WO2020159084A1 - Pathogen diagnostic system integrated with mobile device - Google Patents

Pathogen diagnostic system integrated with mobile device Download PDF

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Publication number
WO2020159084A1
WO2020159084A1 PCT/KR2019/018446 KR2019018446W WO2020159084A1 WO 2020159084 A1 WO2020159084 A1 WO 2020159084A1 KR 2019018446 W KR2019018446 W KR 2019018446W WO 2020159084 A1 WO2020159084 A1 WO 2020159084A1
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Prior art keywords
tube
pathogen
mobile device
test tube
holder
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PCT/KR2019/018446
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French (fr)
Korean (ko)
Inventor
이문근
박유민
김지현
이경균
이태재
배남호
이석재
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한국과학기술원
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/72—Signal processing specially adapted for physiological signals or for diagnostic purposes
    • A61B5/7271—Specific aspects of physiological measurement analysis
    • A61B5/7275—Determining trends in physiological measurement data; Predicting development of a medical condition based on physiological measurements, e.g. determining a risk factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/1455—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using optical sensors, e.g. spectral photometrical oximeters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/72—Signal processing specially adapted for physiological signals or for diagnostic purposes
    • A61B5/7235—Details of waveform analysis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/06—Test-tube stands; Test-tube holders
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B04—CENTRIFUGAL APPARATUS OR MACHINES FOR CARRYING-OUT PHYSICAL OR CHEMICAL PROCESSES
    • B04B—CENTRIFUGES
    • B04B1/00—Centrifuges with rotary bowls provided with solid jackets for separating predominantly liquid mixtures with or without solid particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B04—CENTRIFUGAL APPARATUS OR MACHINES FOR CARRYING-OUT PHYSICAL OR CHEMICAL PROCESSES
    • B04B—CENTRIFUGES
    • B04B5/00—Other centrifuges
    • B04B5/10—Centrifuges combined with other apparatus, e.g. electrostatic separators; Sets or systems of several centrifuges
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B04—CENTRIFUGAL APPARATUS OR MACHINES FOR CARRYING-OUT PHYSICAL OR CHEMICAL PROCESSES
    • B04B—CENTRIFUGES
    • B04B9/00—Drives specially designed for centrifuges; Arrangement or disposition of transmission gearing; Suspending or balancing rotary bowls
    • B04B9/10—Control of the drive; Speed regulating
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
    • 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
    • C12Q2523/00—Reactions characterised by treatment of reaction samples
    • C12Q2523/30—Characterised by physical treatment
    • C12Q2523/32—Centrifugation
    • 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
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/149—Particles, e.g. beads

Definitions

  • the present disclosure relates to a diagnostic system for pathogens integrated with mobile devices. More specifically, the present disclosure provides a mobile method for detecting or diagnosing a target pathogen gene (specifically, an amplification gene of a target pathogen) in a sample in a simple manner using a visualizable mobile phase particle and a fixed phase particle with a specific functional group attached thereto.
  • the present invention relates to a diagnostic system for a pathogen capable of performing on-site analysis at low power by using a centrifugal device that can be driven by a power supplied from a device, and further using an illuminance sensor in a mobile device.
  • pathogens such as bacteria and viruses are inhabited, providing a very good environment.
  • Pathogens are widely distributed in the surrounding environment, and specifically, bacterial pathogens are found in soil, animal organs, water contaminated by animal stools, kitchen utensils, desks, tableware, and other objects in everyday life.
  • the human body also has more than 150 types of bacteria on average, both inside and outside the body, although many microorganisms are harmless to the human body, some types are food poisoning, cholera, diarrhea, and emesis. It causes various infectious diseases including pneumonia and typhoid. In particular, more than 200 diseases can be transmitted through food and drinking water alone.
  • the Escherichia Coli (or E.coli ) family does not cause major problems when ingested, but some of them are known to produce toxins that cause diarrhea and the like.
  • the pathogenic E. coli O157:H7 is an intestinal hemorrhagic E. coli, widely known as a causative agent of food poisoning, and is a worldwide problem.
  • infection causes hemolytic uremic syndrome, hemorrhagic colitis, diarrhea, kidney failure, and seizures, leading to death in severe cases (Reisner, A. et al. , 2006. Journal of Bacteriology. 188, 3572-3581) ; Barrientos, RM et al. , 2009. Brain, Behavior, and Immunity. 23, 450-454; Schrag, SJ et al. , 2006. PEDIATRICS. 118, 570-576).
  • Salmonella choleraesuis , Salmonella bongori , and Salmonella typhimurium are also representative pathogens that cause food poisoning, and are the causative agents of typhoid and paratyphoid, and can be contaminated with various livestock and animals. In addition, if proper washing, processing temperature and storage conditions are not observed, Salmonella can multiply and cross-contaminated with other foods through contaminated drinking water and food.
  • pathogens especially food poisoning bacteria
  • the rate of reproduction of a pathogen is very fast, so even a small number of pathogens, once invading the human body, rapidly grow in the intestine, which is very suitable for their growth environment, to a level that can threaten human health. Therefore, there is a need for a technique capable of accurately diagnosing the presence of a pathogen (especially E. coli ) from a contaminated environment.
  • ATP adenosine triphosphate
  • luciferin/luciferase luciferase
  • PCR polymerase chain reaction
  • clinical diagnostic technology based on genetic analysis mainly recovers nucleic acids from bacteria, fungus, or viruses, performs amplification reactions, and then performs various detection means (eg, optical, It is a method of performing an amplicon analysis using an electrochemical or mechanical biosensor device).
  • POCT point of care testing
  • a pathogen eg, food poisoning fungus
  • a pathogen in a sample may be diagnosed (or analyzed) in the field based on an analysis principle or platform capable of visual detection, and further involved in sample analysis It is intended to provide a method to implement a simple and accurate series of manipulation and/or reading processes using a mobile device.
  • a plurality of mobile phase particles capable of visual identification and modified with a first functional group-containing component, and activated by a chemical functional group having binding ability with the first functional group-containing component A plurality of stationary phase particles, wherein the first functional group-containing component has binding properties with a second functional group-containing component conjugated with a primer specific to the gene of the target pathogen, but the second functional group -When the containing component is conjugated to the gene of the target pathogen amplified through the primer, binding between the mobile phase particle and the stationary phase particle is suppressed due to steric hindrance, and by separating the mobile phase particle that is not bound to the stationary phase particle, An analysis platform for pathogens configured to be identifiable visually or by an illuminance sensor; And
  • (B) (b1) a portable centrifugal separation device that is connected to a power input/output terminal of a mobile device and separates mobile phase particles that are not bound to the stationary phase particles by power supplied therefrom, and (b2) an illuminance sensor embedded in the mobile device
  • a diagnostic system for a pathogen comprising at least one of a sensing unit for analyzing a target pathogen is provided by using the analysis platform.
  • the power input/output terminal may be a power input/output terminal of the USB port.
  • the portable centrifugal separation device the portable centrifugal separation device
  • a motor unit installed on an upper side of the upright support unit and driven by power supplied from a mobile device;
  • a connector terminal provided at a lower side of the upright support portion and electrically connectable to a power input/output terminal of the mobile device
  • At least one ring member mechanically connected to the motor part and configured to rotate in a horizontal direction when the motor part rotates
  • a first tube holder having a locking portion interlocking or interlocking with the at least one ring member and a tube receiving portion formed to insert a tube test tube;
  • the tube test tube inserted into the first tube holder can be rotated while being converted from a vertical position to a horizontal position by centrifugal force.
  • the mobile device may be a smart device.
  • the smart device may be a smartphone, tablet or notebook.
  • the tube test tube the tube test tube
  • test tube body made of a light transmissive material having an opening formed at an upper portion and closed at an lower portion
  • the opening and closing is provided with a locking jaw protruding along the periphery so as to be in a fixed state under the centrifugal force applied when driving the motor part.
  • Dragon lid
  • the sensing unit the sensing unit
  • a mobile device holder having a through groove extending at a lower side to accommodate an exposed surface of the illuminance sensor in the mobile device while being fastened with the mobile device by an insertion method;
  • a second tube holder for inserting a test tube in which the analysis platform is housed in a tube test tube;
  • a light source holder equipped with a light source for irradiating light to the inserted test tube
  • It includes a sensor structure having a,
  • the mobile device holder, the second tube holder, and the light source holder may be arranged so that light irradiated from the light source passes through the test tube and is transmitted to the exposed surface of the illuminance sensor.
  • the sensor structure may be an integral structure.
  • the sensor structure may be manufactured by 3D printing.
  • a genetic diagnostic system is a portable centrifugal separation device operable by a mobile device as a physical separation means, in an analysis platform involving particle-based DNA selective transport through a functionalized interface, and/or
  • the combination of a sensing unit using an illuminance sensor embedded in a mobile device provides an advantage of accurately and positively reading a target pathogen in a sample visually or by a mobile device in a short time.
  • a mobile device carried by a large number can be applied as a power source and/or a sensing unit of a portable centrifugal separator, convenience of on-site diagnosis can be maximized.
  • a mobile device with a built-in illuminance sensor for example, a commercially available smart phone
  • FIG. 1 is a diagram illustratively illustrating a diagnostic principle using a particle-based genetic analysis platform via a functionalized interface according to one embodiment
  • FIG. 2 is a diagram showing elements constituting an analytical platform when N-hydroxysuccinimide (NHS) is used as a chemical functional group for activation of a stationary phase particle in an exemplary embodiment;
  • NHS N-hydroxysuccinimide
  • FIG. 3 is a diagram schematically showing a diagnostic principle in the case of using NHS (N-hydroxysuccinimide) as a chemical functional group for activation of a stationary phase particle in an exemplary embodiment
  • FIG. 4 is a schematic diagram of a particle-based genetic analysis platform via a functionalized interface in one embodiment
  • 5A and 5B are each a diagram showing an exemplary configuration of a portable centrifuge device in a pathogen diagnosis (analysis) system and a tube holder included therein;
  • FIG. 6 is a diagram showing an operating principle of a portable centrifugal device electrically connected to a power input/output terminal of a smartphone as a mobile device;
  • FIG. 7A and 7B each show a modified example of a tube holder in a portable centrifugal device in an exemplary embodiment
  • FIG. 8 is a diagram schematically showing the principle of determining positive and negative by an illuminance sensor on an analysis platform for particle-based genes through a functionalized interface according to an exemplary embodiment
  • FIG. 9 is a view showing a sensor structure constituting a sensing unit in an exemplary embodiment
  • FIGS. 10A and 10B are photographs showing positive and negative test results using a particle-based genetic analysis platform via a functionalized interface in Example 1;
  • FIG. 11 is a diagram showing experimental results and electrophoresis test results for evaluating detection limits of E. Coli O157:H7 using a particle-based genetic analysis platform through a functionalized interface in Example 1;
  • Example 12 is a diagram showing the results of analyzing a milk sample containing E. Coli O157:H7 using a particle-based genetic analysis platform via a functionalized interface in Example 1;
  • Example 13 is a magnetic particle (MP) alone in Example 1, a state in which the amplified gene is conjugated to the magnetic particle (MP+DNA), and a state in which the amplified gene is conjugated by inserting GelRed into the magnetic particle (MP+DNA+) GelRed) is a graph showing the dynamic light scattering (DLS) size for each;
  • Figure 14a is a photograph showing the results of the concentration optimization experiment of the insertion dye (GelRed) according to the GelRed dilution factor in Example 1;
  • Example 14B is a photograph showing the results of optimization experiments of the rotational speed during centrifugation in Example 1;
  • 15A and 15B are each a photograph showing the appearance of a tube holder manufactured by 3D printing used in Example 2 and a portable centrifugal separation device applying the same;
  • Example 16 is a photograph showing a pathogen diagnosis process performed in Example 2.
  • 17A and 17B each use broth and milk containing E. Coli O157:H7 as a sample according to Example 2, and from centrifugation by the portable centrifugation apparatus shown in FIG. 15B A photograph showing the electrophoresis test and visual observation results of the obtained analytical platform;
  • 19A and 19B are each a photograph showing a state in which the sensor structure produced by 3D printing in Example 3 is equipped with a light source, a battery, and a test tube, and the appearance of combining it with a smart phone;
  • 20A and 20B are graphs showing the results of performing a diagnostic test of E. coli in Example 3 using an illuminance sensor embedded in a smartphone.
  • Binding may mean that the surface is coupled or connected in a covalent or non-covalent manner.
  • sample is not limited to a specific type or form as long as it can contain the target pathogen to be detected.
  • the sample can be a biological sample, such as a biological fluid or biological tissue.
  • biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, and the like.
  • Biological tissue is a collection of cells, usually a set of intracellular substances that form one of the structural substances of a human, animal, plant, bacterial, fungal, or viral construct, and a specific kind of connective tissue, epithelial tissue, muscle tissue, and nerves. Organizations, etc. may be this.
  • examples of biological tissue may include organs, tumors, lymph nodes, arteries and individual cell(s).
  • the sample may include an environmental sample containing a biomaterial at a low concentration, and may include various forms and types, such as drinking water and food.
  • Primer can mean an oligonucleotide (synthetic or natural) that can act as an initial point of synthesis or replication of a nucleic acid along a complementary strand when synthesis of the complementary strand is under conditions catalyzed by a polymerase. have.
  • the "target gene” may consist of several hundreds, hundreds, thousands, or millions of nucleotides, and may also be a fragment of DNA, RNA, or the like.
  • Dissolution (lysis) may mean a phenomenon in which cell membranes are ruptured and cell contents are exposed upon cell decomposition, and is commonly used to separate nucleic acids in all stages of amplification processes such as PCR.
  • Bacteria the outer membrane (bacterial envelope) of the Gram (Gram) staining reaction is distinguished according to the structure, the Gram-negative bacteria (thin murine (murein) or peptidoglycan layer and has a lipid bilayer of the outer membrane) and Gram-positive bacteria (thick murine or peptidoglycan layer with crystal violet).
  • the cell membrane of gram-negative bacteria consists of phospholipids and other glycoproteins, and most of the phospholipids have a negative charge (net negative charge).
  • the types include Salmonella, meningitis, spiroheta cholera, plague, typhoid, heterogeneous, E. coli, gonorrhea.
  • the cell wall is mainly composed of peptidoglycan and teichoic acid, and its surface has almost neutral charge characteristics, and staphylococcus, streptococcus, and anthrax , Diphtheria, tetanus, and pneumonia.
  • Amplification can mean a reaction that occurs repeatedly to form multiple copies of at least one segment of a template molecule.
  • PCR refers to a reaction in which a large amount of identical DNA strands are formed from one initial template by a cycle process (heating and cooling are alternately performed).
  • PCR mixtures are (i) sequencing sequences to be amplified.
  • a double-stranded DNA molecule (ii) a primer (a single stranded DNA molecule capable of binding to a complementary DNA sequence in the template DNA), (iii) a mixture of dATP, dTTP, dGTP, and dCTP (in PCR amplification process) Nucleotide subunits joined to form a new DNA molecule) dNTP, and (iv) an enzyme that synthesizes a new DNA molecule using Taq DNA polymerase (dNTP)).
  • dNTP Taq DNA polymerase
  • FIGS. 2 and 3 schematically shows components and diagnostic principles of a diagnostic system in the case of using N-hydroxysuccinimide (NHS) as a chemical functional group for activation of a stationary phase particle.
  • NHS N-hydroxysuccinimide
  • the diagnostic platform 10 of a pathogen is a plurality of highly activated stationary phase particles 2 and visual confirmation or recognition, and a plurality of modified with a first functional group-containing component And mobile phase particles 3.
  • the diagnosis can be performed in the tube test tube (1)
  • the tube test tube (1) may be made of a material having a characteristic that can be observed with the naked eye from the outside, specifically, a transparent material.
  • the material of the tube test tube 1 may be a transparent polymer, glass, etc., and the present invention is not necessarily limited thereto.
  • the plurality of activated stationary phase particles 2 are activated by attaching specific chemical functional groups 12 on the matrix particles 11, respectively, and filled (or packed) in the tube test tube 1 Can be applied in column form.
  • the matrix particles 11 may be made of a material selected from the group consisting of resin, metal and glass.
  • various natural resins for example, agarose (sepharose)
  • synthetic resins for example, polystyrene or polyacrylamide
  • resin moldings, gels, etc. can be applied in the form of
  • the matrix particles 11 may have a regular (eg, spherical, elliptical, etc.) or irregular shape, and may also have a symmetrical or asymmetrical shape. More typically, it can be a sphere.
  • the matrix particles 11 may have a porous structure, specifically a uniform porous structure, and typically, may have a flow pore in the matrix particles.
  • the pore size (diameter) of the porous matrix particles may be, for example, in the range of about 20 to 100 nm, specifically about 30 to 80 nm, and more specifically about 40 to 60 nm, which will be understood in an exemplary sense. Can.
  • the size (diameter) of the stationary phase particles 2 can be varied depending on the material, shape, etc. of the matrix particles, for example, several to hundreds of ⁇ m, specifically about 30 to 400 ⁇ m, more specifically about 40 to 250 ⁇ m , In particular, in the range of about 45 to 200 ⁇ m. However, this may be understood as an exemplary meaning.
  • the matrix particle 11 may be a cross-linked bead-shaped agarose (sepharose) gel, which is known as a polysaccharide (charged and/or neutral)-based material.
  • the content of the agarose in the gel may be, for example, about 1 to 10% by weight, specifically about 2 to 6% by weight, and more specifically about 3 to 5% by weight, as shown in the following general formula 1 It may have a repeating unit displayed.
  • the chemical functional group can be attached to or bonded to the surface of the matrix particle, as long as it can bind or couple (e.g., covalent bond) with the first functional group-containing component, It can be selected from various types.
  • NHS N-hydroxysuccinimide
  • a functional group having can be applied.
  • the matrix particles can be activated using such a chemical functional group, but this can be understood as an example because it can be variously changed according to the first functional group-containing component.
  • the plurality of mobile phase particles 3 may have a form in which the base particles (specifically, beads or spherical particles; 13) are modified with the first functional group-containing component 14. .
