WO2009104890A2 - Polymicrobial detection device using quantum dot nanocrystal complexes and a detection method therefor - Google Patents

Polymicrobial detection device using quantum dot nanocrystal complexes and a detection method therefor Download PDF

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WO2009104890A2
WO2009104890A2 PCT/KR2009/000755 KR2009000755W WO2009104890A2 WO 2009104890 A2 WO2009104890 A2 WO 2009104890A2 KR 2009000755 W KR2009000755 W KR 2009000755W WO 2009104890 A2 WO2009104890 A2 WO 2009104890A2
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attractant
nanocrystals
microorganism
microbe
complex
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PCT/KR2009/000755
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French (fr)
Korean (ko)
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WO2009104890A3 (en
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서경식
김정환
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Seo Kyung-Sik
Kim Jeong-Hwan
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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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • 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
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria

Definitions

  • the present invention relates to a quantum dot nano crystal-microbe attractant complex, a microorganism detection kit using the same, and a detection method.
  • pathogenic microorganisms such as Staphylococcus aureus, Salmonella, Listeria and Escherichia coli O157: H7 are widely spread, and Escherichia coli is a representative species among them, and is mainly used as an indicator of the overall health risk of microorganisms.
  • the degree of contamination by microorganisms can be regarded as starting from the accurate measurement of the population.
  • the methods for measuring the number of microorganisms can be divided into the direct counting method and the indirect method. However, most of the microorganisms have a size of about 1 ⁇ m (micrometer, 1 millionth of a meter) in and out, so there is a limit to the amount of information obtained only through visual observation.
  • SPR sensor is a sensor that uses the surface plasma wave resonance phenomenon that occurs when light energy is absorbed on the surface of the metal thin film.
  • the SPR sensor can be combined with biomolecules or microorganisms to be detected on the metal thin film. Principles of coating materials and biosensors and calculating the number of microorganisms, but the SPR device itself is expensive and the photonic calibration operation is complicated, so it is an early stage for civilian use.
  • the SAW sensor changes the frequency of surface acoustic waves in response to changes in mass when microorganisms adhere to the piezoelectric plate through which surface acoustic waves progress, and by measuring the frequency change, it is possible to secure confidence in the measured values. For this reason, the sensor must be calibrated periodically using calibration equipment related to frequency standards, which is inconvenient.
  • Aquatic microorganisms have a wet, biological chemo-taxis that swims with flagella toward certain chemo-attractants in water (Takafumi M et al. 1986. J. Bacteriology 165 (3) 890-895, Greg MB, James GM 2003. FEMS Microbiology Ecology 43 99-109). Therefore, using the habit of attracting attractants such as amino acids, organic molecules, glucose, etc., it is easy to automatically capture and quickly detect the microorganisms without the treatment of antibodies or other molecular recognition receptors widely used in microorganism recognition.
  • the quantum dot ( QD ) -nanobio -copolymer becomes a bacteria attractant itself, and because the nanoscale is similar in size to nutrients, It is easily mistaken as a nutrient source and easily enters the membrane pore during predation. Nanoparticles invaded into the bacterial cytoplasm are easily detected and diagnosed by electrical or optical methods.
  • E. coli O-157 a representative harmful bacterium, swims with 5 to 10 multiple flagella, so it rushes toward QD treated with attractant serine more selectively than various non-uniform flagella bacteria in water. Due to the chemical stickiness of the flagella surface of the isolated bacteria, the selectivity and sensitivity due to non-specific adhesion with attracted QDs can be greatly increased.
  • Korean Patent Application No. 10-2005-0087315 (Application Date: 2005.09.20) is provided with nanohybrid particles including core particles reacting to magnetic force and nanoparticles coated or adsorbed around the core particles, the nanohybrid particles Reacts with biomolecules to form biomolecules in which biomolecules are immobilized or hybridized, and the biocomposite particles have a characteristic of reacting with a magnetic body, a method of manufacturing nanohybrid particles for detecting biomolecules, and biomolecules using nanohybrid particles A detector, a biomolecule detection method, and an analysis device for biomolecule detection are disclosed. However, there is no mention of a microbial detection method using a microbial attractant.
  • an object of the present invention is to provide a diagnostic kit using a novel nanocrystal complex which is excellent in binding with a microorganism attractant and physicochemically stable.
  • Another object of the present invention is to provide a method for detecting microorganisms using a novel nanocrystal complex having excellent binding properties with microbial attractants and physicochemically stable.
  • the present invention is a nanocrystal selected from the group consisting of metals consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth; Microbial attractants; And a polymer chain having a substituent having a charge property that can be bonded to the nanocrystal on one side, and having a polymer chain having a plurality of water-soluble substituents on the opposite side, which is bonded to the nanocrystal through a substituent on one side, and through the plurality of water-soluble substituents on the opposite side.
  • a nano crystal-microbial attractant complex comprising a stabilization material that stabilizes nanocrystals and forms a bond with the microbial attractant through the plurality of water-soluble substituents.
  • the present invention is a method for producing nanocrystals for labeling microorganism attractant, by reacting hexadecanol, potassium hydroxide and carbon disulfide, hexadecyl xanthate (hereinafter referred to as 'HDX') Preparing a potassium salt; Reacting nanocrystals selected from at least one metal group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth with the obtained HDX potassium salt to produce HDX metal sulfide nanocrystals; Preparing a metal sulfide nanocrystal by reacting an alkyl amine dopant with the HDX metal sulfide nanocrystal; Coupling a bond stabilizing material to the metal sulfide nanocrystals by reacting a bond stabilizing material to the metal sulfide nanocrystals; Activating a metal sulf
  • the present invention is a detection kit for detecting a microorganism using a nano-crystal label, nanocrystals selected from at least one metal group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth, Nano crystal-microorganism comprising a microorganism attracting material for attracting the microorganism and a binding stabilizer that binds to the nanocrystals with substituents of charge characteristics on one side and the microbial attracting materials with a polymer chain having a plurality of water-soluble substituents on the other side Attractant complexes; A collecting electrode configured to capture charge characteristics opposed to nanoparticles of the nanocrystal-microbe attractant material complex by capturing microorganisms; It provides a microbial detection kit comprising a current peak measuring unit for measuring analog current peak corresponding to the collected nano-crystal.
  • the present invention provides a method for detecting a microorganism using a nanocrystalline label, comprising: determining at least one microorganism to be detected and at least one microorganism attracting material capable of attracting microorganisms;
  • One or more nanocrystals are selected from the group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese, and bismuth and combined with each of the one or more microbial attractants to form one or more nanocrystal-microbe attractant complexes.
  • Forming Incorporating the at least one nanocrystal-microbe attractant complex into a sample to be diagnosed and mixing to induce contact of the at least one microcrystal to be detected with the at least one nanocrystal-microbe attractant; Capturing microorganisms in contact with the nanocrystal-microbe attractant material; And measuring an inherent current peak corresponding to the nanocrystals collected from the nanocrystal-microbe attractant material complex in contact with the captured microorganisms.
  • 1 shows a method and flow chart of quantum dot nanocrystal-chemotactic attractant complexes.
  • FIG. 2 shows a quantitative flow chart of microbial capture and anode stripping voltammetric ( ASV ) by quantum dot nanocrystal-chemotactic attractant complexes.
  • 3 shows a microchip type quantitative cartridge sensor module.
  • FIG. 5 shows a microsensor type cartridge sensor module for simple attribute separation.
  • FIG. 6 shows a micro syringe type quantitative cartridge sensor integration module for attribute separation and quantitation.
  • a first aspect of the invention provides a nanocrystal selected from at least one metal group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth; Microbial attractants; And a polymer chain having a substituent having a charge property that can be bonded to the nanocrystal on one side, and having a polymer chain having a plurality of water-soluble substituents on the opposite side, which is bonded to the nanocrystal through a substituent on one side, and through the plurality of water-soluble substituents on the opposite side.
  • a nano crystal-microbial attractant complex comprising a stabilization material that stabilizes nanocrystals and forms a bond with the microbial attractant through the plurality of water-soluble substituents.
  • the nanocrystals used in the present invention are metal-based nanocrystals, which are nanocrystals of metals having excellent resolution and signal selectivity.
  • the nanocrystal used in the present invention is not particularly limited as long as it is a metal having excellent resolution and signal selectivity.
  • Resolution means that the peak width of a signal generated from a metal is narrow so that it is distinguished from the peak of other signals without overlapping, and signal selectivity makes it easy to distinguish a signal peak generated from the metal from signal peaks generated from other metals. Means the degree indicated. In other words, if the resolution is high, the signal selectivity is increased.
  • the nanocrystals obtained by the nanocrystal synthesis method according to the present invention are metal sulfides (MS).
  • Metals used in metal sulfide include zinc (Zn), cadmium (Cd), lead (Pb), copper (Cu), gallium (Ga), arsenic (As), thallium (Tl), nickel (Ni), and manganese ( Mn) or bismuth Bi is preferably used.
  • zinc, cadmium, lead, or copper are preferably used because they produce selective signals with better resolution.
  • microbial attractant refers to an inorganic or organic substance having a chemo-taxis, and thus having an inducible effect on a specific nutrient.
  • specific examples include maltose, aspartate, casamino acid, cysteine, leucine or serine.
  • a nanocrystal-microbe attractant material complex that labels a microbe attractant, hexadecanol, potassium hydroxide, and carbon disulfide are reacted to form a hexadecyl xanthate (hereinafter referred to as 'HDX').
  • a potassium salt Preparing a potassium salt; Reacting nanocrystals selected from at least one metal group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth with the obtained HDX potassium salt to produce HDX metal sulfide nanocrystals; Reacting the HDX metal sulfide nanocrystals with an alkyl amine dopant to prepare metal sulfide nanocrystals; Coupling a bond stabilizing material to the metal sulfide nanocrystals by reacting a bond stabilizing material to the metal sulfide nanocrystals; Activating a metal sulfide nano crystal-bond stabilizer material complex by reacting an activated material with the bonded metal sulfide nano crystal-bond stabilizer material complex; And it provides a method for producing a nano-crystal microbe attractant complex comprising the step of binding a microorganism attractant to the activate
  • the HDX potassium salt preparing step comprises the steps of mixing the hexadecanol and the potassium hydroxide, heating until all the mixed solution is dissolved; Putting the mixed solution into toluene, stirring uniformly, and adding the carbon disulfide; Placing the mixed solution in petroleum ether and further stirring; And filtering the mixed solution with a glass filter and repeating a process of washing with ether.
  • the alkyl amine dopant is a method for producing nanocrystals selected from the group consisting of hexadecyl amine, decyl amine and trioctyl amine.
  • Hexadecyl amine is used as the alkyl amine dopant for HDX zinc sulfide nanocrystals or HDX cadmium sulfide nanocrystals
  • the alkyl amine dopantrodecyl amine or trioctyl amine is used for HDX lead sulfide nanocrystals, and HDX copper sulfide
  • a method for preparing nanocrystal-microbe attractant complexes using hexadecyl amine or trioctyl amine as the alkyl amine dopant a method for preparing nanocrystal-microbe attractant complexes using hexadecyl amine or trioctyl amine as the alkyl amine dop
  • the metal nanoparticles (MS) obtained above are stabilized by reacting with the stabilizing material, activating as an activating material, and then reacting the microbe attractant material to react the nanoparticle-microbe attractant material complex. To obtain.
  • stabilizers chemically and physically stabilize nanoparticles, enhance solubility of nanocrystals to increase compatibility with microbial attractants, contributing to stabilizing nanocrystal-microbe attractant complexes.
  • the stabilizing material has a chemical substituent capable of binding to the nanocrystals on one side, and is composed of a polymer material having a plurality of water-soluble substituents on the opposite side.
  • the nanocrystals are protected from the medium to ensure the stability of the nanocrystals.
  • some of the water-soluble substituents on the opposite side forms a covalent bond with the microorganism attractant to promote the bond between the nanocrystal and the microorganism attractant.
  • the stabilizing material of the present invention preferably uses dithiolthreitol (hereinafter referred to as 'DTT') or dihydrolipoic acid (hereinafter referred to as 'DHLA').
  • DTT or DHLA significantly stabilizes the nanocrystals due to their surface chemistry and solubility in high aqueous solutions.
  • DTT surrounds the nanocrystal surface strongly and uniformly at the nano level by a thiol (-SH) chemical group on its molecular structure, maintaining a stable structure of the nanocrystals, and simultaneously forming a hydroxyl group on the nanocrystal surface. Generated to enhance the water solubility of the nanocrystals.
  • -SH thiol
  • the stabilizing material uniformly surrounds and binds the nanocrystals having positive charge characteristics by using the negative charge characteristics of the thiol (-SH) group. Therefore, as a stabilizing material, a large number of substituents having negative charge characteristics exist on one side to uniformly surround the nanocrystals, thereby stabilizing the nanocrystals, and a plurality of water-soluble substituents exist on the other side to enhance the water solubility of the nanocrystals. do.
  • a thiol (-SH) group is exemplified as a substituent having negative charge characteristics, but a substituent having other negative charge characteristics may be used, and a hydroxyl group (-OH) may also be used as a specific example.
  • a stabilizing material having negative charge characteristics is preferable, but in the case of using nanocrystals having negative charge characteristics, a stabilizing substance having substituents having positive charge characteristics is used. This can be preferably used.
  • an “activating material” allows the activation of a stabilizing material to form carbamate bonds with amino groups of the microorganism attracting material.
  • an "activating material” allows the activation of a stabilizing material to form carbamate bonds with amino groups of the microorganism attracting material.
  • carbonyl diimidazole (1,1-carbonyl diimidazole, hereinafter referred to as 'CDI') is preferably used.
  • a third aspect of the invention is a detection kit for detecting microorganisms using nanocrystalline labels, wherein at least one selected from the group of metals consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth Comprising a nano crystal, a microorganism attracting material for attracting the microorganism and a binding stabilizer for binding to the nanocrystals with a polymer chain having a plurality of water-soluble substituents on the opposite side and the microcrystalline attractant for attracting the microorganism Nano crystal-microbe attractant complexes; A collecting electrode configured to capture charge characteristics opposed to nanoparticles of the nanocrystal-microbe attractant material complex by capturing microorganisms; It provides a microbial detection kit comprising a current peak measuring unit for measuring analog current peak corresponding to the collected nano-crystal.
  • the nanocrystal is a metal sulfide type, at least one selected from the group consisting of zinc, cadmium, lead, and copper, and the microorganism attractant material is maltose, aspartate, casamino acid, At least one selected from the group consisting of cysteine, leucine or serine, wherein the binding stabilizing agent is a microorganism detection kit which is dithiolthitol or dihydro lipoic acid.
  • the detection kit further includes an analog-to-digital converter for converting the current peak measured by the current peak measurement unit into a digital signal, and includes zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth.
  • a microbial detection kit for simultaneously detecting one or more microorganisms using different nanocrystal-microbe attractant complexes prepared using one or more different nanocrystals selected from the metal group.
  • the attractant microorganism capture is a microbial detection kit, characterized in that the separation through the selective semipermeable membrane.
  • the microorganism detection kit applies a predetermined positive potential to the collection electrode.
  • the current peak measurement unit applies a predetermined potential to the nanocrystals collected on the collection electrode to oxidize / reduce the nanoparticles and induces a current unique to each nanocrystal resulting from the oxidation / reduction reaction of the nanocrystals.
  • a detection kit for measuring peaks, and applying a predetermined negative potential or a positive potential to the collection electrode to collect nanocrystals of cationic properties or nanocrystals of anionic properties, respectively, and the current peak measuring unit is provided from the collected nanocrystals. It is a microbial detection kit for measuring the current peaks unique to each of the nanocrystals that occur.
  • the apparatus may further include a digital signal reader unit for interpreting the digital signal and estimating the identity of the microorganisms corresponding to the digital signal or the content of the detected microorganisms, and further including a barcode converter for converting the digital signal into a predetermined barcode.
  • the microbial detection kit further includes a communication unit for transmitting a signal to a predetermined remote diagnosis unit through wired or wireless communication and receiving a result of analyzing the digital signal from the remote diagnosis unit.
