WO2016137236A1 - Procédé de détection de virus utilisant un liposome ou un hybride liposome-polymère - Google Patents

Procédé de détection de virus utilisant un liposome ou un hybride liposome-polymère Download PDF

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WO2016137236A1
WO2016137236A1 PCT/KR2016/001829 KR2016001829W WO2016137236A1 WO 2016137236 A1 WO2016137236 A1 WO 2016137236A1 KR 2016001829 W KR2016001829 W KR 2016001829W WO 2016137236 A1 WO2016137236 A1 WO 2016137236A1
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liposome
virus
glycero
phosphatidylcholine
acid
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Korean (ko)
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정봉현
배판기
박경미
김유정
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재단법인 바이오나노헬스가드연구단
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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
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • 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
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to a virus detection method using a liposome or a liposome-polymer hybrid, and more particularly, a liposome or a liposome-polymer hybrid containing a chromogenic substrate is contacted with a sample presumed to contain a virus.
  • the present invention relates to a virus detection method characterized by detecting the presence or absence of a virus by coloration according to the presence or absence of a chromophore substrate contained in a liposome or a liposome-polymer hybrid by the contact.
  • Liposomes are W / O / W type emulsions proposed by Bangham in the 1960s ( J. Mol . Biol., 13: 238, 1965), in which amphiphilic phospholipids are self-arranged by hydrophobic forces in the water phase.
  • the phospholipid membrane constituting the liposome has the same structure as the cell, and is easily used for cell introduction, and has a large space for supporting a water-soluble substance therein compared to other structures, so that it is used as a carrier and carrier for the water-soluble substance ( Eur . J. Pharm . Biopham, 62: 110, 2006 ; Nat Rev. Drug Discov, 145:... 4, 1979).
  • liposomes are nano-sized (1 ⁇ m or less) capsules as phospholipid delivery and mediators, and they can contain both lipophilic and hydrophilic functional materials, so they are suitable for living organisms similar to human skin cells. It is a substance that, when added to a hydrophilic formulation, remains suspended and has surface stability.
  • Liposomes are spherical vesicles in which the phospholipid bilayer surrounds the water phase.
  • the lipid membrane is an amphiphilic phospholipid consisting of two hydrophobic fatty acid groups and a hydrophilic phosphate group, which forms a double membrane in aqueous solution, which forms closed vesicles like artificial cells.
  • the non-polar fatty acid tail faces the inside of the membrane and the polar head faces outward.
  • Incorporating drugs into such liposomes has been attracting attention as a structure of particle bodies prepared by assembling with polymers, drugs, and antigens, as it can enhance the therapy by reducing the toxicity of drugs and increasing their effects.
  • Liposomes are fully enclosed structures that include a lipid bilayer membrane containing encapsulated aqueous medium. Liposomes may comprise many concentric lipid bilayers (multilamellar vesicles or MLVs) or single membrane bilayers (unilamellar vesicles) separated by an aqueous phase.
  • the lipid bilayer consists of two lipid monolayers with hydrophobic "tail” and hydrophilic "head” regions. In the membrane bilayer, the hydrophobic “tails” in the lipid monolayer are arranged towards the center of the bilayer and the hydrophilic “heads” are arranged towards the aqueous phase.
  • the basic structure of liposomes can be prepared by known methods. For example, lipid molecules suspended in an organic solvent are evaporated under reduced pressure to form a dry film in a vessel, and an appropriate amount of aqueous phase is added to the vessel and the mixture is stirred. The mixture can then be prepared by standing without shaking for a time sufficient to form a multilamellar vesicle. Unilamella vesicles can also be prepared by known techniques (eg, US Pat.
  • Liposome-polymer hybrids are biofilm mimetic amphiphilic structures composed of low amounts of lipids (eg, phospholipids) and high molecular weight polymers (eg, amphiphilic block copolymers).
  • the liposome-polymer hybrid is composed of a lipid component having a biological function (receptor, molecular recognition, etc.) and a polymer having a structural function (structural stability, etc.) can be prepared to bind to the target material (Olubummo A1 et al. , Langmuir, 30 (1): .
  • Liposomes or liposome-polymer hybrids can be designed for diagnostic purposes.
  • Liposomes or liposome-polymer hybrids can be covalently bound to proteins, antibodies and immunoglobulins.
  • thiolated IgG or Protein A can be covalently bound to lipid vesicles and the thiolated antibody or Fab 'fragment can be bound to liposomes or liposome-polymer hybrids.
