as follows.
  • Each part is composed of different membranes, but each membrane is fixed to overlap on a general OHP film (0.4 cm ⁇ 5.5 cm), and the reaction pad is placed at the bottom of the sample to keep the capillary phenomenon of the entire strip sensor constant. It is fixed to be connected to the introduction part and the measurement part pad, and the sample introduction part is positioned at the top for easy absorption.
  • Sample introduction (corresponding to the left assay site where the sample from the upper sensor is introduced): 10 ⁇ L of a solution of 1 ⁇ M (TBS buffer) complex and biothiol combining OhrR tagged with FLAG and double stand DNA (SEQ ID NOs: 1 and 2) After mixing 10 ⁇ L of the sample solution (buffer, blood, urine, etc.) containing 20 ⁇ L of this mixture and 20 ⁇ L of the buffer containing CHP solution (2 ⁇ M), a total of 40 ⁇ L solution was added to the cellulose membrane (0.4 cm ⁇ 1.5 cm). And then adsorbed, the substance dissolved in the sample moves to the sensor reaction part by the principle of chromatography.
  • Reaction part (corresponding to the central gray area where the DNA movement arrow of the upper sensor of FIG. 16 is drawn): Constructed using a nitrocellulose membrane (approximately 0.4 cm x 2.5 cm) and located about 1 cm to the right of the sample introduction direction. Immobilize the anti-FLAG antibody solution (1 mg / mL, phosphorylated buffer solution) at about 1 hr by dropping about 1 ⁇ L. This site is bound by the FLAG antibody immobilized on the surface as the Flag-OhrR-dsDNA complex reacted at the sample introduction part moves. In the presence of biothiol in the sample, dsDNA dissociates from OhrR and continues to move to the right on the membrane. In the absence of biothiol, dsDNA is fixed like OhrR by the antibody and stops moving.
  • the measuring unit is a site for measuring double strand DNA dissociated from the binding and separation unit, and the signal of DNAzyme attached to the dsDNA terminal It is a site that induces.
  • the glass fiber membrane (0.4 cm ⁇ 0.5 cm) can be used to pre-dispense the Hemin solution to remain and confirm the signal response by dropping the final TMB solution or ECL solution.
  • Hemin was prepared by mixing a 5% casein solution in a buffer (40 mM Tris, 200 mM NaCl, 50 mM KCl, and 20 mM MgCl 2 ) solution, and then sufficiently absorbed 100 ⁇ L into a glass fiber membrane and dried at 55 ° C. for 30 minutes. Prepare. After sufficient dissociation of the dsDNA in the reaction part, 10 ⁇ L of the ECL solution and the TMB reaction solution are added for the final signal analysis to induce the reaction. The ECL solution can be analyzed by imaging as soon as the reaction solution is added with the chemiluminescence spectrometer, and the TMB reaction results can be analyzed about 30 minutes after the solution is added to the holder equipped with a mobile phone or digital camera.
  • a buffer 40 mM Tris, 200 mM NaCl, 50 mM KCl, and 20 mM MgCl 2
  • the same sample is divided into two equal volumes, one is treated with DNA-OhrR complex after treatment with DTT for 1 hour or more, and the other is directly reacted with DNA-OhrR complex without DTT addition. That is, the following items can be determined from the same two analysis results.
  • Sample 1 and Sample 2 are analyzed by classifying each sample into characteristics, by disease type, by disease stage, by age, and by gender, and then comparing them.

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Abstract

The present invention relates to a biothiol detecting composition comprising a redox regulation protein, a method for detecting biothiols by using the same, and a biosensor/kit for detecting biothiols. The present invention provides the effect of rapidly measuring free biothiols in body fluids. In addition, relative content ratios and changes of total to free biothiols in body fluids can be detected in real time, which allows biothiols to be available as main indices of diseases through which prediction and warning can be made against various diseases. Further, because various redox stress changes associated with main diseases can be accounted for by variations of biothiols, the present invention can provide important technical, economical, and social values for the investigation of pathogenesis mechanisms and the diagnosis of diseases in the future.

Description

산화환원조절 단백질을 포함하는 바이오티올 검출용 조성물Biothiol detecting composition comprising redox protein
본 발명은 산화환원조절 단백질을 포함하는 바이오티올 검출용 조성물, 상기 조성물을 이용한 바이오티올 검출방법 및 바이오티올 검출용 바이오센서/키트에 관한 것이다. The present invention relates to a biothiol detection composition comprising a redox control protein, a biothiol detection method using the composition, and a biosensor / kit for biothiol detection.
바이오티올(Biothiol)은, 단백질에 존재하는 Cysteine 등의 thiol과는 달리, 저분자량(low molecular weight, LMW) thiol로서 박테리아로부터 인간에 이르기까지 모든 생명체에서 생체 내의 산화적 스트레스 저항 및 생리 활성 조절에 중요한 기능을 수행한다. 이러한 바이오티올은 산화환원 반응에 매우 민감하여 SOH, SO2H, SNO, S-S 등의 기능기로 변화하여 생체분자 활성의 조절스위치 역할을 하고 있고, 세포 내에 다량 존재할 뿐 아니라, 혈액, 소변, 땀, 눈물 등 인간의 주요 체액에서도 광범위하게 감지되는 물질이다. 체액 내에 존재하는 바이오티올로는 시스테인(cysteine, Cys), 호모시스테인(homocysteine, Hcy), 글루타치온(glutathione, GSH), N-아세틸시스테인(N-acetylcysteine, NAC), 시스티아민(cysteamine, CA), γ-글루타밀시스테인(γ-glutamylcysteine, γ-GluCys), 시스테이닐글리신(cysteinylglycine, CysGly), N-아세틸시스테인(N-acetylcysteine, N-AC), 코엔자임 A(Coenzyme A, CoA), 코엔자임 B(Coenzyme B, CoB), 코엔자임 M(Coenzyme M, CoM), 바실리티올(bacillithiol, BacT), 미코티올(mycothiol, MyT), 에르고티오네인(ergothioneine, ErT), 트리판오티온(trypanothione, TrT) 등이 있다. 이들 중 특히 Cys, Hcy, GSH의 생체 내 농도 변화는 다양한 종류의 질병과 깊은 연관이 되어 있고 서로 다른 농도범위에서 존재해 있는 것으로 보고되어 있다[비특허문헌 1,2]. 실제 혈장(plasma) 내에서 Hcy, Cys, GSH의 전체(total) 농도(산화 및 환원된 총량)는 각각 6-20 μM, 150-350 μM, 4-10 μM 농도로 존재하지만 혈장 내의 높은 산화조건으로 Hcy 및 Cys는 거의 산화된 형태로 존재하여 유리 형태(free form)는 극히 작아서 Hcy의 경우 약 0.2 μM 이하 Cys의 경우 약 10 μM 이하로 존재하고 GSH의 경우에도 마찬가지로 세포 내에서는 과량 존재(> 5 mM)하는 것과는 달리 혈장 내에서는 γ-glutamyltransferase 등에 의해 빠르게 전환되어 유리형태는 2 μM 이하 농도로만 존재하는 것으로 알려져 있다. 이와 같은 바이오티올의 총 농도 및 유리 형태의 농도의 상대적인 변화는 심혈관질환(cardiovascular disease), 퇴행성 뇌질환(neurodegenerative disease), 암(cancer), 신장병(kidney dysfunction), 당뇨(diabetes mellitus), 박테리아 및 바이러스 감염(infection) 등을 포함한 주요 질병들과 폭넓은 연관관계를 갖고 있는 것으로 알려져 있다[비특허문헌 3~7]. 그러나 상기와 같이 주요 바이오티올이 질병 공통 바이오마커로서 생체 이상반응을 초기에 감지할 수 있는 지표물질로서 활용될 수 있음에도 불구하고 바이오티올의 빠른 산화 환원 과정으로 그 농도를 효과적으로 탐지할 수 있는 방법의 부재로 인하여 현재까지 폭넓게 사용하지 못하고 있는 실정이다. Biothiol is a low molecular weight (LMW) thiol, unlike the thiols such as Cysteine, which are present in proteins, to regulate oxidative stress resistance and physiological activity in vivo in all living things, from bacteria to humans. Perform important functions. These biothiols are very sensitive to redox reactions, so they switch to functional groups such as SOH, SO 2 H, SNO, and SS, and act as a control switch of biomolecule activity. It is also widely detected in major body fluids such as tears. Biothiols present in body fluids include cysteine (Cys), homocysteine (Hcy), glutathione (GSH), N-acetylcysteine (NAC), cysteamine (CA), γ -Glutamylcysteine (γ-GluCys), Cysteinylglycine (CysGly), N-acetylcysteine (N-AC), Coenzyme A (CoA), Coenzyme B ( Coenzyme B, CoB), Coenzyme M (CoM), bacillithiol (BacT), mycothiol (MyT), ergothioneine (ERT), trypanothione (TrT) Etc. Among them, in particular, changes in the concentrations of Cys, Hcy, and GSH in vivo are closely related to various kinds of diseases and reported to exist in different concentration ranges [Non-Patent Documents 1 and 2]. The total concentrations of Hcy, Cys, and GSH (total amounts oxidized and reduced) in the plasma are present at concentrations of 6-20 μM, 150-350 μM and 4-10 μM, respectively, but high oxidative conditions in plasma Hcy and Cys are present in almost oxidized form, so the free form is extremely small, less than about 0.2 μM for Hcy, and less than about 10 μM for Cys. 5 mM) is rapidly transformed by γ-glutamyltransferase in plasma and is known to exist only at a concentration of 2 μM or less. Relative changes in the total and free form concentrations of such biothiols include cardiovascular disease, degenerative brain disease, cancer, kidney dysfunction, diabetes mellitus, bacteria and It is known to have a wide correlation with major diseases including viral infection (Non-Patent Documents 3-7). However, despite the fact that the main biothiol can be used as an indicator for early detection of biological adverse reactions as a common biomarker for diseases, the rapid redox process of biothiol can effectively detect its concentration. Due to the absence of a wide range of use to date.
바이오티올의 양을 측정할 수 있는 기존 표준분석법은 초고속 액체 크로마토그래피(High Performance Liquid Chromatography, HPLC), 기체 크로마토그래피-질량분석법(Gas Chromatography-Mass Spectrometry, GC-MS), 또는 모세관 전기이동분석법(Capillary Electrophoresis, CE)에 대부분 의존하고 있다. 그러나 이러한 분석법들은 체액 시료 전처리, 분석 소요 시간 과다 등으로 인하여 환원된 총 바이오티올 양을 측정할 수 밖에 없는 기술적 한계점과 비용 문제가 가장 큰 장애요소이다. 다양한 문헌에서 산화방지제, 금속이온 킬레이트제의 첨가와 시료 전처리 과정을 통해 유리 바이오티올을 측정하여 보고하였으나[비특허문헌 8, 9]. 이러한 환경에서도 유리 바이오티올 이 산화되는 속도가 매우 빠르고 분석이 대부분 장시간 소요되는 문제점 때문에 유리 바이오티올의 양을 정확하게 측정하는데 어려움이 있다.Existing standard methods that can measure the amount of biothiol include High Performance Liquid Chromatography (HPLC), Gas Chromatography-Mass Spectrometry (GC-MS), or Capillary Electrophoresis (GC-MS). Capillary Electrophoresis (CE). However, these methods are the biggest obstacles in terms of technical limitations and cost, which can only be measured by reducing the total amount of biothiol reduced due to fluid sample preparation and excessive analysis time. Various literatures have reported and measured free biothiol through the addition of antioxidants, metal ion chelating agents and sample pretreatment [Non-Patent Documents 8, 9]. Even in such an environment, the rate of oxidation of the free biothiol is very fast and the analysis takes a long time, which makes it difficult to accurately measure the amount of free biothiol.
또 다른 바이오티올 측정방식으로서 현재 키트형태로 판매되고 있는 항체기반의 면역측정법(immunoassay)이 널리 사용되고 있으나, 사용된 항체는 모두 유리 바이오티올을 직접 인식하는 것이 아니라 바이오티올 이 산화되어 단백질과 결합된 형태를 인식하게 된다(혈액의 경우 serum albumin 등). 따라서 유리 바이오티올 및 총 바이오티올의 동적 변화를 탐지할 수 없는 한계점이 있다.As another biothiol measurement method, antibody-based immunoassays, which are currently sold in kit form, are widely used. However, all of the used antibodies do not directly recognize free biothiols, but instead oxidize the biothiols to bind proteins. It recognizes the form (such as serum albumin for blood). Thus, there is a limitation in that dynamic changes in free biothiol and total biothiol cannot be detected.
현재 보고된 방법 중 유리 바이오티올의 측정법은 화학결합에 기반을 둔 화학 센서로서, rhodamine, fluorescein, BODIPY, cyanine, flavone, coumarin 등의 형광 dye의 변형체를 활용하여 바이오티올과의 결합 시 유도되는 형광변화 측정을 기초로 하고 있다[비특허문헌 8]. 이 방법들이 유리 바이오티올과 빠르게 반응하지만 이와 같은 형광변화를 유도하는 물질은 대부분 벤젠고리변형체와 기능기에 기초한 합성물질로서 용해성이 매우 낮고 pH 변화에 취약하며, 유리 티올의 -SH기를 이용하여 측정하므로 디설파이드(disulfide) 형태로 단백질에 결합되어 있는 바이오티올 측정이 어렵고, 단백질에 존재하는 Cys의 -SH와도 반응할 수 있어 저분자량 유리 바이오티올만을 특이적으로 측정하는데 큰 한계가 있다. 무엇보다 체액 내 직접 적용 시 광범위한 자가형광(autofluorescence)에 의한 간섭으로 재현성 및 정밀도에 크게 제한이 있어 대부분 세포 내 형광이미징 등의 제한된 용도로서만 사용되고 있다.Among the currently reported methods, the measurement of free biothiol is a chemical sensor based on chemical bonding. Fluorescence induced by binding to biothiol by using variants of fluorescent dyes such as rhodamine, fluorescein, BODIPY, cyanine, flavone, and coumarin It is based on the change measurement [nonpatent literature 8]. Although these methods react rapidly with free biothiol, most of the materials that induce such fluorescence changes are synthetic compounds based on benzene ring variants and functional groups, which have very low solubility, are vulnerable to pH changes, and are measured using the -SH group of free thiol. It is difficult to measure the biothiol bound to the protein in the form of disulfide, and it can react with -SH of Cys present in the protein, so there is a big limitation in measuring only low molecular weight free biothiol. Above all, when applied directly in body fluids, the reproducibility and precision are greatly limited due to the wide range of autofluorescence interferences, and most of them are used only for limited applications such as intracellular fluorescence imaging.
따라서 바이오티올을 질병 경고를 위한 지표인자로 활용하기 위해서는 위와 같은 문제점을 해결하고 체액 내에서 유리 바이오티올 및 총 바이오티올의 함량을 신속하고 정확하게 측정하는 기술의 개발이 절실하다.Therefore, in order to utilize biothiol as an indicator for disease warning, there is an urgent need to solve the above problems and to develop a technique for quickly and accurately measuring the content of free biothiol and total biothiol in body fluids.
[비특허문헌][Non-Patent Documents]
(비특허문헌 1)Persichilli, S., Gervasoni, J., Castagnola, M., Zuppi, C. & Zappacosta, B. A Reversed-Phase HPLC Fluorimetric Method for Simultaneous Determination of Homocysteine-Related Thiols in Different Body Fluids. Labmedicine 42, 657-662 (2011)(Non-Patent Document 1) Persichilli, S., Gervasoni, J., Castagnola, M., Zuppi, C. & Zappacosta, B. A Reversed-Phase HPLC Fluorimetric Method for Simultaneous Determination of Homocysteine-Related Thiols in Different Body Fluids. Labmedicine 42, 657-662 (2011)
(비특허문헌 2)Fiskerstrand, T., Refsum, H., Kvalheim, G. & Ueland, P. M. Homocysteine and Other Thiols in Plasma and Urine - Automated-Determination and Sample Stability. Clin Chem 39, 263-271 (1993))(Non-Patent Document 2) Fiskerstrand, T., Refsum, H., Kvalheim, G. & Ueland, P. M. Homocysteine and Other Thiols in Plasma and Urine-Automated-Determination and Sample Stability. Clin Chem 39, 263-271 (1993))
(비특허문헌 3)Seshadri, S. et al. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. New Engl J Med 346, 476-483, (2002)(Non-Patent Document 3) Seshadri, S. et al. Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. New Engl J Med 346, 476-483, (2002)
(비특허문헌 4)Refsum, H., Ueland, P. M., Nygard, O. & Vollset, S. E. Homocysteine and cardiovascular disease. Annu Rev Med 49, 31-62 (1998)(Non-Patent Document 4) Refsum, H., Ueland, P. M., Nygard, O. & Vollset, S. E. Homocysteine and cardiovascular disease. Annu Rev Med 49, 31-62 (1998)
(비특허문헌 5)Herzenberg, L. A. et al. Glutathione deficiency is associated with impaired survival in HIV disease. P Natl Acad Sci USA 94, 1967-1972 (1997).(Non-Patent Document 5) Herzenberg, L. A. et al. Glutathione deficiency is associated with impaired survival in HIV disease. P Natl Acad Sci USA 94, 1967-1972 (1997).
(비특허문헌 6)El-Khairy, L., Ueland, P. M., Refsum, H., Graham, I. M. & Vollset, S. E. in Circulation Vol. 103 2544-2549 (2001).(Non-Patent Document 6) El-Khairy, L., Ueland, P. M., Refsum, H., Graham, I. M. & Vollset, S. E. in Circulation Vol. 103 2544-2549 (2001).