  • Nanocrystalline label of the microorganism detection kit of the present invention means nanocrystals for detecting a specific microorganism. Specifically, in order to detect a specific microorganism, it means a nanocrystal combined with an attractant capable of attracting the microorganism.
  • the nano-crystal label of the detection kit of the present invention can be replaced and used depending on the type of microorganism to be measured.
  • the digital conversion means of the measurement result of the microbial detection kit of the present invention may be a means for outputting as a bar code, but is not limited thereto, Global System for Mobile (GSM), Bluetooth, Ubiquitous, Code Division Multiple Access (CDMA), etc. Can be integrated into the company's advanced wireless mobile IT technology.
  • GSM Global System for Mobile
  • CDMA Code Division Multiple Access
  • a user of the detection kit can quickly read the output result by reading the output bar code with a bar code reader.
  • the detection result is digitally output in this way, the detection result can be transmitted to the remote place via wired communication or wireless communication.
  • a remote site having a system capable of evaluating detection results can be implemented to receive detection results output from a detection kit to evaluate detection results for a particular sample and send the evaluation results back to the detection kit user.
  • the detection kit of the present invention can be applied to surveillance systems in the fields of environmental monitoring and biological warfare, terrorism (TNT) and crime (drugs), such as water quality and food, as well as medical and clinical applications, as well as replacement of various probes. Can be applied.
  • the nanocrystalline label according to the present invention is easy to synthesize and has a unique redox potential for each metal nanocrystal, and thus it is possible to simultaneously detect a plurality of nanocrystalline labels. Therefore, when using the nanoparticle label according to the invention it is possible to miniaturize a number of microbial detection kit.
  • a fourth aspect of the present invention provides a method for detecting a microorganism using a nanocrystalline label, comprising: determining at least one microorganism to be detected and at least one microorganism attractant capable of attracting microorganisms;
  • One or more nanocrystals are selected from the group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese, and bismuth and combined with each of the one or more microbial attractants to form one or more nanocrystal-microbe attractant complexes.
  • Forming Incorporating the at least one nanocrystal-microbe attractant complex into a sample to be diagnosed and mixing to induce contact of the at least one microcrystal to be detected with the at least one nanocrystal-microbe attractant; Capturing microorganisms in contact with the nanocrystal-microbe attractant material; And measuring an inherent current peak corresponding to the nanocrystals collected from the nanocrystal-microbe attractant material complex in contact with the captured microorganisms.
  • the nanocrystal is a metal sulfide type, at least one selected from the group consisting of zinc, cadmium, lead, and copper, and the microorganism attractant material is maltose, aspartate, casamino acid, It is a microorganism detection method selected from the group consisting of cysteine, leucine or serine.
  • the attracting microorganism capture is a microbial detection method characterized in that the separation through the selective semi-permeable membrane.
  • an electrochemical analysis method is used as a method for detecting a nanocrystalline label.
  • the electrochemical analysis method used in the present invention is carried out in an aqueous solution to measure the potential, current, electrical conductivity, impedance, capacitance or resistance, etc., which enables miniaturization and rapid signal processing and is useful for small array analysis.
  • the present invention particularly employs the square wave negative electrode stripping voltage method among electrochemical analysis methods.
  • the stripping voltage method used in the present invention is largely composed of two steps. First, the microorganism attracting materials labeled with nanocrystals are placed in a predetermined aqueous solution, the electrode is placed, and a specific potential is applied through the electrode. Depending on the applied potential, the nanocrystalline metals move in the direction of the electrode and are collected on the surface of the electrode. Next, a predetermined electric potential is applied to the nanocrystalline metal collected on the electrode to conduct a specific current. At this time, each of the nanocrystalline metals generate a current of a specific peak according to the type of metal of each nanocrystal by the oxidation and reduction reaction, by measuring the current of this particular peak, the presence and concentration of the nanocrystalline label is measured do.
  • the nanocrystal label detection method by stripping voltage method according to the present invention provides a high measurement limit of picomolar levels. These measurement limits are achieved by using high purity nanocrystals according to the present invention. In addition, it is possible to significantly improve the sensitivity of the sensor signal by catalytically adjusting the size of the nanocrystalline particles according to the present invention.
  • each nanocrystal When a plurality of microorganisms are to be detected simultaneously using the nanocrystals according to the present invention, different metal nanoparticles are used for each microorganism. In this case, each nanocrystal exhibits a specific current peak depending on the type of metal, allowing the detection of multiple microorganisms simultaneously.
  • the nanoparticles using the dislocation metal as the nanocrystals derived a current peak signal for easy selective analysis.
  • Selection of a potential metal suitable as a nanoparticle suitable for the present invention selected a metal having a significant current peak without overlapping each other in the cathode voltage section.
  • a bipolar potential metal is preferable.
  • the negative electrode potential metal can be preferably used.
  • the current peak thus obtained can be digitized in accordance with a predetermined digital conversion method in the analog-digital converter. Specifically, the obtained analog type current peak is sampled and output as a corresponding digital signal.
  • the digital conversion method includes a digitization process and a normalization process by substituting signal values of current peak signals. Digitization is performed by statistical optimal threshold and piecewise linear interpolation.
  • the resulting current peak signals can be converted into digital signals according to their magnitude and trend, and transmitted to a remote location via wired or wireless communication means, or stored as predetermined digital characters. Specific examples of such digital characters include bar codes.
  • Hexadecyl xanthate (HDX) a stable capping material of nanocrystalline particles, was synthesized by the following procedure.
  • the mixed solution of 9.69 g of hexadecanol and 2.24 g of potassium hydroxide (KOH) at the same concentration (0.04 mol) mixed was heated at a temperature of 150 ° C until all of the solution was dissolved.
  • the obtained aqueous solution was put into 25 ml of toluene at the temperature of 100 degreeC, and it stirred uniformly.
  • Binary inorganic nanoparticles such as ZnS, PbS, CdS, InAs, GaAs or CuS
  • ZnS, PbS, CdS, InAs, GaAs or CuS can be used to obtain a non-overlapping pool of semiconductor nanoparticle tracers that account for the high selectivity of bioassays. Screened.
  • the electrical codes can be clearly distinguished except for overlapping signals of Cd-In and In-Pb peak pairs. Accordingly, the present invention introduces ZnS and CuS nanoparticles.
  • Synthesis and surface stabilization of high purity metal sulfide nanocrystal grains were carried out as follows. In summary, the synthesis was based on the thermal deposition of metal alkyl xanthate sulfide precursors. 1 ). 3.56 g of HDX, 5 ml of methanol equally equimolar in CdCl 2 , PbCl 2 , ZnCl 2 React with each aqueous solution for 2 minutes. The solution is centrifuged to remove the supernatant, the final M-HDX is washed three times, washed with methanol and dried in a vacuum oven.
  • All nanopotential metal particles are strong electron contributing monosurfactants, which are alkyl amine dopants, decylamine (HDA), decylamine (DA) and trioctylamine (TOA).
  • alkyl amine dopants decylamine (HDA), decylamine (DA) and trioctylamine (TOA).
  • 0.5 g each were mixed with Zn-HDX, Cd-HDX, Pb-HDX, respectively, for capping stabilization and organic solvents of the nanocrystals.
  • the alkyne amine dopants are first heated to 120 ° C. and cooled to 50 ° C., respectively, and 0.05 g of each metal-HDX powder is added during uniform stirring. Then, the heating temperature was stirred for 30 minutes at 100 ° C, and then the temperature was gradually raised to 120 ° C and the reaction was continued for 1.5 hours.
  • the final metal crystal grains obtained were white ZnS, yellow CdS, and black PbS, which were flocculated with methanol and precipitated at the bottom of the tube for easy extraction.
  • the supernatant removal process is carried out several times and the final nanocrystal particles in the form of fine powder are obtained until the final particles are purified.
  • Nanocrystal-microbe attractant complexes were performed according to the following method ( FIG. 1 ). Highly uniform nanocrystalline particles were hydroxylated by dithiolthreitol (DTT) and activated with carbonyl diimidazole (CDI). 100 ⁇ l of CdS, PbS, and ZnS nanocrystal particles are reacted with microorganism attractant. The final aqueous solution pH was adjusted to 1M NaOH to 8.5, and stirred at room temperature for 24 hours. The final water soluble polymers were dispersed in 0.1 M PBS (pH 7.4, 0.05% Tween 20).
  • DTT dithiolthreitol
  • CDI carbonyl diimidazole
  • 3 to 6 specifically illustrate the microbial capture and detection method by the nanocrystal-microbe attractant complex.
  • 3 is a microchip type quantitative cartridge sensor module, in which a sample solution to detect microorganisms is dropped to reach a selective semi-permeable membrane along the buffer solution layer.
  • the selective semipermeable membrane selectively transmits only the microorganisms, and the microorganisms are brought into contact with the nanocrystal-microbe attractant complex.
  • nanocrystal-microbe attractant complexes which are not attracted to the microorganism body are discharged through a drain controlled by a microvalve.
  • the unique current peaks corresponding to the nanocrystals collected from the nanocrystal-microbe attractant complexes in contact with the captured microorganisms were measured and quantitatively analyzed.
  • Figure 4 is a micro-cylinder type quantitative cartridge sensor module, when the sample solution to detect the microorganism passes through a thin pipette tip to reach a selective semi-permeable membrane (Semi-permeable membrane).
  • the selective semipermeable membrane selectively transmits only the microorganisms, and the microorganisms are brought into contact with the nanocrystal-microbe attractant complex.
  • nanocrystal-microbe attractant complexes which are not attracted to the microorganism body are separated through the capillary separation channel.
  • the unique current peaks corresponding to the nanocrystals collected from the nanocrystal-microbe attractant complexes in contact with the captured microorganisms were measured and quantitatively analyzed.
  • micro-syringe type cartridge sensor module for simple attribute separation, wherein the microcrystal-microbe attractant complex is added to a sample solution to detect microorganisms, and the microorganism and nanocrystal-microbe attractant complex for 30 minutes to 1 hour. To make contact. It is then aspirated by a piston in the form of a syringe.
  • the sample solution containing the inhaled nanocrystal-microbe attractant complex passes through a general-purpose filter of 100 nm or less, nanocrystal-microbe attractant complexes that are not attracted to the microorganism body are discharged. Intrinsic current peaks corresponding to the nanocrystals collected from the nanocrystal-microbe attractant complexes in the microorganism trapped inside the filter were measured and quantitatively analyzed.
  • FIG. 6 is a micro syringe type quantitative cartridge sensor integrated module for attribute separation and quantitative analysis, in which a microcrystal-microbe attractant complex is added to a sample solution to detect microorganisms and microorganisms and nanocrystals for 30 minutes to 1 hour. -Contact the microbe attractant complex. It is then aspirated by a piston in the form of a syringe. When the sample solution containing the inhaled nanocrystal-microbe attractant complex passes through a general-purpose filter of 100 nm or less, nanocrystal-microbe attractant complexes that are not attracted to the microorganism body are discharged.
  • the nanoparticles in the microorganism body are provided through an anode stripping voltammetric inserted into the piston. Inherent current peaks corresponding to nanocrystals collected from the crystal-microbe attractant complexes were measured and quantified.
  • Square wave negative electrode stripping voltammetry was performed using a screen printing carbon paste electrode coated with Mercury II ions or Bismuth ions. After QD nanoparticles are dissolved in an aqueous solution of nitric acid, pretreatment (0.6 V) is performed for 1 minute, and a 2 minute accumulation process is performed at -1,4V.
  • the buffer used is 1 ml 0.1 M acetate buffer (pH 4.5). Stripping was performed after a 5 second rest period (without stirring).
  • Specific operating parameters are step potential 50 mV, amplitude 20 mV and frequency 25 Hz in the potential range of -1.2 V to 0.12 V.
  • the microorganism detection method and detection kit using the nanocrystal-microbe attractant complex of the present invention can easily and quantitatively accurately measure the microorganism to be detected by analyzing the current peak inherent in the metal of each nanocrystal.
  • conventional microbial biosensors have very limited selectivity for target microorganisms and are difficult to escape from protein-immunochemistry that recognizes antigens on the surface of microorganisms as specific antibodies or receptors.
  • Various types of bacteria can be easily detected and quantified.
  • IT technologies such as Zigbee and Radio Frequency Identification (RFID), early analysis of water pollution sources, realization of standard alarm system and the cornerstone of advanced national health and safety Can provide.
  • RFID Radio Frequency Identification

Abstract

The invention relates to a polymicrobial detection device using quantum dot nanocrystal complexes and a detection method therefor.  One aspect of the invention provides a nanocrystal-microbial attractant complex, for which a microbial attractant for attracting microbes is coupled to a nanocrystal containing one or more element selected from a metal group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth, and a preparation method therefor.  Further, the invention provides a polymicrobial detection method based on the electrochemical analysis that involves the use of a detection kit for detecting microbes using said nanocrystal-microbial attractant complex, and the SWASV (Square wave anode stripping voltammetric) method using said detection kit.

Description

퀀텀닷 나노결정 복합체를 이용한 다중세균 검출장치 및 이의 검출방법Multi-Bacterial Detection Device Using Quantum Dot Nanocrystal Complex and Its Detection Method
본 발명은 퀀텀닷 나노 결정-미생물 유인 물질 복합체, 이를 이용하는 미생물 검출 키트 및 검출 방법에 관한 것이다. The present invention relates to a quantum dot nano crystal-microbe attractant complex, a microorganism detection kit using the same, and a detection method.
최근 잦은 주요 사회문제로 야기되는 학교 급식 식중독균 사태 및 오염된 식용 어패류에서 세균오염원의 진원루트를 직접 감지하고 근절하기란 현실적으로 매우 어려운 실정이다. 미국의 경우 매년 7천5백만명이 식품 병원균으로부터 고통을 겪고 있으며, 이 중 3십 3만여명이 병원 치료를 받고, 5000여명이 사망하고 있다. 국내에서의 2000년 식중독 발생 환자의 수는 7천 3백여명으로 총 104건이 발생하였으며 이 중 미생물에 의한 발생은 40%가 넘으며 특히 살모넬라균에 의한 식중독이 30건(28.8%)으로 가장 많이 발생하였다. 정화되지 않은 지하수나 하수를 통하여 식재료들을 처리하는 사례가 많아 모든 일상에 걸쳐 수질의 오염문제는 국민 생명과 직결되므로 매우 신중해야 하며 신속히 해결해야 한다.In recent years, it is very difficult to directly detect and eradicate the root of bacterial contaminants from school food poisoning and contaminated edible fish and shellfish caused by major social problems. In the United States, 75 million people suffer from food pathogens each year, of which 330,000 are treated and 5,000 are killed. In 2000, the number of food poisoning cases in Korea was 7,300 people, 104 cases, among which more than 40% were caused by microorganisms, especially 30 cases (28.8%) caused by Salmonella. It was. Since there are many cases of processing raw materials through unpurified groundwater or sewage, the pollution problem of water quality should be very careful and resolved promptly because it is directly connected to the life of the people.