  • Biosensor systems can provide easier and more useful information for analyzing data by detecting signals using the properties of fluorescent nanomaterials at the cellular or in vivo level.
  • Chemical and biosensors are materials or devices that detect and measure information from an object to be measured and change the measurable amount into a usable signal.
  • the sensor acquires information from the target, the sensor converts the signals into recognizable signals such as color, fluorescence, and electrical signals to assist human judgment.
  • the sensor recognizes the target material, it sends a signal through a signal converter for human recognition.
  • sensors used in biosensors require high selectivity and sensitivity to target materials to be detected.
  • Enzymes and antibodies have excellent substrate specificity and high binding capacity, but have the disadvantage of low stability and high price when immobilized in a sensor device.
  • Nanobiosensors are biosensors that are improved by advanced nanotechnology, which converts reactions by binding to biocognitive materials into signals, and refers to sensors that can quickly test specific materials. This is the same principle as the enzyme-substrate complex of the biological concept, in which one ligand is only reactive with one substance having a specific component for the ligand and measures the degree of reactivity.
  • Miniaturized biosensor using nanotechnology minimizes human injury and enables painless human diagnosis and has the advantage of directly analyzing single cells.
  • biosensors with improved operating characteristics such as high stability, fast response time, high sensitivity, and high selectivity using nanotechnology enable continuous measurement of human diagnosis and single-molecule analysis.
  • Korean Patent No. 1561395 treats a hemagglutinin-specific degrading enzyme to activate a virus and contacts the activated virus with amphiphilic particles under acidic conditions of pH 4-8.
  • a virus detection method for detecting the presence or absence of a virus by detecting a change in fluorescence intensity emitted by self-quenching dye present in sex particles by dequenching.
  • detection methods essentially include viral hemagglutinin degrading enzymes and require conditions for bringing the activated virus into contact with the amphiphilic particles under acidic conditions. Therefore, the additional use of a reagent such as hemagglutinin degrading enzyme is inexpensive in terms of cost, and the process of forming and maintaining conditions suitable for detection is complicated and inefficient.
  • the present inventors have made intensive efforts to develop a method for easily detecting a target virus in a short time in a field (onset area), and thus embedding a chromogenic substrate inside a liposome or a liposome-polymer hybrid, and then the liposome or When the virus binds to the liposome-polymer hybrid through the viral membrane protein, the chromosomal substrate contained in the liposome or liposome-polymer hybrid is released, and the color can be easily detected by reacting with the chromophore-inducing substance to visually detect the virus. It confirmed and completed this invention.
  • An object of the present invention is to provide a virus detection composition, virus detection kit, virus detection strip, and virus detection method using the same for easily detecting a virus.
  • the present invention comprises a liposome or liposome-polymer hybrid (chromosome) containing a chromogenic substrate, and a chromosome inducing substance, when the virus binds to the liposome or liposome-polymer hybrid, It provides a composition for detecting a virus, characterized in that the chromogenic substrate contained in the liposome or liposome-polymer hybrid is released while reacting with the chromophore-inducing substance.
  • chromosome a liposome or liposome-polymer hybrid
  • the present invention also provides a virus detection kit comprising the composition and a virus detection strip with a pad coated with the composition.
  • the present invention also provides a method for preparing a liposomal cell containing (a) contacting a liposome or a liposome-polymer hybrid containing a chromophore substrate with an estimated virus-containing sample; And (b) provides a method for detecting a virus comprising the step of adding a coloring inducing substance, to determine whether the color.
  • FIG. 1 shows a process of releasing TMB contained in a liposome or a liposome-polymer hybrid when the liposome or liposome-polymer hybrid containing TMB, which is a chromogenic substrate, is contacted (bound) with a virus.
  • HMB which is a coloring enzyme, oxidizing TMB.
  • Figure 3 is to determine whether the virus (influenza virus) detection using the liposomes containing the color substrate TMB.
  • Figure 4 shows a virus detection strip containing a reagent pad containing liposomes containing the color substrate TMB.
  • FIG. 5 confirms whether a virus (influenza virus) is detected using the virus detecting strip of FIG. 4.
  • FIG. 6 shows whether a virus (influenza virus) is detected by iodine starch reaction using liposomes containing iodine, which is a chromogenic substrate.
  • Figure 7 shows the absorbance measured at 405nm during the color reaction using liposomes containing the color substrate pNPP for virus detection (influenza virus).