  • the base particles 13 as long as it has a visually identifiable, specifically visually identifiable color, beads of various materials can be used.
  • the base particle 13 may be at least one selected from magnetic particles (magnetic beads), polystyrene beads, gold nanoparticles, metal particles, glass beads, and silica beads.
  • the base particles 13 may have a density of a certain level or higher in order to increase separation efficiency in a physical separation (specifically centrifugation) process described later, for example, about 0.01 to 10 g/L, specifically It may be in the range of about 0.5 to 5 g/L, more specifically about 1 to 2 g/L, but the present invention is not limited thereto.
  • magnetic particles can be used as the base particles 13, they may exhibit temporary or permanent magnetism.
  • magnetic particles for example, magnetic particles containing iron, cobalt, nickel, iron oxide, iron hydroxide, and/or other iron alloys, rare earth magnetic particles, and the like can be used.
  • particles coated with a magnetic iron core with a polymer eg, dextran
  • the size of the base particles 13 is not limited to a specific range, but may typically be in a nanoscale to micron scale range, for example, about 0.01 to 10 ⁇ m, specifically 0.1 to 6 ⁇ m, and more specifically 1 to It may be in the range of 3 ⁇ m. According to a specific embodiment, the particle size distribution may be advantageous in securing the efficiency and/or diagnostic accuracy of physical separation (specifically centrifugation) in the future, if possible.
  • the base particle 13 of the mobile phase particle 3 is modified with the first functional group-containing component 14.
  • the first functional group-containing component 14 may be of a type having binding characteristics (ie, high affinity) with the second functional group-containing component 15 described later.
  • the first functional group-containing component 14 is, for example, avidin, streptavidin, biotin, antigen, antibody, aptamer, amine, carboxy, aldehyde, gene (eg For example, DNA capable of complementary binding) may be exemplified, and may be a concept including both naturally occurring and artificially synthesized.
  • the first functional group-containing component 14 may be avidin and/or streptavidin as a biomaterial, and avidin corresponds to a small sized water-soluble biotin and a glycoprotein having high affinity.
  • streptavidin is a protein isolated from bacterial Streptomyces avidinii and is also a component having high affinity for biotin. In particular, it does not bind to lectin because it does not correspond to sugar protein, and may be preferable to avidin in terms of physical properties. .
  • NHS is used as the chemical functional group 12 in the stationary phase particle 2 and avidin or streptavidin is used as the first functional group-containing component 14, there is already a relationship between the NHS and the amine group of avidin. Debonding can be achieved.
  • the functionalized mobile phase particles 3 can be added (or loaded) to the column of activated stationary phase particles 2.
  • the functionalized mobile phase particles 3 can be added, for example, in a liquid medium, specifically in the form dispersed in a water-based medium (specifically a suspension).
  • the concentration of the mobile phase particles 3 in the suspension may be, for example, about 1 to 30 mg/mL, specifically about 5 to 20 mg/mL, and more specifically about 8 to 15 mg/mL.
  • PBS or the like may be further added for the mobile phase's mobility.
  • the gene of the target pathogen in order to diagnose the gene of the target pathogen, it is amplified through the conjugate (if negative) or the primer of the second functional group-containing component and the primer together with the mobile phase particles 2 (for example, in the form of a mixture) or separately. Conjugates of the genes (if positive) can be added.
  • the second functional group-containing component 15 can be selected from the types having binding properties with the first functional group-containing component 14 described above, similarly, avidin, streptavidin, biotin, antigen, Antibodies, aptamers, amines, carboxys, aldehydes, genes (eg, DNA capable of complementary binding) can be used.
  • biotin is a type of vitamin, specifically, a B-complex vitamin (hexahydro-2-oxo-lH-thieno[3,4] consisting of a ureido (tetrahydroimidazalone) ring fused with a tetrahydrothiophene ring.
  • the molecular weight is about 244 g/mol
  • a valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring.
  • Biotin can specifically bind avidin or streptavidin by strong affinity, for example, four biotin molecules can be bound to one streptavidin molecule.
  • a commercially available product in which the first functional group-containing component 14 is modified on the particle 13 may be used, for example, the trade name Dynabeads which is a modified magnetic particle of Life Science. ⁇ MyoneTM Streptavidin C1 can be applied.
  • binding between the first functional group-containing component 14 and the second functional group-containing component 15 can be achieved using an immune response between antigen-antibodies, non-covalent binding, inter-complementary binding, and the like.
  • the second functional group-containing component 15 is conjugated with a primer 16 specific to the gene of the target pathogen, and undergoes an amplification process for the sample in the presence of such conjugate.
  • the target pathogen is a biomaterial such as bacteria, viruses, and the like, and may include Gram-positive bacteria and Gram-negative bacteria.
  • E. coli O157:H7 Salmonella choleraesuis , Salmonella bongori , Salmonella typhimurium ), Staphylococcus aureus, Listeria monocytogenes , Listeria denitrificans , Listeria grayi , Listeria murrayi ), Cholera, Mycobacterium, Pertussis, Diphtheria, Typhoid, Pasteur , Staphylococcus Aureus, more specifically E.coli O157:H7 , Salmonella choleraesuis , Salmonella bongori , Salmonella typhimurium , Listeria monocytogenes , Listeria denitrificans , Listeria grayi , Listeria murrayi, S. enteritidis, Y. enterocolitica, S. aureus , B. cereus , L. mono
  • the amplification reaction immerses the substrate in which the gene (specifically, DNA) of the target pathogen is immobilized in the amplification mixture (i.e., PCR mixture), wherein the immobilized nucleic acid is separated by exposure to high temperatures during the amplification process.
  • the amplification reaction any amplification technique known in the art, such as PCR (DNA amplification method), NASBA (Nucleic Acid Sequence Based Amplification; RNA amplification method under isothermal conditions) as described above, may be used.
  • PCR DNA amplification method
  • NASBA Nucleic Acid Sequence Based Amplification
  • RNA amplification method under isothermal conditions may be used.
  • the PCR method is described in Korean Patent No. 593687
  • the NASBA method is exemplified in EP 0 329 822 B1, U.S.
  • Patent No. 6,110,681, etc. and the aforementioned prior documents are used as references of the present invention. Is included.
  • assembly-PCR assembly-PCR
  • asymmetric PCR digital PCR
  • endpoint PCR endpoint PCR
  • inverse PCR methylation-specific PCR
  • qualitative PCR quantitative PCR
  • quantitative PCR Real-time PCR
  • RT reverse transcription
  • the second functional group-containing component 15 amplifies the gene of the target pathogen by the action of the primer, and consequently the second functional group-containing component (15) is in a state conjugated with the amplification gene 17 of the target pathogen.
  • the gene of the target pathogen amplified through the primer conjugated with the second functional group-containing component 15 has a longer chain than before amplification.
  • the first functional group-containing component 14 on the mobile phase particle 3 is consequently due to the strong binding properties of the second functional group-containing component conjugated with the amplification gene 17 of the target pathogen, resulting in the mobile phase particle 3 ), and as a result, the size (diameter) of the mobile phase particle 3 increases. Due to this increased size, a steric hindrance effect that prevents binding or binding with the stationary phase particle 2 is caused.
  • the size of the amplification gene attached to the mobile phase particle 3 is, for example, about 10 to 150 nm, specifically about 30 to 100 nm, more specifically As it may be in the range of about 50 to 80 nm, the size of the mobile phase particle is increased to a level capable of providing a steric hindrance or a space obstruction effect by bulky amplification gene attachment.
  • the extent of this size increase may be understood as an exemplary meaning as it may be different depending on the type of the base particle 13, the degree of amplification reaction, and the like.
  • the target pathogen when the target pathogen is not present in the sample (when it is negative), some of the first functional group-containing component 14 on the mobile phase particle 3 has a second functional group-containing component 15 and a primer 16 ), while the other part is bound (e.g., covalently) with a chemical functional group present in the stationary phase particle 2, resulting in the mobile phase particle 3 being fixed to the stationary phase particle 2 Will be in.
  • the stationary phase particles 2 and the mobile phase particles 3 may undergo a separation operation step by physical means, and may be exemplified by centrifugation, physical compression, etc. as representative physical separation means, but are not limited thereto. .
  • the centrifugation conditions may be sufficient to transport only the mobile phase particles 3 not bound to the stationary phase particles 2 to the bottom of the container by centrifugal force while suppressing the influence on the column structure in the diagnostic system.
  • the rotational speed in the centrifugation process may vary depending on the length, radius, etc. of the stationary phase column, for example, in the range of about 500 to 2000 rpm, specifically about 600 to 1900 rpm, and more specifically about 800 to 1200 rpm. It can be appropriately adjusted, and the centrifugation time can be determined by considering gravity acceleration, for example, about 0.5 to 5 minutes, specifically about 1 to 4 minutes, and more specifically about 1.5 to 3 minutes. It may be, but it can be understood in an exemplary sense. However, the above-described centrifugation may be repeated one or more times as necessary.
  • the mobile phase particle 3 bound to the gene of the amplified target pathogen tends to be spaced apart from the stationary phase particle 2 spatially by the amplified gene conjugated with the first functional group-containing component present on its surface. It does not bind to the stationary phase particles (2). Therefore, in the centrifugation process, while passing through the columns of the plurality of stationary phase particles 2, it may be precipitated by moving to the lower side, for example, to the bottom of the column.
  • precipitation of the mobile phase particles 3 can be confirmed or identified with the naked eye, and in this case, it can be determined as positive.
  • the mobile phase particles 3 are bound to the stationary phase particles 2, and thus, even by centrifugation, they are distributed in the column of the stationary phase particles, specifically above the column of the stationary phase particles. This can be confirmed with the naked eye. In this case, it can be judged by voice.
  • the mobile phase particles combined with the amplified gene (3) by additionally imparting rigidity to the amplified gene chain using an insertion substance, specifically, an insertion dye The diameter of can be substantially increased to provide a greater steric hindrance effect.
  • the target gene is DNA (specifically, double-stranded DNA)
  • the degree of increase in diameter due to the amplified gene chain increases as the corresponding insert material or insert dye is inserted into the double-helix structure after or after the amplification process. It is more remarkable.
  • the intercalation material or intercalation dye may be mixed or added to the amplification mixture or amplification product.
  • the insertion dye may be inserted between double-stranded DNA (dsDNA).
  • Insertion dye is a dye that can be inserted into a double strand of a gene (specifically DNA) after the amplification process or amplification, GelRed, EtBr (ethidium bromide), GelGreen, SYBR Green, PicoGreen, Hoechst series, BOBO, TOTO, YOYO, JOJO, POPO, LOLO, PO-PRO, BO-PRO, YO-PRO, TO-PRO, JO-PRO, LO-PRO, SYTO series, and the like, and these may be used alone or in combination. In this regard, it may be advantageous to use a kind of insertion dye having specific reactivity to the double helix gene.
  • GelRed may be used as an insertion dye, but is not limited thereto.
  • the present invention is not limited to the above-described insertion material, and various types may be used as long as it can be inserted into a double strand of an amplification reaction process or amplification product to increase the rigidity of the amplification gene chain.
  • the insert material can be applied in a liquid medium, specifically dissolved (diluted) in an aqueous medium.
  • the concentration of the intercalation material in the diluted medium (solution) may range, for example, from about 6 to 19x, specifically from about 7 to 18x, and more specifically from about 8 to 15x.
  • the concentration range of the insert material is variable depending on the type of target pathogen (specifically, bacteria, viruses, etc.), and is not necessarily limited to the above numerical range.
  • the volume ratio of the amplification product (if the gene is contained in the amplification mixture) to the insertion substance is, for example, determined within a range of about 2 to 8, specifically about 3 to 6, and more specifically about 3.5 to 5 It can be lost, but it can be understood as exemplary.
  • the amount of the insert material is used excessively, a cohesive reaction or the like may be caused, and thus, insertion into the intended gene chain may be difficult, whereas when the amount of the insert material is too low, a desired increase in stiffness and/or additional color or As it is difficult to exhibit a fluorescence expression effect, it may be advantageous to use it by appropriately adjusting within the above-described range.
  • the insertion material is incorporated (inserted) into the amplification gene of the target pathogen, and as the stiffness of the gene chain increases, the diameter of the conjugate measured is increased as compared to the case where it is not inserted. Therefore, it is possible to impart a higher steric hindrance, which makes it easier to visually discriminate by moving the mobile phase particles 3 rapidly and separated by physical means such as centrifugation, when a pathogen is present in the sample (positive). You can do it.
  • the rate of increase in the size of the amplification gene attached to the mobile phase particle after incorporation (insertion) of the insert material may be in the range of at least about 20%, specifically about 30 to 80%, more specifically about 40 to 50%, but this can be understood in an exemplary sense.
  • the analysis platform can determine positive and negative visually in the field by a simple physical separation operation (specifically, using only centrifugal force).
  • a simple physical separation operation specifically, using only centrifugal force.
  • an analytical platform may have a low detection limit (LOD) of, for example, a level of about 10 to 10 6 CFU/mL, even 1.0 x 10 1 CFU, but is not necessarily limited thereto. no.
  • LOD low detection limit
  • the stiffness of the amplified gene chain is increased to further increase the separation efficiency of the mobile phase particle due to steric hindrance.
  • the genetic diagnostic system may include a portable centrifugal separation device as a physical separation means required to apply the aforementioned analysis platform.
  • the portable centrifugal separator may be driven using power supplied from a mobile device, and for this purpose, may be detachably connected to a power input/output terminal of the mobile device.
  • a mobile device provided with a power input/output terminal such as a smart phone, a tablet, or a laptop and provides a function to supply power
  • it is not particularly limited, and may include a portable maintenance power supply.
  • a mobile device with a built-in illuminance sensor such as a smart phone or a tablet device, not only functions as a power source for a portable centrifuge, but also as a reading device by light source analysis as described below.
  • the mobile phase particle not bound to the stationary phase particle is physically separated by centrifugation. Can be.
  • FIG. 5A and 5B each show an exemplary configuration of a portable centrifuge device and a first tube holder included therein in a pathogen (its gene) diagnostic system according to an exemplary embodiment.
  • Figure 6 shows the principle of operation of a portable centrifugal device electrically connected to the power input and output terminals of a smart phone as a mobile device.
  • the portable centrifugal separation device 100 is provided with a motor portion A on the upper side of the upright support portion 101.
  • the motor unit may be driven by power supplied from a mobile device, and may include a motor 102 enclosed in a case and a rotating wing 103 that rotates together according to driving of the motor.
  • the motor 103 may use a small motor known in the art, but as low as possible to be suitable for portable power, specifically a range of power supplied from a mobile device or an auxiliary power supply (for example, about 1 To 5 V, specifically about 3 V).
  • a connector terminal (or a power input terminal) is provided at a lower end (opposite end of the motor unit) of the support 101 to be electrically connected to a power input/output terminal of a mobile device or an auxiliary power supply.
  • the power input/output terminal may be a power input/output terminal of a USB port.
  • a commercially available small motor can be used as the motor 102 embedded in the portable centrifugal separation device, and the above-described rotational speed can be realized by using only such a motor.
  • the rotary wing 103 may be formed integrally with the case of the motor, or may be mechanically coupled to each other after each of the case and the rotary wing of the motor are individually manufactured.
  • a pair of rotating wings 103 arranged while facing each other in the motor portion A are fastened to the motor 102, which is exemplary, and includes one or more rotating wings This may be further formed (for example, two pairs of rotating wings may be formed).
  • the rotary wing 103 can be omitted.
  • the ring member 104 is mechanically connected (or fastened) to each rotary wing 103.
  • a fastening groove is formed in the rotating wing 103 so that at least a portion of the ring member 104 can be coupled or fastened by inserting it, and if necessary, a jaw (not shown) is formed in the fastening groove. It is also possible to prevent the ring member from being separated during the centrifugation process. As such, since the ring member 104 is directly or indirectly engaged with the motor 102, it rotates according to the driving of the motor 102, and typically rotates in the horizontal direction.
  • a first tube holder B in which the first cavity 106 and the second cavity 108 are formed is provided.
  • the first cavity 106 is configured to be free from movement of the ring member 104 while having a degree of freedom of movement, by connecting or interlocking the ring member 104 with the ring member 104.
  • the first tube holder B is connected to the first surface 105 and the second surface 107 in an “L” shape, and the first cavity 106 is connected to the first surface 105. ), and a second cavity 108 is formed on the second surface 107, respectively.
  • the first tube holder B may be formed by bending a single metal frame, or may be formed by molding (eg, injection molding) or 3D printing a polymer material.
  • the polymer material constituting the first tube holder (B) is a thermoplastic polymer, polyester, polyolefin (for example, polyethylene, polypropylene, etc.), polyamide, polyvinyl alcohol, polyurethane, Polystyrene, polyvinyl chloride, or a combination thereof.
  • the first tube holder B is hooked or interlocked with the ring member 104 by the first cavity 106 formed on the first surface, and the first cavity 106 is the locking portion Will function as
  • the second cavity 108 formed on the second surface of the first tube holder (B) functions as a tube receiving portion, into which the tube test tube (C) is inserted.
  • the inserted tube test tube (C) is in the vertical position (for example, in the direction of gravity) when the motor 103 is not driven, when driving the motor unit (A), the ring According to the rotation of the member 104, it is converted to a horizontal position by a centrifugal force and rotates.
  • FIG. 7A and 7B each show a variant of the first tube holder in a portable centrifugal device in an exemplary embodiment.
  • the structure of the first tube holder shown in the figure is designed to align the center of gravity of the tube holder in the center to uniformly apply centrifugal force.
  • the first cavity 116 is formed as a locking portion in the first surface 115 corresponding to the upper portion of the first tube holder B.
  • the lower portion of the first tube holder (B) is a bottom surface having a surface direction perpendicular to the surface direction of the first surface 115, for example, to the second surface 117 of a square (specifically square)
  • the second cavity 118 is formed as a tube receiving portion.
  • each of the pair of first connection surfaces 121 having the same width as the first surface 115 is connected (extended) to the lower edge of the first surface 115 while maintaining a predetermined angle, and also a second
  • the upper edge of each of the pair of second connecting surfaces 122 formed to have a trapezoidal shape that gradually decreases in width from the surface 117 is configured to meet the lower edge of the first connecting surface 121.