우리 주위 먹거리에는 포도상구균, 살모넬라, 리스테리아, 대장균 O157:H7 등과 같은 병원성 미생물이 널리 퍼져 있으며, 특히 대장균은 그 중 대표 균종으로, 미생물이 건강에 미치는 위험도를 총체적으로 나타내는 지표로서 주로 사용된다. 미생물에 의한 오염정도는 개체 수를 정확히 측정하는 것으로부터 시작된다고 볼 수 있으며, 미생물 개체수를 측정하기 위한 방법은 미생물 집락 숫자를 직접 세는 방법과 간접방식 두 가지로 나눌 수 있다. 다만 균은 대부분 그 크기가 1 ㎛ (마이크로미터ㆍ100만분의 1 m) 안팎으로 아주 작아 시각적 관찰만을 통해 얻는 정보량에는 한계가 있다. 현재의 미생물 감식법은 대개 종속영양세균을 검시하는 방법으로 표준한천배지법 (standard plate count; SPC)을 사용, 채취한 수중샘플을 35도에서 48 ~ 74 시간이상 배양하여 미생물을 검사하는 전문 연구실의 복잡한 배양분석 절차로서만 가능하며, 최근까지 세균정량분석을 위한 소형 진단키트로 개발되었다 할지라도, 특이 항체나 수용체로써 세균표면의 항원을 인지하는 단백질-면역화학법을 벗어나기 어려우므로 상업화하는데 커다란 난관에 직면하고 있다. 따라서 비전문인도 어디서나 손쉽게 수질을 모니터링할 수 있는 사용자 친화적(user-friendly) 수질 오염원 감시시스템의 개발 및 대중화로 식수 및 식품안전성에 대한 높은 신뢰도를 제공할 수 있는 필요성이 대두되고 있다. 또한 기존 수질진단의 문제점을 근본적으로 해결하기 위하여 종래의 연구실기반의 시간/장비/비용 소모성 세균배양법에서 벗어나 실시간으로 서로 다른 광범위한 특정 신호영역에서 유해미생물에 대한 고해상도 분석능을 갖는 광나노 입자에 기반한 미생물 진단장치 개발이 필요하다.In the food around us, pathogenic microorganisms such as Staphylococcus aureus, Salmonella, Listeria and Escherichia coli O157: H7 are widely spread, and Escherichia coli is a representative species among them, and is mainly used as an indicator of the overall health risk of microorganisms. The degree of contamination by microorganisms can be regarded as starting from the accurate measurement of the population. The methods for measuring the number of microorganisms can be divided into the direct counting method and the indirect method. However, most of the microorganisms have a size of about 1 μm (micrometer, 1 millionth of a meter) in and out, so there is a limit to the amount of information obtained only through visual observation. Current microbial screening methods are usually screened by heterotrophic bacteria, using a standard plate count ( SPC ) method. Only possible as a complex culture assay procedure, but until recently developed as a small diagnostic kit for bacterial quantitative analysis, it is difficult to commercialize because it is difficult to escape protein-immunochemistry that recognizes antigens on bacterial surfaces as specific antibodies or receptors. Face to face. Therefore, there is a need to provide high reliability for drinking water and food safety by developing and popularizing a user-friendly water pollution monitoring system that enables non-specialists to easily monitor water quality anywhere. In addition, in order to fundamentally solve the problems of the existing water quality diagnosis, it is possible to overcome the conventional laboratory-based time / equipment / cost-consuming bacterial culture method based on photo nanoparticles having high resolution analysis of harmful microorganisms in a wide range of specific signal areas in real time. Development of microbial diagnostics is needed.
식품유통 현장에서 실시간으로 미생물을 신속하게 검출할 수 있는 최신기술로 플라즈몬공명(surface plasmon resonance; SPR) 센서나 표면탄성파(surface acoustic wave; SAW) 센서 기술 등이 개발되었으나 다음의 결점을 보인다. SPR 센서는 광(光)에너지가 금속 박막의 표면에 흡수되었을 때 일어나는 표면 플라즈마파(surface plasma wave)의 공명 현상을 이용하는 센서로서, 금속 박막에 검출하고자 하는 생물분자나 미생물과 잘 결합할 수 있는 물질을 코팅, 바이오센서로 삼고 미생물의 숫자를 계산하는 원리지만 SPR 장치 자체가 고가이며 광기술 교정운용이 복잡하여 민간인이 활용하기엔 초기단계이다. SAW 센서는 표면탄성파가 진행하는 압전기판 위에 미생물이 붙었을 때 질량 변화에 따라 표면탄성파의 주파수도 변하게 되는데 이러한 주파수변화를 측정함으로써 붙어있는 미생물의 양을 측정하는 방식으로서, 측정값에 대한 신뢰확보를 위해서 반드시 주파수 표준관련 교정 장비를 이용하여 주기적으로 센서를 교정해야하는데 이는 매우 불편하다. As a state-of-the-art technology for rapidly detecting microorganisms in real time at food distribution sites, surface plasmon resonance ( SPR ) sensors and surface acoustic wave ( SAW ) sensors have been developed. SPR sensor is a sensor that uses the surface plasma wave resonance phenomenon that occurs when light energy is absorbed on the surface of the metal thin film. The SPR sensor can be combined with biomolecules or microorganisms to be detected on the metal thin film. Principles of coating materials and biosensors and calculating the number of microorganisms, but the SPR device itself is expensive and the photonic calibration operation is complicated, so it is an early stage for civilian use. The SAW sensor changes the frequency of surface acoustic waves in response to changes in mass when microorganisms adhere to the piezoelectric plate through which surface acoustic waves progress, and by measuring the frequency change, it is possible to secure confidence in the measured values. For this reason, the sensor must be calibrated periodically using calibration equipment related to frequency standards, which is inconvenient.
수생 미생물들은 수중에 떠도는 특정 화학영양원(chemo-attractants)을 향하여 편모 (flagella)를 이용해 헤엄쳐 이동하는 습성인 생물학적 주화성(chemo-taxis)을 가지고 있다(Takafumi M et al. 1986. J. Bacteriology 165(3) 890-895, Greg M.B., James G.M. 2003. FEMS Microbiology Ecology 43 99-109). 따라서 아미노산, 유기분자, 포도당 등의 유인물질로 모여드는 습성을 이용하면, 현재 미생물 인지시 널리 이용되는 항체나 기타 분자인식 수용체의 처리 없이도 자동포획이 용이하고 미생물을 신속하게 검출할 수 있다. 유인물질을 전기적/광학적으로 민감한 광나노입자 표면에 코팅하게 되면, 퀀텀닷(Quantum Dot; QD)-나노바이오 공중합체로서 세균의 유인물질 자체가 되고, 영양물질과 유사한 크기의 나노스케일이므로 세균이 쉽게 영양원으로 오인하여 포식시 세포막공(membrane pore) 내로 쉽게 유입되며, 이렇게 세균 세포질(cytoplasm) 내로 침입된 나노입자들은 전기적 또는 광학적 방법에 의하여 쉽게 탐지되어 진단된다. 특히 대표격 유해세균종인 대장균 O-157종은 5~10 개의 다중편모로써 헤엄치므로 수중내 여러 비편모 잡세균들에 비해 보다 선택적으로 유인제 세린(Serine)이 처리된 QD를 향해 돌진하며, 유인된 세균들의 편모표면의 화학적 끈적임으로 인하여 유인QD와의 비특이 점착에 의한 선택성 및 민감도를 대폭 높일 수 있다. Aquatic microorganisms have a wet, biological chemo-taxis that swims with flagella toward certain chemo-attractants in water (Takafumi M et al. 1986. J. Bacteriology 165 (3) 890-895, Greg MB, James GM 2003. FEMS Microbiology Ecology 43 99-109). Therefore, using the habit of attracting attractants such as amino acids, organic molecules, glucose, etc., it is easy to automatically capture and quickly detect the microorganisms without the treatment of antibodies or other molecular recognition receptors widely used in microorganism recognition. When the attractant is coated on the surface of the photo-nanoparticles, which are electrically and optically sensitive, the quantum dot ( QD ) -nanobio -copolymer becomes a bacteria attractant itself, and because the nanoscale is similar in size to nutrients, It is easily mistaken as a nutrient source and easily enters the membrane pore during predation. Nanoparticles invaded into the bacterial cytoplasm are easily detected and diagnosed by electrical or optical methods. In particular, E. coli O-157, a representative harmful bacterium, swims with 5 to 10 multiple flagella, so it rushes toward QD treated with attractant serine more selectively than various non-uniform flagella bacteria in water. Due to the chemical stickiness of the flagella surface of the isolated bacteria, the selectivity and sensitivity due to non-specific adhesion with attracted QDs can be greatly increased.
한국특허출원 10-2005-0087315호(출원일: 2005.09.20)는 자력에 반응하는 코어 입자 및 상기 코어 입자의 주위에 코팅되거나 흡착된 나노입자를 포함하는 나노하이브리드 입자를 구비하며, 상기 나노하이브리드 입자는 생체분자와 반응하여 생체분자가 고정 또는 혼성화된 생체 복합 입자를 형성하고, 상기 생체 복합 입자는 자성체에 반응하는 특성을 갖는 생체분자 검출용 나노하이브리드 입자의 제조방법, 나노하이브리드 입자를 이용한 생체분자 검출기, 생체분자 검출 방법 및 생체분자 검출용 분석장치를 개시하고 있다. 하지만 미생물 유인제를 이용한 미생물 검출 방법에 대한 언급은 없다.Korean Patent Application No. 10-2005-0087315 (Application Date: 2005.09.20) is provided with nanohybrid particles including core particles reacting to magnetic force and nanoparticles coated or adsorbed around the core particles, the nanohybrid particles Reacts with biomolecules to form biomolecules in which biomolecules are immobilized or hybridized, and the biocomposite particles have a characteristic of reacting with a magnetic body, a method of manufacturing nanohybrid particles for detecting biomolecules, and biomolecules using nanohybrid particles A detector, a biomolecule detection method, and an analysis device for biomolecule detection are disclosed. However, there is no mention of a microbial detection method using a microbial attractant.
본 발명은 미생물 유인 물질과 결합성이 뛰어나고, 순도가 높으며 물리적으로 안정한 신규한 나노 결정 복합체를 제공하는 것을 목적으로 한다.It is an object of the present invention to provide a novel nanocrystalline composite having excellent binding properties with high microbial attraction and high purity.
또한 본 발명은 미생물 유인 물질과 결합성이 뛰어나고, 물리 화학적으로 안정한 신규한 나노 결정 복합체를 이용하는 진단 키트를 제공하는 것을 목적으로 한다. In addition, an object of the present invention is to provide a diagnostic kit using a novel nanocrystal complex which is excellent in binding with a microorganism attractant and physicochemically stable.
본 발명의 또 다른 목적은 미생물 유인 물질과 결합성이 뛰어나고, 물리 화학적으로 안정한 신규한 나노 결정 복합체를 이용한 미생물 검출 방법을 제공하고자 한다.Another object of the present invention is to provide a method for detecting microorganisms using a novel nanocrystal complex having excellent binding properties with microbial attractants and physicochemically stable.
상기한 목적을 달성하기 위하여, 본 발명은 아연, 카드늄, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 메탈 군으로부터 하나 이상 선택되는 나노 결정; 미생물 유인 물질; 및 일측에 상기 나노 결정과 결합 가능한 전하 특성의 치환기를 가지며, 반대측에 복수의 수용성 치환기를 갖는 고분자 사슬을 가지며, 일측의 치환기를 통해 상기 나노 결정과 결합하고, 반대측의 복수의 수용성 치환기를 통해 상기 나노 결정을 안정화하고, 상기 복수의 수용성 치환기를 통해 상기 미생물 유인 물질과 결합을 구성하는 결합 안정화 물질을 포함하는 나노 결정-미생물 유인 물질 복합체를 제공한다. In order to achieve the above object, the present invention is a nanocrystal selected from the group consisting of metals consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth; Microbial attractants; And a polymer chain having a substituent having a charge property that can be bonded to the nanocrystal on one side, and having a polymer chain having a plurality of water-soluble substituents on the opposite side, which is bonded to the nanocrystal through a substituent on one side, and through the plurality of water-soluble substituents on the opposite side. Provided is a nano crystal-microbial attractant complex comprising a stabilization material that stabilizes nanocrystals and forms a bond with the microbial attractant through the plurality of water-soluble substituents.
또한 본 발명은 미생물 유인 물질을 표지하는 나노 결정을 제조하는 나노 결정 제조 방법에 있어서, 헥사데칸올, 수산화 칼륨 및 카본 디설파이드를 반응시켜, 헥사데실싼테이트(hexadecyl xanthate; 이하 'HDX'라 함) 칼륨 염을 제조하는 단계; 아연, 카드늄, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 메탈 군으로부터 하나 이상 선택되는 나노 결정을 수득된 HDX 칼륨 염과 반응시켜 HDX 메탈 설파이드 나노 결정을 제조하는 단계; 상기 HDX 메탈 설파이드 나노 결정에 알킬 아민 도판트를 반응 시켜, 메탈 설파이드 나노 결정을 제조하는 단계; 상기 메탈 설파이드 나노 결정에 결합 안정화 물질을 반응시켜, 메탈 설파이드 나노 결정에 결합 안정화 물질을 결합시키는 단계; 상기 결합된 메탈 설파이드 나노 결정-결합 안정화 물질 복합체에 활성화 물질을 반응시켜, 메탈 설파이드 나노 결정-결합 안정화 물질 복합체를 활성화시키는 단계; 및 상기 활성화된 메탈 설파이드 나노 결정-결합 안정화 물질 복합체에 미생물 유인물질을 결합하는 단계를 포함하는 나노 결정-미생물 유인물질 복합체 제조 방법을 제공한다.In addition, the present invention is a method for producing nanocrystals for labeling microorganism attractant, by reacting hexadecanol, potassium hydroxide and carbon disulfide, hexadecyl xanthate (hereinafter referred to as 'HDX') Preparing a potassium salt; Reacting nanocrystals selected from at least one metal group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth with the obtained HDX potassium salt to produce HDX metal sulfide nanocrystals; Preparing a metal sulfide nanocrystal by reacting an alkyl amine dopant with the HDX metal sulfide nanocrystal; Coupling a bond stabilizing material to the metal sulfide nanocrystals by reacting a bond stabilizing material to the metal sulfide nanocrystals; Activating a metal sulfide nano crystal-bond stabilizer material complex by reacting an activated material with the bonded metal sulfide nano crystal-bond stabilizer material complex; And it provides a method for producing a nano-crystal microbe attractant complex comprising the step of binding a microorganism attractant to the activated metal sulfide nano crystal-bond stabilizing material complex.
또한 본 발명은 나노 결정 표지를 이용하여 미생물을 검출하는 검출 키트에 있어서, 아연, 카드뮴, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 메탈군으로부터 하나 이상 선택되는 나노 결정, 상기 미생물을 유인하기 위한 미생물 유인 물질 및 일측으로 전하 특성의 치환기로 나노결정과 결합하며 반대측으로 복수의 수용성 치환기를 가지는 고분자 사슬로 상기 미생물 유인 물질과 결합하는 결합안정화물질을 포함하는 나노 결정-미생물 유인 물질 복합체; 유인 미생물 포획에 의한 상기 나노 결정-미생물 유인 물질 복합체의 나노입자와 대항되는 전하적 특성으로 포집하는 포집전극; 포집된 상기 나노 결정에 대응하는 전류 피크를 아날로그로 측정하는 전류 피크 측정부를 포함하는 미생물 검출 키트를 제공한다.In addition, the present invention is a detection kit for detecting a microorganism using a nano-crystal label, nanocrystals selected from at least one metal group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth, Nano crystal-microorganism comprising a microorganism attracting material for attracting the microorganism and a binding stabilizer that binds to the nanocrystals with substituents of charge characteristics on one side and the microbial attracting materials with a polymer chain having a plurality of water-soluble substituents on the other side Attractant complexes; A collecting electrode configured to capture charge characteristics opposed to nanoparticles of the nanocrystal-microbe attractant material complex by capturing microorganisms; It provides a microbial detection kit comprising a current peak measuring unit for measuring analog current peak corresponding to the collected nano-crystal.
또한 본 발명은 나노 결정 표지를 이용하는 미생물 검출 방법에 있어서, 검출하고자 하는 하나 이상의 미생물과 미생물 유인이 가능한 하나 이상의 미생물 유인 물질을 결정하는 단계; 아연, 카드늄, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 군으로부터 하나 이상 나노 결정을 선택하여 상기 하나 이상의 미생물 유인 물질과 각각 결합하여 하나 이상의 나노 결정-미생물 유인 물질 복합체를 형성하는 단계; 상기 하나 이상의 나노 결정-미생물 유인 물질 복합체를 진단하고자 하는 시료 내에 넣고 혼합하여 상기 검출하고자 하는 하나 이상의 미생물과 하나 이상의 나노 결정-미생물 유인 물질의 접촉을 유도하는 단계; 상기 나노 결정-미생물 유인 물질과 접촉한 미생물을 포획하는 단계; 및 상기 포획된 미생물에 접촉한 상기 나노 결정-미생물 유인 물질 복합체로부터 포집된 나노 결정에 대응하는 고유한 전류 피크를 측정하는 단계를 포함하는 미생물 검출 방법을 제공한다.In another aspect, the present invention provides a method for detecting a microorganism using a nanocrystalline label, comprising: determining at least one microorganism to be detected and at least one microorganism attracting material capable of attracting microorganisms; One or more nanocrystals are selected from the group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese, and bismuth and combined with each of the one or more microbial attractants to form one or more nanocrystal-microbe attractant complexes. Forming; Incorporating the at least one nanocrystal-microbe attractant complex into a sample to be diagnosed and mixing to induce contact of the at least one microcrystal to be detected with the at least one nanocrystal-microbe attractant; Capturing microorganisms in contact with the nanocrystal-microbe attractant material; And measuring an inherent current peak corresponding to the nanocrystals collected from the nanocrystal-microbe attractant material complex in contact with the captured microorganisms.