(비특허문헌 7)Andersson, A., Lindgren, A., Arnadottir, M., Prytz, H. & Hultberg, B. Thiols as a measure of plasma redox status in healthy subjects and in patients with renal or liver failure. Clin Chem 45, 1084-1086 (1999).(Non-Patent Document 7) Andersson, A., Lindgren, A., Arnadottir, M., Prytz, H. & Hultberg, B. Thiols as a measure of plasma redox status in healthy subjects and in patients with renal or liver failure. Clin Chem 45, 1084-1086 (1999).
(비특허문헌 8)Jung, H. S., Chen, X. Q., Kim, J. S. & Yoon, J. Recent progress in luminescent and colorimetric chemosensors for detection of thiols. Chem Soc Rev 42, 6019-6031, doi:Doi 10.1039/C3cs60024f (2013).(Non-Patent Document 8) Jung, H. S., Chen, X. Q., Kim, J. S. & Yoon, J. Recent progress in luminescent and colorimetric chemosensors for detection of thiols. Chem Soc Rev 42, 6019-6031, doi: Doi 10.1039 / C3cs60024f (2013).
이에, 본 발명자들은 기존 바이오티올의 검출방법의 문제점을 해결하기 위하여 연구 노력한 결과, 산화환원 조절 단백질(redox-regulating protein)을 이용하여 저분자량의 바이오티올을 검출할 수 있으며, 유리 바이오티올 및 총(Total) 바이오티올의 동시 측정이 가능하며, 미량으로도 검출이 가능하여 민감도를 향상시켰을 뿐만 아니라 신속성, 보관안정성 면에서도 우수한 바이오티올 검출용 조성물을 개발함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors have made a research effort to solve the problem of the conventional detection method of the biothiol, it is possible to detect a low molecular weight biothiol using a redox-regulating protein, free biothiol and total Simultaneous measurement of (Total) biothiol was possible, and the present invention was completed by developing a composition for detecting biothiol which is excellent in terms of rapidity and storage stability as well as improved sensitivity by detecting even a small amount.
따라서, 본 발명은 산화환원 조절 단백질(redox-regulating protein)을 포함하는 바이오티올 검출용 조성물을 제공하는데 목적이 있다.Accordingly, an object of the present invention is to provide a composition for detecting biothiol, which comprises a redox-regulating protein.
또한, 본 발명은 상기 조성물을 이용한 바이오티올의 검출방법을 제공하는데 다른 목적이 있다.Another object of the present invention is to provide a method for detecting biothiol using the composition.
또한, 본 발명은 상기 조성물을 이용한 바이오센서를 제공하는데 또 다른 목적이 있다.In addition, the present invention has another object to provide a biosensor using the composition.
또한, 본 발명은 상기 조성물을 이용한 바이오 칩을 제공하는데 또 다른 목적이 있다.In addition, the present invention has another object to provide a biochip using the composition.
상기 과제를 해결하기 위한 수단으로서, 본 발명은 산화환원 조절 단백질(redox-regulating protein)을 포함하는 바이오티올 검출용 조성물을 제공한다.As a means for solving the above problems, the present invention provides a composition for detecting biothiol comprising a redox-regulating protein.
상기 과제를 해결하기 위한 다른 수단으로서, 본 발명은 상기 조성물을 이용한 바이오티올의 검출방법을 제공한다.As another means for solving the above problems, the present invention provides a method for detecting biothiol using the composition.
상기 과제를 해결하기 위한 또 다른 수단으로서, 본 발명은 상기 조성물을 이용한 바이오센서를 제공한다.As another means for solving the above problems, the present invention provides a biosensor using the composition.
상기 과제를 해결하기 위한 또 다른 수단으로서, 본 발명은 상기 조성물을 이용한 바이오 칩을 제공한다.As another means for solving the above problems, the present invention provides a biochip using the composition.
본 발명에 따른 바이오티올 검출용 조성물은 체액 내에서 유리 형태의 바이오티올을 신속히 측정할 수 있다. 또한, 체액 내 총(total) 및 유리 바이오티올의 상대적 함량비와 변화를 실시간으로 검출할 수 있어 바이오티올을 질병의 주요 지표로 활용 가능하게 하고, 이를 통해 다양한 질병에 대한 예측 및 경고가 가능하다. 또한, 주요 질병과 연관된 각종 산화환원 스트레스 변화를 바이오티올의 변화량으로 설명할 수 있어 향후 질병 발병기전 규명 및 진단을 위한 중요한 기술적, 경제적, 사회적 가치를 제공할 수 있다. The composition for detecting biothiol according to the present invention can rapidly measure the biothiol in free form in the body fluid. In addition, the relative content ratio and change of total and free biothiol in body fluids can be detected in real time, making it possible to use biothiol as a major indicator of disease, thereby enabling prediction and warning of various diseases. . In addition, various redox stress changes associated with major diseases can be explained by the amount of change in biothiol, which can provide important technical, economic and social values for the identification and diagnosis of disease pathogenesis in the future.
도 1은 OhrR과 dsDNA가 바이오티올에 의해 해리되는 원리를 나타낸 모식도이다. 1 is a schematic diagram showing the principle that OhrR and dsDNA are dissociated by biothiol.
도 2는 FA (Fluorescence anisotrophy)법을 이용한 OhrR의 Cys (Cysteine), Hcy (homocysteine), GSH (glutathione) 대한 실시간 결합반응 측정 결과이다.2 is a result of measuring the real-time binding reaction of Cys (Cysteine), Hcy (homocysteine), GSH (glutathione) of OhrR using FA (Fluorescence anisotrophy) method.
도 3은 전기영동법을 이용한 OhrR 단백질과 타겟 DNA의 결합 확인 실험 결과이다.3 is a result of confirming the binding of the OhrR protein and the target DNA using the electrophoresis method.
도 4는 광 센서 표면 위에 형광표지 없이 DNA와 OhrR 단백질이 바이오티올에 의해 해리되는 과정을 광 굴절률을 사용하여 측정한 실험 결과이다.4 is an experimental result of measuring the process of dissociation of DNA and OhrR protein by biothiol without fluorescent label on the optical sensor surface using optical refractive index.
도 5는 MALDI-TOF MS를 이용하여 OhrR과 바이오티올과의 결합을 정량적으로 분석가능한지 확인한 결과이다.5 is a result confirming that the binding of OhrR and biothiol can be quantitatively analyzed using MALDI-TOF MS.
도 6은 MALDI-TOF MS 및 OhrR을 이용하여 마우스 혈액 시료와 사람 혈액 시료에서 대조군 및 비교군에서의 유리 바이오티올의 상대적 양을 분석한 실험결과이다.6 is an experimental result of analyzing the relative amounts of free biothiol in the control and comparison groups in the mouse blood samples and human blood samples using MALDI-TOF MS and OhrR.
도 7은 전체 바이오티올 측정 가능성을 확인하기 위하여 FA 법을 이용한 환원조건에서 OhrR과 Cys의 실시간 반응 조사 결과이다.7 is a result of real-time reaction investigation of OhrR and Cys under reducing conditions using FA method to confirm the possibility of measuring the total biothiol.
도 8은 MALDI-MS 및 OhrR을 이용하여 마우스 혈액에서 시스테인의 농도를 첨가한 이후 유리 시스테인 및 총 시스테인을 검출한 결과이다8 shows the results of detecting free cysteine and total cysteine after adding cysteine concentration in mouse blood using MALDI-MS and OhrR.
도 9는 본 발명의 일 실시예에 따른 바이오티올 검출과정을 나타낸 모식도이다.9 is a schematic diagram showing a biothiol detection process according to an embodiment of the present invention.
도 10은 도 9의 바이오티올의 검출방법에 따른 형광 측정 결과를 나타낸 것이다.FIG. 10 shows fluorescence measurement results according to the biothiol detection method of FIG. 9.
도 11은 도 9의 바이오티올의 검출방법에 따른 화학발광(chemiluminescence) 측정 결과이다.FIG. 11 is a chemiluminescence measurement result according to the biothiol detection method of FIG. 9.
도 12는 DNA의 신호증폭을 나타낸 모식도이다.12 is a schematic diagram showing signal amplification of DNA.
도 13a는 도 12의 DNA 신호증폭을 확인하기 위한 화학발광 측정결과를 나타낸 것이다.Figure 13a shows the chemiluminescence measurement results for confirming the DNA signal amplification of Figure 12.
도 13b는 도 12의 DNA의 신호증폭을 확인하기 위한 전기영동 결과를 나타낸 것이다.Figure 13b shows the results of electrophoresis to confirm the signal amplification of the DNA of Figure 12.
도 14는 OhrR과 DNA간 결합을 이용하여 바이오티올 측정하기 위한 바이오칩 구성의 모식도이다.14 is a schematic diagram of a biochip configuration for measuring biothiol using binding between OhrR and DNA.
도 15는 도 14의 바이오칩을 이용하여 바이오티올을 검출한 결과이다.FIG. 15 is a result of detecting biothiol using the biochip of FIG. 14.
도 16은 도 9의 스트립 형태의 바이오센서의 모식도이다.FIG. 16 is a schematic diagram of the biosensor in strip form of FIG. 9.
본 발명의 일 구현예에 따라, 산화환원 조절 단백질(redox-regulating protein)을 포함하는 바이오티올 검출용 조성물 및 이를 이용한 바이오티올 검출방법을 포함한다. According to one embodiment of the present invention, a biothiol detection composition comprising a redox-regulating protein and a biothiol detection method using the same are included.
본 발명의 일 구현예에 따라, 산화환원 조절 단백질 외에 산화환원 조절 단백질에 결합되는 DNA을 추가로 포함하는 바이오티올 검출용 조성물 및 이의 조성물을 이용한 바이오티올 검출방법을 포함한다.According to one embodiment of the present invention, in addition to the redox control protein, a biothiol detecting composition further comprises a DNA bound to the redox control protein and a biothiol detection method using the composition thereof.
본 발명에서 사용된 용어 "산화환원 조절 단백질(redox-regulating protein)"은 단백질의 산화와 환원 반응에 의해 활성이 조절되는 모든 단백질을 의미하며, 대표적으로 B. subtilis 등의 일부 박테리아에 존재하는 OhrR(organic hydroperoxide regulator), PerR(Peroxide regulator), 그리고 E. coli를 포함한 일부 박테리아에 존재하는 OxyR(Oxygen regulator) 등이 있다. 또한, 단백질 공학을 도입하여 산화환원 조절 단백질 활성 부위 아미노산을 변형시키거나, 다른 종류의 생물체 내에 존재하는 상동분자(orthologue) 단백질을 스크리닝함으로써 특정 바이오티올과 보다 선택적으로 반응하는 산화환원 조절 단백질을 포함할 수 있다.As used herein, the term "redox-regulating protein" refers to all proteins whose activity is regulated by the oxidation and reduction of proteins, typically OhrR present in some bacteria such as B. subtilis . (organic hydroperoxide regulator), PerR (Peroxide regulator), and OxyR (Oxygen regulator) present in some bacteria, including E. coli . In addition, protein engineering may be used to modify redox regulatory protein active site amino acids, or to screen for orthologue proteins present in other types of organisms, including redox regulatory proteins that react more selectively with specific biothiols. can do.
상기 산화환원 조절 단백질은 바이오티올과 DNA와의 결합친화도 조절이 가능한 변이체이거나 표지 단백질이 접합된 형태의 단백질을 포함할 수 있다. 상기 접합 형태(conjugate form)의 예로는 형광단백질-OhrR, 발광단백질-OhrR, FLAG-OhrR, His6-OhrR, GSH-OhrR, Biotin-OhrR 등일 수 있다.The redox regulatory protein may include a variant capable of controlling binding affinity between biothiol and DNA or a protein in which a labeled protein is conjugated. Examples of the conjugate form may include a fluorescent protein-OhrR, a light emitting protein-OhrR, a FLAG-OhrR, His6-OhrR, GSH-OhrR, Biotin-OhrR, and the like.
본 발명에 사용된 용어 "바이오티올"은 분자량이 10 Da 내지 1,000 Da 이하(바람직하게는 10 Da 내지 500 Da)인 저분자량의 티올을 말하며, 구체적으로 시스테인(cysteine, Cys), 호모시스테인(homocysteine, Hcy), 글루타치온(glutathione, GSH), N-아세틸시스테인(N-acetylcysteine, NAC), 시스티아민(cysteamine, CA), γ-글루타밀시스테인(γ-glutamylcysteine, γ-GluCys), 시스테이닐글리신(cysteinylglycine, CysGly), N-아세틸시스테인(N-acetylcysteine, N-AC), 코엔자임 A(Coenzyme A, CoA), 코엔자임 B(Coenzyme B, CoB), 코엔자임 M(Coenzyme M, CoM), 바실리티올(bacillithiol, BacT), 미코티올(mycothiol, MyT), 에르고티오네인(ergothioneine, ErT) 및 트리판오티온(trypanothione, TrT)으로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 제한되지 않는다.As used herein, the term "biothiol" refers to a low molecular weight thiol having a molecular weight of 10 Da to 1,000 Da or less (preferably 10 Da to 500 Da), specifically cysteine (Cys), homocysteine (homocysteine, Hcy), glutathione (GSH), N-acetylcysteine (NAC), cysteamine (CA), γ-glutamylcysteine (γ-GluCys), cysteinylglycine ( cysteinylglycine, CysGly), N-acetylcysteine (N-AC), Coenzyme A (CoA), Coenzyme B (Coenzyme B, CoB), Coenzyme M (Coenzyme M, CoM), bacillithiol , BacT), mycothiol (MyT), ergothioneine (ERT) and trypanothion (trypanothione, TrT) may be one or more selected from the group, but is not limited thereto.
또한, 바이오티올은 심혈관질환(cardiovascular disease), 퇴행성 뇌질환(neurodegenerative disease), 암(cancer), 신장병(kidney dysfunction), 당뇨(diabetes mellitus) 또는 박테리아 및 바이러스 감염(infection) 등과 관련된 다양한 질병의 바이오마커로서 생체 이상 반응을 초기에 감지할 수 있는 지표물질이다In addition, biothiol can be used for various diseases related to cardiovascular disease, neurorodegenerative disease, cancer, cancer, kidney dysfunction, diabetes, diabetes mellitus, or bacterial and viral infections. As a marker, it is an indicator that can detect abnormal biological reactions early.
본 발명은 또한, 산화환원 조절 단백질 및 산화환원 조절 단백질에 결합되는 DNA을 포함하는 바이오티올 검출용 조성물 및 이의 조성물을 이용한 바이오티올 검출방법을 포함한다.The present invention also includes a biothiol detection composition comprising a redox control protein and a DNA bound to the redox control protein and a biothiol detection method using the composition.
본 발명의 일 구현예에 있어서, 산화환원 조절 단백질 및 산화환원 조절 단백질에 결합되는 DNA의 결합/해리 원리를 이용하여 바이오티올을 검출할 수 있다.In one embodiment of the present invention, biothiol can be detected using the principle of binding / dissociation of redox regulatory protein and DNA bound to redox regulatory protein.
상기 DNA는 일 예로, 하기 실시예에서 사용된 서열번호 1 및/또는 서열번호 2로 표시되는 것일 수 있으며, 산화환원 조절 단백질에 결합되는 것이라면 모두 가능하다.The DNA may be, for example, one represented by SEQ ID NO: 1 and / or SEQ ID NO: 2 used in the following Examples, and may be any compound that binds to a redox regulatory protein.
이러한 바이오티올의 검출 원리는 일 구현예로서 첨부도면 도 1에 나타낸 바와 같다.The detection principle of such a biothiol is shown in FIG. 1 as an embodiment.
상기 산화환원 조절 단백질의 대표적인 예인 OhrR은 박테리아에 존재하는 유기과산화물(organic hydroperoxide, ROOH) 감지인자로 OhrR은 homodimer로 존재하며 각 monomer당 1개의 시스테인 잔기를 가지고 있다. 시스테인 잔기가 환원되어 있는 상태(-SH)에서 OhrR은 DNA와 결합한 형태(OhrR와 DNA의 복합체)를 유지하고 있고, 유기과산화물이 존재하는 환경에서 OhrR은 빠르게 산화가 일어난다(-SOH). 산화된 OhrR은 DNA와의 결합을 유지하다가, 바이오티올이 존재하는 환경에서는 바이오티올과 빠르게 반응하여 DNA로부터 해리된다(일반적으로 바이오티올이 10 μM 이상에서 t1/2~0.5 min; t1/ 2은 단백질이 DNA로부터 50% 해리되는데 걸리는 시간을 의미). 해리 속도는 바이오티올의 농도 및 종류에 따라서 다르게 나타낸다. 반면, 바이오티올이 존재하지 않는 조건에서는 ROOH에 의해서 OhrR이 상대적으로 느린 속도로 설펜아미드(-S-N-)를 형성하여 천천히 DNA로부터 해리된다 (t1/2~10 min). OhrR, a representative example of the redox control protein, is an organic perperoxide (ROOH) sensor in bacteria. OhrR is a homodimer and has one cysteine residue per monomer. In the state where the cysteine residue is reduced (-SH), OhrR maintains the form of binding to DNA (complex of OhrR and DNA), and OhrR is rapidly oxidized in the presence of organic peroxide (-SOH). The oxidation is OhrR while maintaining engagement with the DNA, the environment in which the present bio-thiol to quickly react with the bio-thiol is released from the DNA (typically bio-thiol The t 1/2 ~ 0.5 min in at least 10 μM; t 1/2 silver Protein takes 50% dissociation from DNA). Dissociation rates vary depending on the concentration and type of biothiol. On the other hand, in the absence of biothiol, OhrR forms sulfenamide (-SN-) at a relatively slow rate by ROOH and slowly dissociates from DNA (t 1/2 ~ 10 min).