  • FIG. 8 shows whether virus (influenza virus) is detected using liposomes containing pNPP.
  • Figure 9 shows the absorbance measured at 405nm during the color reaction using liposomes containing the chromogenic substrate pNPP for the detection of virus (influenza virus) after trypsin treatment.
  • FIG. 10 shows whether a virus (influenza virus) is detected according to the presence or absence of trypsin using liposomes containing pNPP as a chromogenic substrate.
  • the present invention relates to a virus detection method using a liposome or a liposome-polymer hybrid, and more particularly, a liposome or a liposome-polymer hybrid containing a chromogenic substrate is contacted with a sample presumed to contain a virus.
  • the present invention relates to a virus detection composition, and a virus detection method using the same, which detect the presence or absence of a virus by checking color development according to the release of a chromogenic substrate contained in a liposome or a liposome-polymer hybrid (see FIG. 1). .
  • a liposome containing a color substrate TMB
  • a liposome containing a chromophore substrate pNPP
  • the present invention includes liposomes or liposome-polymer hybrids containing a chromophore substrate, and a chromosome inducing substance, and when the virus binds to the liposomes or liposome-polymer hybrids, the liposomes Or it relates to a virus detection composition characterized in that the color is generated by reacting with the color-inducing substance while the color substrate contained in the liposome-polymer hybrid is released.
  • the virus may bind to liposomes or liposome-polymer hybrids through viral lipid membranes or membrane proteins.
  • the viral membrane protein may be characterized as being HA (hemagglutinin).
  • the lipid membrane may be characterized in that the PC (Phosphatidylcholine), PI (Phosphoinositides), PS (Phosphatidylserine), PE (Phosphatidylethanolamine), SM (Sphingomyelin).
  • the liposome-polymer hybrid is a biomembrane-like amphiphilic structure composed of a low molecular weight lipid (eg, phospholipid) and a high molecular weight polymer (eg, an amphiphilic block copolymer).
  • a low molecular weight lipid eg, phospholipid
  • a high molecular weight polymer eg, an amphiphilic block copolymer
  • the liposome-polymer hybrid may be made of a lipid component having a biological function (receptor, molecular recognition, etc.) and a polymer having a structural function (structural stability, etc.) and may be manufactured to bind to a target material (Olubummo A1 et al., Langmuir , 30 (1): 259-67, 2014; Schulz M et al., Angew Chem Int Ed Engl ., 52 (6): 1829-33, 2013; Miglena I et al., Faraday Discuss. Chem .
  • the liposome is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), egg phosphatidylcholine (EPC) , Egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG) Soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidylinositol (SPI), dipalmito
  • the liposome-polymer hybrid is, for example, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI) ), Egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC) Soy phosphatidylglycerol (SPG), soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidyl inos
  • PC
  • the color development may be carried out through an enzymatic reaction or a non-enzymatic reaction
  • the color development substrate used in the enzymatic reaction is DAB (diaminobenzidine), AEC (3-amino-9-ethylcarbasole), 5-bromo-4-chloro-3-indolyl-phosphate / nitroblue tetrazolium (BCIP / NBT), para-Nitrophenyl phosphate (pNPP), naphthol AS-TR phosphate, BCIP / INT (5-bromo 4-chloro-3-indolyl phosphate / iodonitrotetrazolium (NF), new fuchsin (NF), fast red TR salt (FRT), phenylenediamine, 3,3 ', 5,5'-tetramethylbenzidine (3 , 3 ', 5,5'-tetra methylbenzidine, dianisidine, amino-salicylic acid, 3,3'-diamin
  • the color substrate used in the non-enzymatic reaction is from the group consisting of iodine, calcium (calcium), copper (copper), iron (iron), amino acid (amino acid) and creatinine (creatinine)
  • the chromophore-inducing substance is selected from starch, o-cresolphthalein complexone, bathocuproin disulfonate, and bashophenanthroline disulfonate. It may be characterized in that it is selected from the group consisting of ninhydrin (ninhydrin), o-phthalaldehyde (o-phthalaldehyde: OPA) and picrate (picrate).
  • the color development may be characterized by visual or enzyme-linked immunosorbent assay (ELISA), characterized in that the virus may be characterized in that the influenza virus, the viral membrane protein is It may be characterized as being HA (hemagglutinin).