도 1은 퀀텀닷 나노결정(Quantum Dot Nanocrystal)-미생물 유인물질(chemotactic attractant) 복합체 합성법 및 흐름도를 나타낸다. 1 shows a method and flow chart of quantum dot nanocrystal-chemotactic attractant complexes.
도 2는 퀀텀닷 나노결정(Quantum Dot Nanocrystal)-미생물 유인물질(chemotactic attractant) 복합체에 의한 미생물 포획 및 음전극 스트리핑 전압(anodic stripping voltammetric; ASV)법 정량분석 흐름도를 나타낸다. FIG. 2 shows a quantitative flow chart of microbial capture and anode stripping voltammetric ( ASV ) by quantum dot nanocrystal-chemotactic attractant complexes.
도 3은 마이크로칩 타입의 정량분석 카트리지 센서 모듈을 나타낸다. 3 shows a microchip type quantitative cartridge sensor module.
도 4는 마이크로 실린더 타입의 정량분석 카트리지 센서 모듈을 나타낸다. 4 shows a quantitative cartridge sensor module of the micro cylinder type.
도 5는 단순속성 분리를 위한 마이크로 주사기 타입의 카트리지 센서 모듈을 나타낸다. 5 shows a microsensor type cartridge sensor module for simple attribute separation.
도 6은 속성분리 및 정량분석을 위한 마이크로 주사기 타입의 정량분석 카트리지 센서 통합 모듈을 나타낸다. 6 shows a micro syringe type quantitative cartridge sensor integration module for attribute separation and quantitation.
본 발명의 제 1의 양태는 아연, 카드늄, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 메탈 군으로부터 하나 이상 선택되는 나노 결정; 미생물 유인 물질; 및 일측에 상기 나노 결정과 결합 가능한 전하 특성의 치환기를 가지며, 반대측에 복수의 수용성 치환기를 갖는 고분자 사슬을 가지며, 일측의 치환기를 통해 상기 나노 결정과 결합하고, 반대측의 복수의 수용성 치환기를 통해 상기 나노 결정을 안정화하고, 상기 복수의 수용성 치환기를 통해 상기 미생물 유인 물질과 결합을 구성하는 결합 안정화 물질을 포함하는 나노 결정-미생물 유인 물질 복합체를 제공한다. A first aspect of the invention provides a nanocrystal selected from at least one metal group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth; Microbial attractants; And a polymer chain having a substituent having a charge property that can be bonded to the nanocrystal on one side, and having a polymer chain having a plurality of water-soluble substituents on the opposite side, which is bonded to the nanocrystal through a substituent on one side, and through the plurality of water-soluble substituents on the opposite side. Provided is a nano crystal-microbial attractant complex comprising a stabilization material that stabilizes nanocrystals and forms a bond with the microbial attractant through the plurality of water-soluble substituents.
본 발명에 사용된 나노 결정은 금속계 나노 결정으로서, 해상도 및 신호 선택성이 우수한 금속의 나노 결정이다. 본 발명에서 사용되는 나노 결정은 해상도 및 신호 선택성이 우수한 금속이면 특별히 제한되지 않는다. 해상도는 금속으로부터 발생되는 신호의 피크 폭이 좁아 다른 신호의 피크와 중첩없이 구별되는 특성을 의미하며, 신호 선택성은 해당 금속으로부터 생성되는 신호 피크가 다른 금속들로부터 생성되는 신호 피크와 구별이 용이하게 나타내어 지는 정도를 의미한다. 즉 해상도가 높은 경우 신호 선택성이 증가된다. 본 발명에 따른 나노 결정 합성법에 따라 수득되는 나노결정은 메탈 설파이드(metal sulfide; MS)이다. 메탈 설파이드에 사용되는 금속으로는 아연(Zn), 카드늄(Cd), 납(Pb), 구리(Cu), 갈륨(Ga), 비소(As), 탈륨(Tl), 니켈(Ni), 망간(Mn) 또는 창연(Bi)이 바람직하게 사용된다. 특히, 아연, 카드뮴, 납, 또는 구리가 보다 우수한 해상도를 갖는 선택적 신호를 생성하므로 바람직하게 사용된다. The nanocrystals used in the present invention are metal-based nanocrystals, which are nanocrystals of metals having excellent resolution and signal selectivity. The nanocrystal used in the present invention is not particularly limited as long as it is a metal having excellent resolution and signal selectivity. Resolution means that the peak width of a signal generated from a metal is narrow so that it is distinguished from the peak of other signals without overlapping, and signal selectivity makes it easy to distinguish a signal peak generated from the metal from signal peaks generated from other metals. Means the degree indicated. In other words, if the resolution is high, the signal selectivity is increased. The nanocrystals obtained by the nanocrystal synthesis method according to the present invention are metal sulfides (MS). Metals used in metal sulfide include zinc (Zn), cadmium (Cd), lead (Pb), copper (Cu), gallium (Ga), arsenic (As), thallium (Tl), nickel (Ni), and manganese ( Mn) or bismuth Bi is preferably used. In particular, zinc, cadmium, lead, or copper are preferably used because they produce selective signals with better resolution.
본 발명에서 "미생물 유인물질"은 이동성 미생물이 주화성(chemo-taxis)을 지니고 있어서 특정 화학영양원으로의 유도 효과를 갖는 무기 또는 유기물질을 말한다. 구체적으로는 말토즈(maltose), 아스파테이트(aspartate), 카사미노산(casamino acid), 시스테인(cysteine), 류신(leucine) 또는 세린(serine)을 포함한다.In the present invention, "microbial attractant" refers to an inorganic or organic substance having a chemo-taxis, and thus having an inducible effect on a specific nutrient. Specific examples include maltose, aspartate, casamino acid, cysteine, leucine or serine.
본 발명의 제2의 양태는 미생물 유인 물질을 표지하는 나노 결정-미생물 유인 물질 복합체 제조방법에 있어서, 헥사데칸올, 수산화 칼륨 및 카본 디설파이드를 반응시켜, 헥사데실싼테이트(hexadecyl xanthate; 이하 'HDX'라 함) 칼륨 염을 제조하는 단계; 아연, 카드늄, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 메탈 군으로부터 하나 이상 선택되는 나노 결정을 수득된 HDX 칼륨 염과 반응시켜 HDX 메탈 설파이드 나노 결정을 제조하는 단계; 상기 HDX 메탈 설파이드 나노 결정에 알킬 아민 도판트를 반응시켜, 메탈 설파이드 나노 결정을 제조하는 단계; 상기 메탈 설파이드 나노 결정에 결합 안정화 물질을 반응시켜, 메탈 설파이드 나노 결정에 결합 안정화 물질을 결합시키는 단계; 상기 결합된 메탈 설파이드 나노 결정-결합 안정화 물질 복합체에 활성화 물질을 반응시켜, 메탈 설파이드 나노 결정-결합 안정화 물질 복합체를 활성화시키는 단계; 및 상기 활성화된 메탈 설파이드 나노 결정-결합 안정화 물질 복합체에 미생물 유인물질을 결합하는 단계를 포함하는 나노 결정-미생물 유인물질 복합체 제조 방법을 제공한다. 자세하게는 상기 나노 결정은 아연, 카드뮴, 납 및 구리로 이루어진 메탈 군으로부터 하나 이상 선택되는 나노 결정-미생물 유인 물질 복합체 제조방법이다.According to a second aspect of the present invention, in the method of preparing a nanocrystal-microbe attractant material complex that labels a microbe attractant, hexadecanol, potassium hydroxide, and carbon disulfide are reacted to form a hexadecyl xanthate (hereinafter referred to as 'HDX'). ') Preparing a potassium salt; Reacting nanocrystals selected from at least one metal group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth with the obtained HDX potassium salt to produce HDX metal sulfide nanocrystals; Reacting the HDX metal sulfide nanocrystals with an alkyl amine dopant to prepare metal sulfide nanocrystals; Coupling a bond stabilizing material to the metal sulfide nanocrystals by reacting a bond stabilizing material to the metal sulfide nanocrystals; Activating a metal sulfide nano crystal-bond stabilizer material complex by reacting an activated material with the bonded metal sulfide nano crystal-bond stabilizer material complex; And it provides a method for producing a nano-crystal microbe attractant complex comprising the step of binding a microorganism attractant to the activated metal sulfide nano crystal-bond stabilizing material complex. In detail, the nanocrystal is a method for preparing a nanocrystal-microbe attractant material composite selected from one or more metal groups consisting of zinc, cadmium, lead, and copper.
상기 HDX 칼륨 염 제조 단계는 상기 헥사데칸올과 상기 수산화 칼륨을 혼합하고, 혼합된 용액이 모두 용해될 때까지 가열하는 단계; 상기 혼합 용액을 톨루엔에 넣고 균일하게 교반하고, 상기 카본 디설파이드를 첨가하는 단계; 상기 혼합 용액을 페트로리움 에테르에 넣고 추가 교반하는 단계; 및 상기 혼합 용액을 유리 필터로 필터링하고, 에테르로 세척하는 과정을 반복하는 단계를 포함하는 나노 결정-미생물 유인 물질 복합체 제조방법이다.The HDX potassium salt preparing step comprises the steps of mixing the hexadecanol and the potassium hydroxide, heating until all the mixed solution is dissolved; Putting the mixed solution into toluene, stirring uniformly, and adding the carbon disulfide; Placing the mixed solution in petroleum ether and further stirring; And filtering the mixed solution with a glass filter and repeating a process of washing with ether.
상기 알킬 아민 도판트는 헥사 데실 아민, 데실 아민 및 트리 옥틸 아민으로 이루어지는 군에서 하나 이상 선택되는 나노 결정 제조 방법이다. HDX 아연 설파이드 나노 결정 또는 HDX 카드뮴 설파이드 나노 결정에 대해서는 상기 알킬 아민 도판트로 헥사 데실 아민을 사용하고, HDX 납 설파이드 나노 결정에 대해서는 상기 알킬 아민 도판트로 데실 아민 또는 트리 옥틸 아민을 사용하고, HDX 구리 설파이드 나노 결정에 대해서는 상기 알킬 아민 도판트로 헥사 데실 아민 또는 트리 옥틸 아민을 사용하는 나노 결정-미생물 유인 물질 복합체 제조 방법이다.The alkyl amine dopant is a method for producing nanocrystals selected from the group consisting of hexadecyl amine, decyl amine and trioctyl amine. Hexadecyl amine is used as the alkyl amine dopant for HDX zinc sulfide nanocrystals or HDX cadmium sulfide nanocrystals, and the alkyl amine dopantrodecyl amine or trioctyl amine is used for HDX lead sulfide nanocrystals, and HDX copper sulfide For nanocrystals, a method for preparing nanocrystal-microbe attractant complexes using hexadecyl amine or trioctyl amine as the alkyl amine dopant.
나노 결정-미생물 유인 물질 복합체의 제조 방법은 앞에서 수득한 금속 나노 입자(MS)들을 안정화 물질과 함께 반응시켜 안정화하고, 활성화 물질로 활성화한 후 미생물 유인 물질을 반응시켜 나노 입자-미생물 유인 물질 복합체를 수득한다. In the method of preparing the nanocrystal-microbe attractant complex, the metal nanoparticles (MS) obtained above are stabilized by reacting with the stabilizing material, activating as an activating material, and then reacting the microbe attractant material to react the nanoparticle-microbe attractant material complex. To obtain.
본 발명에서 사용되는 "안정화 물질"은 나노 입자를 화학적 물리적으로 안정화시키고, 나노 결정의 용해도를 증진시켜 미생물 유인 물질에 대한 호환성을 증가시켜, 나노 결정-미생물 유인 물질 복합체를 안정화에 기여한다. 구체적으로 안정화 물질은 일측에 나노 결정과 결합 가능한 화학적 치환기를 가지며, 반대측의 복수의 수용성 치환기를 갖는 고분자 물질로 구성되며, 일측의 화학적 치환기로 나노 결정을 둘러싸 결합하고, 반대측의 수용성 치환기를 통해 수용성 매질로부터 나노 결정을 보호하여 나노 결정의 안정성을 담보한다. 또한, 반대측의 수용성 치환기 중 일부를 통해 미생물 유인 물질과 공유 결합을 이루어 나노 결정과 미생물 유인 물질간의 결합을 촉진한다. As used herein, "stabilizers" chemically and physically stabilize nanoparticles, enhance solubility of nanocrystals to increase compatibility with microbial attractants, contributing to stabilizing nanocrystal-microbe attractant complexes. Specifically, the stabilizing material has a chemical substituent capable of binding to the nanocrystals on one side, and is composed of a polymer material having a plurality of water-soluble substituents on the opposite side. The nanocrystals are protected from the medium to ensure the stability of the nanocrystals. In addition, some of the water-soluble substituents on the opposite side forms a covalent bond with the microorganism attractant to promote the bond between the nanocrystal and the microorganism attractant.
본 발명의 안정화 물질은 바람직하게는 다이티올쓰레이톨 (dithiolthreitol, 이하 'DTT'라 함) 또는 다이하드로 리포익산(dihydrolipoic acid; 이하 'DHLA'라 함)를 사용한다. 이와 같은 DTT나 DHLA 는 그 표면 화학적 특성 및 높은 수용액에 대한 용해도로 나노 결정을 현저히 안정화시킨다. 구체적으로 DTT는 그 분자 구조상의 티올(-SH) 화학 그룹에 의해 나노 결정 표면을 나노 수준에서 강하고 균일하게 둘러싸, 나노 결정의 안정적인 구조를 유지하는 동시에, 나노 결정 표면에 히드록실(hydroxyl) 그룹을 생성시켜, 나노 결정의 수용성을 증진 시킨다. 여기서, 안정화 물질은 티올(-SH) 기의 음 전하적 특성을 이용하여 양 전하적 특성을 갖는 나노 결정을 균일하게 둘러싸 결합하는 것으로 생각된다. 따라서, 안정화 물질로서 일측에 음 전하적 특성을 갖는 치환기가 다수 존재하여 나노 결정을 균일하게 감싸 나노 결정을 안정화하고, 타측에 수용성 치환기를 다수 존재하여 나노 결정의 수용성을 증진시키는 물질이 바람직하게 사용된다. 본 발명에서는 음 전하적 특성을 가지는 치환기로서 티올(-SH)기를 예로 들었으나, 기타 음 전하적 특성을 가지는 치환기도 사용될 수 있으며, 구체적인 예로서 수산화기(-OH)도 사용될 수 있다. 본 발명은 양 전하 특성을 가지는 나노 결정을 사용하고 있으므로, 음 전하적 특성을 가지는 안정화 물질이 바람직하나, 음 전하적 특성을 가지는 나노 결정을 사용하는 경우에는 양 전하 특성을 가지는 치환기를 가지는 안정화 물질이 바람직하게 사용될 수 있다. The stabilizing material of the present invention preferably uses dithiolthreitol (hereinafter referred to as 'DTT') or dihydrolipoic acid (hereinafter referred to as 'DHLA'). Such DTT or DHLA significantly stabilizes the nanocrystals due to their surface chemistry and solubility in high aqueous solutions. Specifically, DTT surrounds the nanocrystal surface strongly and uniformly at the nano level by a thiol (-SH) chemical group on its molecular structure, maintaining a stable structure of the nanocrystals, and simultaneously forming a hydroxyl group on the nanocrystal surface. Generated to enhance the water solubility of the nanocrystals. Here, it is thought that the stabilizing material uniformly surrounds and binds the nanocrystals having positive charge characteristics by using the negative charge characteristics of the thiol (-SH) group. Therefore, as a stabilizing material, a large number of substituents having negative charge characteristics exist on one side to uniformly surround the nanocrystals, thereby stabilizing the nanocrystals, and a plurality of water-soluble substituents exist on the other side to enhance the water solubility of the nanocrystals. do. In the present invention, a thiol (-SH) group is exemplified as a substituent having negative charge characteristics, but a substituent having other negative charge characteristics may be used, and a hydroxyl group (-OH) may also be used as a specific example. Since the present invention uses nanocrystals having positive charge characteristics, a stabilizing material having negative charge characteristics is preferable, but in the case of using nanocrystals having negative charge characteristics, a stabilizing substance having substituents having positive charge characteristics is used. This can be preferably used.