상기 산화환원 조절 단백질에 결합되는 DNA는 형광인자 또는 DNA 기반 효소(DNAzyme)를 결합시켜 형광, 화학발광, 흡광 검출이 가능하게 하거나, DNA서열을 증폭시켜 신호 증폭방법으로 반응 감도를 향상시킬 수 있다.DNA bound to the redox regulatory protein may be coupled to a fluorescence factor or DNA-based enzyme (DNAzyme) to enable fluorescence, chemiluminescence, and absorption detection, or amplify DNA sequences to improve reaction sensitivity. .
본 발명에서 상기 형광인자의 구체적인 종류는 특별히 한정되지 않고, 예를 들면, 로다만과 그의 유도체, 플루오레신과 그의 유도체, 쿠마린과 그의 유도체, 아크리딘과 그의 유도체, 피렌과 그의 유도체, 에리트로신과 그의 유도체, 에오신과 그의 유도체, 및 4-아세트아미도-4′-이소티오시아나토스틸벤-2,2′디설폰산으로 이루어진 군으로부터 선택되는 하나 이상을 들 수 있다. 본 발명에서 사용할 수 있는 상기 형광 물질을 보다 구체적으로 예시하면 하기와 같다. In the present invention, the specific kind of the fluorescent factor is not particularly limited, and examples thereof include rhodaman and its derivatives, fluorescein and its derivatives, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives, and erythrosine. At least one selected from the group consisting of derivatives thereof, eosin and derivatives thereof, and 4-acetamido-4'-isothiocyanatostilben-2,2'disulfonic acid. More specifically illustrating the fluorescent material that can be used in the present invention is as follows.
로다민 및 그의 유도체로서6-카복시-X-로다민(ROX), 6-카복시로다민 (R6G), 리사민 로다민 B 설포닐 클로라이드, 로다민(Rhod), 로다민 B, 로다민 123, 로다민 X 이소티오시아네이트, 설포로다민 B, 설포로다민 101, 설포로다민 101의 설포닐 클로라이드 유도체(Texas Red), N,N,N′,N′-테트라메틸-6-카복시로다민(TAMRA), 테트라메틸 로다민, 테트라메틸 로다민 이소티오시아네이트(TRITC), 리보플라빈, 로졸산, 터븀 킬레이트 유도체, Alexa 유도체, Alexa-350, Alexa-488, Alexa-547 및 Alexa-647 등을 들 수 있고; Rhodamine and its derivatives include 6-carboxy-X-rhodamine (ROX), 6-carboxyrodamine (R6G), lysamine rhodamine B sulfonyl chloride, rhodamine (Rhodamine B, rhodamine 123, Rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivatives (Texas Red) of sulforhodamine 101, N, N, N ', N'-tetramethyl-6-carboxyrodamine (TAMRA), tetramethyl rhodamine, tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid, terbium chelate derivatives, Alexa derivatives, Alexa-350, Alexa-488, Alexa-547 and Alexa-647, etc. May be mentioned;
플루오레세인 및 그의 유도체로서 5-카복시플루오레세인(FAM), 5-(4,6-디클로로트리아진-2-일)아미노플루오레세인(DTAF), 2′7′-디메톡시-4′5′-디클로로-6-카복시플루오레세인(6-FAM), 플루오레세인, 플루오레세인 이소티오시아네이트, QFITC(XRITC), 플루오레스카민(fluorescamine), IR144, IR1446, 말라카이트 그린 이소티오시아네이트, 4-메틸움벨리페론, 오르토 크레졸 프탈레인, 니트로티로신, 파라로자닐린, 페놀 레드, B-피코에리트린 및 o-프탈디알데히드 등을 들 수 있으며; Fluorescein and its derivatives as 5-carboxyfluorescein (FAM), 5- (4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), 2'7'-dimethoxy-4 ' 5′-dichloro-6-carboxyfluorescein (6-FAM), fluorescein, fluorescein isothiocyanate, QFITC (XRITC), fluorescamine, IR144, IR1446, malachite green isothiocia Nate, 4-methylumbelliferone, ortho cresol phthalein, nitrotyrosine, para-rozaniline, phenol red, B-phycoerythrin and o-phthaldialdehyde and the like;
쿠마린 및 그의 유도체로서 쿠마린, 7-아미노-4-메틸쿠마린(AMC, 쿠마린 120), 7-아미노-4-트리플루오로메틸쿠마린(쿠마린 151), 시아노신, 4′-6-디아미니디노-2-페닐인돌(DAPI), 5′,5″-디브로모피로갈롤-설폰프탈레인 (Bromopyrogallol Red), 7-디에틸아미노-3-(4′-이소티오시아나토페닐)-4-메틸쿠마린 디에틸렌트리아민 펜타아세테이트, 4-(4′-디이소티오시아나토디하이드로-스틸벤-2,2′-디설폰산, 4,4′-디이소티오시아나토스틸벤-2,2′-디설폰산, 5-[디메틸아미노]나프탈렌-1-설포닐 클로라이드 (DNS, dansyl chloride), 4-(4′-디메틸아미노페닐아조)벤조산(DABCYL) 및 4-디메틸아미노페닐아조페닐-4′-이소티오시아네이트(DABITC) 등을 들 수 있고; Coumarin and its derivatives as coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151), cyanosine, 4′-6-diaminidino- 2-phenylindole (DAPI), 5 ′, 5 ″ -dibromopyrogallol-sulfonphthalein (Bromopyrogallol Red), 7-diethylamino-3- (4′-isothiocyanatophenyl) -4-methyl Coumarin diethylenetriamine pentaacetate, 4- (4'-diisothiocyanatodihydro-stilben-2,2'-disulfonic acid, 4,4'-diisothiocyanatostilben-2,2 ' Disulfonic acid, 5- [dimethylamino] naphthalene-1-sulfonyl chloride (DNS, dansyl chloride), 4- (4′-dimethylaminophenylazo) benzoic acid (DABCYL) and 4-dimethylaminophenylazophenyl-4 ′ Isothiocyanate (DABITC) and the like;
아크리딘 및 그의 유도체로서 아크리딘, 아크리딘 이소티오시아네이트, 5-(2′-아미노에틸)아미노나프탈렌-1-설폰산(EDANS), 4-아미노-N-[3-비닐설포닐)페닐]나프탈이미드-3,5디설포네이트(LuciferYellow VS), N-(4-아닐리노-1-나프틸)말레이미드, 안트라닐아미드 및 Brilliant Yellow 등을 들 수 있으며; Acridine and its derivatives as acridine, acridine isothiocyanate, 5- (2'-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS), 4-amino-N- [3-vinylsulfur Fonyl) phenyl] naphthalimide-3,5 disulfonate (LuciferYellow VS), N- (4-anilino-1-naphthyl) maleimide, anthranilamide and Brilliant Yellow;
피렌 및 그의 유도체로서 피렌, 피렌 부티레이트, 숙신이미딜 1-피렌 부티레이트, Reactive Red 4 (Cibacron®Brilliant Red 3B-A) 등을 들 수 있고; Pyrene and its derivatives include pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4 (Cibacron® Brilliant Red 3B-A) and the like;
에리트로신 및 그의 유도체로서 에리트로신 B, 에리트로신 이소티오시아네이트 및 에티듐 등을 들 수 있으며; Erythrosine and its derivatives include erythrosin B, erythrosin isothiocyanate and ethidium;
에오신 및 그의 유도체로서 에오신 및 에오신 이소티오시아네이트 등을 들 수 있고; Eosin and Eosin isothiocyanate etc. are mentioned as Eosin and its derivatives ;
4- 아세트아미도 -4′- 이소티오시아나토스틸벤 -2,2′ 디설폰산이 있다. 4- acetamido -4' -isothiocyanatostilbene- 2,2 ' disulfonic acid .
상기 DNA 기반 효소는 다양한 DNA 기반 효소 중에서 퍼옥시다제(Peroxidase) 특징을 가진 퍼옥시다제 모방 DNA 효소(peroxidase-mimicking DNA 효소)[Wang Li et al. Insight into G-quadruplex-hemin DNAzyme/RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity. Nucleic Acids Research 44(15); 7373-7384 (2016)] 및 RNA서열 절단 DNA 효소(RNA-cleaving DNA 효소)[Meng Liu, Dingran Chang, and Yingfu Li. Discovery and Biosensing Applications of Diverse RNA-Cleaving DNAzymes, Accounts of Chemical Research, 50; 2273-2283 (2017)]로 이루어진 군에서 선택된 하나 이상의 서열을 가지는 것일 수 있으나, 이에 제한되지 않는다.The DNA-based enzyme is a peroxidase-mimicking DNA enzyme having peroxidase characteristics among various DNA-based enzymes (Wang Li et al. Insight into G-quadruplex-hemin DNAzyme / RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity. Nucleic Acids Research 44 (15); 7373-7384 (2016)] and RNA-sequencing DNA enzymes (RNA-cleaving DNA enzymes) [Meng Liu, Dingran Chang, and Yingfu Li. Discovery and Biosensing Applications of Diverse RNA-Cleaving DNAzymes, Accounts of Chemical Research, 50; 2273-2283 (2017)] may be, but is not limited to having one or more sequences selected from the group consisting of.
상기 신호 증폭방법은 산화환원조절 단백질 결합 DNA의 한쪽 끝에 짧은(short) DNA 서열을 결합시키고(DNA 증폭이 가능한 single strand DNA template가 결합) Hairpin1 (HP1)과 Hairpin 2 (HP2)를 추가적으로 반응시키게 함으로써 DNA 서열이 PCR 사용 없이도 증폭이 된다. DNA 서열 및 길이, Hairpin의 종류는 하기 실시예(표 1)에 기재된 것으로 한정되지 않는다.The signal amplification method binds a short DNA sequence to one end of the redox regulatory protein binding DNA (a single strand DNA template capable of DNA amplification) and additionally reacts Hairpin1 (HP1) and Hairpin 2 (HP2). DNA sequences are amplified without the use of PCR. DNA sequence and length, and the type of hairpin is not limited to those described in the following Examples (Table 1).
또한 DNA 서열 5' 말단 혹은 3' 말단 부위에 biotin기, alkyne기, azide기, thiol기, amine기 등을 포함한 태그(tag)를 붙여 비드, 나노입자, 칩 표면에 DNA만을 분리하거나 결합하여 이용할 수 있다.In addition, a tag including a biotin group, an alkyne group, an azide group, a thiol group, an amine group, etc. is attached to the 5 'end or 3' end of the DNA sequence to separate or bind DNA only on beads, nanoparticles, and chip surfaces. Can be.
본 발명에 사용된 용어 "바이오티올 검출"은 산화환원조절을 이용하여 바이오티올을 측정하는 것을 의미한다. The term "biothiol detection" as used in the present invention means measuring biothiol using redox control.
상기 바이오티올 측정은 겔 전기영동(gel electrophoresis)법, 형광 비등방성 측정법(fluoresecence anisotropy), MALDI-TOF MS(Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometer), SPR(Surface plasmon resonance), 간섭법(interferometry) 및 비드 측정법으로 이루어진 군에서 선택된 하나 이상으로 수행한다.The biothiol measurement may include gel electrophoresis, fluorescence anisotropy, matrix-assisted laser desorption ionization-time-of-flight mass spectrometer, surface plasmon resonance (SPR) , At least one selected from the group consisting of interferometry and bead measurement.
특히, 전기영동법의 경우에는 특정 장비 없이 손쉽게 단백질과 DNA의 결합 여부를 확인할 수 있다. In particular, in the case of electrophoresis, it is possible to easily check whether the protein is bound to the DNA without any equipment.
또한, MALDI-TOF MS의 경우에는 산화환원 조절 단백질과 바이오티올과의 결합을 바이오티올의 종류에 따라 각각 정량적으로 분석할 수 있으며, 유리 형태의 바이오티올 또는 전체 바이오티올 양을 분석할 수 있다. In addition, in the case of MALDI-TOF MS, the binding of redox regulator protein and biothiol can be quantitatively analyzed according to the type of biothiol, and the amount of free biothiol or total biothiol can be analyzed.
또한, 비드 측정법의 경우에는 첨부도면 도 9에 나타낸 바와 같이 형광 표지 또는 DNA 기반 효소를 산화환원 조절 단백질과 결합하는 DNA에 연결시켜 형광 또는 화합발광으로 바이오티올을 검출할 수 있다. 이때, 산화환원 조절 단백질에 FLAG 태그, His6 태그, GSH 태그, biotin 태그 등이 결합될 수 있으며, 이렇게 결합된 단백질은 상기 태그와 친화성의 비드(FLAG 친화성 비드, NTA-비드, 글루타치온 비드, 아비딘계열의 비드 등)을 사용하여 바이오티올을 검출한다.In addition, in the case of the bead measurement method, as shown in FIG. 9, a biothiol can be detected by fluorescence or chemical luminescence by connecting a fluorescent label or a DNA-based enzyme to a DNA binding to a redox regulatory protein. In this case, the redox regulatory protein may be combined with a FLAG tag, His6 tag, GSH tag, biotin tag, and the like, and the bound protein may be affinity beads (FLAG affinity beads, NTA-beads, glutathione beads, avidin). Series of beads, etc.) to detect biothiol.
본 발명에 따른 바이오티올 검출용 조성물은 유리(free) 형태의 바이오티올과 총(total) 바이오티올을 각각 검출하거나 유리 형태의 바이오티올과 총 바이오티올을 동시에 검출할 수 있다.The composition for detecting biothiol according to the present invention can detect free biothiol and total biothiol in free form or detect free biothiol and total biothiol at the same time.
본 발명의 일 구현예에서, 상기 총 바이오티올을 검출하는 경우에는, 상기 조성물에 환원제를 추가로 포함한다. OhrR은 환원된 유리 바이오티올과만 결합하기 때문에 시료 내 총 바이오티올을 검출하기 위해서는 시료에서 산화된 바이오티올을 환원제를 사용하여 신속히 환원시킴으로써 OhrR에 의한 총 바이오티올의 검출이 가능하다. 상기 환원제는 구체적으로 DTT(dithiothreitol), 2-머캡토에탄올 (2-mercaptoenthanol) 및 TCEP(tris(2-carboxyethyl)phosphine)로 이루어진 군에서 선택된 하나 이상이나, 이에 제한되지 않는다.In one embodiment of the present invention, when detecting the total biothiol, the composition further comprises a reducing agent. Since OhrR binds only to the reduced free biothiol, in order to detect total biothiol in the sample, the total biothiol can be detected by OhrR by rapidly reducing the oxidized biothiol in the sample using a reducing agent. Specifically, the reducing agent is one or more selected from the group consisting of dithiothreitol (DTT), 2-mercaptoethanol (2-mercaptoenthanol), and TCEP (tris (2-carboxyethyl) phosphine), but is not limited thereto.
본 발명은 또한, 상기 조성물을 포함하는 바이오티올 검출용 바이오 칩을 포함한다.The present invention also includes a biothiol detection biochip comprising the composition.
본 발명의 일 구현예로서, 상기 바이오 칩은 첨부도면 도 14에 나타낸 바와 같이, 산화환원 조절 단백질과 결합 가능한 DNA와 복합체를 형성시킬 수 있도록 상기 DNA가 고정화된 플레이트 상에서 산화환원 조절 단백질와 반응시킨다. 이때, DNA에 표지하는 대신 친화성 태그가 결합된 산화환원 조절 단백질을 사용하여 바이오티올 존재 시 바이오칩 표면에 부착된 DNA으로부터 산화환원 조절 단백질이 떨어져 나가므로 검출 신호가 감소되는 원리를 이용하여 측정한다.In one embodiment of the present invention, the biochip is reacted with a redox regulatory protein on a plate onto which the DNA is immobilized to form a complex with a DNA capable of binding to a redox regulatory protein, as shown in FIG. 14. In this case, instead of labeling the DNA, the redox regulatory protein bound to the affinity tag is used to measure the detection signal by reducing the redox regulatory protein from the DNA attached to the surface of the biochip in the presence of biothiol. .
본 발명은 또한, 상기 조성물을 포함하는 바이오티올 검출용 바이오센서를 포함한다.The present invention also includes a biosensor for detecting a biothiol comprising the composition.
본 발명의 일 구현예로서, 스트립(strip) 형태의 바이오티올 검출용 바이오센서를 포함한다.In one embodiment of the present invention, a biosensor for detecting a biothiol in a strip form is included.
본 발명의 일 구현예로서, In one embodiment of the present invention,
산화환원 조절 단백질과 상기 단백질에 결합되는 DNA의 복합체와 결합할 수 있는 고정 부위를 포함하고, 시료와 상기 복합체를 혼합하여 도입하도록 형성된 시료 도입부; 및A sample introduction part including a fixation site capable of binding to a complex of a redox control protein and a DNA bound to the protein, the sample introduction part being configured to introduce a sample and the complex into a mixture; And
상기 시료 도입부와 일 직선 상에서 일정 거리 이격되며, 상기 복합체가 산화 반응 후 DNA와 해리되는 반응부; 및 A reaction part spaced apart from the sample introduction part on a straight line by a predetermined distance, and the complex dissociates with the DNA after the oxidation reaction; And
상기 해리된 DNA가 이동하여 바이오티올을 측정하도록 형성된 측정부;A measurement unit configured to move the dissociated DNA to measure biothiol;
를 포함하는 바이오티올 검출용 바이오센서를 포함한다.It includes a biosensor for detecting biothiol comprising a.