  • the first connecting surface and the second connecting surface may be a single member bent or integrally formed by molding or 3D printing.
  • the second surface 117 is composed of a rectangular planar member, but can be transformed into a polygonal planar member.
  • the second connection surface has a square shape (for example, when the width of the second connection surface is equal to the width of the first connection surface) or an inverted trapezoidal surface (for example, the width of the second connection surface) It may be configured to have a) less than the width of the first connection surface.
  • each of the plurality of beam members 125 from the bottom surface 127 is formed to extend at a predetermined angle so as to face the central upper space of the bottom surface 127 and is joined to each other do.
  • each of the four beam members 125 extends from four vertices or a nearby portion of the bottom surface 127 of a square (specifically a square).
  • the chin is formed at the edge of the bottom surface (a pair of opposite edges in the drawing) so that the bottom surface 127 and the beam member 125 can be easily attached to each other, thereby providing the attachment surface of the beam member.
  • a pair of jaws are formed, but if necessary, attachment jaws may be formed at all corners of the bottom surface.
  • each of the four beam members 125 may have a gradually reduced width in a direction extending from the bottom surface 127.
  • the junction 126 where the four beam members 125 meet each other forms a locking portion that is interlocked or interlocked with the aforementioned ring member 104.
  • the bottom surface is made of a rectangular surface, but in some cases, it may be formed of a polygonal surface other than a circular or rectangular surface.
  • the number of beam members can also be changed so long as the balance can be provided for stable rotation during centrifugation.
  • a cylinder-shaped cavity 128 is formed in the downward direction of the bottom surface 127 to function as a tube receiving portion.
  • the bottom of the cavity 128 may be closed (ie in the form of a pocket) or opened.
  • the first tube holder shown in FIGS. 7A and 7B may be manufactured by molding or 3D printing.
  • the tube holder should hold the test tube stably during centrifugation, and the diameter of the tube receiving portion of the tube holder can be made to be substantially the same as the diameter of the tube test tube used.
  • the tube test tube (C) has a shape of a generally known shape, and largely includes a test tube body 109' and a lid 109" for opening and closing for receiving or discharging the contents.
  • the tube test tube body 109' is not particularly limited as long as it has dimensions suitable for insertion into the tube receiving portion provided in the first tube holder B.
  • it is not limited to the tube test tube C.
  • As an analysis platform i.e., a plurality of mobile phase particles, a plurality of stationary phase particles, and an amplification product (specifically, amplification gene) of a sample, it is necessary to visually check it externally or by an illuminance sensor.
  • ') is preferably made of a transparent material, for example, the tube test tube body 109' may use various inorganic materials (for example, glass) or polymer materials.
  • a locking jaw or protrusion is formed along the perimeter of the lid 109" for opening and closing.
  • the volume or capacity of the tube in vitro (C) may be, for example, in the range of about 200 to 2000 ⁇ l, specifically about 300 to 1000 ⁇ l, more specifically about 350 to 600 ⁇ l, This can be understood in an exemplary sense.
  • a tube test tube with an excessively large receiving volume may not be suitable for centrifugation of a mobile phase (specifically, magnetic particles).
  • the diameter of the tube test tube (C) can be defined, for example, in the range of about 5 to 15 mm, specifically about 6 to 12 mm, which can be understood in an exemplary sense.
  • the mobile phase particles that are not bound to the stationary phase by physical separation means can be visually confirmed by the naked eye, and can be determined using an illuminance sensor. It can be read more accurately in grades and quantity.
  • FIG. 8 schematically illustrates the principle of determining positive and negative from an analytical platform by an illuminance sensor according to an exemplary embodiment.
  • a physical separation between a light source and an illuminance sensor specifically, a test tube (tube test tube containing an analytical platform) that has undergone centrifugal separation is placed, and the transmittance exhibits different values depending on the degree of separation of the mobile phase in the test tube.
  • the reading can be performed by measuring with an illuminance sensor.
  • the light irradiated from the light source it is preferable to arrange the light irradiated from the light source to pass through the test tube (specifically, the lower region of the test tube), and the transmitted light is sensed by the illuminance sensor.
  • the mobile phase particles are concentrated to the lower side of the test tube by principle separation, and the concentrated mobile phase particles reduce light transmittance to reduce the light intensity of the light reaching the illuminance sensor.
  • the concentration of the mobile phase particles in the lower part of the test tube is relatively low. Therefore, since the degree of influence on the transmittance of the irradiated light is small, the illuminance of the light reaching the illuminance sensor is higher than that of the positive case.
  • the target gene in the sample can be qualitatively and quantitatively read (or diagnosed).
  • an illuminance sensor it is possible to accurately and easily check the amount of light reaching the illuminance sensor by using an illuminance sensor embedded in a mobile device, specifically a smart phone or tablet device, and installing an illuminance measurement application on the mobile device.
  • the present embodiment provides a sensing unit capable of implementing the above-described reading principle using the illuminance sensor embedded in the mobile device.
  • the sensing unit is a concept including a sensor structure and an illuminance sensor on which reading is performed.
  • a sensor structure 200 introduces or inserts a light source that provides light required for the illuminance sensor and a test tube to be read, and the sensor structure 200
  • the above-described reading principle can be implemented by attaching the illumination sensor to the mobile device to cover the exposed surface.
  • the light source is used to optically analyze the degree of separation of mobile phase particles, particularly magnetic particles, for example, a white light source can be applied.
  • a white light source it is not limited to a white light source, and various light sources capable of emitting or irradiating light of various wavelengths within a visible light range may be used due to the characteristics of the magnetic particles and the illuminance sensor in the smart device.
  • a light emitting diode (LED) device commonly known as a light source may be used, and the intensity of the irradiated light may be selected, for example, within a range of about 100000 Lux or less, specifically in a range of about 100 to 30000 Lux, ,
  • the wavelength band may be, for example, in the range of about 400 to 1100 nm, specifically about 500 to 800 nm.
  • the sensor structure applicable to constructing the sensing unit in the exemplary embodiment is as illustrated in FIG. 9.
  • the sensor structure 200 is largely for mounting a mobile device holder 201 for attaching to a mobile device with a built-in illuminance sensor, a second tube holder 202 for inserting a test tube, and a light source. It includes a light source holder 203.
  • the sensor structure 200 has a hexagonal shape as a whole.
  • an illumination sensor is usually built in an upper portion of a mobile device, particularly a smart phone, to form an exposed surface of the illumination sensor.
  • the mobile device holder 201 is provided in the form of a through groove extending in a lower portion or a lower surface of the sensor structure 200 so as to insert the upper portion of the mobile device.
  • the through groove is formed across a point or position biased toward the side opposite to the surface where the light source holder 203 is formed, rather than the center of the bottom surface of the sensor structure.
  • the holder 203 may be formed at a position corresponding to about 0.55 to 0.9, specifically about 0.6 to 0.8, from the formed surface.
  • the width of the through groove is not limited to a specific value, as long as it can cover the exposed surface of the illuminance sensor in the mobile device by insertion, and may be determined in consideration of the width of the inserted mobile device, ease of insertion, and the like. .
  • a second tube holder 202 for inserting a test tube is formed on the upper surface of the sensor structure 200.
  • the second tube holder 202 may be formed in a hole shape, which is typically a circular cross-section, so that a test tube can be inserted through the second tube holder 202.
  • the lower side of the hole is open and the bottom is closed, so that it does not escape under the sensor structure while forming the receiving space of the inserted test tube.
  • the end of the lower space of the hole can be opened, in this aspect it can be secured by a locking jaw formed in the lid 109" for the tube's airflow as described above.
  • Second Tube Holder 202 The position and dimensions of the test tube can be appropriately adjusted in consideration of the dimensions of the test tube, the position and spacing of the mobile device holder and the light source holder in the sensor structure.
  • the light source holder 203 is formed as a square-shaped hole on the opposite surface of the mobile device holder 201 side with respect to the second tube holder 202.
  • the light source holder 203 is a space in which a square-shaped hole extends toward the center of the sensor structure 200 and is drilled in a downward direction of the second tube holder 202, and an extension of the mobile device holder 201 It can be communicated with the side of the through groove.
  • This communication structure provides a path through which light emitted from the light source passes through the test tube and can be transmitted to the illumination sensor of the mobile device.
  • the shape of the light source holder has a square shape, but as an example, as long as the light source can be mounted, various cross-sections are possible.
  • the light source mounted on the light source holder 203 can pass through the test tube (specifically, the lower portion of the test tube where the mobile phase not bound to the stationary phase is concentrated by centrifugation) and can be transmitted to the exposed surface of the illuminance sensor.
  • the transmitted light is measured by the illuminance sensor, and the measured value can be processed by an application installed in the mobile device, in particular, to display the measured illuminance on the screen of the mobile device. Therefore, if the illuminance displayed on the screen is low, it can be judged as positive.
  • the sensor structure 200 may be an integral structure, and for this purpose, a mold (or molding) or 3D printing method may be used, but a plurality of holes and communication therebetween in a small-sized three-dimensional structure As it is required to form a structure, it can be easily produced using a 3D printing method.
  • the material of the sensor structure 200 that can be manufactured by 3D printing is known, and typically a synthetic polymer or plastic can be used, more typically polyurethane, carbon fiber, thermoplastic resin, And photo-curable resins or combinations thereof.
  • a light source for example, an LED element
  • a battery for providing electricity required for driving the light source are installed in the sensor structure 200, and the mobile device holder 201 is used for mobile. Insert it into the device. Thereafter, when a test tube to be read is inserted into the second tube holder 202 and irradiated with a light source, the installed application is executed, and illuminance is displayed on the screen of the mobile device.
  • the application for measuring the illuminance installed on the mobile device is not particularly limited, and various types disclosed in the field can be applied without particular limitation.
  • the above-described procedure is exemplary, and some of the procedures may be changed.
  • N-Hydroxysuccinimidyl sepharose ⁇ 4 Fast Flow (4B) was purchased from Sigma Aldrich (Cat # H 8280).
  • PCR mixture (Valencia, USA, Cat # 203643).
  • E.coli was cultured in Luria-Bertani (LB) medium at 37° C. for 16 hours. Next, the colony forming unit (CFU) of E.coli was identified using a colony counting method. E.coli was collected in a test tube using a centrifuge (13000 rpm), and DNA was extracted from E.coli pellets at 98° C. over 15 minutes using Quick ExrtactTM DNA Extraction Solution 1.0.
  • LB Luria-Bertani
  • CFU colony forming unit
  • PCR for E.coli was performed. Primers were designed based on stx 2 gene DNA (ref). The reverse primer was labeled with Cy3 at the 5'end of the base sequence. Biotin was labeled at the 5'end of the forward primer so that it could be captured by streptavidin-labeled magnetic beads. The base sequences of the forward primer and the reverse primer are shown in Table 1 below.
  • E.col For amplification of E.col , it was performed using HotStartTaq aq Plus Master Mix Kit. The reaction mixture contained 2X master mix, 0.08 ⁇ M forward primer, 0.08 ⁇ M reverse primer, 1 ⁇ L extracted DNA and deionized water.
  • a C1000 Touch TM thermal cycler PCR System was used for DNA amplification. The thermal cycle conditions were repeated 35 times for 5 minutes at 95°C, 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension at 72°C for 5 minutes. Was performed to complete the gene amplification reaction.
  • the target DNA was amplified against a 120-bp size amplicon using biotin labeled forward and reverse primers.
  • a visually identifiable sensing principle was designed, and a ligand affinity chromatography-based optical sensing platform was developed.
  • Streptavidin-conjugated magnetic particles (MP) were used as the mobile phase and were also used for molecular signals based on their unique reddish brown color.
  • biotinylated primers were synthesized to directly bind MP and PCR products.
  • NHS-functionalized sepharose was used as the stationary phase.
  • a gene from a pathogen was first amplified using a conventional PCR device and mixed with the prepared magnetic particles, so that the moiety on the MP surface binds the amplified gene from PCR.
  • the target gene could be easily diagnosed by visual observation.
  • E.coli was selected as a biomarker for food-derived pathogens.
  • E.coli of 1.0 ⁇ 10 5 CFU was prepared, and its genetic DNA (gDNA) was extracted and purified.
  • Deionized water was used as a negative control test, and the solution was mixed with magnetic particles for 30 minutes. Then, the GelRed diluted 10-fold was reacted for 10 minutes.
  • the prepared 200 ⁇ L NHS-modified Sepharose was carefully packed in test tubes. Due to the suspended sepharose in solution, the test tube was centrifuged for 30 seconds. Then, each prepared test sample was applied to a Sepharose column, and the column was centrifuged at 1000 rpm for 2 minutes.
  • E.coli Bacteria containing a very small amount of E. coli in foods cause food poisoning, so it is required to exhibit high sensitivity properties.
  • E.coli was sequentially diluted from 1.0 ⁇ 10 6 CFU to 1.0 ⁇ 10 1 CFU, and DNA was extracted as described above.
  • the analysis system according to this example was applied under the same conditions, and the results are shown in FIG. 11 together with the electrophoresis test results. According to the figure, the movement of magnetic particles was observed except for the negative control test, which indicates that when using the detection system according to the present embodiment, E. coli can be diagnosed by visual observation. In addition, the magnetic particles slightly shifted at an E.
  • the detection limit (LOD) was determined to be 1.0 ⁇ 10 1 CFU.
  • E.coli is the main factor of food poisoning
  • the possibility of practical application of the analysis platform according to the present embodiment was evaluated by performing an E.coli analysis experiment based on an actual sample.
  • an actual sample artificially infected was prepared by injecting E.coli from 1.0 ⁇ 10 6 CFU to 1.0 ⁇ 10 1 CFU into 10 ⁇ L of milk.
  • the bacterial-infused milk sample was dissolved and the dissolved solution was subjected to conventional PCR.
  • analytical experiments were performed at least three times under the same conditions to ensure reproducibility. The results are shown in FIG. 12.
  • the movement of the MP was observed in the whole picture, which supports that the E.coli based on the actual food sample can be effectively detected in the system according to the present embodiment.
  • MP clearly shifted 1.0 ⁇ 10 1 CFU at an E.coli concentration of greater than or equal to, whereas no MP change was observed in the negative control.
  • the detection limit can be determined as 1.0 ⁇ 10 1 CFU E.coli .
  • the binding of the amine group of streptavidin and the NHS group on sepharose in the amplified PCR product attached to the magnetic particles in the positive test is interfered by steric hindrance. Therefore, maintaining the structure of the double stranded DNA (dsDNA) in a rigid form is important to increase the reaction efficiency.
  • the double strand of DNA has high flexibility in water system, which prevents accurate analysis.
  • the dsDNA-specific insertion material (molecule) was bound, DNA flexibility was changed. In this example, GelRed was used for evaluation.
  • the distance between the initial magnetic particles was 158 nm.
  • the overall streptavidin size about 20 nm on the magnetic particle surface and the hydrodynamic diameter of the particle, the size is reliable.
  • the DNA-bound MP was measured to be 191 nm.
  • the amplified gene averaged 100 bs, and the theoretical size was about 34 nm.
  • the overall size is increased by 68 nm.
  • the DNA length is variable in aqueous solution, and the results obtained support that the DNA exhibits flexibility.
  • the observed size for GelRed, DNA and MP test results was about 224 nm. The results show that GelRed was successfully inserted between the base pairs of dsDNA, and GelRed effectively reduced the flexibility of the amplified DNA.
  • the MP binding affinity with Sepharose in genetic analysis is closely related to the concentration of GelRed.
  • a sepharose-filled (packed) test tube and 1.0 ⁇ 10 5 CFU E.coli were prepared in the same manner as in the previous test. Genes amplified by PCR were reacted with the same concentration of MP. Then, 5, 10, 20, 50, and 100-fold diluted GelRed were sequentially applied for 10 minutes, and the test tubes thus prepared were centrifuged at 1000 rpm for 2 minutes. As shown in FIG. 14A, in the 5-fold diluted GelRed test, aggregation of MP was observed.
  • the movement level of the magnetic beads was sequentially changed in the positive test among all the test results, while a significant change in the magnetic bead movement was not detected in the negative test, which is a bio-analysis proposed in the present disclosure. It indicates that the working principle of the platform is appropriate. This is due to the difference in surface functional groups according to the conjugation of the PCR product and the magnetic beads. Although the movement change of the magnetic beads according to the positive test and the negative test was observed in the reaction time from 30 seconds to 60 seconds, it was insufficient to determine disease infection because the change of the magnetic bead movement was small.
  • the E.coli analysis platform was prepared according to the same procedure as in Example 1, specifically, the test tube was filled with NHS-modified Sepharose, and streptavidin-conjugated reacted with the amplified gene. The magnetic particles (MP) were added dropwise thereto. At this time, broth and milk were used as actual samples (1.0 ⁇ 10 1 -1.0 ⁇ 10 6 CFU, E. coli O157:H7 ).
  • a test tube containing the analytical platform prepared prior to the portable centrifugal separation apparatus as shown in FIG. 15B was inserted using a first tube holder made of 3D printing as in FIG. 15A.
  • the power connector installed at the bottom of the support of the portable centrifugal separator was connected to a USB terminal of a smart phone (LG product name G4) to perform centrifugation over 2 minutes.
  • the volume of the test tube was 400 ⁇ L, and the rotation speed was 2000 rpm.
  • Example 2 instead of visually observing the centrifuged test tube, positive and negative readings were performed using an illuminance sensor built into the smart phone.
  • the analysis platform performs diagnosis using the difference in absorbance and transmittance between the mobile phase (magnetic particle; MP) and the stationary phase (sepharose), as a pre-experiment, a separation wave on the exposed surface of the illumination sensor embedded in the smartphone After placing a transparent substrate filled with each of rose and magnetic particles (MP), LEDs were irradiated to analyze the difference in the amount of light. The results are shown in FIG. 18.
  • the illuminance displayed on the smartphone screen is low, while Sepharose has a relatively high transmittance of irradiated light, so a high illuminance value is displayed. As such, it is judged that it is based on the present analysis platform and can be diagnosed using the illumination sensor built into the smart phone.