본 발명에서 사용되는 "활성화 물질"은 안정화 물질의 활성화를 유도하여 미생물 유인 물질의 아미노 그룹과 카바메이트(carbamate) 결합을 형성할 수 있게 한다. 본 발명에서 사용한 활성화 물질은 바람직하게는 카보닐 다이이미다졸(1,1-carbonyl diimidazole, 이하 'CDI'라 함)을 사용하였다. As used herein, an "activating material" allows the activation of a stabilizing material to form carbamate bonds with amino groups of the microorganism attracting material. As the activating material used in the present invention, carbonyl diimidazole (1,1-carbonyl diimidazole, hereinafter referred to as 'CDI') is preferably used.
본 발명의 제3의 양태는 나노 결정 표지를 이용하여 미생물을 검출하는 검출 키트에 있어서, 아연, 카드뮴, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 메탈군으로부터 하나 이상 선택되는 나노 결정, 상기 미생물을 유인하기 위한 미생물 유인 물질 및 일측으로 전하 특성의 치환기로 나노결정과 결합하며 반대측으로 복수의 수용성 치환기를 가지는 고분자 사슬로 상기 미생물 유인 물질과 결합하는 결합안정화물질을 포함하는 나노 결정-미생물 유인 물질 복합체; 유인 미생물 포획에 의한 상기 나노 결정-미생물 유인 물질 복합체의 나노입자와 대항되는 전하적 특성으로 포집하는 포집전극; 포집된 상기 나노 결정에 대응하는 전류 피크를 아날로그로 측정하는 전류 피크 측정부를 포함하는 미생물 검출 키트를 제공한다. A third aspect of the invention is a detection kit for detecting microorganisms using nanocrystalline labels, wherein at least one selected from the group of metals consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth Comprising a nano crystal, a microorganism attracting material for attracting the microorganism and a binding stabilizer for binding to the nanocrystals with a polymer chain having a plurality of water-soluble substituents on the opposite side and the microcrystalline attractant for attracting the microorganism Nano crystal-microbe attractant complexes; A collecting electrode configured to capture charge characteristics opposed to nanoparticles of the nanocrystal-microbe attractant material complex by capturing microorganisms; It provides a microbial detection kit comprising a current peak measuring unit for measuring analog current peak corresponding to the collected nano-crystal.
상기 나노 결정은 메탈 설파이드 형이고, 아연, 카드뮴, 납 및 구리로 이루어진 메탈 군으로부터 하나 이상 선택되고, 상기 미생물 유인 물질은 말토즈(maltose), 아스파테이트(aspartate), 카사미노산(casamino acid), 시스테인(cysteine), 류신(leucine) 또는 세린(serine)으로 이루어진 그룹으로부터 하나 이상 선택되며, 상기 결합 안정화 물질은 다이티올쓰레이톨 또는 다이하드로 리포익산인 미생물 검출 키트이다.The nanocrystal is a metal sulfide type, at least one selected from the group consisting of zinc, cadmium, lead, and copper, and the microorganism attractant material is maltose, aspartate, casamino acid, At least one selected from the group consisting of cysteine, leucine or serine, wherein the binding stabilizing agent is a microorganism detection kit which is dithiolthitol or dihydro lipoic acid.
상기 검출 키트는 상기 전류 피크 측정부에 의해 측정된 전류피크를 디지털 신호로 변환하기 위한 아날로그 디지털 변환부를 추가 포함하고, 아연, 카드늄, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 메탈그룹에서 선택된 하나 이상의 각기 다른 나노 결정을 이용하여 제조된 각기 다른 나노결정-미생물 유인 물질 복합체를 사용하여 하나 이상의 미생물을 동시에 검출하는 미생물 검출 키트이다.The detection kit further includes an analog-to-digital converter for converting the current peak measured by the current peak measurement unit into a digital signal, and includes zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth. A microbial detection kit for simultaneously detecting one or more microorganisms using different nanocrystal-microbe attractant complexes prepared using one or more different nanocrystals selected from the metal group.
상기 유인 미생물 포획은 선택적 반투과막을 통해 분리하는 것을 특징으로 하는 미생물 검출 키트이다.The attractant microorganism capture is a microbial detection kit, characterized in that the separation through the selective semipermeable membrane.
상기 나노 결정이 양이온 특성을 갖는 경우 상기 포집 전극에 소정의 음 전위를 인가하고, 상기 나노 결정이 음이온 특성을 갖는 경우 상기 포집 전극에 소정의 양 전위를 인가하는 미생물 검출 키트이다. When the nanocrystals have a cationic property, a predetermined negative potential is applied to the collection electrode, and when the nanocrystals have anionic properties, the microorganism detection kit applies a predetermined positive potential to the collection electrode.
상기 전류 피크 측정부는 상기 포집 전극에 포집된 상기 나노 결정에 소정의 전위를 인가하여, 상기 나노 입자를 산화/환원 반응시키고, 상기 나노 결정의 산화/환원 반응으로부터 발생하는 각 나노 결정에 고유한 전류 피크를 측정하는 검출 키트이며, 상기 포집 전극에 소정의 음 전위 또는 양 전위를 인가하여, 각각 양이온 특성의 나노 결정 또는 음이온 특성의 나노 결정을 포집하고, 상기 전류 피크 측정부는 포집된 상기 나노 결정으로부터 각각 발생하는 상기 나노 결정에 고유한 전류 피크를 각각 측정하는 미생물 검출 키트이다. 또한 상기 디지털 신호를 해석하여 상기 디지털 신호에 대응하는 미생물의 정체 또는 검출된 미생물의 함량을 추정하는 디지털 신호 리더부를 추가 포함하며 상기 디지털 신호를 소정의 바코드로 변환하는 바코드 변환부를 추가 포함하고 상기 디지털 신호를 유선 또는 무선 통신을 통해 소정의 원격 진단부로 전송하고 상기 원격 진단부로부터 상기 디지털 신호의 해석 결과를 수신하는 통신부를 추가 포함하는 미생물 검출 키트이다.The current peak measurement unit applies a predetermined potential to the nanocrystals collected on the collection electrode to oxidize / reduce the nanoparticles and induces a current unique to each nanocrystal resulting from the oxidation / reduction reaction of the nanocrystals. A detection kit for measuring peaks, and applying a predetermined negative potential or a positive potential to the collection electrode to collect nanocrystals of cationic properties or nanocrystals of anionic properties, respectively, and the current peak measuring unit is provided from the collected nanocrystals. It is a microbial detection kit for measuring the current peaks unique to each of the nanocrystals that occur. The apparatus may further include a digital signal reader unit for interpreting the digital signal and estimating the identity of the microorganisms corresponding to the digital signal or the content of the detected microorganisms, and further including a barcode converter for converting the digital signal into a predetermined barcode. The microbial detection kit further includes a communication unit for transmitting a signal to a predetermined remote diagnosis unit through wired or wireless communication and receiving a result of analyzing the digital signal from the remote diagnosis unit.
본 발명의 미생물 검출 키트의 나노 결정 표지는 특정 미생물을 검출하기 위한 나노 결정을 의미한다. 구체적으로 특정 미생물을 검출하기 위해, 그 미생물을 유인할 수 있는 유인 물질과 결합된 나노 결정을 의미한다. 이러한 본 발명의 검출 키트의 나노 결정 표지는 측정하고자 하는 미생물의 종류에 따라 교체되어 사용될 수 있다.Nanocrystalline label of the microorganism detection kit of the present invention means nanocrystals for detecting a specific microorganism. Specifically, in order to detect a specific microorganism, it means a nanocrystal combined with an attractant capable of attracting the microorganism. The nano-crystal label of the detection kit of the present invention can be replaced and used depending on the type of microorganism to be measured.
그리고, 본 발명의 미생물 검출 키트의 측정 결과의 디지털 변환 수단은 바코드로 출력하는 수단일 수 있으나 이에 한정되는 것은 아니며, GSM(Global System for Mobile), 블루투스, 유비쿼터스, CDMA(Code Division Multiple Access) 등의 첨단 무선 모바일 IT 기술에 통합될 수 있다. In addition, the digital conversion means of the measurement result of the microbial detection kit of the present invention may be a means for outputting as a bar code, but is not limited thereto, Global System for Mobile (GSM), Bluetooth, Ubiquitous, Code Division Multiple Access (CDMA), etc. Can be integrated into the company's advanced wireless mobile IT technology.
이와 같이 특정 시료에 대한 진단 결과가 바코드로 출력되는 경우, 검출 키트의 이용자는 출력된 바코드를 바코드 리더기로 읽어 진단 결과를 신속하게 알 수 있다. 이렇게 검출 결과가 디지털로 출력되는 경우, 검출 결과는 유선 통신 또는 무선 통신을 통해 원격지로 송신될 수 있다. 따라서, 검출 결과를 평가할 수 있는 시스템을 갖는 원격지는 검출 키트으로부터 출력된 검출 결과를 수신하여 특정 시료에 대한 검출 결과를 평가하고, 그 평가 결과를 다시 검출 키트 사용자에게 송신하도록 구현될 수 있다. 이렇게 되는 경우, 본 발명의 검출 키트는 의학적, 임상적 응용뿐만 아니라, 다양한 프로브의 교체를 통해 수질, 식품 등의 환경모니터링 및 생물학전, 테러(TNT) 와 범죄(마약) 등의 분야에서 감시 시스템에 적용될 수 있다. As described above, when the diagnosis result for a specific sample is output as a bar code, a user of the detection kit can quickly read the output result by reading the output bar code with a bar code reader. When the detection result is digitally output in this way, the detection result can be transmitted to the remote place via wired communication or wireless communication. Thus, a remote site having a system capable of evaluating detection results can be implemented to receive detection results output from a detection kit to evaluate detection results for a particular sample and send the evaluation results back to the detection kit user. In this case, the detection kit of the present invention can be applied to surveillance systems in the fields of environmental monitoring and biological warfare, terrorism (TNT) and crime (drugs), such as water quality and food, as well as medical and clinical applications, as well as replacement of various probes. Can be applied.
이상 살펴본 바와 같이 본 발명에 따른 나노 결정 표지는 합성이 간편하고 각 금속 나노 결정마다 고유한 산화 환원 전위를 갖고 있어 동시에 다수의 나노 결정 표지를 검출하는 것이 가능하다. 따라서, 본 발명에 따른 나노 입자 표지를 이용하는 경우 다수의 미생물 검출 키트의 소형화가 가능하게 된다. As described above, the nanocrystalline label according to the present invention is easy to synthesize and has a unique redox potential for each metal nanocrystal, and thus it is possible to simultaneously detect a plurality of nanocrystalline labels. Therefore, when using the nanoparticle label according to the invention it is possible to miniaturize a number of microbial detection kit.
본 발명의 제4의 양태는 나노 결정 표지를 이용하는 미생물 검출 방법에 있어서, 검출하고자 하는 하나 이상의 미생물과 미생물 유인이 가능한 하나 이상의 미생물 유인 물질을 결정하는 단계; 아연, 카드늄, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 군으로부터 하나 이상 나노 결정을 선택하여 상기 하나 이상의 미생물 유인 물질과 각각 결합하여 하나 이상의 나노 결정-미생물 유인 물질 복합체를 형성하는 단계; 상기 하나 이상의 나노 결정-미생물 유인 물질 복합체를 진단하고자 하는 시료 내에 넣고 혼합하여 상기 검출하고자 하는 하나 이상의 미생물과 하나 이상의 나노 결정-미생물 유인 물질의 접촉을 유도하는 단계; 상기 나노 결정-미생물 유인 물질과 접촉한 미생물을 포획하는 단계; 및 상기 포획된 미생물에 접촉한 상기 나노 결정-미생물 유인 물질 복합체로부터 포집된 나노 결정에 대응하는 고유한 전류 피크를 측정하는 단계를 포함하는 미생물 검출 방법을 제공한다. A fourth aspect of the present invention provides a method for detecting a microorganism using a nanocrystalline label, comprising: determining at least one microorganism to be detected and at least one microorganism attractant capable of attracting microorganisms; One or more nanocrystals are selected from the group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese, and bismuth and combined with each of the one or more microbial attractants to form one or more nanocrystal-microbe attractant complexes. Forming; Incorporating the at least one nanocrystal-microbe attractant complex into a sample to be diagnosed and mixing to induce contact of the at least one microcrystal to be detected with the at least one nanocrystal-microbe attractant; Capturing microorganisms in contact with the nanocrystal-microbe attractant material; And measuring an inherent current peak corresponding to the nanocrystals collected from the nanocrystal-microbe attractant material complex in contact with the captured microorganisms.
상기 나노 결정은 메탈 설파이드 형이고, 아연, 카드뮴, 납 및 구리로 이루어진 메탈 군으로부터 하나 이상 선택되고, 상기 미생물 유인 물질은 말토즈(maltose), 아스파테이트(aspartate), 카사미노산(casamino acid), 시스테인(cysteine), 류신(leucine) 또는 세린(serine)으로 이루어진 그룹으로부터 하나 이상 선택되는 미생물 검출방법이다.The nanocrystal is a metal sulfide type, at least one selected from the group consisting of zinc, cadmium, lead, and copper, and the microorganism attractant material is maltose, aspartate, casamino acid, It is a microorganism detection method selected from the group consisting of cysteine, leucine or serine.
상기 유인 미생물 포획은 선택적 반투과막을 통해 분리하는 것을 특징으로 하는 미생물 검출 방법이다.The attracting microorganism capture is a microbial detection method characterized in that the separation through the selective semi-permeable membrane.
본 발명에서는 나노 결정 표지의 검출 방법으로 전기 화학적 분석 방법을 이용한다. 본 발명에서 사용되는 전기 화학적 분석 방법은 수용액 상에서 수행되어, 전위, 전류, 전기 전도도, 임피던스, 커패시턴스 또는 저항등을 측정하는 것으로서, 소형화 및 신속한 신호처리가 가능하여 소규모 어레이 분석에 유용하다. In the present invention, an electrochemical analysis method is used as a method for detecting a nanocrystalline label. The electrochemical analysis method used in the present invention is carried out in an aqueous solution to measure the potential, current, electrical conductivity, impedance, capacitance or resistance, etc., which enables miniaturization and rapid signal processing and is useful for small array analysis.
본 발명는 전기 화학적 분석 방법 중에서 특히 스퀘어 웨이브 음전극 스트리핑 전압법을 이용하였다. 본 발명에서 사용된 스트리핑 전압법은 크게 두 단계로 구성된다. 먼저, 나노 결정으로 표지된 미생물 유인 물질들을 소정의 수용액에 넣고, 전극을 위치시키고, 전극을 통해 특정 전위를 인가한다. 인가된 전위에 따라 나노 결정 금속들은 해당 전극 방향으로 이동하여 해당 전극 표면에 포집된다. 다음으로, 해당 전극에 포집된 나노 결정 금속에 소정의 전위를 가하여 특정 전류를 도통하게 한다. 이때, 각 나노 결정 금속들은 산화 및 환원 반응에 의해 각 나노 결정의 금속의 종류에 따라 특정 피크의 전류를 발생하게 되는데, 이러한 특정 피크의 전류를 측정하여, 나노 결정 표지의 존재 및 그 농도를 측정한다. The present invention particularly employs the square wave negative electrode stripping voltage method among electrochemical analysis methods. The stripping voltage method used in the present invention is largely composed of two steps. First, the microorganism attracting materials labeled with nanocrystals are placed in a predetermined aqueous solution, the electrode is placed, and a specific potential is applied through the electrode. Depending on the applied potential, the nanocrystalline metals move in the direction of the electrode and are collected on the surface of the electrode. Next, a predetermined electric potential is applied to the nanocrystalline metal collected on the electrode to conduct a specific current. At this time, each of the nanocrystalline metals generate a current of a specific peak according to the type of metal of each nanocrystal by the oxidation and reduction reaction, by measuring the current of this particular peak, the presence and concentration of the nanocrystalline label is measured do.