상기 고정 부위는 항체(예. FLAG tag-OhrR, Anti-FLAG antibody, anti-His6 antibody 등) 또는 수용체(예. His6 tag-OhrR, NTA 혹은 Biotin-OhrR, Strepavidin (avidin, NeutrAvidin 포함) 등)가 결합된 것일 수 있다.The fixation site may be an antibody (eg FLAG tag-OhrR, Anti-FLAG antibody, anti-His6 antibody, etc.) or a receptor (eg His6 tag-OhrR, NTA or Biotin-OhrR, Strepavidin (including avidin, NeutrAvidin), etc.). It may be combined.
이러한 스트립 형태의 바이오티올 검출용 바이오센서는 예를 들면 첨부도면 도 16에 나타낸 바와 같다.The biosensor for detecting a biothiol in the form of a strip is, for example, as shown in FIG. 16.
혈액 내에 존재하는 저분자 바이오티올의 산화/환원된 양을 상대적으로 측정할 수 있다. 시료 도입부에는 OhrR-dsDNA 복합체와 결합할 수 있는 고정부위로, 항체(예. anti-FLAG antibody 혹은 anti-His6 antibody) 또는 친화성 수용체(예, avidin 계열, NTA 등)를 고정화시키고 시료(예. 혈장)에 OhrR-dsDNA를 혼합하여 시료 도입에 떨어뜨리면 상기 혼합 용액이 크로마토그래피 원리에 의해 오른쪽으로 흘려가면서 고정 부위를 지나가게 되어 OhrR-dsDNA는 더 이상 이동하지 않고 고정 부위에 결합하게 된다. 이때 시료 내 바이오티올이 존재 시 OhrR에 결합되어 있는 dsDNA가 빠르게 해리되어 오른쪽으로 dsDNA만 이동하게 되고, 센서의 오른쪽 측정부에서 dsDNA에 있는 DNAzyme 등에 의해 기질과 반응하여 화학발광으로 검출할 수 있다.The oxidized / reduced amount of low molecular weight biothiol present in the blood can be measured relatively. The sample introduction part is a fixed site capable of binding to the OhrR-dsDNA complex, and immobilizes an antibody (e.g., an anti-FLAG antibody or an anti-His6 antibody) or an affinity receptor (e.g., an avidin family, NTA, etc.) Plasma) OhrR-dsDNA is mixed and dropped in the sample introduction, the mixed solution flows to the right by the chromatographic principle to pass through the fixed site, the OhrR-dsDNA is bound to the fixed site without moving anymore. At this time, dsDNA bound to OhrR is rapidly dissociated in the presence of biothiol in the sample, and only dsDNA is moved to the right side, and it is detected by chemiluminescence by reacting with the substrate by DNAzyme in dsDNA at the right measuring part of the sensor.
본 발명에 따라 산화환원 조절 단백질을 사용하여 바이오티올을 검출하는 경우 기존 바이오티올 검출법과 비교하여 다음과 같은 차별성 및 우수성을 가진다.In the case of detecting biothiol using the redox control protein according to the present invention, it has the following differentiation and superiority compared to the conventional biothiol detection method.
첫 번째, 민감도가 현저히 향상되고 측정 시료량을 최소한으로 줄일 수 있다. OhrR의 monomer는 바이오티올과 1:1 (몰수비)로 결합되기 때문에 바이오티올과 반응하는 거대분자의 단백질 (예, OhrR) 을 측정함으로써 반응 민감도를 향상시킬 수 있다(예를 들어 OhrR과 OhrR+바이오티올의 질량분석법). 또한 단백질와 결합하는 DNA 부위에 신호인자 및 증폭인자를 도입하고, 단백질과의 결합 또는 해리과정에 의해 DNA 신호가 조절되도록 디자인할 경우 기존 측정방법(크로마토그래피법, 면역분석법, 화학센서기반 분석법) 대비 높은 민감도를 얻을 수 있다. 또한, 현재 OhrR 단백질을 사용할 경우 약 1-2 μL의 혈액만으로도 전체 바이오티올 뿐만 아니라 유리 형태의 바이오티올 측정이 가능하여 분석시료량을 최소한으로 줄일 수 있다.First, the sensitivity is significantly improved and the sample volume to be measured can be reduced to a minimum. Since monomers of OhrR are 1: 1 (molar ratio) with biothiol, reaction sensitivity can be improved by measuring proteins (eg, OhrR) of macromolecules that react with biothiol (eg, OhrR and OhrR + bio). Mass spectrometry of thiols). In addition, when the signal and amplification factors are introduced into the DNA site that binds to the protein and the DNA signal is controlled to be controlled by the binding or dissociation process with the protein, compared with the conventional methods (chromatography, immunoassay, chemical sensor-based analysis) High sensitivity can be obtained. In addition, when using the OhrR protein, it is possible to measure not only the whole biothiol but also the free form of the biothiol using only 1-2 μL of blood, thereby reducing the amount of sample to be analyzed.
두 번째, 안정성이 우수하다. 산화환원 조절 단백질인 OhrR은 대량 발현이 가능하며 단백질임에도 불구하고 상대적으로 작은 크기 (17 kD)에 상온에서 쉽게 분해되지 않아 장기간 보관이 가능하다. 일반적인 peroxide 센서 단백질과는 달리 organic hydroperoxide에 특이적인 반응을 하므로 체액 내 산화조건(예, 산소 및 과산화수소)가 있는 조건에서 쉽게 변형되지 않아 안정성이 뛰어나며, 일반적인 전사촉진인자 보다 DNA 결합력(Kd =10-9 M 이하)이 매우 높아 정상적인 조건에서 쉽게 해리되지 않으므로 단백질-DNA 결합력을 이용할 경우 background 신호를 매우 낮게 유지할 수 있다.Second, the stability is excellent. OhrR, a redox control protein, can be expressed in large quantities and can be stored for a long time because it is not easily degraded at room temperature in a relatively small size (17 kD). Unlike general peroxide sensor protein, it reacts specifically to organic hydroperoxide, so it is not easily deformed under oxidizing conditions (eg oxygen and hydrogen peroxide) in body fluids, so it has excellent stability and DNA binding ability (K d = 10). -9 M or less) is so high that it is not easily dissociated under normal conditions, so the background signal can be kept very low when using protein-DNA binding.
세 번째, 반응특이성이 높다. 산화환원 조절 단백질인 OhrR은 유기산화물의 존재 하에 저분자량의 바이오티올과만 반응하고 거대분자 단백질에 존재하는 티올 기와는 반응하지 않아 저분자 바이오티올만 특이적으로 검출 가능하다.Third, the reaction specificity is high. OhrR, a redox regulator protein, reacts only with low molecular weight biothiol in the presence of organic oxides and does not react with thiol groups present in macromolecular protein, so that only low molecular weight biothiol can be detected.
네 번째, 유리 바이오티올과 총 바이오티올을 동시에 측정할 수 있다. 유리 바이오티올과의 빠른 반응성은 물론이고 높은 농도의 환원제가 존재하는 환경에서도 바이오티올과 안정적으로 mixed disulfide를 형성시킬 수 있기 때문에 고농도의 환원제를 시료에 처리할 경우 모두 환원된 바이오티올의 전체 양을 산화환원조절 단백질을 이용하여 신속하게 검출할 수 있다. Fourth, free biothiol and total biothiol can be measured simultaneously. In addition to rapid reactivity with free biothiol, it is possible to stably form mixed disulfide with biothiol even in the presence of a high concentration of reducing agent. Therefore, when a high concentration of reducing agent is applied to a sample, the total amount of reduced biothiol is reduced. Redox proteins can be used for rapid detection.
다섯 번째, 반응 시간을 현저히 줄일 수 있다. 산화환원 조절 단백질인 OhrR에는 하나의 Cys 잔기가 존재하는데, 이 Cys 잔기와 peroxide 간의 2차 반응 속도는 약 104 - 105 M-1s-1로서 일반적인 단백질에 존재하는 Cys 잔기 혹은 유리 바이오티올의 peroxide 반응 속도(약 1 M-1s-1) 보다 수만배 혹은 수십만배 빠르고, 이후 바이오티올과 산화환원 조절 단백질의 2차 반응 속도는 약 103 M-1s-1로 화학적 프로브(10-2 -101 M-1s-1) 보다 수천 배가 빠르다. 또한, 바이오티올을 센싱한 산화환원 조절 단백질은 DNA로부터 즉시 해리(50% 해리시간, t1/2 < 0.5 min)되기 때문에 시료와 수 분 이내 반응하여 결과신호를 신속히 유도할 수 있다.Fifth, the reaction time can be significantly reduced. To one of the Cys residues, the reduction control protein OhrR oxide present, the second reaction rate between the Cys residues and the peroxide is from about 10 4 - 10 5 M -1 Cys residues present in or as a common protein s -1 glass Bio-thiol Tens of thousands or hundreds of thousands of times faster than the peroxide reaction rate (about 1 M -1 s -1 ), and the secondary reaction rate of biothiol and redox regulatory protein was about 10 3 M -1 s -1 . -2 -10 1 M -1 s -1 ) thousands of times faster. In addition, the redox regulatory protein, which senses the biothiol, is immediately dissociated from the DNA (50% dissociation time, t 1/2 <0.5 min), thereby reacting with the sample within a few minutes to rapidly induce a resultant signal.
마지막으로, 대장균에서 쉽게 대량 발현시킬 수 있는 단백질과 짧은 올리고 서열만으로 구성할 수 있어 저가용 칩이나 바이오센서 구성에 매우 효과적이다. Finally, it can be composed only of proteins and short oligo sequences that can be easily expressed in E. coli, which is very effective for low-cost chip or biosensor construction.
이하, 본 발명의 실시예를 통해 상세히 설명한다. 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 실시예들은 본 발명의 개시가 완전하도록 하고, 본 발명이 속하는 기술 분야에서 통상의 지식이 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다.Hereinafter, the embodiment of the present invention will be described in detail. The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples. These embodiments are provided so that this disclosure will be thorough, and will fully convey the scope of the invention to those skilled in the art, and the invention is defined by the scope of the claims. It is only.
[[ 실시예Example ]]
실시예Example 1:  One: OhrROhrR 단백질과  Protein and CysCys , , HycHyc , , GSH의Of GSH 반응성 확인 Reactivity Check
FA(Fluorescence anisotropy)를 이용하여 OhrR의 실시간 DNA 결합 활성을 측정하였다. Fluorescence anisotropy (FA) was used to measure real-time DNA binding activity of OhrR.
<실험 조건><Experimental conditions>
버퍼: 20 mM Tris (pH 8.0) 150 mM NaCl, 5% Glycerol (vol/vol)Buffer: 20 mM Tris (pH 8.0) 150 mM NaCl, 5% Glycerol (vol / vol)
DNA 농도: 50 nMDNA concentration: 50 nM
OhrR 농도: 300 nMOhrR concentration: 300 nM
CHP 농도: 3 μMCHP concentration: 3 μM
측정 시간: every 10s 측정 Measurement time: every 10s measurement
측정 조건: ex 492 nm; slit width 15 nm, em 520 nm; slit width 20 nm, integration time 1 sMeasurement conditions: ex 492 nm; slit width 15 nm, em 520 nm; slit width 20 nm, integration time 1 s
DNA 서열: DNA sequence:
Template DNA: 5'-TAC AAT TAA ATT GTA TAC AAT TAA ATT GTA-3' (서열번호 1)Template DNA: 5'-TAC AAT TAA ATT GTA TAC AAT TAA ATT GTA-3 '(SEQ ID NO: 1)
Complemenatary DNA: 5'- TAC AAT TTA ATT GTA TAC AAT TTA ATT GTA-3' (서열번호 2)Complemenatary DNA: 5'- TAC AAT TTA ATT GTA TAC AAT TTA ATT GTA-3 '(SEQ ID NO: 2)
OhrR 서열: B. subtilis 균주에서 직접 cloning함. MENKFDHMKLENQLCFLLYASSREMTKQYKPLLDKLNITYPQYLALLLLWEHETLTVKKM GEQLYLDSGTLTPMLKRMEQQGLITRKRSEEDERSVLISLTEDGALLKEKAVDIPGTILGLSKQSGEDLKQLKSALYTLL ETLHQKN (서열번호 3)OhrR sequence: Direct cloning in B. subtilis strains. MENKFDHMKLENQLCFLLYASSREMTKQYKPLLDKLNITYPQYLALLLLWEHETLTVKKM GEQLYLDSGTLTPMLKRMEQQGLITRKRSEEDERSVLISLTEDGALLKEKAVDIPGTILGLSKQSGEDLKQLKSALYTLL ETLHQKN (SEQ ID NO: 3)
<실험과정>Experimental Process
LS55 luminescence spectrometer (PerkinElmer) 장비를 사용하여 OhrR과 형광(6FAM, 6-carboxyfluorescein) 표지된 OhrR 결합 DNA의 결합 활성을 3 mL의 버퍼에 상기 실험조건의 농도로 녹인 후 10초 단위로 측정하였다. OhrR과 DNA가 결합하면 Anisotropy (Anis) 값이 증가하고 DNA와 해리가 되면 Anisotropy 값이 감소한다. 각 실험에서는 대표적인 3개의 바이오티올인 Cys(Cysteine), Hcy (Homocysteine), GSH(Glutathione)을 다양한 농도 (0, 1, 2, 4, 8, 16, 32, 64 μM)로 처리하였다. 유리 바이오티올만 있는 상태에서 OhrR은 DNA와 결합하고 있다가 대표적인 유기과산화물의 한 종류인 CHP(cumene hydroperoxide)를 처리하여 (300sec에 처리) OhrR의 DNA 해리 속도를 실시간으로 측정하였다.The binding activity of OhrR and fluorescence (6FAM, 6-carboxyfluorescein) labeled OhrR binding DNA was dissolved in 3 mL of buffer using a LS55 luminescence spectrometer (PerkinElmer). When OhrR and DNA are combined, Anisotropy (Anis) increases, and when it dissociates with DNA, Anisotropy decreases. In each experiment, three representative biothiols, Cys (Cysteine), Hcy (Homocysteine), and GSH (Glutathione) were treated at various concentrations (0, 1, 2, 4, 8, 16, 32, 64 μM). In the presence of only free biothiol, OhrR binds to DNA and then measures the dissociation rate of OhrR in real time by treating CHP (cumene hydroperoxide), one of the representative organic peroxides (at 300 sec).
<실험결과><Experiment Result>
도 2 바이오티올의 농도가 높을수록 OhrR의 DNA 해리 속도가 빨라지는 것을 확인하였다. 도 2의 막대그래프는 바이오티올의 농도에 따라 OhrR이 DNA와 해리될 때 Anisotropy 값이
Figure PCTKR2017012344-appb-I000001
로 줄어드는데 걸리는 시간(OhrR이 DNA로 부터
Figure PCTKR2017012344-appb-I000002
해리되는데 걸리는 시간)을 나타낸 것이다.Figure 2 confirms that the higher the biothiol concentration, the faster the DNA dissociation rate of OhrR. 2 shows anisotropy value when OhrR dissociates with DNA depending on the concentration of biothiol.
Figure PCTKR2017012344-appb-I000001
The time it takes to decrease from OhrR to DNA
Figure PCTKR2017012344-appb-I000002
Time to dissociate).
실시예Example 2: 전기영동법을 이용한  2: using electrophoresis OhrROhrR 단백질과  Protein and 타겟target DNA의DNA 결합 확인 Join check
형광프로브(FAM)가 결합된 double strand DNA (200 nM)[서열번호 1과 2]와 OhrR을 도 3에 표시된 농도별로 혼합 후 상온에서 30분 동안 반응시킨 다음 폴리아크릴아마이드 겔(7%)을 이용한 전기영동을 통해(25 mA, 30 min) dsDNA 밴드를 형광측정장비 (모델명 KIF-300, Korea Lab Tech, Korea)로 측정하였다. OhrR의 농도가 약 1.6 μM (DNA 농도 대비 약 8배의 농도)부터 dsDNA의 형광 밴드가 위쪽으로 이동(shift)되었음을 알 수 있다. Fluorescent probe (FAM) -coupled double strand DNA (200 nM) [SEQ ID NOs: 1 and 2] and OhrR were mixed at the concentrations shown in FIG. 3 and reacted at room temperature for 30 minutes, followed by polyacrylamide gel (7%). By using electrophoresis (25 mA, 30 min), the dsDNA band was measured with a fluorescence measuring instrument (Model KIF-300, Korea Lab Tech, Korea). It can be seen that the fluorescence band of dsDNA shifted upward from the concentration of OhrR of about 1.6 μM (about 8 times the concentration of DNA).
상기 실시예 1에서 측정한 FA 측정법과는 달리 PAGE 전기영동의 경우 단백질-DNA간 결합을 실시간으로 측정한 결과가 아니고 OhrR과 DNA간의 결합을 위해서는 다량의 단백질이 요구되고 있으나 (따라서 실제 결합상수를 이 방법으로는 측정할 수는 없음) 특정장비 없이 손쉽게 결합 여부를 확인할 수 있는 방법으로는 이용할 수 있다.Unlike the FA measurement measured in Example 1, PAGE electrophoresis is not a result of measuring protein-DNA binding in real time, and a large amount of protein is required for OhrR-to-DNA binding (hence the actual binding constant It is not possible to measure with this method.) It can be used as a method to easily check the coupling without a specific device.