  • the lipid membrane may be characterized in that the PC (Phosphatidylcholine), PI (Phosphoinositides), PS (Phosphatidylserine), PE (Phosphatidylethanolamine), SM (Sphingomyelin).
  • the liposome is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), egg phosphatidylcholine (EPC) , Egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG) Soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidylinositol (SPI), dipalmito
  • the liposome-polymer hybrid is, for example, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI) ), Egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC) Soy phosphatidylglycerol (SPG), soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidyl inos
  • PC
  • the liposome or liposome-polymer hybrid may be characterized by having a negative charge.
  • Influenza viruses are inserted into host cells through receptor-mediated endocytosis, in which the virus converts HA 0 to HA 1 / HA 2 through trypsin-mediated proteolytic cleavage at low pH, resulting in a host cell membrane. Enable fusion (Skehel, JJ et al., Annu . Rev. Biochem ., 69: 531-569, 2000; White, J. et al., J. Cell. Biol ., 89: 674-679, 1981 ).
  • the influenza virus contacts and releases the chromophore substrate contained without contacting the enzymes that degrade HA of the influenza virus.
  • the negative charge of the liposome or liposome-polymer hybrid is sufficient to maintain the negative charge before detection by color development, for example, to maintain the negative charge in the sample state for detecting color development, or to carry out the negative charge under various experimental conditions possible before color detection. It is enough to maintain, but is not limited thereto.
  • a virus influenza virus
  • TMB chromosome substrate
  • the present invention relates to a virus detection kit comprising the composition and a virus detection strip with a pad coated with the composition.
  • the present invention provides a method for preparing a liposomal cell containing (a) contacting a liposome or a liposome-polymer hybrid containing a chromogenic substrate with a putative virus-containing sample; And (b) by adding a coloring inducing substance, relates to a virus detection method comprising the step of confirming the color development.
  • the color development of step (b) is when the virus binds to the liposomes or liposome-polymer hybrids through the viral membrane protein, while the chromophore-containing substances contained in the liposomes or liposome-polymer hybrids are released, It may be characterized in that the color is expressed by the reaction, it can be characterized in that the color development of step (b) is characterized in that it is carried out through an enzyme reaction or a non-enzymatic reaction.
  • the color development of step (b) is when the virus binds to the liposomes or liposome-polymer hybrids through the viral membrane protein, while the chromophore-containing substances contained in the liposomes or liposome-polymer hybrids are released, It may be characterized in that the color is expressed by the reaction, it can be characterized in that the color development of step (b) is characterized in that it is carried out through an enzyme reaction or a non-enzymatic reaction.
  • the chromogenic substrate used for the enzyme reaction is DAB (diaminobenzidine), AEC (3-amino-9-ethylcarbasole), BCIP / NBT (5-bromo-4-chloro-3-indolyl-phosphate / nitroblue tetrazolium ), para-Nitrophenyl phosphate (pNPP), naphthol AS-TR phosphate, BCIP / INT (5-bromo-4-chloro-3-indolyl phosphate / iodonitrotetrazolium), NF (new fuchsin), FRT (Fast Red TR Salt), phenylenediamine, 3,3 ', 5,5'-tetramethylbenzidine, 3,3', 5,5'-tetra methylbenzidine, dianisidine, Amino-salicylic acid, 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole, 4- 4-
  • the color substrate used in the non-enzymatic reaction is from the group consisting of iodine, calcium (calcium), copper (copper), iron (iron), amino acid (amino acid) and creatinine (creatinine)
  • the chromophore-inducing substance is selected from starch, o-cresolphthalein complexone, bathocuproin disulfonate, and bashophenanthroline disulfonate. It may be characterized in that it is selected from the group consisting of ninhydrin (ninhydrin), o-phthalaldehyde (o-phthalaldehyde: OPA) and picrate (picrate).
  • the color development of step (b) may be characterized by visual or enzyme-linked immunosorbent assay (ELISA), the virus may be characterized in that the influenza virus,
  • the viral membrane protein may be characterized as being HA (hemagglutinin).
  • the liposome is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), egg phosphatidylcholine (EPC) , Egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG) Soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidylinositol (SPI), dipalmito
  • the liposome-polymer hybrid is, for example, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI) ), Egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC) Soy phosphatidylglycerol (SPG), soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidyl inos
  • PC
  • the liposome or liposome-polymer hybrid may be characterized by having a negative charge.