본 발명에 따른 스트리핑 전압법에 의한 나노 결정 표지 검출 방법은 피코몰(picomolar) 수준의 높은 측정 한계를 제공한다. 이러한 측정한계는 본 발명에 따른 고순도의 나노 결정을 사용함으로써 달성된다. 또한, 본 발명에 따른 나노결정 입자의 크기를 촉매적으로 조절하여 센서 신호의 민감도를 월등히 향상시키는 것이 가능하다. The nanocrystal label detection method by stripping voltage method according to the present invention provides a high measurement limit of picomolar levels. These measurement limits are achieved by using high purity nanocrystals according to the present invention. In addition, it is possible to significantly improve the sensitivity of the sensor signal by catalytically adjusting the size of the nanocrystalline particles according to the present invention.
본 발명에 따른 나노 결정을 이용하여 다수의 미생물을 동시에 검출하고자 하는 경우, 각 미생물에 대하여 각각 상이한 금속 나노 입자를 사용한다. 이 경우, 각 나노 결정은 금속의 종류에 따라 특정한 전류 피크를 나타내어, 다수의 미생물을 동시에 검출할 수 있게 한다. When a plurality of microorganisms are to be detected simultaneously using the nanocrystals according to the present invention, different metal nanoparticles are used for each microorganism. In this case, each nanocrystal exhibits a specific current peak depending on the type of metal, allowing the detection of multiple microorganisms simultaneously.
이와 같이 본 발명에서 적용된 음전극 스트리핑을 이용하는 전압 스트리핑법에서는 나노 결정으로서 전위 금속을 이용한 나노 입자들이 선택적 분석이 용이한 전류 피크 신호를 도출하였다. 본 발명에 적합한 나노 입자로서 적합한 전위 금속의 선정은 음극 전압 구간에서 서로 중첩되지 않으며 현저한 전류 피크를 갖는 금속을 선정하였다. 음전극 스트리핑 법을 이용하기 위해서는 양극성 전위 금속이 바람직하였다. 그러나, 양전극 스트리핑 법을 이용하는 경우, 음극성 전위 금속이 바람직하게 사용할 수 있게 된다. As described above, in the voltage stripping method using the negative electrode stripping applied in the present invention, the nanoparticles using the dislocation metal as the nanocrystals derived a current peak signal for easy selective analysis. Selection of a potential metal suitable as a nanoparticle suitable for the present invention selected a metal having a significant current peak without overlapping each other in the cathode voltage section. In order to use the negative electrode stripping method, a bipolar potential metal is preferable. However, in the case of using the positive electrode stripping method, the negative electrode potential metal can be preferably used.
이렇게 양극 및 음극을 동시에 이용하여 각 전극마다 고유의 전류 피크를 생성하는 나노 금속을 이용하는 경우 더욱 많은 금속을 나노 결정으로 이용할 수 있게 된다. 결과적으로 더 많은 수의 미생물을 동시에 검출하는 것이 가능하게 된다. In the case of using a nano metal that uses a positive electrode and a negative electrode to generate a unique current peak for each electrode, more metals can be used as nano crystals. As a result, it is possible to detect a larger number of microorganisms simultaneously.
이렇게 수득된 전류 피크는 아날로그 디지털 변환부에서 소정의 디지털 변환 방법에 따라 디지털화될 수 있다. 구체적으로, 수득된 아날로그 타입의 전류 피크는 샘플링되어 대응하는 디지털 신호로 출력된다. 디지털 변환 방법으로 전류 피크 신호들의 신호 값의 대입을 통한 디지털화(digitization) 과정과 노말리제이션(normalization) 과정을 포함한다. 디지털화는 통계적 다봉 분할(statistical optimal threshold)과 피스와이스 선형 인터폴레이션(piecewise linear interpolation)에 의해 수행된다. The current peak thus obtained can be digitized in accordance with a predetermined digital conversion method in the analog-digital converter. Specifically, the obtained analog type current peak is sampled and output as a corresponding digital signal. The digital conversion method includes a digitization process and a normalization process by substituting signal values of current peak signals. Digitization is performed by statistical optimal threshold and piecewise linear interpolation.
그 결과 획득된 전류 피크 신호들은 그 크기와 트렌드에 따라 디지털 신호로 변환되어, 유선 또는 무선 통신 수단을 통해 원격지로 전송되거나, 소정의 디지털 문자로 저장될 수 있다. 이러한 디지털 문자의 구체적인 예로서 바코드를 들 수 있다.The resulting current peak signals can be converted into digital signals according to their magnitude and trend, and transmitted to a remote location via wired or wireless communication means, or stored as predetermined digital characters. Specific examples of such digital characters include bar codes.
이하, 실시예에 의하여 본 발명을 더욱 상세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail with reference to Examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
< 실시예 1 > M(Zn, Cd, Pb)-HDX (C16 xanthate) 칼륨(potassium) 염의 제조 Example 1 Preparation of M (Zn, Cd, Pb) -HDX ( C 16 xanthate) Potassium Salts
나노결정입자들의 안정적 캡핑 물질인 헥사데실 싼테이트(Hexadecyl xanthate:HDX)는 다음과 같은 절차로 합성되었다. 금속 싼테이트 염(metal xanthates salts:M-HDX, M= Zn, Cd, Pb)은 도 1의 방법을 따랐다. 혼합된 동일 농도(0.04 몰)의 헥사데칸올(hexadecanol) 9.69 g과 수산화 칼륨(KOH) 2.24 g 이 혼합된 용액을 전부 용해될 때까지 150℃의 온도에서 가열시켰다. 얻어진 수용액은 100℃의 온도에서 25ml의 톨루엔(toluene)에 넣고 균일하게 교반하였다. 카본 디설파이드(carbon disulfide) 4.41g을 상온에서 미량으로 조금씩 첨가하면 두꺼운 노란색용액이 얻어지며, 이를 냉각시킨후 다시 1 시간동안 강하게 교반시키고, 용액은 100 ml의 페트로리움 에테르(petroleum ether)에 넣고 2 시간동안 추가교반을 실시한다. 최종 산물은 유리 필터(glass funnel)로 걸러지고 에테르로 여러 번 세척된 후 모아졌다. 싼테이트는 진공 오븐에서 완전히 건조시키고 다시 20 ml의 차가운 증류수로 세척한후 다시 필터시키고 다시 건조시키며 에테르로 세척된다. 그러한 최종 HDX는 메탄올로 3번 세척되고 다시 건조된다. Hexadecyl xanthate (HDX), a stable capping material of nanocrystalline particles, was synthesized by the following procedure. Metal xanthates salts (M-HDX, M = Zn, Cd, Pb) followed the method of FIG. 1 . The mixed solution of 9.69 g of hexadecanol and 2.24 g of potassium hydroxide (KOH) at the same concentration (0.04 mol) mixed was heated at a temperature of 150 ° C until all of the solution was dissolved. The obtained aqueous solution was put into 25 ml of toluene at the temperature of 100 degreeC, and it stirred uniformly. 4.41 g of carbon disulfide is added in small portions at room temperature to obtain a thick yellow solution. After cooling, the solution is stirred vigorously for 1 hour, and the solution is added to 100 ml of petroleum ether. Carry out additional stirring during the time. The final product was filtered through a glass funnel, washed several times with ether and collected. The acetate is completely dried in a vacuum oven, washed again with 20 ml of cold distilled water, filtered again, dried and washed with ether. Such final HDX is washed three times with methanol and dried again.
< 실시예 2 > 원팟(One-pot) 고순도 메탈 썰파이드 MS(M=Zn, Cd, Pb) 나노결정입자의 합성 Example 2 Synthesis of One-pot High Purity Metal Sulfide MS (M = Zn, Cd, Pb) Nanocrystalline Particles
2.1. 금속 나노입자들의 전압서명(signature) 2.1. Voltage signature of metal nanoparticles
바이오 에세이의 높은 선택성을 고려한 반도체 나노입자 트레이서(tracer)의 비-오버래핑 풀(nonoverlapping pool)을 얻기위해, ZnS, PbS, CdS, InAs, GaAs 또는 CuS와 같은 이원 무기화합물(binary inorganic) 나노입자들을 선별하였다. Cd-In 과 In-Pb 피크쌍의 중첩된 신호를 제외한 전기 코드들은 확연히 구별될 수 있다. 따라서 본 발명서는 ZnS 및 CuS 나노입자를 도입하게 되었다. Binary inorganic nanoparticles, such as ZnS, PbS, CdS, InAs, GaAs or CuS, can be used to obtain a non-overlapping pool of semiconductor nanoparticle tracers that account for the high selectivity of bioassays. Screened. The electrical codes can be clearly distinguished except for overlapping signals of Cd-In and In-Pb peak pairs. Accordingly, the present invention introduces ZnS and CuS nanoparticles.
2.2. 고순도 메탈 썰파이드 나노결정입자들의 합성2.2. Synthesis of High Purity Metal Sulfide Nanocrystalline Particles
고순도 메탈 썰파이드 나노결정입자들의 합성과 표면안정화는 다음과 같은 방법으로 수행되었다. 요약하면, 메탈 알킬 싼테이트(xanthate) 황화 전구체의 저온(thermal) 침적(decomposition)법에 근간한 합성법으로 이뤄졌다(도 1). HDX 3.56 g, 5 ml의 메탄올이 동일량의 몰의(equimolar) CdCl2, PbCl2, ZnCl2 수용액 각각에 2분간 반응된다. 용액은 원심분리로 상층액은 제거되고 최종 M-HDX은 세번정도 세척되고 메탄올로 세척 후 진공 오븐에서 건조된다. 모든 나노 전위금속입자들은 강력한 전자 기여 모노써펙턴트(monosurfactant)로써 알킬 아민 도판트(alkyl amine dopants)인 헥사데실아민(decylamine:HDA), 데실아민(decylamine:DA), 트리옥틸아민(trioctylamine:TOA) 0.5 g씩을 캡핑 안정화 및 나노결정들의 유기용매목적으로 Zn-HDX, Cd-HDX, Pb-HDX에 각각 혼합시켰다. 먼저 알킨 아민도판트들은 각각 120℃까지 가열되고 50℃까지 냉각된 후, 균일하게 교반시키는 동안 각 금속-HDX 분말 0.05g이 첨가된다. 그리고 난 후, 가열온도는 100℃에서 30분간 교반된후, 다시 온도를 조금씩 120℃까지 올리고 1.5시간동안 반응을 지속시킨다. 반응종료 전, 140℃에서 2분간 최종반응을 마친 후, 천천히 70℃까지 온도를 낮춘다. 얻어진 최종 금속 결정입자들은 각각 흰색의 ZnS, 황색의 CdS, 그리고 검정색의 PbS이며, 이들은 메탄올에 의해 프로컬레이션(flocculation)되어 손쉬운 추출을 위해 시험관바닥에 침전된다. 최종입자들이 깨끗이 정제될때 까지 상층액 제거과정이 여러번 수행되고 상온에서 바짝 건조되어 고운 분말형태의 최종 나노결정입자가 얻어진다.Synthesis and surface stabilization of high purity metal sulfide nanocrystal grains were carried out as follows. In summary, the synthesis was based on the thermal deposition of metal alkyl xanthate sulfide precursors.1). 3.56 g of HDX, 5 ml of methanol equally equimolar in CdCl2, PbCl2, ZnCl2 React with each aqueous solution for 2 minutes. The solution is centrifuged to remove the supernatant, the final M-HDX is washed three times, washed with methanol and dried in a vacuum oven. All nanopotential metal particles are strong electron contributing monosurfactants, which are alkyl amine dopants, decylamine (HDA), decylamine (DA) and trioctylamine (TOA). ) 0.5 g each were mixed with Zn-HDX, Cd-HDX, Pb-HDX, respectively, for capping stabilization and organic solvents of the nanocrystals. The alkyne amine dopants are first heated to 120 ° C. and cooled to 50 ° C., respectively, and 0.05 g of each metal-HDX powder is added during uniform stirring. Then, the heating temperature was stirred for 30 minutes at 100 ° C, and then the temperature was gradually raised to 120 ° C and the reaction was continued for 1.5 hours. After completion of the reaction at 140 ° C. for 2 minutes before completion of the reaction, slowly lower the temperature to 70 ° C. The final metal crystal grains obtained were white ZnS, yellow CdS, and black PbS, which were flocculated with methanol and precipitated at the bottom of the tube for easy extraction. The supernatant removal process is carried out several times and the final nanocrystal particles in the form of fine powder are obtained until the final particles are purified.
< 실시예 3 > 메탈 썰파이드 MSQD 나노결정-미생물유인물질 복합체의 제조 Example 3 Preparation of Metal Sulfide MSQD Nanocrystal-Microbe Attractant Complex
나노결정-미생물 유인물질 복합체는 다음의 방법대로 수행되었다(도 1). 매우 균일한 나노 결정 입자들은 다이티올쓰레이톨(dithiolthreitol:DTT)에 의해 수산화(hydroxylated)되고 카보닐 다이이밀다졸(1,1-carbonyl diimidazole:CDI)으로 활성화(activate)되었다. 100㎕의 CdS, PbS, ZnS 나노결정입자들은 미생물 유인물질과 각각 반응 되어진다. 최종수용액 pH는 8.5까지 1M NaOH로 조절되었고, 상온에서 24시간 동안 교반 반응된다. 최종 수용성 중합체들은 0.1 M PBS(pH 7.4, 0.05% Tween 20)에 디스퍼스되었다.Nanocrystal-microbe attractant complexes were performed according to the following method ( FIG. 1 ). Highly uniform nanocrystalline particles were hydroxylated by dithiolthreitol (DTT) and activated with carbonyl diimidazole (CDI). 100 μl of CdS, PbS, and ZnS nanocrystal particles are reacted with microorganism attractant. The final aqueous solution pH was adjusted to 1M NaOH to 8.5, and stirred at room temperature for 24 hours. The final water soluble polymers were dispersed in 0.1 M PBS (pH 7.4, 0.05% Tween 20).
< 실시예 4 > 나노결정-미생물 유인물질 복합체에 의한 미생물 포획 및 검출 Example 4 Microbial Capture and Detection by Nanocrystal-Microbe Attractant Complexes
도 3 내지 도 6은 나노결정-미생물 유인물질 복합체 의한 미생물 포획 및 검출 방법을 구체적으로 설명한다. 3 to 6 specifically illustrate the microbial capture and detection method by the nanocrystal-microbe attractant complex.
도 3은 마이크로칩 타입의 정량분석 카트리지 센서 모듈로서, 미생물을 검출하고자 하는 샘플용액을 떨어뜨리면 완충용액층을 따라 선택적 반투과막(Semi-permeable membrane)에 도달하게 된다. 선택적 반투과막은 미생물만 선택적으로 투과하게 되며, 투과된 미생물들은 나노결정-미생물 유인물질 복합체와 접촉하게 된다. 이 과정에서 미생물 체내에 유인되지 못한 나노결정-미생물 유인물질 복합체들은 마이크로 밸브로 조절되는 배출구(drain)를 통해 배출된다. 이렇게 포획된 미생물에 접촉한 나노 결정-미생물 유인 물질 복합체로부터 포집된 나노 결정에 대응하는 고유한 전류 피크를 측정하여 정량분석 하였다. 3 is a microchip type quantitative cartridge sensor module, in which a sample solution to detect microorganisms is dropped to reach a selective semi-permeable membrane along the buffer solution layer. The selective semipermeable membrane selectively transmits only the microorganisms, and the microorganisms are brought into contact with the nanocrystal-microbe attractant complex. In this process, nanocrystal-microbe attractant complexes which are not attracted to the microorganism body are discharged through a drain controlled by a microvalve. The unique current peaks corresponding to the nanocrystals collected from the nanocrystal-microbe attractant complexes in contact with the captured microorganisms were measured and quantitatively analyzed.