  • a sensor structure (mold) was fabricated using 3D printing as shown in FIG. 19A.
  • the LED element was attached as a light source to the light source holder of the sensor structure thus manufactured, and the light source was operated by a 3V battery attached to the side.
  • an analysis platform was applied to two samples (( E. coli O157:H7 in the range of 10 1 to 10 6 CFU)) and a test tube subjected to centrifugation was inserted into the second tube holder of the sensor structure. .
  • a sensor structure in which a test tube is inserted is configured to cover the exposed surface of the illumination sensor of the smart phone, and the illuminance sensor is used to measure the illuminance of the LED light passing through the test tube and drive the installed application.
  • the illuminance value displayed on the screen of the smart phone was confirmed. The results are shown in FIGS. 20A and 20B.
  • the sensor structure used for this has a simple structure, so it can be manufactured inexpensively, and thus, it is considered that there is a high possibility of commercialization.

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Abstract

According to a specific embodiment of the present disclosure, a pathogen diagnostic system is disclosed, wherein a centrifuge that can be driven by power supplied from a mobile device can be used in detecting or diagnosing a target pathogen gene (specifically, an amplified gene of a target pathogen) in a sample through a simple method using visualizable mobile phase particles and stationary phase particles having specific functional groups attached thereto, and on-site analysis can be conveniently performed with low power by using an illuminance sensor in the mobile device.

Description

모바일 기기와 통합된 병원체의 진단 시스템Pathogen diagnostic system integrated with mobile devices
본 개시 내용은 모바일 기기와 통합된 병원체의 진단 시스템에 관한 것이다. 보다 구체적으로, 본 개시 내용은 시각화 가능한 이동상 입자 및 특이적 기능기가 부착된 고정상 입자를 이용하여 간단한 방식으로 시료 내 타겟 병원체의 유전자(구체적으로, 타겟 병원체의 증폭 유전자)를 검출 또는 진단함에 있어서 모바일 기기로부터 공급된 전원에 의하여 구동 가능한 원심 분리 장치를 적용하고, 더 나아가 모바일 기기 내 조도 센서를 이용하여 저전력으로 간편하게 현장 분석을 수행할 수 있는 병원체의 진단 시스템에 관한 것이다.The present disclosure relates to a diagnostic system for pathogens integrated with mobile devices. More specifically, the present disclosure provides a mobile method for detecting or diagnosing a target pathogen gene (specifically, an amplification gene of a target pathogen) in a sample in a simple manner using a visualizable mobile phase particle and a fixed phase particle with a specific functional group attached thereto. The present invention relates to a diagnostic system for a pathogen capable of performing on-site analysis at low power by using a centrifugal device that can be driven by a power supplied from a device, and further using an illuminance sensor in a mobile device.
인간을 포함한 모든 온혈동물의 장 내에는 박테리아, 바이러스 등과 같은 다양한 병원체가 서식하는데 매우 좋은 환경이 제공되고 있다. 병원체는 주변 환경에서 널리 분포되어 있는 바, 구체적으로 박테리아 병원체는 흙, 동물 장기, 동물의 변에 의하여 오염된 물 주방, 주방도구, 책상, 식기 등 일상생활 속의 물건등에서 발견되고 있다. 인체 역시 평균적으로 인체 내외에 걸쳐 150 타입 이상의 박테리아를 갖고 있으며, 이중 많은 미생물들이 인체에 무해하기는 하나, 몇몇 종류는 식중독(botulism), 콜레라(cholera), 설사(diarrhea), 구토(emesis), 폐렴(pneumonia), 장티푸스(typhoid) 등을 포함하는 다양한 감염성 질환을 유발한다. 특히, 200 종류 이상의 질병이 음식 및 음용수 단독을 통하여 전염될 수 있다. 예를 들면, Escherichia Coli(또는 E.coli) 계열은 섭취시 큰 문제를 일으키지 않지만, 이중 몇몇은 설사 등을 유발하는 독소를 생성하는 병원체로 알려져 있다. In the intestines of all warm-blooded animals, including humans, various pathogens such as bacteria and viruses are inhabited, providing a very good environment. Pathogens are widely distributed in the surrounding environment, and specifically, bacterial pathogens are found in soil, animal organs, water contaminated by animal stools, kitchen utensils, desks, tableware, and other objects in everyday life. The human body also has more than 150 types of bacteria on average, both inside and outside the body, although many microorganisms are harmless to the human body, some types are food poisoning, cholera, diarrhea, and emesis. It causes various infectious diseases including pneumonia and typhoid. In particular, more than 200 diseases can be transmitted through food and drinking water alone. For example , the Escherichia Coli (or E.coli ) family does not cause major problems when ingested, but some of them are known to produce toxins that cause diarrhea and the like.
특히, 병원성 대장균 E.coli O157:H7은 장 출혈성 대장균으로서 식중독의 원인균으로 널리 알려져 있으며, 전세계적으로 문제시되고 있다. 또한, 감염 시 용혈성 요독 증후군, 출혈성 대장염, 설사, 신장부전, 발작 등을 유발하며, 심한 경우에는 사망에 이르도록 한다(Reisner, A. et al., 2006. Journal of Bacteriology. 188, 3572-3581; Barrientos, R. M. et al., 2009. Brain, Behavior, and Immunity. 23, 450-454; Schrag, S. J. et al., 2006. PEDIATRICS. 118, 570-576). 살모넬라속균(Salmonella choleraesuis, Salmonella bongori, Salmonella typhimurium) 역시 식중독을 일으키는 대표적인 병원체로서 장티푸스 및 파라티푸스의 원인균이며, 다양한 가축 및 동물 등에 오염될 수 있다. 또한, 적절한 세척, 가공온도 및 저장조건이 지켜지지 못한다면, 살모넬라균이 번식할 수 있으며, 이렇게 오염된 식수, 식품 등을 통하여 다른 식품과 교차 오염될 수 있다.In particular, the pathogenic E. coli O157:H7 is an intestinal hemorrhagic E. coli, widely known as a causative agent of food poisoning, and is a worldwide problem. In addition, infection causes hemolytic uremic syndrome, hemorrhagic colitis, diarrhea, kidney failure, and seizures, leading to death in severe cases (Reisner, A. et al. , 2006. Journal of Bacteriology. 188, 3572-3581) ; Barrientos, RM et al. , 2009. Brain, Behavior, and Immunity. 23, 450-454; Schrag, SJ et al. , 2006. PEDIATRICS. 118, 570-576). Salmonella choleraesuis , Salmonella bongori , and Salmonella typhimurium are also representative pathogens that cause food poisoning, and are the causative agents of typhoid and paratyphoid, and can be contaminated with various livestock and animals. In addition, if proper washing, processing temperature and storage conditions are not observed, Salmonella can multiply and cross-contaminated with other foods through contaminated drinking water and food.
한편, 병원체, 특히 식중독균은 일반적으로 오염된 식품을 통하여 쉽게 인간에게 감염될 수 있다. 통상의 조건 하에서 병원체의 번식속도는 매우 빠르기 때문에 아무리 적은 수의 병원체라도 일단 인체 내에 침입할 경우, 이의 생장환경에 매우 적합한 장 속에서 빠르게 성장하여 인간의 건강을 위협할 수 있는 수준까지 이르게 된다. 따라서, 오염된 환경으로부터 병원체(특히, E.coli)의 존재 여부를 정확하게 진단할 수 있는 기술이 요구되고 있다. On the other hand, pathogens, especially food poisoning bacteria, can be easily infected by humans through generally contaminated food. Under normal conditions, the rate of reproduction of a pathogen is very fast, so even a small number of pathogens, once invading the human body, rapidly grow in the intestine, which is very suitable for their growth environment, to a level that can threaten human health. Therefore, there is a need for a technique capable of accurately diagnosing the presence of a pathogen (especially E. coli ) from a contaminated environment.
이와 관련하여, 시료(예를 들면, 음식물, 식수 등) 내 병원체의 존재 유무를 진단하는 방법에 관한 다양한 연구가 진행되고 있는 바, 예를 들면 ATP(adenosine triphosphate) 및 루시페린(luciferin)/루시페라아제(luciferase)가 반응하여 발광하는 원리를 이용하는 ATP 측정법을 들 수 있다. 이러한 방법은 정확한 진단을 위하여는 음식물 등으로부터 유래하는 ATP를 제거하고, 검출 대상인 미생물-유래 ATP만을 측정해야 하는 만큼, 유해 미생물만을 포집하여 정확한 진단을 수행하는데 근본적인 한계를 갖고 있다. 다른 방법으로 DNA 및 RNA와 같은 핵산을 기반으로 하는 검출법이 알려져 있는 바, 핵산, 특히 DNA의 경우에는 PCR(polymerase chain reaction)이라는 강력한 증폭 기술에 의하여 진단 감도를 높일 수 있어 널리 이용되고 있다. 일반적으로, PCR은 체외에서 DNA 유전자 시료를 증폭하는 분자 생물학적인 방법으로서, DNA에 대한 감도를 증가시키기 위하여 DNA 시료의 량 및 농도를 높이는 기술이다. 이처럼, 유전학적 분석을 기반으로 하는 임상 진단 기술은 주로 박테리아(bacteria), 균류(fungus) 또는 바이러스(virus)로부터 핵산을 회수하여 증폭 반응을 수행한 다음, 다양한 검출 수단(예를 들면, 광학적, 전기화학적 또는 기계적 바이오센서 디바이스)을 이용하여 앰플리콘(amplicon) 분석을 수행하는 방식이다.In this regard, various studies have been conducted on a method of diagnosing the presence or absence of a pathogen in a sample (e.g., food, drinking water, etc.), for example, ATP (adenosine triphosphate) and luciferin/luciferase ( luciferase) is an ATP measurement method using the principle of light emission. This method has a fundamental limitation in collecting only harmful microorganisms and performing accurate diagnosis, since it is necessary to remove ATP derived from food and the like for accurate diagnosis and to measure only the microorganism-derived ATP to be detected. As other methods, detection methods based on nucleic acids such as DNA and RNA are known, and in the case of nucleic acids, especially DNA, they are widely used because they can enhance diagnostic sensitivity by a powerful amplification technique called polymerase chain reaction (PCR). In general, PCR is a molecular biological method for amplifying a DNA gene sample in vitro, and is a technique of increasing the amount and concentration of a DNA sample in order to increase sensitivity to DNA. As such, clinical diagnostic technology based on genetic analysis mainly recovers nucleic acids from bacteria, fungus, or viruses, performs amplification reactions, and then performs various detection means (eg, optical, It is a method of performing an amplicon analysis using an electrochemical or mechanical biosensor device).
한편, 자연발생적으로 입수되는 환경 시료의 경우, 진단 대상인 특정(타겟) 박테리아 또는 바이러스가 미량으로 존재하는 경우가 일반적이다. 특히, E.coli를 함유하는 환경 시료의 경우에 있어서, 진단하고자 하는 타겟 병원체가 제한된 량으로 존재하기 때문에 정확한 검출을 위하여는 시료 내 바이오물질의 핵산을 효과적으로 회수하는 것이 요구된다. 이에 대하여, 신속 정확하며 소량의 시료를 이용하여 검출할 수 있는 현장진료 테스트(point of care testing; POCT) 관련 기술에 대한 연구가 활발히 진행되고 있다. POCT 기술은 현재 각종 병원, 연구기관 등에서 널리 적용되고 있는 바, 특히 높은 감도 특성을 갖고 정확한 진단이 가능한 POCT를 구현하는 것은 임상 및 상용 의료 분야에서는 주된 관심사 중 하나이다.On the other hand, in the case of an environmental sample that is obtained naturally, it is common for a specific (target) bacteria or virus to be diagnosed to be present in a trace amount. In particular, in the case of an environmental sample containing E. coli , since the target pathogen to be diagnosed exists in a limited amount, it is required to effectively recover the nucleic acid of the biomaterial in the sample for accurate detection. On the other hand, research on a technology related to point of care testing (POCT) that is quick and accurate and can be detected using a small amount of samples is actively being conducted. POCT technology is currently widely applied in various hospitals, research institutes, etc. In particular, it is one of the main concerns in clinical and commercial medical fields to realize POCT with high sensitivity and accurate diagnosis.
현재, 다양한 바이오 분석 플랫폼이 제시되고 있는 바, 바이오센싱 절차 및 장치 측면에서 POCT가 실제 활용될 수 있도록 관련 기술을 개선하고 있다. 최근 개발된 바이 분석 플랫폼의 원리 및 장치로 인하여 종래에 비하여 진단 신뢰성을 높일 수 있음에도 불구하고, 전체 프로세스를 정확하게 수행하기 위하여는 복잡한 실험 방법 및 숙련된 전문가에 의한 수행이 요구되므로 여전히 병원 및 연구 설비 수준에서 이루어지고 있는 실정이다. 이러한 높은 기술 수준이 필요한 바이오센서는 일반 환자 및 사용자에게는 접근이 용이하지 않은 한계를 갖고 있다(Mabey et al., 2004; Peeling and Mabey, 2010). Currently, various bio-analysis platforms have been proposed, and related technologies are being improved so that POCT can be actually utilized in terms of bio-sensing procedures and devices. Despite the fact that the diagnostic reliability of the bi-analytical platform developed recently can be increased compared to the conventional one, it is still required for complex experiment methods and skilled experts to perform the entire process, so it is still used in hospitals and research facilities. This is happening at the level. Biosensors that require such a high level of technology have limitations that are not easily accessible to ordinary patients and users (Mabey et al., 2004; Peeling and Mabey, 2010).
이처럼, 복잡한 바이오센싱 시스템의 간편화 및 비용 절감과 관련한 이슈로 인하여, 보다 간편한 분석 플랫폼이 요구되고 있다. 그러나, 종래 기술에서는 입수된 시료의 처리부터 분석 또는 진단까지의 일련의 과정을 현장에서 간편하게 수행하기 곤란한 바, 시료 분석을 위하여는 원심 분리와 같은 최소한의 물리적 조작이 수반되기 때문이다. 따라서, 현장에서 입수되는 시료 내 유전자를 간편하면서도 정확하게 진단할 수 있고, 더 나아가 진단에 수반되는 일련의 조작을 간편하게 수행할 수 있는 방안이 절실히 요구된다.As such, due to issues related to the simplification of the complex bio-sensing system and cost reduction, a simple analysis platform is required. However, in the prior art, it is difficult to easily perform a series of processes from the processing of an obtained sample to analysis or diagnosis in the field, because for analysis of the sample, minimal physical manipulation such as centrifugation is involved. Therefore, there is an urgent need for a method capable of easily and accurately diagnosing a gene in a sample obtained in the field, and further performing a series of operations accompanying the diagnosis.
본 개시 내용에 따른 구체예에서는 육안 검출이 가능한 분석 원리 또는 플랫폼을 기반으로 하여 시료 내 병원체(예를 들면, 식중독 균)를 현장에서 진단(또는 분석)할 수 있고, 더 나아가 시료 분석에 수반되는 일련의 조작 및/또는 판독 과정을 모바일 기기를 이용하여 간편하면서도 정확하게 구현할 수 있는 방안을 제공하고자 한다.In a specific example according to the present disclosure, a pathogen (eg, food poisoning fungus) in a sample may be diagnosed (or analyzed) in the field based on an analysis principle or platform capable of visual detection, and further involved in sample analysis It is intended to provide a method to implement a simple and accurate series of manipulation and/or reading processes using a mobile device.
본 개시 내용의 일 구체예에 따르면,According to one embodiment of the present disclosure,
(A) 시각적 확인(identification)이 가능하고, 제1 기능기-함유 성분으로 수식된 복수의 이동상(mobile phase) 입자, 및 상기 제1 기능기-함유 성분과 결합능을 갖는 화학적 기능기에 의하여 활성화된 복수의 고정상(stationary phase) 입자를 포함하고, 상기 제1 기능기-함유 성분이 타겟 병원체의 유전자에 특이적인 프라이머와 접합된 제2 기능기-함유 성분과 바인딩 특성을 갖되, 상기 제2 기능기-함유 성분이 프라이머를 통하여 증폭된 타겟 병원체의 유전자와 접합되어 있는 경우에는 입체 장애로 인하여 상기 이동상 입자와 상기 고정상 입자 간의 바인딩이 억제되며, 그리고 상기 고정상 입자에 바인딩되지 않은 이동상 입자를 분리함으로써, 시각적으로 또는 조도 센서에 의하여 확인 가능하도록 구성된 병원체의 분석 플랫폼; 그리고(A) A plurality of mobile phase particles capable of visual identification and modified with a first functional group-containing component, and activated by a chemical functional group having binding ability with the first functional group-containing component A plurality of stationary phase particles, wherein the first functional group-containing component has binding properties with a second functional group-containing component conjugated with a primer specific to the gene of the target pathogen, but the second functional group -When the containing component is conjugated to the gene of the target pathogen amplified through the primer, binding between the mobile phase particle and the stationary phase particle is suppressed due to steric hindrance, and by separating the mobile phase particle that is not bound to the stationary phase particle, An analysis platform for pathogens configured to be identifiable visually or by an illuminance sensor; And
(B) (b1) 모바일 기기의 전원 입출력 단자와 연결되고 이로부터 공급된 전원에 의하여 상기 고정상 입자에 바인딩되지 않은 이동상 입자를 분리하는 휴대용 원심 분리 장치, 및 (b2) 모바일 기기에 내장된 조도 센서를 이용하여 상기 분석 플랫폼에 의하여 타겟 병원체를 분석하기 위한 센싱부 중 적어도 하나를 포함하는 병원체의 진단 시스템이 제공된다.(B) (b1) a portable centrifugal separation device that is connected to a power input/output terminal of a mobile device and separates mobile phase particles that are not bound to the stationary phase particles by power supplied therefrom, and (b2) an illuminance sensor embedded in the mobile device A diagnostic system for a pathogen comprising at least one of a sensing unit for analyzing a target pathogen is provided by using the analysis platform.
예시적 구체예에 따르면, 상기 전원 입출력 단자는 USB 포트의 전원 입출력 단자일 수 있다. According to an exemplary embodiment, the power input/output terminal may be a power input/output terminal of the USB port.