  • Influenza viruses are inserted into host cells through receptor-mediated endocytosis, in which the virus converts HA 0 to HA 1 / HA 2 through trypsin-mediated proteolytic cleavage at low pH, resulting in a host cell membrane. Enable fusion (Skehel, JJ et al., Annu . Rev. Biochem . , 69: 531-569, 2000; White, J. et al., J. Cell. Biol ., 89: 674-679, 1981 ).
  • the influenza virus can be contacted to release the chromophore substrate contained therein without including an enzyme that degrades HA of the influenza virus.
  • the negative charge of the liposome or liposome-polymer hybrid is sufficient to maintain the negative charge before detection by color development, for example, to maintain the negative charge in the sample state for detecting color development, or to carry out the negative charge under various experimental conditions possible before color detection. It is enough to maintain, but is not limited thereto.
  • liposomes or liposome-polymer hybrids may contain (capture) various molecules such as absorbers, fluorescent materials, or chemiluminescent materials in the inner aqueous phase.
  • Example 1 Preparation of liposomes or liposome-polymer hybrids containing TMB
  • a liposome suspension was prepared by adding 0-50 ⁇ M TMB solution (Sigma-Aldrich, USA) to the lipid membrane and dispersing it for 10 minutes with an ultrasonicator (Jeiotech, Korea). Here, the process of freezing and thawing for the preparation of liposomes of a single lipid layer was repeated five times.
  • liposomes of the same size were prepared using a Mini-Extruder (Avanti Polar Lipids Inc., USA) to prepare liposomes of uniform size.
  • Liposomes containing the TMB prepared above were removed using a desalting column (GE Healthcare, UK) and stored at 4 ° C. in cold storage. The size of liposomes containing TMB was measured using DLS (dynamic light scattering, Otsuka, Japan).
  • liposomes containing TMB and having a diameter of about 50 to 200 nm were prepared.
  • a method for preparing a liposome-polymer hybrid is prepared by dissolving an appropriate amount of lipid components (PC, PE, PS, etc.) in chloroform-methanol, depositing it on an anode, then applying an electric field and adding distilled water.
  • liposome electroformation (Olubummo A1 et al., Langmuir , 30 (1): 259-67, 2014; Schulz M et al., Angew) Chem Int Ed Engl ., 52 (6): 1829-33, 2013; Miglena I et al., Faraday Discuss. Chem .
  • Liposomes and influenza viruses containing TMB were prepared by conventional methods in the art, and used at appropriate concentrations and exposure times.
  • the liposomes containing the TMB can be replaced with liposome-polymer hybrids containing the TMB.
  • Example 2 50 ⁇ l of the liposome containing TMB prepared by the method of Example 1, 10 ⁇ l of influenza virus (A / California / 04/2009 (H1N1)) (1 * 10 7 TCID / ml), 100 mM CP buffer (citrate-phosphate buffer, pH) 5.0) 10 ⁇ l was mixed and reacted at 37 ° C. for 30 minutes to allow virus and liposomes to contact (couple). Then, 25 ⁇ l of hydrogen peroxide (Hig, Horseradish peroxidase, Sigma-Aldrich, USA) and 1 U (unit) of hydrogen peroxide (hydrogen peroxide, Sigma-Aldrich, USA) were added to the reaction to perform a color reaction. After the reaction, the reaction stop solution (H 2 SO 4 , Sigma-Aldrich, USA) was added to stop the reaction and the absorbance at 450 nm was measured by ELISA Reader (SpectraMax, Molecular Devices).
  • the virus influenza virus
  • TMB chromophore substrate
  • As the material of the pad polyester, glass fiber, cellulose, or the like was used.
  • the reagent pads to which the coating solution was adsorbed were attached to the upper strips cut into widths of 1-4 mm ⁇ length of 10-40 mm to prepare detection (diagnosis) strips.
  • the liposomes containing the TMB can be replaced with liposome-polymer hybrids containing the TMB.
  • Influenza detection test was carried out using a strip coated with the TMB inclusion liposome and peroxidase mixture prepared by the method of Example 3.
  • the color of the pad changes when the strip is immersed in the virus solution and a reaction occurs between the virus and the mixed solution on the strip pad. That is, when the virus contacts (binds) the liposomes on the pads, the TMB released from the liposomes and the peroxidase react with the color to develop a color change of the pads on the strip to detect the presence of the virus.
  • the liposomes containing the TMB can be replaced with liposome-polymer hybrids containing the TMB.