도 4는 마이크로 실린더 타입의 정량분석 카트리지 센서 모듈로서, 미생물을 검출하고자 하는 샘플용액이 얇은 피펫 팁을 통과하면 선택적 반투과막(Semi-permeable membrane)에 도달하게 된다. 선택적 반투과막은 미생물만 선택적으로 투과하게 되며, 투과된 미생물들은 나노결정-미생물 유인물질 복합체와 접촉하게 된다. 이 과정에서 미생물 체내에 유인되지 못한 나노결정-미생물 유인물질 복합체들은 모세관 분리채널을 통해 분리된다. 이렇게 포획된 미생물에 접촉한 나노 결정-미생물 유인 물질 복합체로부터 포집된 나노 결정에 대응하는 고유한 전류 피크를 측정하여 정량분석 하였다. Figure 4 is a micro-cylinder type quantitative cartridge sensor module, when the sample solution to detect the microorganism passes through a thin pipette tip to reach a selective semi-permeable membrane (Semi-permeable membrane). The selective semipermeable membrane selectively transmits only the microorganisms, and the microorganisms are brought into contact with the nanocrystal-microbe attractant complex. In this process, nanocrystal-microbe attractant complexes which are not attracted to the microorganism body are separated through the capillary separation channel. The unique current peaks corresponding to the nanocrystals collected from the nanocrystal-microbe attractant complexes in contact with the captured microorganisms were measured and quantitatively analyzed.
도 5는 단순속성 분리를 위한 마이크로 주사기 타입의 카트리지 센서 모듈로서, 미생물을 검출하고자 하는 샘플용액에 나노결정-미생물 유인물질 복합체를 첨가하고 30분 내지 1시간 동안 미생물과 나노결정-미생물 유인물질 복합체가 접촉하도록 한다. 그 후 주사기 형태의 피스톤으로 이를 흡입한다. 흡입된 나노결정-미생물 유인물질 복합체가 포함된 샘플용액이 100nm 이하의 범용필터를 통과하는 과정을 거치면 미생물 체내에 유인되지 못한 나노결정-미생물 유인물질 복합체들은 배출된다. 필터 내부에 포획된 미생물 체내의 나노 결정-미생물 유인 물질 복합체로부터 포집된 나노 결정에 대응하는 고유한 전류 피크를 측정하여 정량분석 하였다. 5 is a micro-syringe type cartridge sensor module for simple attribute separation, wherein the microcrystal-microbe attractant complex is added to a sample solution to detect microorganisms, and the microorganism and nanocrystal-microbe attractant complex for 30 minutes to 1 hour. To make contact. It is then aspirated by a piston in the form of a syringe. When the sample solution containing the inhaled nanocrystal-microbe attractant complex passes through a general-purpose filter of 100 nm or less, nanocrystal-microbe attractant complexes that are not attracted to the microorganism body are discharged. Intrinsic current peaks corresponding to the nanocrystals collected from the nanocrystal-microbe attractant complexes in the microorganism trapped inside the filter were measured and quantitatively analyzed.
도 6은 속성분리 및 정량분석을 위한 마이크로 주사기 타입의 정량분석 카트리지 센서 통합 모듈로서, 미생물을 검출하고자 하는 샘플용액에 나노결정-미생물 유인물질 복합체를 첨가하고 30분 내지 1시간 동안 미생물과 나노결정-미생물 유인물질 복합체가 접촉하도록 한다. 그 후 주사기 형태의 피스톤으로 이를 흡입한다. 흡입된 나노결정-미생물 유인물질 복합체가 포함된 샘플용액이 100nm 이하의 범용필터를 통과하는 과정을 거치면 미생물 체내에 유인되지 못한 나노결정-미생물 유인물질 복합체들은 배출된다. 필터 내부에 포획된 나노 결정-미생물 유인 물질 복합체를 포함한 미생물을 필터로부터 분리한 후, 이를 주사기 형태의 피스톤으로 재흡입하면 피스톤 내부에 삽입된 음전극 스트리핑 전압(anodic stripping voltammetric)을 통하여 미생물 체내의 나노 결정-미생물 유인 물질 복합체로부터 포집된 나노 결정에 대응하는 고유한 전류 피크를 측정하여 정량분석 하였다. FIG. 6 is a micro syringe type quantitative cartridge sensor integrated module for attribute separation and quantitative analysis, in which a microcrystal-microbe attractant complex is added to a sample solution to detect microorganisms and microorganisms and nanocrystals for 30 minutes to 1 hour. -Contact the microbe attractant complex. It is then aspirated by a piston in the form of a syringe. When the sample solution containing the inhaled nanocrystal-microbe attractant complex passes through a general-purpose filter of 100 nm or less, nanocrystal-microbe attractant complexes that are not attracted to the microorganism body are discharged. After the microorganisms containing the nanocrystal-microbe attractant complex trapped inside the filter are separated from the filter and re-inhaled by the syringe-type piston, the nanoparticles in the microorganism body are provided through an anode stripping voltammetric inserted into the piston. Inherent current peaks corresponding to nanocrystals collected from the crystal-microbe attractant complexes were measured and quantified.
<실시예 5 > 전기화학적 다중 분석법Example 5 Electrochemical Multiplex Assay
스퀘어 웨이브 음전극 스트리핑 전압(Square wave anodic stripping voltammetric:SWASV)법에 따른 분석은 1.5ml glass cell에서 2x4mm 스크린 프린팅 카본 작업전극(screen-printed carbon (Acheson-ink) working electrodes)과 Ag/AgCl 기준(reference) 전극(CH Instruments, Austin, TX), 그리고 백금 전극을 카운터 전극으로 이용하였다.Analysis based on the square wave anodic stripping voltammetric (SWASV) method was performed using a 2x4 mm screen-printed carbon (Acheson-ink) working electrode and Ag / AgCl reference on 1.5 ml glass cells. ) Electrodes (CH Instruments, Austin, TX), and platinum electrodes were used as counter electrodes.
스퀘어 웨이브 음전극 스트리핑 전압법(SWASV)은 수은(Mercury)II 이온 또는 비스무스(Bismuth) 이온이 코팅된 스크린 프린팅 카본 페이스트 전극을 이용하여 수행되었다. QD 나노입자들을 질산수용액에 녹인후, 1분간 전처리(pretreatment) (0.6 V)하고, -1,4V에서 2분 축적(accumulation) 과정을 수행한다. 사용된 버퍼는 1ml의 0.1M 아세테이트(acetate) 버퍼(pH 4.5)이다. 스트리핑은 5초 휴지기(rest period (without stirring)) 후에 실행되어졌다. 구체적인 기기운용변수들은 -1.2 V ~ 0.12 V의 전위범위에서 스텝 전위(step potential) 50 mV, 전폭(amplitude) 20 mV 그리고 주파수(frequency) 25 Hz이다.    Square wave negative electrode stripping voltammetry (SWASV) was performed using a screen printing carbon paste electrode coated with Mercury II ions or Bismuth ions. After QD nanoparticles are dissolved in an aqueous solution of nitric acid, pretreatment (0.6 V) is performed for 1 minute, and a 2 minute accumulation process is performed at -1,4V. The buffer used is 1 ml 0.1 M acetate buffer (pH 4.5). Stripping was performed after a 5 second rest period (without stirring). Specific operating parameters are step potential 50 mV, amplitude 20 mV and frequency 25 Hz in the potential range of -1.2 V to 0.12 V.
본 발명의 나노 결정-미생물 유인물질 복합체를 이용한 미생물 검출방법 및 검출키트는 각 나노 결정의 금속에 고유한 전류 피크를 해석하여 검출하고자 하는 미생물을 간편하고, 정량적으로 정확하게 측정하는 것이 가능하다. 또한 종래의 미생물 바이오센서들은 타겟 미생물들에 대한 선택성이 매우 제한적이며 특이 항체나 수용체로써 미생물 표면의 항원을 인지하는 단백질-면역화학법을 벗어나기 어려운데, 본 발명은 생물학적 주화성(chemo-taxis)을 이용하여 다양한 종류의 세균을 간편하게 검출하고 정량화할 수 있다. 또한 지그비(Zigbee) 및 무선주파수인식(Radio Frequency Identification; RFID) 등의 IT기술이 접목된 대중적 미생물진단장치를 개발함으로써 수질오염원의 조기 속성분석과 표준경보체제의 실현 및 선진 국민 건강 안전의 초석을 제공할 수 있다.The microorganism detection method and detection kit using the nanocrystal-microbe attractant complex of the present invention can easily and quantitatively accurately measure the microorganism to be detected by analyzing the current peak inherent in the metal of each nanocrystal. In addition, conventional microbial biosensors have very limited selectivity for target microorganisms and are difficult to escape from protein-immunochemistry that recognizes antigens on the surface of microorganisms as specific antibodies or receptors. Various types of bacteria can be easily detected and quantified. In addition, by developing popular microbiological diagnostic devices incorporating IT technologies, such as Zigbee and Radio Frequency Identification (RFID), early analysis of water pollution sources, realization of standard alarm system and the cornerstone of advanced national health and safety Can provide.

Claims (39)

  1. 아연, 카드늄, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 메탈 군으로부터 하나 이상 선택되는 나노 결정; Nanocrystals selected from at least one metal group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth;
    미생물 유인 물질; 및Microbial attractants; And
    일측에 상기 나노 결정과 결합 가능한 전하 특성의 치환기를 가지며, 반대측에 복수의 수용성 치환기를 갖는 고분자 사슬을 가지며, 일측의 치환기를 통해 상기 나노 결정과 결합하고, 반대측의 복수의 수용성 치환기를 통해 상기 나노 결정을 안정화하고, 상기 복수의 수용성 치환기를 통해 상기 미생물 유인 물질과 결합을 구성하는 결합 안정화 물질을 포함하는 나노 결정-미생물 유인 물질 복합체. One side has a substituent having a charge characteristic capable of binding to the nanocrystals, the other side has a polymer chain having a plurality of water-soluble substituents, and combines with the nanocrystals through a substituent on one side, the nano through a plurality of water-soluble substituents on the opposite side A nano-crystal-microbe attractant complex comprising a bond stabilizing material that stabilizes crystals and forms a bond with the microbe attractant material through the plurality of water soluble substituents.
  2. 제1항에 있어서, The method of claim 1,
    상기 나노결정은 메탈 설파이드 형인 나노 결정-미생물 유인 물질 복합체. The nanocrystals are metal sulfide type nano crystal-microbe attractant material complex.
  3. 제1항에 있어서, The method of claim 1,
    상기 나노 결정은 아연, 카드뮴, 납 및 구리로 이루어진 메탈 군으로부터 하나 이상 선택되는 나노 결정-미생물 유인 물질 복합체. The nanocrystals are nanocrystalline-microbial attractant composites selected from at least one metal group consisting of zinc, cadmium, lead and copper.
  4. 제1항에 있어서, The method of claim 1,
    상기 나노 결정은 둘 이상의 나노 결정이 결합하여 형성된 나노 결정 복합체를 추가 포함하는 나노 결정-미생물 유인 물질 복합체. The nano-crystal further comprises a nano-crystal complex formed by combining two or more nano-crystals nano-crystal-microbe attractant material complex.
  5. 제1항에 있어서, The method of claim 1,
    상기 미생물 유인 물질은 말토즈(maltose), 아스파테이트(aspartate), 카사미노산(casamino acid), 시스테인(cysteine), 류신(leucine) 또는 세린(serine)으로 이루어진 그룹으로부터 하나 이상 선택되는 나노 결정-미생물 유인 물질 복합체. The microorganism attractant is nanocrystal-microorganism selected from the group consisting of maltose, aspartate, casamino acid, cysteine, leucine or serine. Attractant complex.
  6. 제1항에 있어서, The method of claim 1,
    상기 결합 안정화 물질이 다이티올쓰레이톨(dithiolthreitol) 또는 다이하드로 리포익산(dihydrolipoic acid)를 포함하는 나노 결정-미생물 유인 물질 복합체. The nano-crystal-microbe attractant complex, wherein the binding stabilizing material comprises dithiolthreitol or dihydrolipoic acid.
  7. 제1항에 있어서, The method of claim 1,
    상기 결합 안정화 물질과 상기 미생물 유인 물질과의 결합이 카바메이트(carbamate) 결합인 나노 결정-미생물 유인 물질 복합체. The nano crystal-microbe attractant complex of the bond stabilizing material and the microorganism attractant is a carbamate bond.
  8. 미생물 유인 물질을 표지하는 나노 결정-미생물 유인 물질 복합체 제조 방법에 있어서, In the method for producing a nano crystal-microbial attractant complex for labeling a microorganism attractant,
    헥사데칸올, 수산화 칼륨 및 카본 디설파이드를 반응시켜, 헥사데실싼테이트(hexadecyl xanthate; 이하 'HDX'라 함) 칼륨 염을 제조하는 단계;Reacting hexadecanol, potassium hydroxide and carbon disulfide to prepare a hexadecyl xanthate potassium salt (hereinafter referred to as 'HDX');
    아연, 카드늄, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 메탈 군으로부터 하나 이상 선택되는 나노 결정을 수득된 HDX 칼륨 염과 반응시켜 HDX 메탈 설파이드 나노 결정을 제조하는 단계;Reacting nanocrystals selected from at least one metal group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth with the obtained HDX potassium salt to produce HDX metal sulfide nanocrystals;
    상기 HDX 메탈 설파이드 나노 결정에 알킬 아민 도판트를 반응시켜, 메탈 설파이드 나노 결정을 제조하는 단계;Reacting the HDX metal sulfide nanocrystals with an alkyl amine dopant to prepare metal sulfide nanocrystals;
    상기 메탈 설파이드 나노 결정에 결합 안정화 물질을 반응시켜, 메탈 설파이드 나노 결정에 결합 안정화 물질을 결합시키는 단계;Coupling a bond stabilizing material to the metal sulfide nanocrystals by reacting a bond stabilizing material to the metal sulfide nanocrystals;
    상기 결합된 메탈 설파이드 나노 결정-결합 안정화 물질 복합체에 활성화 물질을 반응시켜, 메탈 설파이드 나노 결정-결합 안정화 물질 복합체를 활성화시키는 단계; 및Activating a metal sulfide nano crystal-bond stabilizer material complex by reacting an activated material with the bonded metal sulfide nano crystal-bond stabilizer material complex; And
    상기 활성화된 메탈 설파이드 나노 결정-결합 안정화 물질 복합체에 미생물 유인물질을 결합하는 단계를 포함하는 나노 결정-미생물 유인물질 복합체 제조 방법.A method of manufacturing a nano crystal-microbe attractant complex, comprising coupling a microorganism attractant to the activated metal sulfide nano crystal-binding stabilizing material complex.
  9. 제8항에 있어서, The method of claim 8,
    상기 나노 결정은 아연, 카드뮴, 납 및 구리로 이루어진 메탈 군으로부터 하나 이상 선택되는 나노 결정-미생물 유인물질 복합체 제조 방법. The nanocrystals are one or more selected from the group of metals consisting of zinc, cadmium, lead and copper nanocrystal-microbe attractant composite.
  10. 제8항에 있어서, The method of claim 8,
    상기 HDX 칼륨 염 제조 단계는 The HDX potassium salt manufacturing step
    상기 헥사데칸올과 상기 수산화 칼륨을 혼합하고, 혼합된 용액이 모두 용해될 때까지 가열하는 단계;Mixing the hexadecanol and the potassium hydroxide and heating until all of the mixed solution is dissolved;
    상기 혼합 용액을 톨루엔에 넣고 균일하게 교반하고, 상기 카본 디설파이드를 첨가하는 단계;Putting the mixed solution into toluene, stirring uniformly, and adding the carbon disulfide;
    상기 혼합 용액을 페트로리움 에테르에 넣고 추가 교반하는 단계; 및Placing the mixed solution in petroleum ether and further stirring; And
    상기 혼합 용액을 유리 필터로 필터링하고, 에테르로 세척하는 과정을 반복하는 단계를 포함하는 나노 결정-미생물 유인물질 복합체 제조 방법. Filtering the mixed solution with a glass filter and repeating the process of washing with ether.