실시예Example 3: 형광표지 없이  3: without fluorescent label DNA와DNA and OhrROhrR 단백질 간의 결합이  The bond between proteins 바이오티올에In biothiol 의해 해리되는 과정을  The process of being dissociated by 광굴절률로At photorefractive index 측정한 실험 Measured experiment
Biolayer interferometry를 측정할 수 있는 장비(Blitz, Fortebio사, USA)를 사용하여 광 센서(optical fiber) 표면 위에서의 DNA와 단백질 간의 결합 정도를 측정하였다. A device capable of measuring biolayer interferometry (Blitz, Fortebio, USA) was used to measure the degree of binding between DNA and protein on the optical fiber surface.
<실험조건><Experimental conditions>
binding buffer, washing buffer: TBS (Tris 20 mM, NaCl 150 mM)binding buffer, washing buffer: TBS (Tris 20 mM, NaCl 150 mM)
DNA binding time, OhrR binding time: 2 minDNA binding time, OhrR binding time: 2 min
Washing time: 30 secWashing time: 30 sec
DNA 농도: 2.5 μMDNA concentration: 2.5 μM
OhrR 농도: 50 μMOhrR concentration: 50 μM
CHP 농도: 100 μM CHP concentration: 100 μM
<실험과정>Experimental Process
우선 streptavidin이 코팅된 광 센서(optical fiber) 위에 바이오틴이 결합된 double strand DNA[서열번호 1과 2]를 TBS 버퍼에 상기 농도로 녹이고 120초 동안 흘려주어 결합시킨 후 버퍼로 세척하고 OhrR 단백질 TBS 버퍼에서 상기 농도로 120초 동안 추가로 흘려주어 광센서에 결합된 double strand DNA와 결합시켰다(association). 상기의 결합반응(association)이 완성된 이후에도 120초 동안 버퍼만을 추가적으로 흘려줌으로써 상기 DNA-OhrR의 complex의 해리과정을 확인하였다(dissociation).First, biotin-coupled double strand DNA [SEQ ID NOS: 1 and 2] was dissolved in the TBS buffer at the above concentration and flowed for 120 seconds on the streptavidin-coated optical fiber, and then washed with a buffer and washed with OhrR protein TBS buffer. At 120 ° C., the solution was further flowed for 120 seconds to associate with double strand DNA bound to an optical sensor. Even after the association was completed, dissociation of the DNA-OhrR complex was confirmed by additionally flowing only the buffer for 120 seconds.
<실험결과><Experiment Result>
Biolayer interferometry 측정은 광센서 표면에서의 생체물질 (DNA 혹은 OhrR)이 결합하게 될 경우, 광센서 표면 굴절률의 변화를 필수적으로 수반하게 되고 (굴절률은 생체물질의 농도와 양의 상관관계) 이 굴절률의 변화값을 두께 값(thickness)으로 변환한 원리를 적용한 것이다. OhrR의 경우 dsDNA와 강하게 결합되어 약 4.3 nm의 binding thickness 값(OhrR의 dissociation equilibrium 수치에서 dsDNA binding equilibrium 값을 뺀 수치) 을 유지하였고, 이와 유사하게 CHP 혹은 바이오티올의 어느 하나만 OhrR 단백질과 섞어준 후 DNA에 흘려준 경우에도 OhrR이 DNA에 효과적으로 결합함을 알 수가 있다. 이와는 반대로, CHP와 바이오티올이 모두 존재 시 OhrR 단백질에서 DNA 결합 부위의 바이오티올에 의한 산화과정이 수반됨으로써 DNA와의 결합력이 급속하게 감소됨을 확인하였다(약 1 nm의 thickness). 즉, COhrR와 DNA의 결합반응을 이용하여 CHP 존재 시에 바이오티올을 농도를 손쉽게 예측할 수 있음을 확인할 수 있다. Biolayer interferometry measurements inevitably involve changes in the refractive index of the optical sensor when the biomaterial (DNA or OhrR) is bound to the optical sensor surface (refractive index is positively correlated with the concentration of the biomaterial). It applies the principle of converting the change value into thickness. OhrR was strongly bound to dsDNA to maintain a binding thickness value of about 4.3 nm (minus the value of the dissociation equilibrium minus dsDNA binding equilibrium), and similarly, after mixing only one of CHP or biothiol with OhrR protein Even when spilled into DNA, it can be seen that OhrR binds to DNA effectively. On the contrary, in the presence of both CHP and biothiol, it was confirmed that the binding force to DNA was rapidly decreased by the oxidation of the OhrR protein by the biothiol at the DNA binding site (thickness of about 1 nm). That is, it can be confirmed that the biothiol concentration can be easily predicted in the presence of CHP using the coupling reaction of COhrR and DNA.
이 방법은 광 센서 표면 위에서 dsDNA나 OhrR를 형광체 등으로 별도로 표지(labeling)하지 않고도 다양한 바이오티올의 종류 및 농도에 대한 반응성을 효과적으로 비교할 수 있는 방법으로 사용할 수 있다.This method can be used as a method to effectively compare the reactivity to various types and concentrations of biothiol without separately labeling dsDNA or OhrR with phosphors on the optical sensor surface.
실시예Example 4:  4: MALDIMALDI -- TOFTOF MS를MS 이용한  Used OhrR과OhrR 바이오티올Biothiol 결합 확인 Join check
MALDI-TOF MS(말티토프 질량 분석기, Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometer)를 이용하여 OhrR과 바이오티올과의 결합을 정량적으로 분석 가능한지 확인하였다.MALDI-TOF MS (Maltitop Mass Spectrometer, Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometer) was used to confirm the quantitative analysis of the binding of OhrR to biothiol.
300 nM OhrR이 존재하는 버퍼(20 mM Tris (pH 8.0) 100 mM NaCl, 5% Glycerol) 1 mL에 바이오티올(Cys, Hcy, GSH)을 농도 별(0, 1, 2, 4, 8, 16, 32, 64 μM)로 처리하였다. 각각에 3 μM CHP를 2분간 반응시킨 후에 100% TCA를 110 μL 처리하여 산화반응을 멈추고 OhrR을 침전(precipitation)시켰다. 침전된 OhrR을 알킬화시키기 위하여 50 mM Iodoacetamide를 처리하여 환원되어있는 OhrR의 시스테인 잔기를 Blocking시켰다. SDS-PGAE로 단백질을 분리한 후에 OhrR에 해당하는 Band를 자른 후 트립신 (37℃, 12시간 이상)을 처리한 후에 gel에서 추출하였다. 추출된 펩타이드 0.5 μL와 CHCA(α-Cyano-4-hydroxycinnamic acid) (in 50% Acetonitrile, 1% TFA) 0.5 μL를 처리하여 말린 후에 MALDI-TOF 분석을 진행하였다. MADI-TOF 분석은 4700 Proteomics Analyzer instrument (Applied Biosystems) 장비를 사용하였다.Biothiol (Cys, Hcy, GSH) was added to 1 mL of buffer (20 mM Tris (pH 8.0) 100 mM NaCl, 5% Glycerol) in 300 nM OhrR (0, 1, 2, 4, 8, 16). , 32, 64 μM). After reacting 3 μM CHP for 2 minutes to each, 110 μL of 100% TCA was used to stop the oxidation reaction and precipitated OhrR. In order to alkylate the precipitated OhrR, 50 mM Iodoacetamide was treated to block the reduced cysteine residue of OhrR. After separating the protein by SDS-PGAE, after cutting the band corresponding to OhrR and trypsin (37 ℃, 12 hours or more) and extracted from the gel. 0.5 μL of the extracted peptide and 0.5 μL of CHCA (α-Cyano-4-hydroxycinnamic acid) (in 50% Acetonitrile, 1% TFA) were treated and dried before MALDI-TOF analysis. MADI-TOF analysis was performed using a 4700 Proteomics Analyzer instrument (Applied Biosystems).
도 5에 나타낸 바와 같이, Cys 및 Hcy, GSH의 농도가 증가함에 따라 각각 [OhrR+biothiol]에 해당하는 m/z 값이 증가하여 검출되었으며 바이오티올에 따라서 질량 검출능이 다소 차이가 있으나 대략 약 1 μM의 농도 이상으로 존재 시 MALDI-TOF MS 법에 의해 검출이 가능함을 확인하였고 Cys의 경우 측정 바이오티올 중에서 가능 높은 검출신호값을 보여주었다.As shown in FIG. 5, as the concentrations of Cys, Hcy, and GSH increased, m / z values corresponding to [OhrR + biothiol] were increased, respectively, and the mass detection ability was slightly different depending on biothiol, but about 1 In the presence of more than the concentration of μM it was confirmed that the detection by the MALDI-TOF MS method, Cys showed the highest detection signal value among the measured biothiol.
실시예 5: MALDI - TOF MS와 OhrR를 이용한 마우스/인간 혈액 시료에서 유리 바이오티올 분석한 실험 Example 5: MALDI - TOF With MS Free Biothiol Analysis in Mouse / Human Blood Samples Using OhrR
정상 마우스와 동맥경화 마우스 모델(ΔLDLR 마우스) 혹은 인간 혈액 샘플 (25세 비흡연 남성의 혈액(human 1)과 45세 흡연 남성의 혈액(human 2)) 사이에 존재하는 유리 바이오티올 양의 차이가 OhrR을 이용하여 분석 가능함을 MALDI-TOF MS 분석을 통하여 확인하였다. Differences in the amount of free biothiol present between normal and atherosclerotic mouse models (ΔLDLR mice) or human blood samples (blood from 25-year-old non-smoking men (human 1) and 45-year-old smoking men (human 2)) Analysis was possible using OhrR through MALDI-TOF MS analysis.
마우스와 인간에서 채취한 Plasma 2 μL에 25 μM의 OhrR과 50 μM의 CHP를 10분간 반응시켰다(총 2.5 μL에서 반응). 반응 후에 10% TCA를 1 ml 처리하여 OhrR을 포함한 단백질을 침전시켰다. 침전된 OhrR의 환원되어있는 시스테인의 추가적인 산화를 막기 위하여 알킬화제인 Iodoacetamide를 50 mM의 농도로 처리하여 환원되어있는 시스테인과 반응 시켰다. SDS-PGAE로 단백질을 분리한 후에 OhrR에 해당하는 단백질 Band를 자른 후 0.2 μg의 트립신을 37℃에서 12시간 이상 처리하였다. 트립신에 의해 잘린 펩타이드들은 원심분리를 통하여 gel에서 추출시켰다. 추출된 펩타이드 0.5 μL와 CHCA(in 50% Acetonitrile, 1% TFA) 0.5 μL를 처리하여 말린 후에 MALDI-TOF 분석을 수행하였다. MADI-TOF 분석은 4700 Proteomics Analyzer instrument (Applied Biosystems) 장비를 사용하였다.2 μL of Plasma collected from mice and humans was reacted with 25 μM of OhrR and 50 μM of CHP for 10 min (2.5 μL in total). After the reaction, 1 ml of 10% TCA was treated to precipitate proteins including OhrR. In order to prevent further oxidation of the reduced cysteine of precipitated OhrR, the alkylating agent Iodoacetamide was treated at a concentration of 50 mM to react with the reduced cysteine. After protein separation by SDS-PGAE, the protein band corresponding to OhrR was cut and treated with 0.2 μg of trypsin at 37 ° C. for 12 hours or more. Peptides cut by trypsin were extracted from the gel by centrifugation. 0.5 μL of the extracted peptide and 0.5 μL of CHCA (in 50% Acetonitrile, 1% TFA) were treated and dried, followed by MALDI-TOF analysis. MADI-TOF analysis was performed using a 4700 Proteomics Analyzer instrument (Applied Biosystems).
도 6에 나타낸 바와 같이, 동맥경화 모델 마우스 혈액에서는 OhrR-Cys의 질량신호값이 정상 마우스 혈액에 비해 감소하였고, 45세 남성흡연자 혈액에서도 25세 비흡연자 혈액에 비해 OhrR-Cys의 질량 신호값이 감소함을 알 수 있다. 즉, OhrR이 유리 바이오티올과 빠른 시간에 반응함을 감안할 때, 상기 혈액에 존재하는 유리 바이오티올(특히 여기서는 유리 시스테인)의 농도는 마우스 동맥경화 모델 및 흡연자 모델에서 상대적으로 감소해 있음을 관찰할 수 있다. 이와 같이 다양한 질병 환자군의 소량의 혈액만으로도 OhrR의 질량분석을 통해 효과적으로 유리 바이오티올의 양을 비교 분석할 수 있음을 확인하였다.As shown in FIG. 6, the mass signal value of OhrR-Cys was reduced in arteriosclerosis model mouse blood compared to normal mouse blood, and the mass signal value of OhrR-Cys was higher in 45 year old male smoker blood than in 25 year old non-smoker blood. It can be seen that the decrease. In other words, given that OhrR reacts with free biothiol in a fast time, it can be observed that the concentration of free biothiol (particularly free cysteine) present in the blood is relatively decreased in the mouse atherosclerosis model and in the smoker model. Can be. As such, it was confirmed that the amount of free biothiol can be effectively analyzed by mass spectrometry of OhrR even with a small amount of blood of various disease patients.
실시예Example 6: 총  6: gun 바이오티올Biothiol 측정 확인 Measurement Check
1) FA 측정법으로 확인 1) Confirmation by FA measurement
총 바이오티올의 측정 가능성 확인을 위하여, 환원제(dithiothreitol, DTT) 처리 하에서의 OhrR와 시스테인과의 반응성을 조사하였다. FA 측정은 Cys 농도를 0, 1 mM으로 CHP의 농도를 30 μM으로 실험하는 과정을 제외하고는 상기 실시예 1의 실험과정과 동일하게 진행하였다. To confirm the measurability of total biothiol, the reactivity of OhrR and cysteine under reducing agent (dithiothreitol (DTT) treatment was investigated. FA measurement Cys concentration was 0, 1 mM and the same procedure as in Example 1 except for the process of experiment to the concentration of CHP 30 μM.
DTT를 처리하면 (산화된) 모든 종류의 바이오티올의 디설파이드 결합은 환원되어 유리 형태(free form)를 형성하게 된다. 이후에 산화물인 CHP를 처리하면 산화된 OhrR이 다시 빠르게 환원되어있는 유리 형태의 바이오티올과 반응하여 OhrR의 DNA로부터의 해리속도를 가속화시키고, 이를 통해 총 바이오티올의 양을 측정할 수 있디(DTT는 이후 DTT가 가진 티올 분자간 internal 결합에 의해 cyclization이 되어 추가적인 바이오티올의 환원반응에 참여하지 않고 OhrR과도 반응하지 않게 된다)[도 7의 좌측 도면 참조]. Treatment of DTT results in the reduction of the disulfide bonds of all kinds of (oxidized) biothiols to form a free form. Subsequent treatment of the oxide, CHP, accelerates the dissociation rate of the OhrR DNA from the oxidized OhrR again with the rapidly reduced free form of biothiol, thereby measuring the total amount of biothiol (DTT). Then, cyclization is performed by internal binding between thiol molecules of DTT and thus does not participate in the reduction reaction of additional biothiol and does not react with OhrR (see left figure of FIG. 7).
2) MALDI - TOF 분석법으로 확인 2) Confirmed by MALDI - TOF method
<실험 조건 (Mouse Plasma Biothiol detection assay)><Mouse Plasma Biothiol Detection Assay>
OhrR 농도: 25 μMOhrR concentration: 25 μM
CHP 농도: 50 μMCHP concentration: 50 μM
Plasma 양: 2 μL (Norma Mouse serum, Jackson ImmunoResearch에서 구입)Plasma volume: 2 μL (purchased from Norma Mouse serum, Jackson ImmunoResearch)
DTT 농도: -/+ 1 mM (1시간 동안 선처리 후 실험 진행)DTT concentration:-/ + 1 mM (run experiment after 1 hour pretreatment)
<실험 과정(Mouse Plasma Biothiol detection assay)><Mouse Plasma Biothiol Detection Assay>
마우스에서 채취한 Plasma에 -/+ 1mM DTT를 1시간 동안 RT (23-25 ℃)에서 반응시켰다. 이 후에 Plasma 2 μL에 25 μM의 OhrR과 50 μM의 CHP를 10분간 반응시켰다 (총 2.5 μL에서 반응). 반응 후에 10% TCA를 1 mL 처리하여 OhrR을 포함한 단백질을 침전시켰다. 침전된 OhrR의 환원되어있는 시스테인의 추가적인 산화를 막기 위하여 알킬화제인 Iodoacetamide를 50 mM의 농도로 처리하여 환원되어있는 시스테인과 반응시켰다. SDS-PGAE로 단백질을 분리한 후에 OhrR에 해당하는 단백질 Band를 자른 후 0.2 μg의 트립신을 37℃에서 12시간 이상 처리함. 트립신에 의해 잘린 펩타이드들은 원심분리를 통하여 gel에서 추출시켰다. 추출된 펩타이드 0.5 μL와 CHCA(in 50% Acetonitrile, 1% TFA) 0.5 μL를 처리하여 말린 후에 MALDI-TOF 분석을 수행하였다.Plasma collected from the mice was reacted with-/ + 1 mM DTT at RT (23-25 ° C.) for 1 hour. Thereafter, 2 μL of Plasma was reacted with 25 μM of OhrR and 50 μM of CHP for 10 minutes (total reaction at 2.5 μL). After the reaction, 1 mL of 10% TCA was treated to precipitate proteins containing OhrR. In order to prevent further oxidation of the reduced cysteine of precipitated OhrR, the alkylating agent Iodoacetamide was treated at a concentration of 50 mM to react with the reduced cysteine. After separating the protein by SDS-PGAE, cut the protein band corresponding to OhrR, and then treated 0.2 μg of trypsin at 37 ° C. for 12 hours or more. Peptides cut by trypsin were extracted from the gel by centrifugation. 0.5 μL of the extracted peptide and 0.5 μL of CHCA (in 50% Acetonitrile, 1% TFA) were treated and dried, followed by MALDI-TOF analysis.