  • the virus influenza virus
  • TMB chromosome substrate
  • Liposomes containing iodine include POPC (1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine, Avanti Polar Lipids Inc., USA) and POPG (1-palmitoyl-2-oleoyl- sn -glycero-3- It consists of phospho- (1'- rac- glycero), Avanti Polar Lipids Inc., USA, and used Mini-Extruder (Avanti Polar Lipids Inc., USA) to prepare liposomes of uniform size. Liposomes containing the prepared iodine were removed using a desalting column (GE Healthcare, UK) to remove residual iodine and stored at 4 ° C. The size of liposomes containing iodine was measured using DLS (dynamic light scattering, Otsuka, Japan).
  • liposomes containing iodine were prepared to have a diameter of about 50 to 200 nm.
  • a method for preparing a liposome-polymer hybrid is prepared by dissolving an appropriate amount of lipid components (PC, PE, PS, etc.) in chloroform-methanol, depositing it on an anode, then applying an electric field and adding distilled water.
  • liposome electroformation (Olubummo A1 et al., Langmuir , 30 (1): 259-67, 2014; Schulz M et al., Angew) Chem Int Ed Engl ., 52 (6): 1829-33, 2013; Miglena I et al., Faraday Discuss. Chem .
  • Liposomes and influenza viruses containing iodine were prepared by routine methods in the art and used at appropriate concentrations and exposure times. Non-enzymatic assays used conventional methods in the art (Sarkar BC et al., Anal Biochem . , 20 (1): 155-66, 1967; Zak B., Clin Chim Acta ., 3 (4): 328-34, 1958; Hawk, Philip B. et al., Practical Physiological Chemistry , Churchill, London, pp 839-844, 1947).
  • the liposomes containing iodine can be replaced with liposome-polymer hybrids containing iodine.
  • Example 5 50 ⁇ l of the liposome containing iodine prepared in Example 5, 10 ⁇ l of influenza virus (A / California / 04/2009 (H1N1)) (1 ⁇ 10 7 TCID / ml), 100 mM CP buffer (citrate-phosphate buffer, pH 5.0) ) 10 ⁇ l was mixed and reacted at 37 ° C. for 30 minutes to allow virus and liposomes to contact (couple). Then, starch solution (Starch, Acros organics, USA) was added to the reaction to iodine starch reaction to confirm the presence of virus.
  • H1N1 influenza virus
  • 100 CP buffer citrate-phosphate buffer, pH 5.0
  • the virus influenza virus
  • the chromogenic substrate iodine
  • the sample containing the virus was blue violet, and the sample containing no virus did not show color change, and the presence of the virus was visually confirmed.
  • Example 7 Preparation of liposomes or liposome-polymer hybrids containing pNPP
  • a liposome suspension was prepared by dispersing 270 mM pNPP solution (4-Nitrophenyl phosphate disodium salt hexahydrate, Sigma-Aldrich, USA) for 10 minutes with an ultrasonicator (Jeiotech, Korea). Here, the process of freezing and thawing for the preparation of liposomes of a single lipid layer was repeated five times.
  • liposomes of the same size were prepared using a Mini-Extruder (Avanti Polar Lipids Inc., USA) to prepare liposomes of uniform size.
  • the prepared liposomes containing pNPP were removed using residual Sephacryl S-1000 column (sephacryl S-1000 column, 1.5 x 12 cm, GE Healthcare, UK) and refrigerated at 4 °C.
  • the size of liposomes containing pNPP was measured using DLS (dynamic light scattering, Otsuka, Japan).
  • liposomes containing pNPP and having a diameter of about 50 to 200 nm were prepared.
  • a method for preparing a liposome-polymer hybrid is prepared by dissolving an appropriate amount of lipid components (PC, PE, PS, etc.) in chloroform-methanol, depositing it on an anode, then applying an electric field and adding distilled water.
  • liposome electroformation (Olubummo A1 et al., Langmuir , 30 (1): 259-67, 2014; Schulz M et al., Angew) Chem Int Ed Engl ., 52 (6): 1829-33, 2013; Miglena I et al., Faraday Discuss. Chem .
  • Influenza viruses were prepared by routine methods in the art and used at appropriate concentrations and exposure times.
  • liposomes containing pNPP prepared by the method of Example 7, 10 ⁇ l of influenza virus (A / chicken / korea / S1 / 2003 (H9N2)) (1 * 10 7 TCID 50 / ml), 100 mM CP buffer (citrate-phosphate) buffer, pH 4.0) 10 ⁇ l was mixed and reacted for 30 minutes at 37 °C to contact (bind) virus and liposomes.