  11. 제8항에 있어서,The method of claim 8,
    상기 알킬 아민 도판트는 헥사 데실 아민, 데실 아민 및 트리 옥틸 아민으로 이루어지는 군에서 하나 이상 선택되는 나노 결정-미생물 유인물질 복합체 제조 방법. Wherein said alkyl amine dopant is at least one selected from the group consisting of hexadecyl amine, decyl amine, and trioctyl amine.
  12. 제11항에 있어서,The method of claim 11,
    HDX 아연 설파이드 나노 결정 또는 HDX 카드뮴 설파이드 나노 결정에 대해서는 상기 알킬 아민 도판트로 헥사 데실 아민을 사용하고, For the HDX zinc sulfide nanocrystals or HDX cadmium sulfide nanocrystals, hexadecyl amine is used as the alkyl amine dopant,
    HDX 납 설파이드 나노 결정에 대해서는 상기 알킬 아민 도판트로 데실 아민 또는 트리 옥틸 아민을 사용하고, For the HDX lead sulfide nanocrystals, the above alkyl amine dopantrodecyl amine or trioctyl amine is used,
    HDX 구리 설파이드 나노 결정에 대해서는 상기 알킬 아민 도판트로 헥사 데실 아민 또는 트리 옥틸 아민을 사용하는 나노 결정-미생물 유인물질 복합체 제조 방법. A method for preparing a nano crystal-microbe attractant complex using hexadecyl amine or trioctyl amine as the alkyl amine dopant for HDX copper sulfide nano crystals.
  13. 제8항에 있어서, The method of claim 8,
    상기 미생물 유인 물질은 말토즈(maltose), 아스파테이트(aspartate), 카사미노산(casamino acid), 시스테인(cysteine), 류신(leucine) 또는 세린(serine)으로 이루어진 그룹으로부터 하나 이상 선택되는 나노 결정-미생물 유인 물질 복합체 제조방법. The microorganism attractant is nanocrystal-microorganism selected from the group consisting of maltose, aspartate, casamino acid, cysteine, leucine or serine. Method for preparing attractant complex.
  14. 제8항에 있어서, The method of claim 8,
    상기 결합 안정화 물질이 다이티올쓰레이톨(dithiolthreitol) 또는 다이하드로 리포익산(dihydrolipoic acid)를 포함하는 나노 결정-미생물 유인 물질 복합체 제조방법. The method of manufacturing a nano crystal-microbe attractant complex, wherein the binding stabilizing material comprises dithiolthreitol or dihydrolipoic acid.
  15. 제8항에 있어서, The method of claim 8,
    상기 활성화 물질이 카보닐 다이이미다졸(1,1-carbonyl diimidazole)인 나노 결정-미생물 유인 물질 복합체 제조방법. The activating material is carbonyl diimidazole (1,1-carbonyl diimidazole) nanocrystal-microbe attractant material composite method.
  16. 제8항에 있어서, The method of claim 8,
    상기 결합 안정화 물질이 상기 활성화 물질로 인해 활성화되고, 활성화된 결합 안정화 물질이 상기 미생물 유인 물질과 결합을 구성하는 나노 결정-미생물 유인 물질 복합체 제조방법.And the binding stabilizing material is activated by the activating material, and the activated binding stabilizing material forms a bond with the microorganism attracting material.
  17. 제8항에 있어서, The method of claim 8,
    상기 결합 안정화 물질과 상기 미생물 유인 물질과의 결합이 카바메이트(carbamate) 결합인 나노 결정-미생물 유인 물질 복합체 제조방법. The method of manufacturing a nano crystal-microbial attractant complex of claim 1, wherein the binding stabilizing material and the microbial attractant are carbamate bonds.
  18. 나노 결정 표지를 이용하여 미생물을 검출하는 검출 키트에 있어서,In the detection kit for detecting a microorganism using a nano-crystal label,
    아연, 카드뮴, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 메탈군으로부터 하나 이상 선택되는 나노 결정, 상기 미생물을 유인하기 위한 미생물 유인 물질 및 일측으로 전하 특성의 치환기로 나노결정과 결합하며 반대측으로 복수의 수용성 치환기를 가지는 고분자 사슬로 상기 미생물 유인 물질과 결합하는 결합안정화물질을 포함하는 나노 결정-미생물 유인 물질 복합체;Nanocrystals selected from at least one metal group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth; A nano crystal-microbial attractant complex comprising a bond stabilizer that binds to and binds the microbial attractant to a polymer chain having a plurality of water-soluble substituents on the opposite side;
    유인 미생물 포획에 의한 상기 나노 결정-미생물 유인 물질 복합체의 나노입자와 대항되는 전하적 특성으로 포집하는 포집전극;A collecting electrode configured to capture charge characteristics opposed to nanoparticles of the nanocrystal-microbe attractant material complex by capturing microorganisms;
    포집된 상기 나노 결정에 대응하는 전류 피크를 아날로그로 측정하는 전류 피크 측정부를 포함하는 미생물 검출 키트. Microbial detection kit comprising a current peak measuring unit for measuring analog current peak corresponding to the collected nano-crystals.
  19. 제18항에 있어서, The method of claim 18,
    상기 나노 결정은 메탈 설파이드 형인 미생물 검출 키트. The nanocrystals are metal sulfide type microbial detection kit.
  20. 제18항에 있어서, The method of claim 18,
    상기 나노 결정은 아연, 카드뮴, 납 및 구리로 이루어진 메탈 군으로부터 하나 이상 선택되는 미생물 검출 키트. The nano-crystal is at least one microbial detection kit selected from the group consisting of metal, zinc, cadmium, lead and copper.
  21. 제18항에 있어서, The method of claim 18,
    상기 미생물 유인 물질은 말토즈(maltose), 아스파테이트(aspartate), 카사미노산(casamino acid), 시스테인(cysteine), 류신(leucine) 또는 세린(serine)으로 이루어진 그룹으로부터 하나 이상 선택되는 미생물 검출 키트. The microorganism attracting material is at least one selected from the group consisting of maltose, aspartate, casamino acid, casamino acid, cysteine, leucine or serine.
  22. 제18항에 있어서, The method of claim 18,
    상기 결합 안정화 물질이 다이티올쓰레이톨 또는 다이하드로 리포익산인 미생물 검출 키트. The microorganism detection kit, wherein the binding stabilizing material is lithipoic acid as dithiolthitol or dihard.
  23. 제18항에 있어서, The method of claim 18,
    상기 전류 피크 측정부에 의해 측정된 전류피크를 디지털 신호로 변환하기 위한 아날로그 디지털 변환부를 추가 포함하는 미생물 검출 키트. Microbial detection kit further comprises an analog-to-digital converter for converting the current peak measured by the current peak measuring unit into a digital signal.
  24. 제18항에 있어서, The method of claim 18,
    아연, 카드늄, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 메탈그룹에서 선택된 하나 이상의 각기 다른 나노 결정을 이용하여 제조된 각기 다른 나노결정-미생물 유인 물질 복합체를 사용하여 하나 이상의 미생물을 동시에 검출하는 미생물 검출 키트.One or more different nanocrystal-microbe attractant complexes prepared using one or more different nanocrystals selected from metal groups consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese and bismuth Microbial detection kit for detecting microorganisms at the same time.
  25. 제18항에 있어서, The method of claim 18,
    상기 유인 미생물 포획은 선택적 반투과막을 통해 분리하는 것을 특징으로 하는 미생물 검출 키트. The microorganism detection kit, characterized in that the attracting microorganism capture is separated through a selective semipermeable membrane.
  26. 제18항에 있어서, The method of claim 18,
    상기 나노 결정이 양이온 특성을 갖는 경우 상기 포집 전극에 소정의 음 전위를 인가하고, When the nanocrystals have a cationic property, a predetermined negative potential is applied to the collection electrode,
    상기 나노 결정이 음이온 특성을 갖는 경우 상기 포집 전극에 소정의 양 전위를 인가하는 미생물 검출 키트. The microorganism detection kit to apply a predetermined positive potential to the collection electrode when the nanocrystals have anionic properties.
  27. 제18항에 있어서, The method of claim 18,
    상기 전류 피크 측정부는 The current peak measuring unit
    상기 포집 전극에 포집된 상기 나노 결정에 소정의 전위를 인가하여, 상기 나노 입자를 산화/환원 반응시키고, A predetermined potential is applied to the nanocrystals collected on the collection electrode to oxidize / reduce the nanoparticles,
    상기 나노 결정의 산화/환원 반응으로부터 발생하는 각 나노 결정에 고유한 전류 피크를 측정하는 미생물 검출 키트. Microbial detection kit for measuring the current peak unique to each nanocrystal resulting from the oxidation / reduction reaction of the nanocrystals.
  28. 제27항에 있어서, The method of claim 27,
    상기 포집 전극에 소정의 음 전위 또는 양 전위를 인가하여, 각각 양이온 특성의 나노 결정 또는 음이온 특성의 나노 결정을 포집하고, A predetermined negative potential or a positive potential is applied to the collection electrode to collect nanocrystals having cationic properties or nanocrystals having anionic properties, respectively.
    상기 전류 피크 측정부는 포집된 상기 나노 결정으로부터 각각 발생하는 상기 나노 결정에 고유한 전류 피크를 각각 측정하는 미생물 검출 키트. And the current peak measuring unit measures a current peak unique to the nanocrystals, respectively, generated from the collected nanocrystals.
  29. 제23항에 있어서, The method of claim 23, wherein
    상기 디지털 신호를 해석하여 상기 디지털 신호에 대응하는 미생물의 정체 또는 검출된 미생물의 함량을 추정하는 디지털 신호 리더부를 추가 포함하는 미생물 검출 키트. And a digital signal reader unit for interpreting the digital signal to estimate the identity of the microorganisms corresponding to the digital signal or the content of the detected microorganisms.
  30. 제23항에 있어서, The method of claim 23, wherein
    상기 디지털 신호를 소정의 바코드로 변환하는 바코드 변환부를 추가 포함하는 미생물 검출 키트. Microbial detection kit further comprises a barcode conversion unit for converting the digital signal into a predetermined barcode.
  31. 제23항에 있어서, The method of claim 23, wherein
    상기 디지털 신호를 유선 또는 무선 통신을 통해 소정의 원격 진단부로 전송하고 상기 원격 진단부로부터 상기 디지털 신호의 해석 결과를 수신하는 통신부를 추가 포함하는 미생물 검출 키트. And a communication unit for transmitting the digital signal to a predetermined remote diagnosis unit through wired or wireless communication and receiving a result of the analysis of the digital signal from the remote diagnosis unit.
  32. 나노 결정 표지를 이용하는 미생물 검출 방법에 있어서, In the microorganism detection method using a nano-crystal label,
    검출하고자 하는 하나 이상의 미생물과 미생물 유인이 가능한 하나 이상의 미생물 유인 물질을 결정하는 단계;Determining one or more microorganisms to be detected and one or more microorganism attractants capable of attracting microorganisms;
    아연, 카드늄, 납, 구리, 갈륨, 비소, 탈륨, 니켈, 망간 및 창연으로 이루어진 군으로부터 하나 이상 나노 결정을 선택하여 상기 하나 이상의 미생물 유인 물질과 각각 결합하여 하나 이상의 나노 결정-미생물 유인 물질 복합체를 형성하는 단계;One or more nanocrystals are selected from the group consisting of zinc, cadmium, lead, copper, gallium, arsenic, thallium, nickel, manganese, and bismuth and combined with each of the one or more microbial attractants to form one or more nanocrystal-microbe attractant complexes. Forming;
    상기 하나 이상의 나노 결정-미생물 유인 물질 복합체를 검출하고자 하는 시료 내에 넣고 혼합하여 상기 검출하고자 하는 하나 이상의 미생물과 하나 이상의 나노 결정-미생물 유인 물질의 접촉을 유도하는 단계;Incorporating the one or more nanocrystal-microbe attractant complexes into a sample to be detected and mixing to induce contact of the one or more microcrystals to be detected with the one or more nanocrystal-microbe attractant materials;
    상기 나노 결정-미생물 유인 물질과 접촉한 미생물을 포획하는 단계; 및Capturing microorganisms in contact with the nanocrystal-microbe attractant material; And
    상기 포획된 미생물에 접촉한 상기 나노 결정-미생물 유인 물질 복합체로부터 포집된 나노 결정에 대응하는 고유한 전류 피크를 측정하는 단계를 포함하는 미생물 검출 방법. Measuring a unique current peak corresponding to the nanocrystals collected from the nanocrystal-microbe attractant complex in contact with the captured microorganism.
  33. 제32항에 있어서, 33. The method of claim 32,
    상기 나노 결정은 메탈 설파이드 형인 미생물 검출 방법. The nanocrystal is a metal sulfide type microorganism detection method.
  34. 제32항에 있어서, 33. The method of claim 32,
    상기 나노 결정은 아연, 카드뮴, 납 및 구리로 이루어진 메탈 군으로부터 하나 이상 선택되는 미생물 검출 방법. The nano-crystal is one or more microbial detection method from the metal group consisting of zinc, cadmium, lead and copper.
  35. 제32항에 있어서, 33. The method of claim 32,
    상기 미생물 유인 물질은 말토즈(maltose), 아스파테이트(aspartate), 카사미노산(casamino acid), 시스테인(cysteine), 류신(leucine) 또는 세린(serine)으로 이루어진 그룹으로부터 하나 이상 선택되는 미생물 검출 방법. The microorganism attracting substance is at least one selected from the group consisting of maltose, aspartate, casamino acid, cysteine, leucine or serine.
  36. 제32항에 있어서, 33. The method of claim 32,
    상기 나노 결정-미생물 유인 물질 복합체의 형성 단계가 The step of forming the nano-crystal microbe attractant complex
    일측에 나노 결정과 결합 가능한 전하 특성의 치환기를 가지며, 반대측에 복수의 수용성 치환기를 갖는 고분자 사슬형의 결합 안정화 물질을 상기 나노 결정과 결합시켜 상기 나노 결정 안정화하는 단계;Stabilizing the nanocrystals by binding a polymer chain-type bond stabilizing material having a substituent having charge properties capable of binding to the nanocrystals on one side and a plurality of water-soluble substituents on the opposite side with the nanocrystals;
    상기 안정화된 나노 결정에 결합된 결합 안정화 물질을 활성화시키는 단계; 및 Activating a bond stabilizing material bound to the stabilized nanocrystals; And
    상기 활성화된 결합 안정화 물질과 상기 미생물 유인 물질을 결합하는 단계;Coupling the activated binding stabilizing material and the microorganism attracting material;
    를 포함하는 미생물 검출 방법. Microbial detection method comprising a.
  37. 제36항에 있어서, The method of claim 36,
    상기 활성화 단계는 상기 나노 결정-결합 안정화 물질 복합체에 카보닐 다이이미다졸을 반응시켜 상기 결합 안정화 물질을 활성화시키는 미생물 검출 방법.The activation step is a microorganism detection method for activating the binding stabilizing material by reacting the carbonyl diimidazole to the nano-crystal-stabilizing material complex.
  38. 제32항에 있어서, 33. The method of claim 32,
    상기 측정된 전류 피크를 디지털 신호로 변환하는 단계를 추가 포함하는 미생물 검출 방법. And converting the measured current peak into a digital signal.
  39. 제38항에 있어서, The method of claim 38,
    상기 변환된 디지털 신호를 유선 또는 무선 통신을 통해 소정의 원격 진단부로 전송하는 단계; 및 Transmitting the converted digital signal to a predetermined remote diagnosis unit through wired or wireless communication; And
    상기 원격 진단부로부터 상기 디지털 신호에 대한 검출 결과를 수신하는 단계를 추가 포함하는 미생물 검출 방법. And receiving a detection result of the digital signal from the remote diagnosis unit.
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