<실험 결과><Experiment Result>
상기 실험을 통하여 약 1 mM의 DTT가 포함된 조건에서도 환원된 바이오티올은 CHP 존재 시 OhrR과 빠르게 반응하고 (약 5분 이내에) 이후에는 시간이 경과할수록 DTT에 의해 바이오티올이 OhrR에 해리될 수 있음을 알 수 있다. 이로부터, DTT를 처리 후 총 바이오티올의 양을 검출하기 위해서는 OhrR과 CHP를 처리 후 약 5분 이내에 측정하는 것이 바람직하다. 이와 같은 조건을 MALDI-TOF MS로 분석한 결과, 마우스 혈장 (plasma)에서 DTT를 처리하지 않을 경우 유리 시스테인의 양을 측정할 수 있고 DTT를 처리할 경우 총 시스테인의 양을 측정할 수 있음을 확인하였다(도 7의 우측 도면).Through the above experiment, the reduced biothiol reacts rapidly with OhrR in the presence of CHP (within about 5 minutes) even in the presence of about 1 mM of DTT. Afterwards, biothiol can be dissociated into OhrR by DTT. It can be seen that. From this, it is preferable to measure OhrR and CHP within about 5 minutes after treatment to detect the amount of total biothiol after treatment with DTT. These conditions were analyzed by MALDI-TOF MS, and it was confirmed that the amount of free cysteine can be measured when DTT is not treated in mouse plasma and the amount of total cysteine when DTT is treated. (Right figure of FIG. 7).
실시예Example 7: 유리  7: glass 바이오티올Biothiol 및 총  And gun 바이오티올의Biothiol 동시 검출 확인 Simultaneous detection confirmation
MALDI-TOF MS 및 OhrR을 이용하여 마우스 혈액에서 시스테인의 농도를 첨가한 이후 free cysteine 및 total cysteine을 검출하였다. 즉, 마우스 혈청에 DTT를 처리하여 산화된 바이오티올을 환원시켰을 때, OhrR에 의해 분석되는 바이오티올의 양이 증가함을 MALDI-TOF MS 분석을 통하여 확인하였다. Free cysteine and total cysteine were detected after addition of cysteine concentrations in mouse blood using MALDI-TOF MS and OhrR. That is, it was confirmed through MALDI-TOF MS analysis that the amount of biothiol analyzed by OhrR is increased when the oxidized biothiol is reduced by treating DTT with mouse serum.
<실험 조건 (Mouse Plasma Biothiol detection assay)><Mouse Plasma Biothiol Detection Assay>
OhrR 농도: 25 μMOhrR concentration: 25 μM
CHP 농도: 50 μMCHP concentration: 50 μM
Cys 농도: 0 / 10 / 100 μMCys concentration: 0/10/100 μM
Plasma 양: 2 μL(Norma Mouse serum, Jackson ImmunoResearch에서 구입)Plasma volume: 2 μL (from Norma Mouse serum, Jackson ImmunoResearch)
DTT 농도: -/+ 1 mM (1시간 동안 선처리 후 실험 진행)DTT concentration:-/ + 1 mM (run experiment after 1 hour pretreatment)
<실험 과정 (Mouse Plasma Biothiol detection assay)><Mouse Plasma Biothiol Detection Assay>
0/10/100 μM의 Free Cysteine을 사용한 것을 제외하고는 상기 실시예 6의 Mouse Plasma Biothiol detection assay과 동일하게 실시하였다.Except for using the Free Cysteine of 0/10/100 μM was carried out in the same manner as in the Mouse Plasma Biothiol detection assay of Example 6.
<실험결과><Experiment Result>
정상 마우스 plasma에서의 DTT를 사용하지 않은 유리 시스테인 분석 결과는 도 8의 좌측 도면에 나타내었으며, DTT를 사용한 이후 총 시스테인 분석 결과는 도 8의 우측 도면에 나타내었다. 도 8은 유리 시스테인 분석을 위한 OhrR+cys (T3-Cys) 질량 분석을 비교한 것이다. DTT를 처리하지 않은 실험군에 비해 DTT를 처리한 실험군에서 총 시스테인이 증가하므로 OhrR-Cys 신호가 상대적으로 높게 나왔다. 또한 시스테인을 임의로 넣어주었을 경우 OhrR+Cys의 질량신호값은 넣어준 시스테인의 농도에 비례함을 알 수 있다.The results of free cysteine analysis without DTT in normal mouse plasma are shown in the left figure of FIG. 8, and the total cysteine analysis results after using DTT are shown in the right figure of FIG. 8. 8 compares OhrR + cys (T3-Cys) mass spectrometry for free cysteine analysis. Compared with the experimental group without the DTT, the total cysteine increased in the experimental group treated with the DTT, resulting in a relatively high OhrR-Cys signal. In addition, when the cysteine is added arbitrarily, it can be seen that the mass signal value of OhrR + Cys is proportional to the concentration of cysteine.
실시예Example 8:  8: 비드Bead 측정법을 이용한  Measurement 바이오티올Biothiol 검출 확인 Detection confirmation
본 실험은 실험실 시험관 수준에서 바이오티올에 대한 신속 간편 측정법 구현을 위해 OhrR와 DNA를 이용하여 바이오티올을 측정한 실시예로서, 하기 실시예 9 (dsDNA에 형광체 표지), 실시예 10 (dsDNA에 ssDNAzyme 표지)는 본 실험과정을 공통적으로 적용하고 측정방법만 달리한 실시예이다.This experiment is an example of measuring the biothiol using OhrR and DNA to implement a quick and simple measurement method for biothiol at the laboratory in vitro level, Example 9 (phosphor labeling on dsDNA), Example 10 (ssDNAzyme on dsDNA) The label) is an example in which this experimental procedure is commonly applied and only the measuring method is different.
우선, 각 샘플수만큼 튜브에 M2 FLAG affinity bead(sigma, A2220) 30 μL씩을 넣은 뒤, 총 부피 200 μL가 되도록, FLAG tagged OhrR (2 μM)를 1시간 동안 결합시켰다 (OhrR이 His6 tag로 결합되어 있을 경우 NTA-Bead를 사용해도 무방함). 결합되지 않은 OhrR을 제거하기 위해 Spin down 후 상층액을 제거하고 형광체 혹은 DNAzyme이 결합된 OhrR 결합 dsDNA (100 nM, 서열번호 1과 2)를 30분 동안 OhrR이 결합된 비드에 결합시켰다. spin down으로 결합되지 않은 dsDNA를 제거한 다음, 각 튜브에 버퍼(Tris Buffered Saline, TBS) 160 μL-> biothiols (농도별) 20 μL -> CHP (500 μM) 20 μL 순서로 넣어준 후, 5분 동안 쉐이킹하였다. 이후 spin down 하여 얻은 상층액 200 μL를 96 웰 플레이트에 옮겨 형광 혹은 화학발광을 측정하였다. 환원제(DTT)를 처리하지 않은 시료는 유리 바이오티올을 측정할 수 있으며, 환원제를 처리한 시료는 전체 바이오티올을 측정할 수 있다[도 9]. First, 30 μL of M2 FLAG affinity bead (sigma, A2220) was added to the tube by the number of samples, and then FLAG tagged OhrR (2 μM) was bound for 1 hour so as to have a total volume of 200 μL (OhrR bound to His6 tag). If so, you can use NTA-Bead). In order to remove unbound OhrR, the supernatant was removed after spin down, and OhrR binding dsDNA (100 nM, SEQ ID NO: 1 and 2) to which phosphor or DNAzyme was bound was bound to OhrR bound beads for 30 minutes. Remove unbound dsDNA by spin down, then add 160 μL-> biothiols (by concentration) 20 μL to 20 μL of buffer (Tris Buffered Saline, TBS) in each tube, and then 5 minutes. Shaken. Subsequently, 200 μL of the supernatant obtained by spin down was transferred to a 96 well plate, and fluorescence or chemiluminescence was measured. Samples not treated with the reducing agent (DTT) may measure free biothiol, and samples with the reducing agent may measure total biothiol [FIG. 9].
실시예Example 9: 형광 표지된  9: fluorescently labeled dsDNA을dsDNA 이용한 형광 측정 Fluorescence Measurement
OhrR-binding dsDNA에 형광인자로 FAM을 결합하여 최종 상층액 200 μL를 96 well plate로 옮긴 후 multi plate reader (Variokan, Thermo Scientific 사)를 통해 형광 측정하였다. 형광측정은 480 nm 여기파장에서 얻은 525 nm 방출파장에서의 형광신호값을 얻은 후 형광인자가 들어있지 않고 버퍼만 들어 있는 well을 기준값으로 하여 각 well의 형광값을 보정한 이후 비교하였다.FAM was combined with OhrR-binding dsDNA as a fluorescence factor and 200 μL of the final supernatant was transferred to a 96 well plate, and fluorescence was measured using a multi plate reader (Variokan, Thermo Scientific). Fluorescence measurements were made after obtaining fluorescence signal values at 525 nm emission wavelengths obtained from 480 nm excitation wavelengths, and then comparing the fluorescence values of each well with the wells containing no buffers but only buffers as reference values.
FAM이 표지된 dsDNA[서열번호 1과 2]과 OhrR을 결합시킨 후, CHP와 다양한 농도의 바이오티올을 처리하였을 때 OhrR과 분리된 형광 표지 dsDNA의 신호를 측정하였다. 동일한 시간동안 CHP만을 처리하였을 때(대조군)와 비교해 볼 때 CHP/바이오티올의 농도에 따라 형광신호가 정량적으로 증가하는 것을 확인하였다. After binding FAM-labeled dsDNA [SEQ ID NOS: 1 and 2] to OhrR, the signal of fluorescently labeled dsDNA separated from OhrR was measured when CHP and various concentrations of biothiol were treated. It was confirmed that the fluorescence signal was quantitatively increased according to the concentration of CHP / biothiol when compared with the CHP treatment (control) for the same time.
도 10은 임의의 바이오티올의 농도를 넣어주어 검출한 결과로서 바이오티올 종류에 따른 검출량은 예측할 수 없으나, 본 실시예를 통해 시료 내의 저분자 바이오티올 총량(DTT 미처리 시 free biothiol양, DTT 처리 시 total biothiol 양)을 효과적으로 검출할 수 있음을 보여주었다.10 shows the detection amount according to the type of biothiol as a result of detecting the concentration of an arbitrary biothiol, but the total amount of low-molecular biothiol in the sample (free biothiol amount without DTT treatment, and total value when DTT treatment was obtained through this embodiment). biothiol amount) can be detected effectively.
실시예Example 10:  10: DNAzyme이DNAzyme 결합된Combined dsDNA을dsDNA 이용한 화학발광 측정 Chemiluminescence Measurement
<실험조건><Experimental conditions>
buffer: TBS (Tris 20 mM, NaCl 150 mM)buffer: TBS (Tris 20 mM, NaCl 150 mM)
OhrR 농도: 5 μMOhrR concentration: 5 μM
DNA 농도: 100 nMDNA concentration: 100 nM
CHP, thiol reaction time: 5 minCHP, thiol reaction time: 5 min
dsDNA 서열 (IDT사 합성, USA)dsDNA sequence (synthesis of IDT, USA)
Template DNA 서열: 5' - GG GTT GGG CGG GAT GGG TTT TTT TTT TAC AAT TAA ATT GTA TAC AAT TAA ATT GTA-3' [서열번호 4](Italic/bold 부분은 DNA 효소, Italic부분은 T9-linker)Template DNA Sequence: 5 ' -GG GTT GGG CGG GAT GGG TTT TTT TTT TAC AAT TAA ATT GTA TAC AAT TAA ATT GTA-3 '[SEQ ID NO: 4] (Italic / bold part is DNA enzyme, Italic part is T9-linker)
Complementary DNA 서열: 5'- biotin - TAC AAT TTA ATT GTA TAC AAT TTA ATT GTA-3' [서열번호 5]Complementary DNA Sequence: 5'- biotin-TAC AAT TTA ATT GTA TAC AAT TTA ATT GTA-3 '[SEQ ID NO: 5]
<실험과정>Experimental Process
DNAzyme (5'-GG GTT GGG CGG GAT GGG-3'[서열번호 6], IDT 사에서 합성) 이 결합된 OhrR-binding dsDNA를 상층액으로부터 200 μL를 회수한 이후 여기에 NeutrAvidin®이 코팅된 비드(Thermo Scientific사, USA)의 현탁액 20 μL를 넣고 상온에서 30분간 shaking하였다. 이후 TBS(Tris Buffered Saline, 20 mM Tris, NaCl 150 mM, pH 7.4) 버퍼로 4회 세척하였다. 다음 DNA 효소의 활성을 유도하기 위해 TBS 버퍼 180 μL와 1 μM의 Hemin(Calbiochem, USA) (in TBS) 용액 20 μL를 섞어준 후 총 200 μL에 비드를 침전 시킨 다음 상온 암 조건 하에서 15분 이상 방치시켰다. 이후 비드가 담긴 혼합액 200 μL를 튜브에 넣고 루미노미터(모델명 Glo-Max 20/20, Promega사, USA)에 장착한 다음 ECL 반응용액(G-healthcare 사, USA) A(Luminol+H2O2 용액, 50 μL)와 B (enhancer, 50 μL)의 혼합액 100 μL를 넣은 후 즉시 화학발광세기를 측정하였다.DNAzyme (5'-GG GTT GGG CGG GAT GGG-3 '[SEQ ID NO: 6], synthesized by IDT) The combined OhrR-binding since the number of times the 200 μL of dsDNA from the supernatant Here NeutrAvidin ® is put into a suspension 20 μL of the coated beads (Thermo Scientific Inc., USA) was shaking 30 min at room temperature. After washing 4 times with TBS (Tris Buffered Saline, 20 mM Tris, NaCl 150 mM, pH 7.4) buffer. Next, 180 μL of TBS buffer and 20 μL of 1 μM Hemin (Calbiochem, USA) (in TBS) solution were mixed to induce the activity of DNA enzymes. Then, beads were precipitated in a total of 200 μL. It was left to stand. Then, 200 μL of the mixed solution containing beads was placed in a tube, mounted on a luminometer (Model Glo-Max 20/20, Promega, USA), and then ECL reaction solution (G-healthcare, USA) A (Luminol + H 2 O 2 Solution, 50 μL) and 100 μL of a mixture of B (enhancer, 50 μL) were added and the chemiluminescence intensity was measured immediately.
OhrR-binding dsDNA 서열 중 한쪽 strand 말단에는 ssDNAzyme (Horseradish peroxidase 의 성질을 가진 DNAzyme) 서열을 T9 linker에 의해 연결하고[서열번호 4], 다른 strand 말단에는 biotin을 도입하여 DNA 프로브[서열번호 5]를 구성하였다. OhrR 단백질은 Flag peptide (DYKDDDDK, 재조합 DNA를 제작 후 직접 균주에서 발현) [서열번호 7]를 결합하여 대장균에서 발현/정제 후 실험에 사용하였다. 실시예 8에서 언급하였듯이, anti-Flag antibody가 결합된 agarose bead로 OhrR 단백질을 capture하여 상기 DNA probe를 넣고 결합한 이후에, 시료 내 CHP와 시스테인을 넣어주면 DNA 프로브 부분이 빠르게 해리된다. 해리된 DNA 프로브는 원심분리 이후 상층액에 존재하는데, 이 상층액을 다시 Avidin이 결합된 bead와 반응시킨 뒤 세척과정을 거쳐 bead가 담긴 용액에 존재하는 dsDNA와 hemin과 결합을 시킨 다음 luminol 반응을 하면 강한 화학발광을 유도할 수 있다. One strand of the OhrR-binding dsDNA sequence is linked to the ssDNAzyme (a DNAzyme having the property of Horseradish peroxidase) by the T9 linker [SEQ ID NO: 4], and the other strand is introduced with a biotin to introduce the DNA probe (SEQ ID NO: 5). Configured. OhrR protein was used in the experiment after expression / purification in Escherichia coli by combining the flag peptide (DYKDDDDK, expressed in direct strain after making recombinant DNA). As mentioned in Example 8, after capturing the OhrR protein with the agarose bead to which the anti-Flag antibody is bound and inserting the DNA probe, the DNA probe portion is rapidly dissociated by adding CHP and cysteine in the sample. The dissociated DNA probe is present in the supernatant after centrifugation. The supernatant is reacted with the avidin-bound bead, washed, and then combined with dsDNA and hemin in the bead-containing solution, followed by luminol reaction. This can induce strong chemiluminescence.
<실험결과><Experiment Result>
측정 결과, 시스테인 반응 후 약 20분 후에 anti-Flag Ab bead가 존재하는 상층액에서 효과적으로 화학발광 신호가 검출됨을 확인하였다(도 11). Anti-Flag Ab bead에는 반대로 시스테인을 처리하지 않는 조건에서 과량의 화학신호가 검출된 것으로 보아 시스테인 존재 시 효과적으로 Flag-OhrR 단백질과 DNA probe가 효과적으로 해리되고, 형광보다 높은 신호로 검출될 수 있음을 확인하였다.As a result, about 20 minutes after the cysteine reaction, it was confirmed that the chemiluminescence signal was effectively detected in the supernatant containing anti-Flag Ab bead (FIG. 11). Anti-Flag Ab bead, on the contrary, was found that excessive chemical signal was detected under cysteine-free condition, so that Flag-OhrR protein and DNA probe can be effectively dissociated in the presence of cysteine and detected with higher signal than fluorescence. It was.