  • chromatase phosphatase (phosphatase, Sigma-Aldrich, USA) was added to the reaction, followed by coloring by adjusting the appropriate pH with 0.1 M glycine buffer containing 1 mM ZnCl 2 and 1 mM MgCl 2.
  • the reaction proceeded.
  • Absorbance at 405 nm during color development was measured by ELISA Reader (SpectraMax, Molecular Devices) (FIG. 7).
  • the liposomes containing pNPP are replaceable with liposome-polymer hybrids containing pNPP.
  • the virus influenza virus
  • pNPP chromophore substrate
  • Influenza viruses were prepared by routine methods in the art and used at appropriate concentrations and exposure times.
  • chromatase phosphatase (phosphatase, Sigma-Aldrich, USA) was added to the reaction, and then titrated with 0.1M Glycine buffer containing 1 mM ZnCl 2 and 1 mM MgCl 2 .
  • the color reaction was carried out. Absorbance at 405 nm during the color reaction was measured by ELISA Reader (SpectraMax, Molecular Devices) (Fig. 9).
  • the liposomes containing pNPP are replaceable with liposome-polymer hybrids containing pNPP.
  • the virus influenza virus
  • pNPP chromogenic substrate
  • the virus detection method using liposomes or liposome-polymer hybrids according to the present invention is easily detectable by the naked eye, and in particular, hydrophobic TMB (3,3 ', 5,5'-tetramethyl), which is a chromogenic substrate.
  • TMB hydrophobic TMB
  • iodine When using liposomes or liposome-polymer hybrids containing bezidine) or iodine, TMB or iodine, does not leak out of the liposomes or liposome-polymer hybrids, and thus have high stability against natural coloration by oxygen or chemical reaction.
  • the TMB or iodine contained in the liposomes or liposome-polymer hybrids are released and reacted with HRP or starch, a color-inducing substance.
  • the virus detection signal appearing was significantly higher.

Abstract

La présente invention concerne un procédé de détection d'un virus en utilisant un liposome ou un hybride liposome-polymère et, plus spécifiquement, un procédé de détection d'un virus par fabrication d'un liposome ou d'un hybride liposome-polymère, les deux contenant un substrat chromogène, entrant en contact avec un échantillon supposé contenir un virus, puis détecter la présence ou l'absence d'un virus, à travers le développement de couleurs selon qu'il existe ou non une émission du substrat chromogène, qui est contenu dans le liposome ou l'hybride liposome-polymère, par le contact réalisé entre les deux. Selon la présente invention, le procédé de détection d'un virus en utilisant un liposome ou un hybride liposome-polymère permet la détection facile d'un virus à l'œil nu. En particulier, lors de l'utilisation d'un liposome ou d'un hybride liposome-polymère, les deux contenant de la 3,3',5,5'-tétraméthyl bézidine (TMB) ou de l'iode hydrophobe, qui sont des substrats chromogènes, la stabilité de développement de couleur naturelle par l'oxygène ou une réaction chimique est élevée étant donné que la TMB ou l'iode ne fuit pas à l'extérieur du liposome ou de l'hybride liposome-polymère. De plus, on a démontré que la TMB ou l'iode contenu dans le liposome ou l'hybride liposome-polymère est déchargé et que le signal de détection de virus devant être produit par une réaction avec HRP ou de l'amidon, qui sont tous deux des matériaux inducteurs de développement de couleur, est remarquablement élevé lorsqu'un virus se lie au liposome ou à l'hybride liposome-polymère par le biais d'une protéine de membrane virale.
PCT/KR2016/001829 2015-02-25 2016-02-25 Procédé de détection de virus utilisant un liposome ou un hybride liposome-polymère WO2016137236A1 (fr)

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KR19980702179A (ko) * 1995-02-13 1998-07-15 포올 알. 마르틴 3차원 비색 검정 어셈블리
JP2002517728A (ja) * 1998-05-29 2002-06-18 デイド・ベーリング・インコーポレイテッド 分析対象物質を検出する方法
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KR19980702179A (ko) * 1995-02-13 1998-07-15 포올 알. 마르틴 3차원 비색 검정 어셈블리
JP2002517728A (ja) * 1998-05-29 2002-06-18 デイド・ベーリング・インコーポレイテッド 分析対象物質を検出する方法
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