예시적 구체예에 따르면, 상기 휴대용 원심 분리 장치는,According to an exemplary embodiment, the portable centrifugal separation device,
직립형 지지부의 상측에 설치되며, 모바일 기기로부터 공급된 전원에 의하여 구동되는 모터부;A motor unit installed on an upper side of the upright support unit and driven by power supplied from a mobile device;
상기 직립형 지지부의 하측에 구비되어 상기 모바일 기기의 전원 입출력 단자에 전기적으로 연결 가능한 커넥터 단자;A connector terminal provided at a lower side of the upright support portion and electrically connectable to a power input/output terminal of the mobile device;
상기 모터부와 기계적으로 연결되어 모터부의 회전 시 수평 방향으로 회전하도록 구성된 적어도 하나의 고리 부재; 및At least one ring member mechanically connected to the motor part and configured to rotate in a horizontal direction when the motor part rotates; And
상기 적어도 하나의 고리 부재와 고리 결합 또는 인터로킹(interlocking)되는 걸림부 및 튜브 시험관을 삽입하도록 형성된 튜브 수용부를 구비하는 제1 튜브 홀더;A first tube holder having a locking portion interlocking or interlocking with the at least one ring member and a tube receiving portion formed to insert a tube test tube;
를 포함하며,It includes,
상기 모터부의 회전 시 제1 튜브 홀더에 삽입된 튜브 시험관이 원심력에 의하여 수직 위치에서 수평 위치로 전환되면서 회전할 수 있다.When the motor part rotates, the tube test tube inserted into the first tube holder can be rotated while being converted from a vertical position to a horizontal position by centrifugal force.
예시적 구체예에 따르면, 상기 모바일 기기는 스마트 기기일 수 있다.According to an exemplary embodiment, the mobile device may be a smart device.
예시적 구체예에 따르면, 상기 스마트 기기는 스마트폰, 태블릿 또는 노트북일 수 있다.According to an exemplary embodiment, the smart device may be a smartphone, tablet or notebook.
예시적 구체예에 따르면, 상기 튜브 시험관은,According to an exemplary embodiment, the tube test tube,
상단부에 개구가 형성되고 하단부는 폐쇄되어 있는 광 투과성 재질의 시험관 본체; 및A test tube body made of a light transmissive material having an opening formed at an upper portion and closed at an lower portion; And
상기 시험관 본체의 개구에 체결되도록 구성되며, 상기 튜브 홀더의 튜브 수용부 내로 튜브 시험관이 삽입된 후에는 모터부의 구동 시 가해지는 원심력 하에서 고정 상태에 있도록 둘레를 따라 돌출되어 있는 걸림 턱이 구비된 개폐용 뚜껑;It is configured to be fastened to the opening of the test tube body, and after the tube test tube is inserted into the tube receiving part of the tube holder, the opening and closing is provided with a locking jaw protruding along the periphery so as to be in a fixed state under the centrifugal force applied when driving the motor part. Dragon lid;
을 포함할 수 있다.It may include.
예시적 구체예에 따르면, 상기 센싱부는, According to an exemplary embodiment, the sensing unit,
삽입 방식으로 모바일 기기와 체결되면서 상기 모바일 기기 내 조도 센서의 노출면을 수용하도록 하측에 연장된 관통 홈이 형성된 모바일 기기 홀더;A mobile device holder having a through groove extending at a lower side to accommodate an exposed surface of the illuminance sensor in the mobile device while being fastened with the mobile device by an insertion method;
튜브 시험관 내에 분석 플랫폼이 수용되어 있는 테스트 시험관을 삽입하기 위한 제2 튜브 홀더; 및A second tube holder for inserting a test tube in which the analysis platform is housed in a tube test tube; And
상기 삽입된 테스트 시험관으로 광을 조사하기 위한 광원이 장착되는 광원 홀더;A light source holder equipped with a light source for irradiating light to the inserted test tube;
를 구비하는 센서 구조물을 포함하고, It includes a sensor structure having a,
상기 센서 구조물 내에서 모바일 기기 홀더, 제2 튜브 홀더 및 광원 홀더는 광원으로부터 조사된 광이 테스트 시험관을 투과하여 조도 센서의 노출면으로 전달되도록 배열될 수 있다. In the sensor structure, the mobile device holder, the second tube holder, and the light source holder may be arranged so that light irradiated from the light source passes through the test tube and is transmitted to the exposed surface of the illuminance sensor.
예시적 구체예에 따르면, 상기 센서 구조물은 일체형 구조물일 수 있다.According to an exemplary embodiment, the sensor structure may be an integral structure.
예시적 구체예에 따르면, 상기 센서 구조물은 3D 프린팅 방식으로 제작된 것일 수 있다.According to an exemplary embodiment, the sensor structure may be manufactured by 3D printing.
본 개시 내용의 구체예에 따른 유전자 진단 시스템은, 기능화된 인터페이스를 통한 입자-기반의 DNA 선택적 운반을 수반하는 분석 플랫폼에, 물리적 분리 수단으로서 모바일 기기에 의하여 구동 가능한 휴대용 원심 분리 장치, 및/또는 모바일 기기에 내장된 조도 센서를 이용한 센싱부를 조합함으로써 시료 내 타겟 병원체의 양성/음성을 육안으로 또는 모바일 기기에 의하여 단시간에 정확하게 판독할 수 있는 장점을 제공한다. 특히, 다수가 휴대하고 있는 모바일 기기를 휴대용 원심분리 장치의 전원 공급원 및/또는 센싱부로 적용할 수 있기 때문에 현장 진단의 편의성을 극대화할 수 있다. 더 나아가, 조도 센서가 내장된 모바일 기기(예를 들면, 시판 중인 스마트 폰)를 사용할 경우, 단일 기기만으로도 원심분리의 전원 공급원 및 센싱부의 2가지 기능을 동시에 구현할 수 있다. A genetic diagnostic system according to an embodiment of the present disclosure is a portable centrifugal separation device operable by a mobile device as a physical separation means, in an analysis platform involving particle-based DNA selective transport through a functionalized interface, and/or The combination of a sensing unit using an illuminance sensor embedded in a mobile device provides an advantage of accurately and positively reading a target pathogen in a sample visually or by a mobile device in a short time. In particular, since a mobile device carried by a large number can be applied as a power source and/or a sensing unit of a portable centrifugal separator, convenience of on-site diagnosis can be maximized. Furthermore, when using a mobile device with a built-in illuminance sensor (for example, a commercially available smart phone), it is possible to simultaneously implement two functions of a centrifugal power supply and a sensing unit using only a single device.
따라서, 향후 임상 진단, 환경 모니터링 등의 다양한 분야에서 광범위한 활용이 기대된다.Therefore, it is expected to be widely used in various fields such as clinical diagnosis and environmental monitoring in the future.
도 1은 일 구체예에 따라 기능화된 인터페이스를 통한 입자-기반의 유전자 분석 플랫폼을 이용한 진단 원리를 예시적으로 도시하는 도면이고;1 is a diagram illustratively illustrating a diagnostic principle using a particle-based genetic analysis platform via a functionalized interface according to one embodiment;
도 2는 예시적 구체예에서 고정상 입자의 활성화를 위한 화학적 기능기로 NHS(N-hydroxysuccinimide)를 사용하는 경우의 분석 플랫폼을 구성하는 요소를 도시하는 도면이고;FIG. 2 is a diagram showing elements constituting an analytical platform when N-hydroxysuccinimide (NHS) is used as a chemical functional group for activation of a stationary phase particle in an exemplary embodiment;
도 3은 예시적 구체예에 있어서 고정상 입자의 활성화를 위한 화학적 기능기로 NHS(N-hydroxysuccinimide)를 사용하는 경우에 있어서 진단 원리를 개략적으로 보여주는 도면이고;3 is a diagram schematically showing a diagnostic principle in the case of using NHS (N-hydroxysuccinimide) as a chemical functional group for activation of a stationary phase particle in an exemplary embodiment;
도 4는 일 구체예에 있어서 기능화된 인터페이스를 통한 입자-기반의 유전자 분석 플랫폼을 개략적으로 도시하는 도면이고;4 is a schematic diagram of a particle-based genetic analysis platform via a functionalized interface in one embodiment;
도 5a 및 도 5b 각각은 병원체 진단(분석) 시스템 내 휴대용 원심 분리 장치 및 이에 포함된 튜브 홀더의 예시적인 구성을 도시하는 도면이고; 5A and 5B are each a diagram showing an exemplary configuration of a portable centrifuge device in a pathogen diagnosis (analysis) system and a tube holder included therein;
도 6은 모바일 기기로서 스마트폰의 전원 입출력 단자와 전기적으로 연결된 휴대용 원심 분리 장치의 작동 원리를 도시하는 도면이고;FIG. 6 is a diagram showing an operating principle of a portable centrifugal device electrically connected to a power input/output terminal of a smartphone as a mobile device;
도 7a 및 도 7b 각각은 예시적 구체예에 있어서 휴대용 원심 분리 장치 내 튜브 홀더의 변형 예를 도시하는 도면이고;7A and 7B each show a modified example of a tube holder in a portable centrifugal device in an exemplary embodiment;
도 8은 예시적 구체예에 따라 기능화된 인터페이스를 통한 입자-기반의 유전자의 분석 플랫폼을 조도 센서에 의하여 양성 및 음성을 판정하는 원리를 개략적으로 도시하는 도면이고;8 is a diagram schematically showing the principle of determining positive and negative by an illuminance sensor on an analysis platform for particle-based genes through a functionalized interface according to an exemplary embodiment;
도 9는 예시적 구체예에 있어서 센싱부를 구성하는 센서 구조물을 도시하는 도면이고;9 is a view showing a sensor structure constituting a sensing unit in an exemplary embodiment;
도 10a 및 도 10b 각각은 실시예 1에서 기능화된 인터페이스를 통한 입자-기반의 유전자 분석 플랫폼을 이용한 양성 및 음성 테스트 결과를 나타내는 사진이고; 10A and 10B, respectively, are photographs showing positive and negative test results using a particle-based genetic analysis platform via a functionalized interface in Example 1;
도 11은 실시예 1에서 기능화된 인터페이스를 통한 입자-기반의 유전자 분석 플랫폼을 이용한 E. Coli O157:H7의 검출 한계를 평가하기 위한 실험 결과 및 전기영동 테스트 결과를 나타내는 도면이고;FIG. 11 is a diagram showing experimental results and electrophoresis test results for evaluating detection limits of E. Coli O157:H7 using a particle-based genetic analysis platform through a functionalized interface in Example 1;
도 12는 실시예 1에서 기능화된 인터페이스를 통한 입자-기반의 유전자 분석 플랫폼을 이용하여 E. Coli O157:H7를 함유하는 우유 시료를 분석한 결과를 나타내는 도면이고;12 is a diagram showing the results of analyzing a milk sample containing E. Coli O157:H7 using a particle-based genetic analysis platform via a functionalized interface in Example 1;
도 13은 실시예 1에서 자성 입자(MP) 단독, 자성 입자에 증폭된 유전자가 접합된 상태(MP+DNA), 그리고 자성 입자에 GelRed가 삽입되어 증폭된 유전자가 접합된 상태(MP+DNA+GelRed) 각각에 대한 DLS(dynamic light scattering) 사이즈를 보여주는 그래프이고;13 is a magnetic particle (MP) alone in Example 1, a state in which the amplified gene is conjugated to the magnetic particle (MP+DNA), and a state in which the amplified gene is conjugated by inserting GelRed into the magnetic particle (MP+DNA+) GelRed) is a graph showing the dynamic light scattering (DLS) size for each;
도 14a는 실시예 1에서 GelRed 희석 인자(dilution factor)에 따른 삽입 염료(GelRed)의 농도 최적화 실험 결과를 나타내는 사진이고; Figure 14a is a photograph showing the results of the concentration optimization experiment of the insertion dye (GelRed) according to the GelRed dilution factor in Example 1;
도 14b는 실시예 1에서 원심 분리 중 회전 속도의 최적화 실험 결과를 나타내는 사진이고;14B is a photograph showing the results of optimization experiments of the rotational speed during centrifugation in Example 1;
도 15a 및 도 15b 각각은 실시예 2에서 사용된 3D 프린팅에 의하여 제작된 튜브 홀더 및 이를 적용한 휴대용 원심 분리 장치의 외관을 보여주는 사진이고;15A and 15B are each a photograph showing the appearance of a tube holder manufactured by 3D printing used in Example 2 and a portable centrifugal separation device applying the same;
도 16은 실시예 2에서 수행된 병원체 진단 과정을 보여주는 사진이고; 16 is a photograph showing a pathogen diagnosis process performed in Example 2;
도 17a 및 도 17b 각각은 실시예 2에 따라 시료로서 E. Coli O157:H7를 함유하는 브로스(broth) 및 우유(milk)를 사용하고, 도 15b에 도시된 휴대용 원심분리 장치에 의한 원심 분리로부터 얻은 분석 플랫폼의 전기영동 테스트 및 육안 관찰 결과를 보여주는 사진이고; 17A and 17B each use broth and milk containing E. Coli O157:H7 as a sample according to Example 2, and from centrifugation by the portable centrifugation apparatus shown in FIG. 15B A photograph showing the electrophoresis test and visual observation results of the obtained analytical platform;
도 18은 사전 테스트로서 이동상 입자(자성 입자: MP) 및 고정상 입자(아가로오스) 각각에 대하여 스마트 폰에 내장된 조도 센서를 이용하여 센서를 이용하여 측정한 조도를 스마트 폰에 설치된 어플리케이션을 통하여 표시한 결과를 나타내는 사진이고; 18 is a preliminary test for mobile phase particles (magnetic particles: MP) and fixed phase particles (agarose) for each of the illuminance measured using a sensor using an illuminance sensor built into a smart phone through an application installed on the smart phone. A photograph showing the displayed result;
도 19a 및 도 19b 각각은 실시예 3에서 3D 프린팅으로 제작된 센서 구조물에 광원, 배터리 및 테스트 시험관을 장착한 상태, 및 이를 스마트 폰에 결합한 외관을 보여주는 사진이고; 그리고19A and 19B are each a photograph showing a state in which the sensor structure produced by 3D printing in Example 3 is equipped with a light source, a battery, and a test tube, and the appearance of combining it with a smart phone; And
도 20a 및 도 20b 각각은 실시예 3에서 스마트 폰에 내장된 조도 센서를 이용하여 대장균의 진단 테스트를 수행한 결과를 보여주는 그래프이다. 20A and 20B are graphs showing the results of performing a diagnostic test of E. coli in Example 3 using an illuminance sensor embedded in a smartphone.
본 발명은 첨부된 도면을 참고로 하여 하기의 설명에 의하여 모두 달성될 수 있다. 하기의 설명은 본 발명의 바람직한 구체예를 기술하는 것으로 이해되어야 하며, 본 발명이 반드시 이에 한정되는 것은 아님을 이해해야 한다. The present invention can be achieved by the following description with reference to the accompanying drawings. It should be understood that the following description is intended to describe preferred embodiments of the invention, and that the invention is not necessarily limited thereto.
또한, 첨부된 도면은 이해를 돕기 위하여 실제 층의 두께(또는 높이) 또는 다른 층과의 비율에 비하여 다소 과장되게 표현된 것일 수 있으며, 그 의미는 후술하는 관련 기재의 구체적 취지에 의하여 적절히 이해될 수 있다.In addition, the accompanying drawings may be expressed somewhat exaggerated compared to the thickness (or height) of the actual layer or a ratio with other layers in order to help understanding, and its meaning will be properly understood by the specific purpose of the related description to be described later. Can.
본 명세서에서 사용되는 용어는 하기와 같이 정의될 수 있다.Terms used in the present specification may be defined as follows.
"바인딩(binding)"은 표면에 공유 또는 비공유 방식으로 결합 또는 연결되는 것을 의미할 수 있다."Binding" may mean that the surface is coupled or connected in a covalent or non-covalent manner.
"시료"는 검출하고자 하는 타겟 병원체를 함유할 수 있는 한, 특정 종류 또는 형태로 한정되는 것은 아니다. 예시적으로, 시료는 생물학적 시료, 예를 들면 생물학적 유체(fluid) 또는 생물학적 조직일 수 있다. 생물학적 유체의 예로서, 뇨, 혈액, 혈장, 혈청, 타액, 정액, 대변, 가래, 뇌척수액, 눈물, 점액, 양수 등을 들 수 있다. 생물학적 조직은 세포의 집합으로서, 대체적으로 인간, 동물, 식물, 세균, 진균 또는 바이러스 구조물의 구조적 물질의 하나를 형성하는 세포 내 물질들과 특정 종류의 집합으로서 연결 조직, 상피 조직, 근육 조직 및 신경 조직 등이 이에 해당될 수 있다. 또한, 생물학적 조직의 예에는 장기, 종양, 림프절, 동맥 및 개별적인 세포(들)도 포함될 수 있다. 이외에도, 시료는 바이오물질을 저농도로 함유하는 환경 시료(environmental sample)를 포함할 수 있으며, 예를 들면 식수, 음식물 등과 같이 다양한 형태 및 종류를 포함할 수 있다."Sample" is not limited to a specific type or form as long as it can contain the target pathogen to be detected. Illustratively, the sample can be a biological sample, such as a biological fluid or biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, and the like. Biological tissue is a collection of cells, usually a set of intracellular substances that form one of the structural substances of a human, animal, plant, bacterial, fungal, or viral construct, and a specific kind of connective tissue, epithelial tissue, muscle tissue, and nerves. Organizations, etc. may be this. Also, examples of biological tissue may include organs, tumors, lymph nodes, arteries and individual cell(s). In addition, the sample may include an environmental sample containing a biomaterial at a low concentration, and may include various forms and types, such as drinking water and food.
"프라이머"는 상보적 스트랜드의 합성이 폴리머라아제에 의하여 촉매화되는 조건 하에 있는 경우, 상보적 스트랜드를 따라 핵산의 합성 또는 복제 초기 지점으로 작용할 수 있는 올리고뉴클레오티드(합성 또는 천연)를 의미할 수 있다.“Primer” can mean an oligonucleotide (synthetic or natural) that can act as an initial point of synthesis or replication of a nucleic acid along a complementary strand when synthesis of the complementary strand is under conditions catalyzed by a polymerase. have.