즉, 용액 내 시스테인이 존재할 때에만 특이적으로 DNAzyme이 결합된 타겟 DNA가 OhrR 단백질로부터 해리되어 DNAzyme 신호를 내는 것을 확인하였다(DNA probe, OhrR, 시스테인의 최종 농도는 각각 1 μM, 1 μM, 20 nM임, 시스테인을 넣은 후 20분 후에 화학발광 측정함).That is, it was confirmed that only DNAzyme-bound target DNA dissociated from the OhrR protein to give a DNAzyme signal only when cysteine was present in the solution (DNA probe, OhrR, and final concentrations of cysteine were 1 μM, 1 μM, and 20, respectively). nM, chemiluminescence measurement 20 minutes after cysteine).
실시예Example 11:  11: DNADNA 신호 증폭 방법 및 확인 Signal amplification method and confirmation
1) 신호 증폭1) signal amplification
OhrR+F (template forward DNA)와 OhrR_R (complementary reverse DNA)는 OhrR-binding DNA를 구성하는 dsDNA이고 Modified OhrR-F는 증폭을 위한 짧은 DNA 서열을 추가적으로 도입한 것을 나타낸다(표 1). Hairpin 1과 Hairpin 2를 최종 반응산물에 넣을 경우 DNA 서열이 증폭되어 다양한 방법(전기영동, 형광측정법, 발광측정법)으로 이를 검출할 수 있다[도 12].OhrR + F (template forward DNA) and OhrR_R (complementary reverse DNA) are dsDNA constituting OhrR-binding DNA and Modified OhrR-F shows the addition of a short DNA sequence for amplification (Table 1). When hairpin 1 and hairpin 2 are added to the final reaction product, DNA sequences are amplified and can be detected by various methods (electrophoresis, fluorescence, luminescence) [FIG. 12].
Figure PCTKR2017012344-appb-T000001
Figure PCTKR2017012344-appb-T000001
TBS에 희석되어 있는 2.5 μM의 template과 40 μM의 Hairpin 1,2 DNA를 각각 2 μL씩 넣고, TBS 버퍼로 총 부피가 20 μL가 되도록 만들었다. 그런 다음, 각 샘플을 상온에서 30분 이상 반응시키고 샘플 중 10 μL를 새로운 튜브로 옮긴 후에 1 μM 농도의 Hemin을 10 μL, TBS를 80 μL를 넣어 총 부피 100 μL을 만들었다. 그런 다음, 빛이 없는 조건으로 상온에서 30분 동안 반응을 시켰다. 또한 상대적인 신호크기의 비교를 위해 hemin과 TBS로만 만든 샘플을 추가로 준비하였다. 이후 상기 실시예 10에서 서술한 것과 같은 방법으로 Promega사의 Glo-Max 20/20 single tube luminometer를 사용하여 DNAzyme의 활성을 측정하였다. 측정 결과는 hemin만을 ECL 용액과 반응시킨 값을 기준으로 하여 상대적인 값으로 표현하였다.2.5 μM of template diluted in TBS and 2 μL of 40 μM of Hairpin 1,2 DNA were added, and the total volume was made to 20 μL with TBS buffer. Then, each sample was allowed to react at room temperature for at least 30 minutes, and 10 μL of the sample was transferred to a new tube. Then, 100 μL of the total volume was made by adding 10 μL of 1 μM Hemin and 80 μL of TBS. Then, the reaction was performed for 30 minutes at room temperature under no light conditions. In addition, a sample made only of hemin and TBS was further prepared for comparison of relative signal sizes. Thereafter, DNAzyme activity was measured using a Pro-Mega Glo-Max 20/20 single tube luminometer in the same manner as described in Example 10 above. The measurement results are expressed in relative values based on the value of reacting only hemin with the ECL solution.
상기 실시예 10과 같이 DNAzyme 서열 하나만을 double strand DNA 한쪽 말단에 결합하여 활용하는 것에 비해서 임의의 ssDNA 서열 결합한 이후 HP1과 HP2의 첨가반응에 의한 결과로 DNAzyme을 효과적으로 생성시킴으로써 hemin의 background 신호에 비해서 16.5배 이상, 그리고 기본 DNAzyme이 직접 결합된 template 보다 약 2.6 배 이상으로 화학발광 및 비색신호를 증폭시킬 수 있음을 확인하였다[도 13a]Compared to using only one DNAzyme sequence at one end of a double strand DNA as in Example 10, 16.5 compared to hemin's background signal by effectively generating DNAzyme as a result of HP1 and HP2 addition reaction after binding to any ssDNA sequence. It was confirmed that amplification of the chemiluminescence and colorimetric signals by more than twice and about 2.6 times more than the template DNAzyme directly bonded [Fig. 13a]
2) 신호 증폭 확인2) Check signal amplification
<실험조건><Experimental conditions>
DNA template (Modified OhrR_F+OhrR_R) 농도: 500 nMDNA template (Modified OhrR_F + OhrR_R) concentration: 500 nM
H1, H2 농도: 각 2 μMH1, H2 concentration: 2 μM each
반응시간, 전기영동 시간: 각 30 minResponse time, electrophoresis time: 30 min each
<실험과정>Experimental Process
모든 DNA oligo는 IDT를 통해서 제작주문 후 사용하였음. 100 μM 의 Modified OhrR_F와 OhrR-R을 20 μL씩 섞은 뒤, 95℃의 온도에서 10분간 가열 후 상온에서 천천히 식힘으로써 OhrR과 결합하는 double strand 서열을 준비하였다. 이 double strand DNA를 template로 사용하여, 5 μM의 template을 2 μL, 20 μM의 hairpin 1과 20 μM의 hairpin 2를 2 μL씩 사용하고 총 부피는 20 μL가 되도록 상온에서 30분 반응시켰다. 이후 8 μL씩의 샘플을 사용하여 아가로스 젤 전기영동을 통해 증폭되어 길이가 늘어난 DNA 밴드를 UV에서 육안으로 확인하였다.All DNA oligos were used after production order through IDT. 20 μL of 100 μM Modified OhrR_F and OhrR-R were mixed, and a double strand sequence was prepared by binding to OhrR by heating at 95 ° C. for 10 minutes and then slowly cooling at room temperature. Using this double strand DNA as a template, 2 μL of 5 μM template, 2 μL of 20 μM hairpin 1 and 2 μL of 20 μM hairpin 2 were reacted at room temperature for 30 minutes at a total volume of 20 μL. Thereafter, 8 μL of each sample was amplified by agarose gel electrophoresis, and the length of the DNA band was visually confirmed by UV.
<실험결과><Experiment Result>
전기영동의 결과에 나타난 바와 같이 HP1과 HP2는 single strand 형태로 50 bp 미만의 위치에 밴드 크기가 나타나는데 template이 없는 상태에서는 연쇄반응이 일어나지 않는다 (DNA 겔 상의 밴드위치의 변화 없음). 그러나 template가 존재 시 H1이 먼저 template과 결합하여 H1의 hairpin 구조가 풀리게 되고, 이렇게 열려진 H1 서열은 이후 H2 서열과 결합하면서 H2를 열린 구조로 만들게 된다. Template과 H1만을 반응한 결과에서 보듯이 template DNA 및 H1 각각의 DNA 밴드크기 보다 더 큰 크기에서 관찰되며, template DNA 보다 상대적으로 많은 양이 존재하기에 결합하지 못하고 남은 H1이 아래에 여전히 남아있는 것을 볼 수 있다. 그러나 template DNA와 H2만을 반응시킨 경우는 서로 결합하지 못하기 때문에 template과 H2 모두 밴드 크기에 변화가 없음을 확인할 수 있었다. 그리고 template DNA와 H1, H2가 동시에 존재 시 template DNA가 H1/H2에 의해 연쇄적으로 결합하여 DNA 밴드 크기가 효과적으로 증폭이 됨을 확인하였다[도 13b]. 이와 같이 증폭된 DNA 밴드 크기 서열에 DNA 효소가 포함되어 있기 때문에 도 13a에서 보듯이 DNA 효소활성이 크게 나타나는 것을 관찰하였다. 이와 같은 방법을 활용하면 바이오티올에 의한 DNA-OhrR의 상호작용에 의해서 DNA효소 활성을 증폭함으로써 저농도의 바이오티올을 효과적으로 측정할 수 있다.As shown in the results of electrophoresis, HP1 and HP2 are single stranded and have a band size of less than 50 bp. In the absence of the template, no chain reaction occurs (no change in the band position on the DNA gel). However, when the template exists, H1 is first combined with the template to release the hairpin structure of H1. Thus, the opened H1 sequence is then combined with the H2 sequence to make H2 an open structure. As shown in the result of reacting only template and H1, it is observed at a size larger than the DNA band size of each template DNA and H1, and the remaining amount of H1 still remains below because it is relatively larger than template DNA. can see. However, when only template DNA and H2 were reacted, they could not bind to each other, so both template and H2 showed no change in band size. And when template DNA and H1, H2 are present at the same time it was confirmed that the template DNA is chained by H1 / H2 chain size is effectively amplified DNA (Fig. 13b). As shown in FIG. 13A, the DNA enzyme activity was large because the DNA band size sequence amplified was included. Using such a method, it is possible to effectively measure low concentration of biothiol by amplifying DNA enzyme activity by interaction of DNA-OhrR with biothiol.
실시예Example 12: 바이오 칩 제작  12: biochip fabrication
DNA에 형광 또는 DNAzyme으로 표지하는 대신 OhrR의 His6-tag을 이용하여 측정하는 바이오 칩 방식으로서 바이오티올이 존재 시 바이오 칩 표면에 부착된 dsDNA으로부터 OhrR이 떨어져 나가므로 검출신호가 감소되는 원리이다.Instead of labeling DNA with fluorescence or DNAzyme, a biochip method is measured by using His6-tag of OhrR. In the presence of biothiol, OhrR is separated from dsDNA attached to the surface of biochip.
투명한 96-well plate의 각 well에 coating buffer(100 mM Na2HPO4, 50 mM Citric Acid)로 희석된 5 mg/mL 농도의 NeutrAvidin®(Thermo Scientific사, USA)을 150 μL씩 넣은 후 37℃에서 4-5시간을 반응 후 결합하지 않은 잔여물을 TBS로 4회 세척하였다. 세척 후 Seablock blocking buffer(Thermo scientific사, USA)를 각 well에 300 μL씩 넣고 상온에서 2시간 동안 shaking하면서 blocking (비특이적 반응을 억제하기 위해 NeutrAvidin과 결합하지 않은 표면을 코팅하는 과정)을 한 다음 다시 TBS로 4회 세척하였다. Blocking이 완료된 마이크로 플레이트 각 웰에 TBS 버퍼에 1 μM 농도로 희석된 biotin-DNA 용액을 100 μL씩 넣고 상온에서 1시간 반응시킨 후 TBS로 4회 세척하였다. 이후 his6-tag이 결합된 OhrR을 5 μM 농도로 100 μL씩 상온에서 1시간 결합시킨 뒤 TBS로 4회 세척과정을 거쳐 바이오티올 측정을 위한 바이오 칩을 준비하였다.150 μL of 5 mg / mL NeutrAvidin ® (Thermo Scientific, USA) diluted with coating buffer (100 mM Na 2 HPO 4 , 50 mM Citric Acid) was added to each well of a clear 96-well plate. The unbound residue was washed four times with TBS after 4-5 h reaction at. After washing, put 300 μL of Seablock blocking buffer (Thermo scientific, USA) into each well and shake for 2 hours at room temperature. Wash 4 times with TBS. 100 μL of the biotin-DNA solution diluted to 1 μM concentration in TBS buffer was added to each well of blocked microplates, and reacted at room temperature for 1 hour, and then washed 4 times with TBS. After binding his6-tag bound OhrR to 5 μM concentration at 100 μL for 1 hour at room temperature, the biochip was measured for biothiol by four washes with TBS.
상기 NeutrAvidin이 코팅된 플레이트에 Biotin-dsDNA와 His6-tag OhrR이 고정된 바이오칩을 사용하여 각 well에 CHP(final 5 μM)와 바이오티올 중 하나 (L-cysteine, homocysteine, GSH 각 final 100 μM)을 각각 50 μL씩 동시에 넣고 상온에서 10분 동안 반응시킨 후 TBS로 4회 세척하였다. 이후 HisProbe-horseradish peroxidase conjugate (HisProbe-HRP, ThermoScientific, USA; HRP는 Horseredox peroxidase)용액을 100 μL 넣고 상온에서 1시간 동안 결합시킨 후 TBS로 4회 세척하고 이후 TMB (3,3', 5,5'-tetramethylbenzidine 용액을 처리 후 5분 뒤 2M 황산으로 반응을 중지시켜 450 nm에서의 흡광도를 측정 후 결과를 비교하였다[도 14].Using a biochip fixed with Biotin-dsDNA and His6-tag OhrR on the NeutrAvidin-coated plate, one of CHP (final 5 μM) and biothiol (L-cysteine, homocysteine, GSH each final 100 μM) was added to each well. 50 μL each was added at the same time, and reacted at room temperature for 10 minutes, and then washed four times with TBS. Then, put 100 μL of HisProbe-horseradish peroxidase conjugate (HisProbe-HRP, ThermoScientific, USA; HRP, Horseredox peroxidase) solution, bind at room temperature for 1 hour, wash 4 times with TBS, and then TMB (3,3 ', 5,5) 5 minutes after treatment with the '-tetramethylbenzidine solution, the reaction was stopped with 2M sulfuric acid, and the absorbance at 450 nm was measured and the results were compared.
바이오티올이 존재하지 않은 well에서는 OhrR이 well에 고정되어 있는 dsDNA와 결합하여 OhrR의 His-tag을 표적으로 하는 Hisprobe-HRP가 결합되어 HRP와 반응하는 TMB의 흡광신호가 가장 높게 나타남. 그러나 CHP와 함께 바이오티올이 존재하면 OhrR이 바이오 칩 표면에 고정된 dsDNA에서부터 해리되면서 세척과정을 통해 제거된다. 따라서 Hisprobe-HRP가 결합되어 HRP와 반응하는 TMB의 흡광신호가 감소하게 된다. 실시예 9에서와 같이 바이오티올은 같은 농도조건에서 L-cysteine이 Homocysteine이나 GSH 보다 흡광신호의 감소 효과가 더 크게 관찰되었다[도 15].In wells without biothiols, the OabR-immobilized Hisprobe-HRP, which targets the His-tag of OhrR, binds to dsDNA immobilized in the well, resulting in the highest absorption of TMB. However, if biothiol is present with CHP, OhrR is dissociated from dsDNA immobilized on the surface of the biochip and is removed by washing. Therefore, hisprobe-HRP is combined to reduce the absorption signal of TMB reacting with HRP. As in Example 9, the biothiol was observed to have a greater effect of reducing the absorption signal than L-cysteine and Homocysteine or GSH under the same concentration conditions [FIG. 15].
실시예 13: 스트립 형태의 바이오센서 제작 Example 13 Preparation of Strip-shaped Biosensor
도 16에 예시된 스트립 센서는 3개의 주요 부위(시료 도입부, 반응부, 측정부)로 구성될 수 있으며 스트립 센서의 제작을 위한 멤브레인으로 셀룰로오스(cellulose), 니트로셀룰로오스(nitrocellulose)와 유리섬유(glass-fiber) 멤브레인 등을 사용할 수 있다. 각 부위별 구조 및 특징을 다음과 같다. The strip sensor illustrated in FIG. 16 may be composed of three main parts (sample introduction part, reaction part, and measuring part), and cellulose, nitrocellulose and glass fiber as membranes for fabricating the strip sensor. -fiber) membrane and the like can be used. The structure and characteristics of each site are as follows.
각 부위는 서로 다른 멤브레인으로 구성하되 각 멤브레인은 범용 OHP 필름 (0.4 cm × 5.5 cm) 위에 중첩되게 고정하고, 전체 스트립센서의 모세관 현상이 일정하게 유지될 수 있도록 반응부 패드를 가장 아래쪽에 놓아 시료 도입부와 측정부 패드까지 연결될 수 있도록 고정하고, 시료 도입부는 흡수가 용이하도록 맨 위쪽으로 위치를 시킨다. Each part is composed of different membranes, but each membrane is fixed to overlap on a general OHP film (0.4 cm × 5.5 cm), and the reaction pad is placed at the bottom of the sample to keep the capillary phenomenon of the entire strip sensor constant. It is fixed to be connected to the introduction part and the measurement part pad, and the sample introduction part is positioned at the top for easy absorption.