"타겟 유전자"는 수개, 수백 개, 수천 개 또는 수백만 개의 뉴클레오티드로 이루어질 수 있으며, 또한 DNA, RNA 등의 절편(fragment)도 해당될 수 있다.The "target gene" may consist of several hundreds, hundreds, thousands, or millions of nucleotides, and may also be a fragment of DNA, RNA, or the like.
"용해(lysis)"는 세포의 분해에 따라서 세포막이 파열되는 동시에 세포 내용물이 노출되는 현상을 의미할 수 있는 바, 통상적으로 PCR과 같은 증폭 과정의 전 단계에서 핵산을 분리하기 위하여 많이 사용되고 있다. "Dissolution (lysis)" may mean a phenomenon in which cell membranes are ruptured and cell contents are exposed upon cell decomposition, and is commonly used to separate nucleic acids in all stages of amplification processes such as PCR.
"박테리아"는 외막(bacterial envelope)은 구조에 따라 그람(Gram) 염색반응이 구별되는 바, 그람 음성 박테리아(얇은 뮤레인(murein) 또는 펩티도글리칸 층을 가지며 외막의 지질 이중층을 가짐)와 그람 양성 박테리아(두꺼운 뮤레인 또는 펩티도글리칸 층을 가지며 이로써 Crystal violet을 보유함)로 구분된다. 그람 음성 박테리아의 세포막은 포스포리피드(phospholipid)와 기타 글리코프로테인(glycoprotein)으로 이루어져 있으며, 포스포리피드의 대부분은 (-)의 하전을 띄고 있다(net negative charge). 그 종류로는 살모넬라균, 수막염균, 스피로헤타 콜레라균, 페스트균, 티푸스균, 이질균, 대장균, 임균 등이 있다. 반면, 그람 양성 박테리아의 경우, 세포벽은 주로 펩티도글리칸(peptidoglycan) 및 타이코산(teichoic acid)으로 구성되는 바, 그 표면은 거의 중성에 가까운 하전 특성을 갖고 있으며, 포도상구균, 연쇄상구균, 탄저균, 디프테리아균, 파상풍균, 폐렴균 등을 들 수 있다."Bacteria", the outer membrane (bacterial envelope) of the Gram (Gram) staining reaction is distinguished according to the structure, the Gram-negative bacteria (thin murine (murein) or peptidoglycan layer and has a lipid bilayer of the outer membrane) and Gram-positive bacteria (thick murine or peptidoglycan layer with crystal violet). The cell membrane of gram-negative bacteria consists of phospholipids and other glycoproteins, and most of the phospholipids have a negative charge (net negative charge). The types include Salmonella, meningitis, spiroheta cholera, plague, typhoid, heterogeneous, E. coli, gonorrhea. On the other hand, in the case of Gram-positive bacteria, the cell wall is mainly composed of peptidoglycan and teichoic acid, and its surface has almost neutral charge characteristics, and staphylococcus, streptococcus, and anthrax , Diphtheria, tetanus, and pneumonia.
"증폭(amplification)"은 주형 분자의 적어도 하나의 세그먼트의 복수개 복제물을 형성하기 위하여 반복적으로 일어나는 반응을 의미할 수 있다.“Amplification” can mean a reaction that occurs repeatedly to form multiple copies of at least one segment of a template molecule.
"PCR"은 사이클 프로세스(가열 및 냉각이 교대로 이루어짐)에 의하여 하나의 최초 주형으로부터 다량의 동일한 DNA 스트랜드가 형성되는 반응을 의미하는 바, 통상적으로 PCR 혼합물은 (i) 증폭하고자 하는 염기서열을 갖는 주형인 이중나선형 DNA 분자, (ii) 프라이머(주형 DNA 내 상보적 DNA 염기서열과 결합할 수 있는 단일 스트랜드 DNA 분자), (iii) dATP, dTTP, dGTP, 및 dCTP의 혼합물(PCR 증폭 과정에서 새로운 DNA 분자를 형성하도록 합쳐지는 뉴클레오티드 서브유닛)인 dNTP, 및 (iv) Taq DNA 폴리머라아제(dNTP)를 사용하여 새로운 DNA 분자를 합성하는 효소)를 포함할 수 있다.“PCR” refers to a reaction in which a large amount of identical DNA strands are formed from one initial template by a cycle process (heating and cooling are alternately performed). Typically, PCR mixtures are (i) sequencing sequences to be amplified. A double-stranded DNA molecule, (ii) a primer (a single stranded DNA molecule capable of binding to a complementary DNA sequence in the template DNA), (iii) a mixture of dATP, dTTP, dGTP, and dCTP (in PCR amplification process) Nucleotide subunits joined to form a new DNA molecule) dNTP, and (iv) an enzyme that synthesizes a new DNA molecule using Taq DNA polymerase (dNTP)).
"상에" 및 "위에"라는 표현은 상대적인 위치 개념을 언급하기 위하여 사용되는 것으로 이해될 수 있다. 따라서, 언급된 층에 다른 구성 요소 또는 층이 직접적으로 존재하는 경우뿐만 아니라, 그 사이에 다른 층(중간층) 또는 구성 요소가 개재되거나 존재할 수도 있다. 이와 유사하게, "하측에", "하부에" 및 "아래에"라는 표현 및 "사이에"라는 표현 역시 위치에 대한 상대적 개념으로 이해될 수 있을 것이다. 또한, "순차적으로"라는 표현 역시 상대적인 위치 개념으로 이해될 수 있다. The expressions “on” and “on” can be understood to be used to refer to the concept of relative position. Thus, as well as when other components or layers are present directly in the recited layer, other layers (intermediate layers) or components may be interposed or present therebetween. Similarly, the expressions “below”, “below” and “below” and “between” may also be understood as relative concepts of location. In addition, the expression "sequentially" can also be understood as a concept of relative location.
병원체의 분석 플랫폼의 원리Principles of the pathogen analysis platform
도 1은 본 개시 내용의 일 구체예에 따라 기능화된 인터페이스를 통한 입자-기반의 유전자 분석 플랫폼을 이용한 진단 원리를 예시적으로 도시한다. 또한, 도 2 및 도 3 각각은 고정상 입자의 활성화를 위한 화학적 기능기로 NHS(N-hydroxysuccinimide)를 사용하는 경우에 있어서 진단 시스템의 구성 요소 및 진단 원리를 개략적으로 도시한다. 1 illustratively illustrates a diagnostic principle using a particle-based genetic analysis platform through a functionalized interface according to one embodiment of the present disclosure. In addition, each of FIGS. 2 and 3 schematically shows components and diagnostic principles of a diagnostic system in the case of using N-hydroxysuccinimide (NHS) as a chemical functional group for activation of a stationary phase particle.
상기 도면을 참조하면, 일 구체예에 따른 병원체의 진단 플랫폼(10)은 크게 활성화된 복수의 고정상 입자(2) 및 시각적 확인 또는 인식이 가능하고, 제1 기능기-함유 성분으로 수식된 복수의 이동상 입자(3)를 포함한다. 이때, 진단은 튜브 시험관(1) 내에서 수행될 수 있는 바, 상기 튜브 시험관(1)은 가급적 외부에서 육안으로 관찰 가능한 특성을 갖는 재질, 구체적으로 투명성 재질로 구성될 수 있다. 일 예로서, 튜브 시험관(1)의 재질은 투명성 고분자, 글라스 등일 수 있는 바, 본 발명은 반드시 이에 한정되지 않는다. Referring to the drawings, the diagnostic platform 10 of a pathogen according to one embodiment is a plurality of highly activated stationary phase particles 2 and visual confirmation or recognition, and a plurality of modified with a first functional group-containing component And mobile phase particles 3. At this time, the diagnosis can be performed in the tube test tube (1), the tube test tube (1) may be made of a material having a characteristic that can be observed with the naked eye from the outside, specifically, a transparent material. As an example, the material of the tube test tube 1 may be a transparent polymer, glass, etc., and the present invention is not necessarily limited thereto.
도시된 바와 같이, 복수의 활성화된 고정상 입자(2)는 각각 매트릭스 입자(11) 상에 특정 화학적 기능기(12)가 부착되어 활성화된 형태로서, 튜브 시험관(1) 내에 충진(또는 팩킹)되어 컬럼 형태로 적용될 수 있다. As shown, the plurality of activated stationary phase particles 2 are activated by attaching specific chemical functional groups 12 on the matrix particles 11, respectively, and filled (or packed) in the tube test tube 1 Can be applied in column form.
예시적 구체예에 따르면, 매트릭스 입자(11)는 수지, 금속 및 글라스로 이루어지는 군으로부터 선택되는 재질로 이루어질 수 있다. 이와 관련하여, 수지를 사용할 경우, 각종 천연수지(예를 들면, 아가로오스(세파로오스)) 또는 합성수지(예를 들면, 폴리스티렌 또는 폴리아크릴아미드)를 사용할 수 있으며, 또한 수지 성형물, 겔 등의 형태로 적용될 수 있다. 또한, 매트릭스 입자(11)는 정형(예를 들면, 구형, 타원형 등) 또는 비정형 형상을 가질 수 있고, 또한 대칭 또는 비대칭 형상을 가질 수 있다. 보다 전형적으로, 구체일 수 있다. According to an exemplary embodiment, the matrix particles 11 may be made of a material selected from the group consisting of resin, metal and glass. In this connection, when using a resin, various natural resins (for example, agarose (sepharose)) or synthetic resins (for example, polystyrene or polyacrylamide) can be used, and resin moldings, gels, etc. Can be applied in the form of Further, the matrix particles 11 may have a regular (eg, spherical, elliptical, etc.) or irregular shape, and may also have a symmetrical or asymmetrical shape. More typically, it can be a sphere.
특정 구체예에 따르면, 매트릭스 입자(11)는 다공성 구조, 구체적으로 균일한 다공성 구조를 가질 수 있는 바, 전형적으로는 매트릭스 입자 내에 플로우 포어를 갖는 것일 수 있다. 이때, 다공성 매트릭스 입자의 포어 사이즈(직경)는, 예를 들면 약 20 내지 100 nm, 구체적으로 약 30 내지 80 nm, 보다 구체적으로 약 40 내지 60 nm 범위일 수 있으나, 이는 예시적인 의미로 이해될 수 있다.According to a specific embodiment, the matrix particles 11 may have a porous structure, specifically a uniform porous structure, and typically, may have a flow pore in the matrix particles. At this time, the pore size (diameter) of the porous matrix particles may be, for example, in the range of about 20 to 100 nm, specifically about 30 to 80 nm, and more specifically about 40 to 60 nm, which will be understood in an exemplary sense. Can.
이외에도, 고정상 입자(2)의 사이즈(직경)는, 매트릭스 입자의 재질, 형상 등에 따라 변화 가능하며, 예를 들면 수 내지 수백 ㎛, 구체적으로 약 30 내지 400 ㎛, 보다 구체적으로 약 40 내지 250 ㎛, 특히 구체적으로 약 45 내지 200 ㎛ 범위일 수 있다. 다만, 이는 예시적인 의미로 이해될 수 있다.In addition, the size (diameter) of the stationary phase particles 2 can be varied depending on the material, shape, etc. of the matrix particles, for example, several to hundreds of μm, specifically about 30 to 400 μm, more specifically about 40 to 250 μm , In particular, in the range of about 45 to 200 μm. However, this may be understood as an exemplary meaning.
예시적 구체예에 따르면, 매트릭스 입자(11)는 가교된 비드 형상의 아가로오스(세파로오스) 겔일 수 있는 바, 아가로오스는 폴리사카라이드(하전 및/또는 중성)계 물질로 알려져 있다. 이때, 겔 내의 아가로오스의 함량은, 예를 들면 약 1 내지 10 중량%, 구체적으로 약 2 내지 6 중량%, 보다 구체적으로 약 3 내지 5 중량% 범위일 수 있는 바, 하기 일반식 1로 표시되는 반복 단위를 가질 수 있다.According to an exemplary embodiment, the matrix particle 11 may be a cross-linked bead-shaped agarose (sepharose) gel, which is known as a polysaccharide (charged and/or neutral)-based material. . At this time, the content of the agarose in the gel may be, for example, about 1 to 10% by weight, specifically about 2 to 6% by weight, and more specifically about 3 to 5% by weight, as shown in the following general formula 1 It may have a repeating unit displayed.
[일반식 1][Formula 1]
Figure PCTKR2019018446-appb-I000001
Figure PCTKR2019018446-appb-I000001
한편, 예시적 구체예에 있어서, 화학적 기능기는 매트릭스 입자의 표면에 부착 또는 결합될 수 있는 것으로서 제1 기능기-함유 성분과 결합하거나 커플링(예를 들면, 공유결합 등)할 수 있는 한, 다양한 종류로부터 선정될 수 있다.On the other hand, in an exemplary embodiment, as long as the chemical functional group can be attached to or bonded to the surface of the matrix particle, as long as it can bind or couple (e.g., covalent bond) with the first functional group-containing component, It can be selected from various types.
이와 관련하여, 상술한 화학적 기능기로서 대표적으로 NHS(N-hydroxysuccinimide)를 적용할 수 있다. 택일적으로, 아민기, 카르복시기, 히드록시기, 실란올기(예를 들면, 메톡시기, 에톡시기 등), 말레이미드기, 티올기, 알데히드기, PEG(polyethylene glycol) 등으로부터 선택되는 적어도 하나의 모이티를 갖는 기능기를 적용할 수 있다. 이러한 화학적 기능기를 이용하여 매트릭스 입자를 활성화시킬 수 있으나, 이는 제1 기능기-함유 성분에 따라 다양하게 변경할 수 있기 때문에 예시적으로 이해될 수 있을 것이다.In this regard, NHS (N-hydroxysuccinimide) can be typically applied as the above-described chemical functional group. Alternatively, at least one moiety selected from amine groups, carboxy groups, hydroxy groups, silanol groups (eg, methoxy groups, ethoxy groups, etc.), maleimide groups, thiol groups, aldehyde groups, PEG (polyethylene glycol), etc. A functional group having can be applied. The matrix particles can be activated using such a chemical functional group, but this can be understood as an example because it can be variously changed according to the first functional group-containing component.
한편, 도 2에 도시된 바와 같이 복수의 이동상 입자(3)는, 베이스 입자(구체적으로, 비드 또는 구형의 입자; 13)가 제1 기능기-함유 성분(14)으로 수식된 형태일 수 있다. 이때, 베이스 입자(13)는, 시각적 확인이 가능한, 구체적으로 육안으로 식별 가능한 색을 갖는 한, 다양한 재질의 비드를 사용할 수 있다. 베이스 입자(13)의 예로서 자성 입자(자성 비드), 폴리스티렌 비드, 금 나노입자, 금속 입자, 글라스 비드, 실리카 비드 등으로부터 선택된 적어도 하나일 수 있다. 다만, 베이스 입자(13)는 후술하는 물리적 분리(구체적으로 원심분리) 과정에서 분리 효율성을 높이기 위하여 일정 수준 이상의 밀도를 갖는 것이 바람직할 수 있는 바, 예를 들면 약 0.01 내지 10 g/L, 구체적으로 약 0.5 내지 5 g/L, 보다 구체적으로 약 1 내지 2 g/L 범위일 수 있으나, 본 발명이 이에 한정되는 것은 아니다.Meanwhile, as illustrated in FIG. 2, the plurality of mobile phase particles 3 may have a form in which the base particles (specifically, beads or spherical particles; 13) are modified with the first functional group-containing component 14. . At this time, the base particles 13, as long as it has a visually identifiable, specifically visually identifiable color, beads of various materials can be used. Examples of the base particle 13 may be at least one selected from magnetic particles (magnetic beads), polystyrene beads, gold nanoparticles, metal particles, glass beads, and silica beads. However, the base particles 13 may have a density of a certain level or higher in order to increase separation efficiency in a physical separation (specifically centrifugation) process described later, for example, about 0.01 to 10 g/L, specifically It may be in the range of about 0.5 to 5 g/L, more specifically about 1 to 2 g/L, but the present invention is not limited thereto.
예시적 구체예에 따르면, 베이스 입자(13)로서 자성 입자를 사용할 수 있는 바, 일시적 또는 영구적 자성을 나타낼 수 있다. 자성 입자로서, 예를 들면 철, 코발트, 니켈, 산화철, 수산화철 및/또는 기타 철 합금을 함유하는 자성 입자, 희토류 자성 입자 등을 사용할 수 있다. 택일적으로, 자성 철 코어를 고분자(예를 들면, 덱스트란)로 코팅한 입자를 사용할 수 있다.According to an exemplary embodiment, since magnetic particles can be used as the base particles 13, they may exhibit temporary or permanent magnetism. As the magnetic particles, for example, magnetic particles containing iron, cobalt, nickel, iron oxide, iron hydroxide, and/or other iron alloys, rare earth magnetic particles, and the like can be used. Alternatively, particles coated with a magnetic iron core with a polymer (eg, dextran) can be used.
베이스 입자(13)의 사이즈는 특정 범위로 한정되는 것은 아니지만, 전형적으로 나노스케일에서 마이크론 스케일 범위 내일 수 있는 바, 예를 들면 약 0.01 내지 10 ㎛, 구체적으로 0.1 내지 6 ㎛, 보다 구체적으로 1 내지 3 ㎛ 범위일 수 있다. 특정 구체예에 따르면, 입자(13) 사이즈 분포는 가급적 단일분산성을 갖는 것이 향후 물리적 분리(구체적으로 원심분리)의 효율성 및/또는 진단 정확도 확보에 있어서 유리할 수 있다.The size of the base particles 13 is not limited to a specific range, but may typically be in a nanoscale to micron scale range, for example, about 0.01 to 10 μm, specifically 0.1 to 6 μm, and more specifically 1 to It may be in the range of 3 μm. According to a specific embodiment, the particle size distribution may be advantageous in securing the efficiency and/or diagnostic accuracy of physical separation (specifically centrifugation) in the future, if possible.
도시된 바와 같이, 이동상 입자(3)는 베이스 입자(13)가 제1 기능기-함유 성분(14)으로 수식되어 있다. 이때, 제1 기능기-함유 성분(14)은 후술하는 제2 기능기-함유 성분(15)과 바인딩 특성(즉, 높은 친화성(affinity))을 갖는 종류일 수 있다. 예시적 구체예에 있어서, 제1 기능기-함유 성분(14)은, 예를 들면 아비딘, 스트렙트아비딘, 바이오틴, 항원, 항체, 압타머(aptamer), 아민, 카르복시, 알데하이드, 유전자(예를 들면, 상보적 결합이 가능한 DNA) 등을 예시할 수 있고, 천연적으로 발생하거나 인공적으로 합성된 것 모두 포함하는 개념일 수 있다. 보다 구체적으로, 제1 기능기-함유 성분(14)은 바이오물질로서 아비딘 및/또는 스트렙트아비딘일 수 있는 바, 아비딘은 작은 사이즈의 수용성 바이오틴과 높은 친화성을 갖는 당 단백질(glycoprotein)에 해당하며, 스트렙트아비딘은 박테리아 Streptomyces avidinii로부터 분리된 단백질로서 역시 바이오틴에 대한 친화성이 높은 성분이며, 특히 당 단백질에 해당되지 않기 때문에 렉틴과 바인딩되지 않고, 또한 물리적 특성 면에서 아비딘보다 바람직할 수 있다. 이와 관련하여, 고정상 입자(2)에서 화학적 기능기(12)로서 NHS를 사용하고, 제1 기능기-함유 성분(14)으로서 아비딘 또는 스트렙트아비딘을 사용할 경우에는 NHS와 아비딘의 아민기 간의 이미드 결합이 이루어질 수 있다.As shown, the base particle 13 of the mobile phase particle 3 is modified with the first functional group-containing component 14. In this case, the first functional group-containing component 14 may be of a type having binding characteristics (ie, high affinity) with the second functional group-containing component 15 described later. In an exemplary embodiment, the first functional group-containing component 14 is, for example, avidin, streptavidin, biotin, antigen, antibody, aptamer, amine, carboxy, aldehyde, gene (eg For example, DNA capable of complementary binding) may be exemplified, and may be a concept including both naturally occurring and artificially synthesized. More specifically, the first functional group-containing component 14 may be avidin and/or streptavidin as a biomaterial, and avidin corresponds to a small sized water-soluble biotin and a glycoprotein having high affinity. In addition, streptavidin is a protein isolated from bacterial Streptomyces avidinii and is also a component having high affinity for biotin. In particular, it does not bind to lectin because it does not correspond to sugar protein, and may be preferable to avidin in terms of physical properties. . In this regard, when NHS is used as the chemical functional group 12 in the stationary phase particle 2 and avidin or streptavidin is used as the first functional group-containing component 14, there is already a relationship between the NHS and the amine group of avidin. Debonding can be achieved.
기능화된 이동상 입자(3)는 활성화된 고정상 입자(2)의 컬럼에 첨가(또는 로딩)될 수 있다. 예시적 구체예에 따르면, 기능화된 이동상 입자(3)는, 예를 들면 액상 매질, 구체적으로 수계 매질 내에 분산된 형태(구체적으로 서스펜션)로 첨가될 수 있다. 이때, 서스펜션 내 이동상 입자(3)의 농도는, 예를 들면 약 1 내지 30 mg/mL, 구체적으로 약 5 내지 20 mg/mL, 보다 구체적으로 약 8 내지 15 mg/mL 범위일 수 있다. 또한, 이동상의 이동효율을 위하여 PBS 등을 더 첨가할 수 있다.The functionalized mobile phase particles 3 can be added (or loaded) to the column of activated stationary phase particles 2. According to an exemplary embodiment, the functionalized mobile phase particles 3 can be added, for example, in a liquid medium, specifically in the form dispersed in a water-based medium (specifically a suspension). At this time, the concentration of the mobile phase particles 3 in the suspension may be, for example, about 1 to 30 mg/mL, specifically about 5 to 20 mg/mL, and more specifically about 8 to 15 mg/mL. In addition, PBS or the like may be further added for the mobile phase's mobility.
이때, 타겟 병원체의 유전자를 진단하기 위하여, 이동상 입자(2)와 함께(예를 들면, 혼합물 형태로) 또는 별도로 제2 기능기-함유 성분과 프라이머의 접합체(음성인 경우) 또는 프라이머를 통하여 증폭된 유전자(양성인 경우)의 접합체가 첨가될 수 있다. 이때, 제2 기능기-함유 성분(15)은 전술한 제1 기능기-함유 성분(14)과 바인딩 특성을 갖는 종류에서 선택할 수 있는 바, 이와 유사하게 아비딘, 스트렙트아비딘, 바이오틴, 항원, 항체, 압타머(aptamer), 아민, 카르복시, 알데하이드, 유전자(예를 들면, 상보적 결합이 가능한 DNA) 등을 사용할 수 있다. 다만, 제1 기능기-함유 성분(14) 및 제2 기능기-함유 성분(15)은 서로 상이한 종류일 수 있는 바, 예시적 구체예에서는 제1 기능기-함유 성분(14)으로 아비딘 또는 스트렙트아비딘을 사용하고, 제2 기능기-함유 성분(15)으로 바이오틴을 사용할 수 있다. 이와 관련하여, 바이오틴은 비타민의 일종으로, 구체적으로 테트라하이드로티오펜 고리와 융합된 우레이도(테트라하이드로이미디잘론) 고리로 이루어지는 B-착체 비타민(hexahydro-2-oxo-lH-thieno[3,4-d]imidazoline-4-valeric acid)으로, 분자량은 약 244 g/mol이며, 테트라하이드로티오펜 고리의 탄소 원자 중 하나에 발레르산 치환기가 부착되어 있다. 바이오틴은 아비딘 또는 스트렙트아비딘과 강한 친화성에 의하여 특이적으로 결합할 수 있는 바, 예를 들면 1개의 스트렙타비딘 분자에 4개의 바이오틴 분자가 결합할 수 있다.At this time, in order to diagnose the gene of the target pathogen, it is amplified through the conjugate (if negative) or the primer of the second functional group-containing component and the primer together with the mobile phase particles 2 (for example, in the form of a mixture) or separately. Conjugates of the genes (if positive) can be added. At this time, the second functional group-containing component 15 can be selected from the types having binding properties with the first functional group-containing component 14 described above, similarly, avidin, streptavidin, biotin, antigen, Antibodies, aptamers, amines, carboxys, aldehydes, genes (eg, DNA capable of complementary binding) can be used. However, since the first functional group-containing component 14 and the second functional group-containing component 15 may be of different types, in an exemplary embodiment, avidin or the first functional group-containing component 14 may be used. Streptavidin may be used, and biotin may be used as the second functional group-containing component 15. In this regard, biotin is a type of vitamin, specifically, a B-complex vitamin (hexahydro-2-oxo-lH-thieno[3,4] consisting of a ureido (tetrahydroimidazalone) ring fused with a tetrahydrothiophene ring. -d]imidazoline-4-valeric acid), the molecular weight is about 244 g/mol, and a valeric acid substituent is attached to one of the carbon atoms of the tetrahydrothiophene ring. Biotin can specifically bind avidin or streptavidin by strong affinity, for example, four biotin molecules can be bound to one streptavidin molecule.
택일적 구체예에 따르면, 제1 기능기-함유 성분(14)이 입자(13) 상에 수식된 형태로 시판 중인 제품을 사용할 수도 있는 바, 예를 들면 Life Science사의 수식된 자성 입자인 상품명 Dynabeadsㄾ Myone™ Streptavidin C1 등을 적용할 수 있다.According to an alternative embodiment, a commercially available product in which the first functional group-containing component 14 is modified on the particle 13 may be used, for example, the trade name Dynabeads which is a modified magnetic particle of Life Science. ㄾ Myone™ Streptavidin C1 can be applied.
이외에도, 제1 기능기-함유 성분(14)과 제2 기능기-함유 성분(15) 간의 바인딩은 항원-항체 간의 면역 반응, 비공유 결합, 유전자간 상보결합 등을 이용하여 달성될 수 있다. In addition, binding between the first functional group-containing component 14 and the second functional group-containing component 15 can be achieved using an immune response between antigen-antibodies, non-covalent binding, inter-complementary binding, and the like.
예시적 구체예에 따르면, 제2 기능기-함유 성분(15)은 타겟 병원체의 유전자에 특이적인 프라이머(16)와 접합되어 있으며, 이러한 접합체의 존재 하에서 시료에 대한 증폭 과정을 거치게 된다. According to an exemplary embodiment, the second functional group-containing component 15 is conjugated with a primer 16 specific to the gene of the target pathogen, and undergoes an amplification process for the sample in the presence of such conjugate.
이와 관련하여, 타겟 병원체는, 전형적으로 박테리아, 바이러스 등의 바이오물질로서, 그람-양성균 및 그람-음성균을 포함할 수 있는 바, 구체적으로 대장균 E. coli O157:H7, 살모넬라속균(Salmonella choleraesuis, Salmonella bongori, Salmonella typhimurium), 황색포도상구균, 리스테리아속균(Listeria monocytogenes, Listeria denitrificans, Listeria grayi, Listeria murrayi), 콜레라균, 적리균, 백일해균, 디프테리아균, 장티푸스균, 페스트균, 용혈성 연쇄구균 또는 스타필로코커스 아우레우스일 수 있으며, 보다 구체적으로는 E.coli O157:H7, Salmonella choleraesuis, Salmonella bongori, Salmonella typhimurium, Listeria monocytogenes, Listeria denitrificans, Listeria grayi, Listeria murrayi, S. enteritidis, Y. enterocolitica, S. aureus, B. cereus, L. monocytognes 등일 수 있다. In this regard, the target pathogen is a biomaterial such as bacteria, viruses, and the like, and may include Gram-positive bacteria and Gram-negative bacteria. Specifically, E. coli O157:H7 , Salmonella choleraesuis , Salmonella bongori , Salmonella typhimurium ), Staphylococcus aureus, Listeria monocytogenes , Listeria denitrificans , Listeria grayi , Listeria murrayi ), Cholera, Mycobacterium, Pertussis, Diphtheria, Typhoid, Pasteur , Staphylococcus Aureus, more specifically E.coli O157:H7 , Salmonella choleraesuis , Salmonella bongori , Salmonella typhimurium , Listeria monocytogenes , Listeria denitrificans , Listeria grayi , Listeria murrayi, S. enteritidis, Y. enterocolitica, S. aureus , B. cereus , L. monocytognes , and the like.
한편, 증폭 반응은 타겟 병원체의 유전자(구체적으로, DNA)가 고정된 기재를 증폭용 혼합물(즉, PCR 혼합물) 내에 침지(immersion)시키는데, 이때 고정된 핵산은 증폭 과정 중 고온에 노출되어 분리되고, 유전 서열은 복제 준비 상태에 있게 된다. 증폭 반응을 위하여, 전술한 바와 같이 PCR(DNA 증폭 방식), NASBA(Nucleic Acid Sequence Based Amplification; RNA를 이용한 등온 조건 하에서의 증폭 방식) 등과 같이 당업계에서 공지된 임의의 증폭 기술을 활용할 수 있다. 예를 들면, PCR 방식은 국내특허번호 제593687호 등에 기재되어 있고, NASBA 방식은 EP 0 329 822 B1, 미국특허번호 제6,110,681호 등에 예시되어 있는 바, 전술한 선행문헌들은 본 발명의 참고자료로 포함된다. 또한, PCR 방식으로서, 어셈블리-PCR, 비대칭(asymmetric) PCR, 디지털(digital) PCR, 종점(endpoint) PCR, 인버스(inverse) PCR, 메틸화-특이성 PCR, 정성(qualitiative) PCR, 정량화(quantitative) PCR, 실시간(real-time) PCR, RT(reverse transcription)-PCR, 등온-PCR 등을 예시할 수 있다.On the other hand, the amplification reaction immerses the substrate in which the gene (specifically, DNA) of the target pathogen is immobilized in the amplification mixture (i.e., PCR mixture), wherein the immobilized nucleic acid is separated by exposure to high temperatures during the amplification process. , The genetic sequence is ready for replication. For the amplification reaction, any amplification technique known in the art, such as PCR (DNA amplification method), NASBA (Nucleic Acid Sequence Based Amplification; RNA amplification method under isothermal conditions) as described above, may be used. For example, the PCR method is described in Korean Patent No. 593687, and the NASBA method is exemplified in EP 0 329 822 B1, U.S. Patent No. 6,110,681, etc., and the aforementioned prior documents are used as references of the present invention. Is included. In addition, as a PCR method, assembly-PCR, asymmetric PCR, digital PCR, endpoint PCR, inverse PCR, methylation-specific PCR, qualitative PCR, quantitative PCR , Real-time PCR, reverse transcription (RT)-PCR, isothermal-PCR, and the like.
이때, 시료 내에 타겟 병원체가 존재하여 증폭된 경우(양성인 경우)에는 제2 기능기-함유 성분(15)은 프라이머의 작용에 의하여 타겟 병원체의 유전자가 증폭되고, 결과적으로 제2 기능기-함유 성분(15)은 타겟 병원체의 증폭 유전자(17)와 접합된 상태에 있게 된다. At this time, if the target pathogen is present in the sample and amplified (if positive), the second functional group-containing component 15 amplifies the gene of the target pathogen by the action of the primer, and consequently the second functional group-containing component (15) is in a state conjugated with the amplification gene 17 of the target pathogen.
이처럼, 제2 기능기-함유 성분(15)과 접합된 프라이머를 통하여 증폭된 타겟 병원체의 유전자는 증폭 전에 비하여 긴 사슬을 갖게 된다. 이때, 이동상 입자(3) 상의 제1 기능기-함유 성분(14)은 타겟 병원체의 증폭 유전자(17)와 접합된 제2 기능기-함유 성분과의 강한 바인딩 특성으로 인하여 결과적으로 이동상 입자(3)에 고정되고, 그 결과 이동상 입자(3)의 사이즈(직경)가 증가하게 된다. 이와 같이 증가된 사이즈로 인하여 고정상 입자(2)와의 결합 또는 바인딩을 방해하는 입체 장애 효과가 유발된다. 이와 관련하여, DLS(dynamic light scattering)에 의한 측정 기준으로, 이동상 입자(3) 상에 부착되는 증폭 유전자의 사이즈는, 예를 들면 약 10 내지 150 nm, 구체적으로 약 30 내지 100 nm, 보다 구체적으로 약 50 내지 80 nm 범위일 수 있는 바, 이와 같이 벌키한 증폭 유전자 부착에 의하여 이동상 입자의 사이즈는 입체 장애 또는 공간 방해 효과를 제공할 수 있는 수준으로 증가하게 된다. 다만, 이러한 사이즈 증가 정도는 베이스 입자(13)의 종류, 증폭 반응의 정도 등에 따라 상이할 수 있는 만큼, 예시적인 의미로 이해될 수 있다. As described above, the gene of the target pathogen amplified through the primer conjugated with the second functional group-containing component 15 has a longer chain than before amplification. At this time, the first functional group-containing component 14 on the mobile phase particle 3 is consequently due to the strong binding properties of the second functional group-containing component conjugated with the amplification gene 17 of the target pathogen, resulting in the mobile phase particle 3 ), and as a result, the size (diameter) of the mobile phase particle 3 increases. Due to this increased size, a steric hindrance effect that prevents binding or binding with the stationary phase particle 2 is caused. In this regard, as a measurement standard by dynamic light scattering (DLS), the size of the amplification gene attached to the mobile phase particle 3 is, for example, about 10 to 150 nm, specifically about 30 to 100 nm, more specifically As it may be in the range of about 50 to 80 nm, the size of the mobile phase particle is increased to a level capable of providing a steric hindrance or a space obstruction effect by bulky amplification gene attachment. However, the extent of this size increase may be understood as an exemplary meaning as it may be different depending on the type of the base particle 13, the degree of amplification reaction, and the like.
반면, 시료 내에 타겟 병원체가 존재하지 않을 경우(음성인 경우), 이동상 입자(3) 상의 제1 기능기-함유 성분(14) 중 일부가 제2 기능기-함유 성분(15)과 프라이머(16)의 접합체와 바인딩되는 한편, 나머지 일부는 고정상 입자(2)에 존재하는 화학적 기능기와 결합(예를 들면, 공유 결합)됨으로써, 결과적으로 이동상 입자(3)가 고정상 입자(2)에 고정된 상태에 있게 된다. On the other hand, when the target pathogen is not present in the sample (when it is negative), some of the first functional group-containing component 14 on the mobile phase particle 3 has a second functional group-containing component 15 and a primer 16 ), while the other part is bound (e.g., covalently) with a chemical functional group present in the stationary phase particle 2, resulting in the mobile phase particle 3 being fixed to the stationary phase particle 2 Will be in.
이후, 고정상 입자(2)와 이동상 입자(3)는 물리적 수단에 의한 분리 조작 단계를 거칠 수 있는 바, 대표적인 물리적 분리 수단으로서 원심분리, 물리압착 등을 예시할 수 있으나, 반드시 이에 한정되는 것은 아니다. 다만, 본 구체예의 경우, 간편한 조작에 의한 진단 과정을 수행할 수 있도록 하는 만큼, 원심 분리 방식을 적용하는 것이 유리할 수 있다. Thereafter, the stationary phase particles 2 and the mobile phase particles 3 may undergo a separation operation step by physical means, and may be exemplified by centrifugation, physical compression, etc. as representative physical separation means, but are not limited thereto. . However, in the case of the present embodiment, it may be advantageous to apply a centrifugal separation method so that a diagnostic process by simple manipulation can be performed.