시료 도입부(도 16 상단 센서의 시료가 도입되는 좌측 검정 부위에 해당): FLAG tag이 된 OhrR과 double stand DNA(서열번호 1과 2)를 결합한 복합체 1 μM (TBS 버퍼) 용액 10 μL와 바이오티올이 함유된 시료용액 (버퍼, 혈액, 소변 등) 10 μL를 섞은 후 이 혼합액 20 μL와 CHP 용액 (2 μM)이 든 버퍼를 20 μL 반응하여 총 40 μL 용액을 셀룰로오스 멤브레인 (0.4 cm × 1.5 cm) 에 떨어뜨린 후 흡착시키면, 크로마토그래피에 의한 원리에 의해 시료에 용해된 물질이 센서 반응부로 이동하게 된다. Sample introduction (corresponding to the left assay site where the sample from the upper sensor is introduced): 10 μL of a solution of 1 μM (TBS buffer) complex and biothiol combining OhrR tagged with FLAG and double stand DNA (SEQ ID NOs: 1 and 2) After mixing 10 μL of the sample solution (buffer, blood, urine, etc.) containing 20 μL of this mixture and 20 μL of the buffer containing CHP solution (2 μM), a total of 40 μL solution was added to the cellulose membrane (0.4 cm × 1.5 cm). And then adsorbed, the substance dissolved in the sample moves to the sensor reaction part by the principle of chromatography.
반응부(도 16 상단 센서의 DNA 이동 화살표가 그려져 있는 중앙 회색 부위에 해당): 니트로셀룰로오스 멤브레인(약 0.4 cm × 2.5 cm)을 사용하여 구성하고, 시료도입부 방향에부터 약 1 cm 오른쪽에 위치한 곳에 anti-FLAG antibody 용액 (1 mg/mL, 인산화 완충용액)을 약 1 μL를 떨어뜨려 37 ℃에서 1시간 방치하여 고정화한다. 이 부위는 시료 도입부에서 반응한 Flag-OhrR-dsDNA 복합체가 이동하면서 표면에 고정된 FLAG 항체에 의해 결합이 된다. 시료 내 바이오티올이 존재 시 dsDNA는 OhrR으로부터 해리가 되어 멤브레인 상에서 오른쪽으로 계속 이동해가고, biothiol이 존재하지 않을 경우 dsDNA는 항체에 의해 OhrR과 같이 고정되어 이동이 멈추게 된다. Reaction part (corresponding to the central gray area where the DNA movement arrow of the upper sensor of FIG. 16 is drawn): Constructed using a nitrocellulose membrane (approximately 0.4 cm x 2.5 cm) and located about 1 cm to the right of the sample introduction direction. Immobilize the anti-FLAG antibody solution (1 mg / mL, phosphorylated buffer solution) at about 1 hr by dropping about 1 μL. This site is bound by the FLAG antibody immobilized on the surface as the Flag-OhrR-dsDNA complex reacted at the sample introduction part moves. In the presence of biothiol in the sample, dsDNA dissociates from OhrR and continues to move to the right on the membrane. In the absence of biothiol, dsDNA is fixed like OhrR by the antibody and stops moving.
측정부(도 16 상단 센서의 바이오티올 검출 여부를 확인하는 우측 검정 부위에 해당): 측정부는 결합 및 분리부로부터 해리되어 이동한 double strand DNA를 측정하는 부위로서, dsDNA 말단에 부착된 DNAzyme의 신호를 유도하는 부위이다. 즉, 유리섬유 멤브레인(0.4 cm × 0.5 cm)을 사용하여 Hemin 용액을 사전 분주하여 잔류하게 하고 최종 TMB 용액 혹은 ECL 용액을 떨어뜨림으로써 신호반응을 확인할 수 있게 된다. Hemin은 버퍼(40 mM Tris, 200 mM NaCl, 50 mM KCl 및 20 mM MgCl2) 용액에 5% casein 용액을 혼합하여 제조한 후 유리섬유 멤브레인에 100 μL를 충분히 흡수시켜 55℃에서 30분간 건조하여 준비한다. 반응부에서 해리된 dsDNA가 충분히 이동한 이후 최종 신호 분석을 위해 ECL 용액 및 TMB 반응 용액 10 μL를 첨가하여 반응을 유도한다. ECL 용액은 화학발광분석장치로 반응용액 투입 즉시 이미징으로 분석 가능하며 TMB 반응 결과는 mobile phone 혹은 digital camera가 장착된 고정대에서 용액 투입 후 약 30분 후에 분석 가능하다.Measuring unit (corresponding to the right assay site for detecting the biothiol of the upper sensor of FIG. 16): The measuring unit is a site for measuring double strand DNA dissociated from the binding and separation unit, and the signal of DNAzyme attached to the dsDNA terminal It is a site that induces. In other words, the glass fiber membrane (0.4 cm × 0.5 cm) can be used to pre-dispense the Hemin solution to remain and confirm the signal response by dropping the final TMB solution or ECL solution. Hemin was prepared by mixing a 5% casein solution in a buffer (40 mM Tris, 200 mM NaCl, 50 mM KCl, and 20 mM MgCl 2 ) solution, and then sufficiently absorbed 100 μL into a glass fiber membrane and dried at 55 ° C. for 30 minutes. Prepare. After sufficient dissociation of the dsDNA in the reaction part, 10 μL of the ECL solution and the TMB reaction solution are added for the final signal analysis to induce the reaction. The ECL solution can be analyzed by imaging as soon as the reaction solution is added with the chemiluminescence spectrometer, and the TMB reaction results can be analyzed about 30 minutes after the solution is added to the holder equipped with a mobile phone or digital camera.
동일 시료는 동일한 부피를 둘로 나누어 하나는 DTT와 1시간 이상 처리 후 DNA-OhrR 복합체와 반응하고, 다른 하나는 DTT 첨가 없이 바로 DNA-OhrR 복합체와 반응을 한다. 즉 동일한 2개의 분석결과로부터 아래 사항을 판단할 수 있게 된다.The same sample is divided into two equal volumes, one is treated with DNA-OhrR complex after treatment with DTT for 1 hour or more, and the other is directly reacted with DNA-OhrR complex without DTT addition. That is, the following items can be determined from the same two analysis results.
시료 1: DTT 미첨가, 유리 바이오티올 측정Sample 1: No DTT, Free Biothiol Determination
시료 2: DTT 첨가, 총 바이오티올 측정Sample 2: DTT addition, total biothiol determination
시료 1과 시료 2의 반응 결과를 각 시료 성상별, 질병종류별, 질병단계별, 나이별, 성별 등으로 분류해서 분석한 후 비교한다.The reaction results of Sample 1 and Sample 2 are analyzed by classifying each sample into characteristics, by disease type, by disease stage, by age, and by gender, and then comparing them.

Claims (22)

  1. 산화환원 조절 단백질(redox-regulating protein)을 포함하는 바이오티올 검출용 조성물. Biothiol detection composition comprising a redox-regulating protein.
  2. 제 1 항에 있어서,The method of claim 1,
    상기 산화환원 조절 단백질은 OhrR(organic hydroperoxide regulator protein), PerR(Peroxide regulator) 및 OxyR(Oxygen regulator)으로 이루어진 군에서 선택된 하나 이상인, 바이오티올 검출용 조성물.The redox regulator protein is at least one selected from the group consisting of OhrR (organic hydroperoxide regulator protein), PerR (Peroxide regulator) and OxyR (Oxygen regulator), the composition for detecting a biothiol.
  3. 제 1 항에 있어서,The method of claim 1,
    상기 산화환원 조절 단백질은 바이오티올과 DNA와의 결합친화도 조절이 가능한 변이체 또는 표지 단백질이 접합된 형태의 단백질을 포함하는, 바이오티올 검출용 조성물.The redox regulatory protein comprises a biothiol-detecting composition comprising a protein in which a variant or labeling protein capable of controlling binding affinity between biothiol and DNA is conjugated.
  4. 제 1 항에 있어서,The method of claim 1,
    산화환원 조절 단백질에 결합되는 DNA를 추가로 포함하는 바이오티올 검출용 조성물.Biothiol detection composition further comprises a DNA bound to the redox regulatory protein.
  5. 제 4 항에 있어서,The method of claim 4, wherein
    상기 DNA는 형광인자 또는 DNA 기반 효소(DNAzyme)이 결합되거나 신호 증폭방법으로 DNA 서열을 증폭시킨 것인, 바이오티올 검출용 조성물. Wherein the DNA is a fluorescent factor or DNA-based enzyme (DNAzyme) is bound or amplified DNA sequence by a signal amplification method, biothiol detection composition.
  6. 제 5 항에 있어서,The method of claim 5, wherein
    상기 형광인자는 로다민과 그의 유도체, 플루오레신과 그의 유도체, 쿠마린과 그의 유도체, 아크리딘과 그의 유도체, 피렌과 그의 유도체, 에리트로신과 그의 유도체, 에오신과 그의 유도체, 및 4-아세트아미도-4′-이소티오시아나토스틸벤-2,2′디설폰산으로 이루어진 군에서 선택된 하나 이상인, 바이오티올 검출용 조성물.The fluorescent factors include rhodamine and its derivatives, fluorescein and its derivatives, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives, erythrocin and its derivatives, eosin and its derivatives, and 4-acetamido- At least one selected from the group consisting of 4'-isothiocyanatostilbene-2,2 'disulfonic acid, the composition for detecting a biothiol.
  7. 제 5 항에 있어서,The method of claim 5, wherein
    상기 DNA 기반 효소는 퍼옥시다제 모방(peroxidase-mimicking) DNA 효소 및 RNA-절단(RNA-cleaving) DNA 효소로 이루어진 군에서 선택된 하나 이상의 서열을 가지는 바이오티올 검출용 조성물.The DNA-based enzyme is a composition for detecting biothiol having one or more sequences selected from the group consisting of peroxidase-mimicking DNA enzyme and RNA-cleaving DNA enzyme.
  8. 제 1 항에 있어서,The method of claim 1,
    유리(free) 형태의 바이오티올과 총(total) 바이오티올을 각각 검출하거나 유리 형태의 바이오티올과 총 바이오티올을 동시에 검출하는, 바이오티올 검출용 조성물.A composition for detecting biothiol, which detects free biotol and total biothiol, respectively, or simultaneously detects free biotol and total biothiol.
  9. 제 8 항에 있어서,The method of claim 8,
    상기 총 바이오티올을 검출하는 경우, 상기 조성물에 환원제를 추가로 포함하는, 바이오티올 검출용 조성물. When detecting the total biothiol, biothiol detection composition further comprises a reducing agent in the composition.
  10. 제 9 항에 있어서,The method of claim 9,
    상기 환원제는 DTT(dithiothreitol), 2-머캡토에탄올 및 TCEP(tris(2-carboxyethyl)phosphine)로 이루어진 군에서 선택된 하나 이상인, 바이오티올 검출용 조성물. The reducing agent is at least one selected from the group consisting of DTT (dithiothreitol), 2-mercaptoethanol and TCEP (tris (2-carboxyethyl) phosphine), the composition for detecting a biothiol.
  11. 제 1 항에 있어서,The method of claim 1,
    상기 바이오티올은 10 Da 내지 1000 Da의 분자량을 갖는, 바이오티올 검출용 조성물.The biothiol has a molecular weight of 10 Da to 1000 Da, the composition for detecting biothiol.
  12. 제 1 항에 있어서,The method of claim 1,
    상기 바이오티올은 시스테인(cysteine, Cys), 호모시스테인(homocysteine, Hcy), 글루타치온(glutathione, GSH), N-아세틸시스테인(N-acetylcysteine, NAC), 시스티아민(cysteamine, CA), γ-글루타밀시스테인(γ-glutamylcysteine, γ-GluCys), 시스테이닐글리신(cysteinylglycine, CysGly), N-아세틸시스테인(N-acetylcysteine, N-AC), 코엔자임 A(Coenzyme A, CoA), 코엔자임 B(Coenzyme B, CoB), 코엔자임 M(Coenzyme M, CoM), 바실리티올(bacillithiol, BacT), 미코티올(mycothiol, MyT), 에르고티오네인(ergothioneine, ErT) 및 트리판오티온(trypanothione, TrT)으로 이루어진 군에서 선택된 하나 이상인, 바이오티올 검출용 조성물.The biothiol is cysteine (Cys), homocysteine (Hcy), glutathione (GSH), N-acetylcysteine (NAC), cysteamine (CA), γ-glutamylcysteine (γ-glutamylcysteine, γ-GluCys), cysteinylglycine (CysGly), N-acetylcysteine (N-AC), coenzyme A (Coenzyme A, CoA), coenzyme B (Coenzyme B, CoB ), Coenzyme M (CoM), bacillithiol (BacT), mycothiol (MyT), ergothioneine (ErT) and trypanothione (TrT) At least one selected, biothiol detection composition.
  13. 제 1 항에 있어서,The method of claim 1,
    상기 바이오티올은 심혈관질환(cardiovascular disease), 퇴행성 뇌질환(neurodegenerative disease), 암(cancer), 신장병(kidney dysfunction), 당뇨(diabetes mellitus) 또는 박테리아 및 바이러스 감염(infection)과 관련된 것인, 바이오티올 검출용 조성물. The biothiol is associated with cardiovascular disease, degenerative brain disease, neurodegenerative disease, cancer, kidney dysfunction, diabetes, diabetes mellitus or bacterial and viral infections. Detection composition.
  14. 제 1 항의 조성물을 이용한 바이오티올 검출 방법.Biothiol detection method using the composition of claim 1.
  15. 제 14 항에 있어서,The method of claim 14,
    겔 전기영동(gel electrophoresis)법, 형광 비등방성 측정법(fluoresecence anisotropy), MALDI-TOF MS(Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometer), SPR(Surface plasmon resonance), 간섭법(interferometry) 및 비드 측정법으로 이루어진 군에서 선택된 하나 이상으로 바이오티올을 검출하는 방법.Gel electrophoresis, fluorescence anisotropy, matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (SPR), surface plasmon resonance (SPR), interferometry And detecting the biothiol with at least one selected from the group consisting of bead measurement.
  16. 제 1 항의 조성물을 포함하는 바이오티올 검출용 바이오센서.Biosensor for detecting biothiol comprising the composition of claim 1.
  17. 제 16 항에 있어서,The method of claim 16,
    스트립(strip) 형태인 바이오센서.Biosensor in strip form.
  18. 제 1 항의 조성물을 포함하는 바이오티올 검출용 바이오 칩.Biochip for detecting a biothiol comprising the composition of claim 1.
  19. 제 18 항에 있어서, The method of claim 18,
    상기 조성물 내 산화환원 조절 단백질은 친화성 태그가 포함된 것인 바이오 칩.The redox regulatory protein in the composition is a biochip containing an affinity tag.
  20. 제 18 항에 있어서,The method of claim 18,
    상기 조성물 내 산화환원 조절 단백질은 상기 단백질과 결합 가능한 DNA가 고정화된 플레이트 상에서 DNA와 단백질 복합체를 형성하는, 바이오 칩The redox regulatory protein in the composition is a biochip, which forms a protein complex with DNA on a plate on which DNA bindable to the protein is immobilized.
  21. 산화환원 조절 단백질과 상기 단백질에 결합되는 DNA의 복합체와 결합할 수 있는 고정 부위를 포함하고, 시료와 상기 복합체를 혼합하여 도입하도록 형성된 시료 도입부; 및A sample introduction part including a fixation site capable of binding to a complex of a redox control protein and a DNA bound to the protein, the sample introduction part being configured to introduce a sample and the complex into a mixture; And
    상기 시료 도입부와 일 직선 상에서 일정 거리 이격되며, 상기 복합체가 산화 반응 후 DNA와 해리되는 반응부; 및 A reaction part spaced apart from the sample introduction part on a straight line by a predetermined distance, and the complex dissociates with the DNA after the oxidation reaction; And
    상기 해리된 DNA가 이동하여 바이오티올을 측정하도록 형성된 측정부;A measurement unit configured to move the dissociated DNA to measure biothiol;
    를 포함하는 스트립(strip) 형태의 바이오티올 검출용 바이오센서.Biosensor for detecting a biothiol in the form of a strip comprising a.
  22. 제 21 항에 있어서,The method of claim 21,
    상기 고정 부위는 산화환원 조절 단백질의 부위를 인식할 수 있는 항체 또는 수용체가 결합된, 바이오티올 검출용 바이오센서.The fixing site is a biosensor for detecting a biothiol is coupled to an antibody or a receptor that can recognize the site of the redox regulatory protein.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4288334B2 (en) * 2002-09-26 2009-07-01 株式会社産学連携機構九州 Method for measuring homocysteine and reagent for measurement
KR101535750B1 (en) * 2014-08-11 2015-07-09 경북대학교 산학협력단 Chemical sensor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4288334B2 (en) * 2002-09-26 2009-07-01 株式会社産学連携機構九州 Method for measuring homocysteine and reagent for measurement
KR101535750B1 (en) * 2014-08-11 2015-07-09 경북대학교 산학협력단 Chemical sensor

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GROITL, B. ET AL.: "Thiol-based Redox Switches", BIOCHIMICA ET BIOPHYSICA ACTA , (ELECTRONIC PUBLISHING, vol. 1844, no. 8, 19 March 2014 (2014-03-19), pages 1335 - 1343, XP028849468 *
PARK, K. S. ET AL.: "A Label-free Method for Detecting Biological Thiols Based on Blocking of Hg2+-Quenching of Fluorescent Gold Nanoclusters", BIOSENSORS AND BIOELECTRONICS, (ELECTRONIC PUBLISHING, vol. 45, 4 February 2013 (2013-02-04), pages 65 - 69, XP055482543 *
POOLE, L. B.: "The Basics of Thiols and Cysteines in Redox Biology and Chemistry", FREE RADICAL BIOLOGY AND MEDICINE, (ELECTRONIC PUBLISHING, vol. 80, 27 November 2014 (2014-11-27), pages 148 - 157, XP055482538 *
SUZUKI, Y. J. ET AL.: "18]Redox Regulation of DNA-Protein interactions by Biothiois", METHODS IN ENZYMOLOGY, vol. 252, 1995, pages 175 - 180 *

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