예시적 구체예에 따르면, 원심 분리 조건은 진단 시스템 내의 컬럼 구조에 영향을 억제하면서 고정상 입자(2)에 결합되지 않은 이동상 입자(3)만을 원심력에 의하여 용기 하부로 운반할 수 있는 정도이면 충분할 수 있다. 이와 관련하여, 원심 분리 과정에서 회전 속도는 고정상 컬럼의 길이, 반경 등에 따라 변화 가능한 바, 예를 들면 약 500 내지 2000 rpm, 구체적으로 약 600 내지 1900 rpm, 보다 구체적으로 약 800 내지 1200 rpm 범위 내에서 적절히 조절 가능하며, 또한 원심 분리 시간은, 중력 가속도 등을 고려하여 정하여질 수 있는 바, 예를 들면 약 0.5 내지 5분, 구체적으로 약 1 내지 4분, 보다 구체적으로 약 1.5 내지 3분 범위일 수 있으나, 이는 예시적인 의미로 이해될 수 있다. 다만, 상술한 원심분리는 필요에 따라 1회 이상 반복 수행할 수도 있다.According to an exemplary embodiment, the centrifugation conditions may be sufficient to transport only the mobile phase particles 3 not bound to the stationary phase particles 2 to the bottom of the container by centrifugal force while suppressing the influence on the column structure in the diagnostic system. have. In this regard, the rotational speed in the centrifugation process may vary depending on the length, radius, etc. of the stationary phase column, for example, in the range of about 500 to 2000 rpm, specifically about 600 to 1900 rpm, and more specifically about 800 to 1200 rpm. It can be appropriately adjusted, and the centrifugation time can be determined by considering gravity acceleration, for example, about 0.5 to 5 minutes, specifically about 1 to 4 minutes, and more specifically about 1.5 to 3 minutes. It may be, but it can be understood in an exemplary sense. However, the above-described centrifugation may be repeated one or more times as necessary.
이처럼, 증폭된 타겟 병원체의 유전자와 결합된 이동상 입자(3)는 이의 표면에 존재하는 제1 기능기-함유 성분과 접합된 증폭 유전자에 의하여 공간적으로 고정상 입자(2)와 이격된 상태에 있는 경향을 나타내므로 고정상 입자(2)와는 결합하지 않게 된다. 따라서, 원심 분리 과정에서 복수의 고정상 입자(2)의 컬럼을 통과하면서 하측, 예를 들면 컬럼의 바닥까지 이동하여 침전될 수 있다. As such, the mobile phase particle 3 bound to the gene of the amplified target pathogen tends to be spaced apart from the stationary phase particle 2 spatially by the amplified gene conjugated with the first functional group-containing component present on its surface. It does not bind to the stationary phase particles (2). Therefore, in the centrifugation process, while passing through the columns of the plurality of stationary phase particles 2, it may be precipitated by moving to the lower side, for example, to the bottom of the column.
그 결과, 육안으로 이동상 입자(3)의 침전을 확인 또는 식별할 수 있으며, 이 경우 양성으로 판정할 수 있다. 반면, 시료가 타겟 병원체의 유전자를 함유하지 않을 경우, 이동상 입자(3)는 고정상 입자(2)와 결합되어 있기 때문에 원심 분리에 의하여도 고정상 입자의 컬럼 내, 구체적으로 고정상 입자의 컬럼 상부에 분포하게 되며, 이를 육안으로 확인할 수 있다. 이 경우는 음성으로 판단할 수 있다. As a result, precipitation of the mobile phase particles 3 can be confirmed or identified with the naked eye, and in this case, it can be determined as positive. On the other hand, when the sample does not contain the gene of the target pathogen, the mobile phase particles 3 are bound to the stationary phase particles 2, and thus, even by centrifugation, they are distributed in the column of the stationary phase particles, specifically above the column of the stationary phase particles. This can be confirmed with the naked eye. In this case, it can be judged by voice.
한편, 본 개시 내용의 다른 구체예에 따르면, 증폭 반응 시 또는 증폭 반응 후 삽입 물질, 구체적으로 삽입 염료를 사용하여 증폭된 유전자 사슬에 강직성을 추가적으로 부여함으로써 증폭된 유전자와 결합된 이동상 입자(3)의 직경을 실질적으로 증가시켜 보다 큰 입체 장애 효과를 제공할 수 있다. On the other hand, according to another embodiment of the present disclosure, during the amplification reaction or after the amplification reaction, the mobile phase particles combined with the amplified gene (3) by additionally imparting rigidity to the amplified gene chain using an insertion substance, specifically, an insertion dye The diameter of can be substantially increased to provide a greater steric hindrance effect.
특히, 타겟 유전자가 DNA(구체적으로 이중 스트랜드 DNA)인 경우, 증폭 과정 또는 후에 이중나선 구조 내에 해당 삽입 물질 또는 삽입 염료가 삽입되어 증가된 강성을 나타내는 만큼, 증폭된 유전자 사슬로 인한 직경 증가 정도가 더욱 현저하다.Particularly, when the target gene is DNA (specifically, double-stranded DNA), the degree of increase in diameter due to the amplified gene chain increases as the corresponding insert material or insert dye is inserted into the double-helix structure after or after the amplification process. It is more remarkable.
예시적 구체예에 따르면, 증폭 단계에 앞서, 증폭 과정 중 또는 증폭 반응 후에 삽입 물질 또는 삽입 염료가 증폭 혼합물 또는 증폭 생성물과 혼합되거나 이에첨가될 수 있다. 이때, 증폭 대상이 이중 스트랜드 DNA인 경우, 삽입 염료는 이중 스트랜드 DNA(dsDNA) 사이에 삽입될 수 있다. According to an exemplary embodiment, prior to the amplification step, during the amplification process or after the amplification reaction, the intercalation material or intercalation dye may be mixed or added to the amplification mixture or amplification product. In this case, when the target to be amplified is double-stranded DNA, the insertion dye may be inserted between double-stranded DNA (dsDNA).
삽입 염료는 증폭 과정 또는 증폭 후에 유전자(구체적으로 DNA)의 이중 스트랜드 내로 삽입 가능한 염료로서, GelRed, EtBr(ethidium bromide), GelGreen, SYBR Green, PicoGreen, Hoechst 시리즈(series), BOBO, TOTO, YOYO, JOJO, POPO, LOLO, PO-PRO, BO-PRO, YO-PRO, TO-PRO, JO-PRO, LO-PRO, SYTO 시리즈 등을 포함할 수 있으며, 이들을 단독으로 또는 조합하여 사용할 수 있다. 이와 관련하여, 삽입 염료 중 이중나선 유전자에 대한 특이적 반응성을 갖는 종류를 사용하는 것이 유리할 수 있다. 상기의 점을 고려할 때, 삽입 염료로서 GelRed를 사용할 수 있으나, 반드시 이에 한정되는 것은 아니다. 본 발명이 상술한 삽입 물질로 한정되는 것은 아니며, 증폭 반응 과정 또는 증폭 생성물의 이중 스트랜드에 삽입되어 증폭 유전자 사슬의 강성을 증가시킬 수 있는 한, 다양한 종류를 사용할 수도 있다. 이외에도, 삽입 물질은 액상 매질, 구체적으로 수계 매질에 용해(희석)된 상태로 적용 가능하다. 예시적으로, 희석된 매질(용액) 내 삽입 물질의 농도는, 예를 들면 약 6 내지 19x, 구체적으로 약 7 내지 18x, 보다 구체적으로 약 8 내지 15x 범위일 수 있다. 다만, 삽입 물질의 농도 범위는 타겟 병원체(구체적으로 박테리아, 바이러스 등)의 종류에 따라 가변적인 만큼, 반드시 상기 수치 범위로 한정되는 것은 아니다.Insertion dye is a dye that can be inserted into a double strand of a gene (specifically DNA) after the amplification process or amplification, GelRed, EtBr (ethidium bromide), GelGreen, SYBR Green, PicoGreen, Hoechst series, BOBO, TOTO, YOYO, JOJO, POPO, LOLO, PO-PRO, BO-PRO, YO-PRO, TO-PRO, JO-PRO, LO-PRO, SYTO series, and the like, and these may be used alone or in combination. In this regard, it may be advantageous to use a kind of insertion dye having specific reactivity to the double helix gene. Considering the above points, GelRed may be used as an insertion dye, but is not limited thereto. The present invention is not limited to the above-described insertion material, and various types may be used as long as it can be inserted into a double strand of an amplification reaction process or amplification product to increase the rigidity of the amplification gene chain. In addition, the insert material can be applied in a liquid medium, specifically dissolved (diluted) in an aqueous medium. Illustratively, the concentration of the intercalation material in the diluted medium (solution) may range, for example, from about 6 to 19x, specifically from about 7 to 18x, and more specifically from about 8 to 15x. However, the concentration range of the insert material is variable depending on the type of target pathogen (specifically, bacteria, viruses, etc.), and is not necessarily limited to the above numerical range.
또한, 삽입 물질에 대한 증폭 생성물(증폭 혼합물 내에 유전자가 함유된 경우)의 체적 비는, 예를 들면 약 2 내지 8, 구체적으로 약 3 내지 6, 보다 구체적으로 약 3.5 내지 5의 범위 내에서 정하여질 수 있으나, 이는 예시적인 것으로 이해될 수 있다. 이와 관련하여, 삽입 물질의 사용량이 지나치게 많은 경우에는 응집 반응 등이 유발되어 의도하는 유전자 사슬 내 삽입이 곤란할 수 있는 반면, 삽입 물질의 사용량이 지나치게 낮은 경우에는 원하는 강성 증가 효과 및/또는 추가적인 색상 또는 형광 발현 효과를 나타내기 곤란한 만큼, 전술한 범위 내에서 적절히 조절하여 사용하는 것이 유리할 수 있다. In addition, the volume ratio of the amplification product (if the gene is contained in the amplification mixture) to the insertion substance is, for example, determined within a range of about 2 to 8, specifically about 3 to 6, and more specifically about 3.5 to 5 It can be lost, but it can be understood as exemplary. In this regard, when the amount of the insert material is used excessively, a cohesive reaction or the like may be caused, and thus, insertion into the intended gene chain may be difficult, whereas when the amount of the insert material is too low, a desired increase in stiffness and/or additional color or As it is difficult to exhibit a fluorescence expression effect, it may be advantageous to use it by appropriately adjusting within the above-described range.
이와 같이, 타겟 병원체의 증폭 유전자 내에 삽입 물질이 혼입(삽입)되고, 유전자 사슬의 강성이 커짐에 따라 삽입되지 않는 경우에 비하여 측정되는 접합체의 직경이 증가하게 된다. 따라서, 보다 높은 입체 장애를 부여할 수 있고, 이는 시료 내에 병원체가 존재하는 경우(양성인 경우), 이동상 입자(3)가 원심 분리 등과 같은 물리적 수단에 의하여 신속하게 분리되어 이동함으로써 육안 식별을 더욱 용이하게 할 수 있다. 예시적으로, DLS에 의한 측정 기준으로, 삽입 물질의 혼입(삽입) 없이 이동상 입자에 부착된 증폭 유전자의 사이즈에 비하여 삽입 물질의 혼입(삽입) 후 이동상 입자에 부착된 증폭 유전자의 사이즈 증가율은, 예를 들면 적어도 약 20%, 구체적으로 약 30 내지 80%, 보다 구체적으로 약 40 내지 50%의 범위일 수 있으나, 이는 예시적인 의미로 이해될 수 있다. As described above, the insertion material is incorporated (inserted) into the amplification gene of the target pathogen, and as the stiffness of the gene chain increases, the diameter of the conjugate measured is increased as compared to the case where it is not inserted. Therefore, it is possible to impart a higher steric hindrance, which makes it easier to visually discriminate by moving the mobile phase particles 3 rapidly and separated by physical means such as centrifugation, when a pathogen is present in the sample (positive). You can do it. Exemplarily, as measured by DLS, compared to the size of the amplification gene attached to the mobile phase particle without incorporation (insertion) of the insert material, the rate of increase in the size of the amplification gene attached to the mobile phase particle after incorporation (insertion) of the insert material, For example, it may be in the range of at least about 20%, specifically about 30 to 80%, more specifically about 40 to 50%, but this can be understood in an exemplary sense.
상술한 바와 같이, 본 개시 내용의 구체예에 따른 분석 플랫폼은, 간단한 물리적 분리 조작(구체적으로 원심력만을 이용함)에 의하여 현장에서 육안으로 양성 및 음성을 판정할 수 있다. 특히, 형광 또는 전기영동 기반의 종래 방법이 갖는 문제점(즉, 양호한 민감도를 갖고 있으나, 장치 규모 및 절차가 복잡하고 상대적으로 긴 진단 시간을 요함)을 완화할 수 있고, 식중독 균 등의 병원체에 의한 감염을 현장에서 신속하게 진단할 수 있다. As described above, the analysis platform according to an embodiment of the present disclosure can determine positive and negative visually in the field by a simple physical separation operation (specifically, using only centrifugal force). In particular, it is possible to alleviate the problems of the conventional method based on fluorescence or electrophoresis (that is, it has good sensitivity, but the device size and procedure is complex and requires relatively long diagnostic time), and may be caused by pathogens such as food poisoning bacteria. Infections can be quickly diagnosed in the field.
이와 관련하여, 예시적 구체예에 따른 분석 플랫폼은, 예를 들면 약 10 내지 106 CFU/mL, 심지어 1.0 x 101 CFU 수준의 낮은 검출 한계(LOD)를 가질 수 있으나, 반드시 이에 한정되는 것은 아니다.In this regard, an analytical platform according to an exemplary embodiment may have a low detection limit (LOD) of, for example, a level of about 10 to 10 6 CFU/mL, even 1.0 x 10 1 CFU, but is not necessarily limited thereto. no.
더 나아가, 삽입 물질을 이용할 경우, 타겟 병원체의 증폭된 유전자와 이동상 입자의 접합 시 증폭 유전자 사슬의 강성을 증가시켜 입체 장애에 따른 이동상 입자의 분리 효율을 추가적으로 높일 수 있다.Furthermore, when the insertion material is used, when the amplified gene of the target pathogen and the mobile phase particle are joined, the stiffness of the amplified gene chain is increased to further increase the separation efficiency of the mobile phase particle due to steric hindrance.
휴대용 원심 분리 장치Portable centrifugal separator
본 개시 내용의 일 구체예에 따른 유전자 진단 시스템은 전술한 분석 플랫폼을 적용하는데 요구되는 물리적 분리 수단으로서 휴대용 원심 분리 장치를 포함할 수 있다. 이와 관련하여, 휴대용 원심 분리 장치는 모바일 기기로부터 공급되는 전원을 이용하여 구동될 수 있는 바, 이를 위하여 모바일 기기의 전원 입출력 단자와 착탈식으로 연결될 수 있다. 예시적으로, 스마트 폰, 태블릿, 노트북 등과 같이 전원 입출력 단자가 구비되어 전원을 공급할 수 있는 기능을 제공하는 모바일 기기인 한 특별히 한정되는 것은 아니며, 휴대 가능한 보전 전원장치를 포함할 수 있다. 특히, 스마트폰, 태블릿 기기 등과 같이 조도 센서가 내장된 모바일 기기는 휴대용 원심 분리기의 전원 공급원으로 기능할 뿐만 아니라, 후술하는 바와 같이 광원 분석에 의한 판독 기기로서 기능할 수 있다는 점은 주목할 만하다. The genetic diagnostic system according to one embodiment of the present disclosure may include a portable centrifugal separation device as a physical separation means required to apply the aforementioned analysis platform. In this regard, the portable centrifugal separator may be driven using power supplied from a mobile device, and for this purpose, may be detachably connected to a power input/output terminal of the mobile device. For example, as long as it is a mobile device provided with a power input/output terminal such as a smart phone, a tablet, or a laptop and provides a function to supply power, it is not particularly limited, and may include a portable maintenance power supply. In particular, it is noteworthy that a mobile device with a built-in illuminance sensor, such as a smart phone or a tablet device, not only functions as a power source for a portable centrifuge, but also as a reading device by light source analysis as described below.
이처럼, 앞서 기술한 분석 플랫폼에 있어서 제2 기능기-함유 성분이 프라이머를 통하여 증폭된 유전자(타겟 병원체의 유전자)와 접합된 결과, 고정상 입자에 바인딩되지 않은 이동상 입자가 원심 분리에 의하여 물리적으로 분리될 수 있다. As described above, in the above-described analysis platform, as a result of conjugation of the second functional group-containing component with a gene amplified through the primer (the gene of the target pathogen), the mobile phase particle not bound to the stationary phase particle is physically separated by centrifugation. Can be.
도 5a 및 도 5b 각각은 예시적 구체예에 따른 병원체(이의 유전자) 진단 시스템 내 휴대용 원심 분리 장치 및 이에 포함된 제1 튜브 홀더의 예시적인 구성을 도시한다. 또한, 도 6은 모바일 기기로서 스마트 폰의 전원 입출력 단자와 전기적으로 연결된 휴대용 원심 분리 장치의 작동 원리를 도시한다.5A and 5B each show an exemplary configuration of a portable centrifuge device and a first tube holder included therein in a pathogen (its gene) diagnostic system according to an exemplary embodiment. In addition, Figure 6 shows the principle of operation of a portable centrifugal device electrically connected to the power input and output terminals of a smart phone as a mobile device.
상기 도면을 참조하면, 휴대용 원심 분리 장치(100)는 직립형 지지부(101)의 상측에는 모터부(A)가 설치되어 있다. 모터부는 모바일 기기로부터 공급된 전원으로 구동될 수 있으며, 크게 케이스에 둘러싸인 모터(102) 및 모터의 구동에 따라 함께 회전하는 회전 윙(103)을 포함할 수 있다. 이와 관련하여, 모터(103)는 당업계에서 공지된 소형 모터를 사용할 수 있고, 다만 휴대용에 적합하도록 가급적 낮은 전원, 구체적으로 모바일 기기 또는 보조 전원장치로부터 공급되는 전원 범위(예를 들면, 약 1 내지 5 V, 구체적으로 약 3 V 내외)에서 구동 가능한 종류를 사용할 수 있다. 이와 관련하여, 지지부(101)의 하측 단부(모터부의 반대측 단부; 110)에는 커넥터 단자(또는 전원 입력 단자)가 구비되어 모바일 기기 또는 보조 전원장치의 전원 입출력 단자와 전기적으로 연결될 수 있다. 이때, 전원 입출력 단자는, 전형적으로 USB 포트의 전원 입출력 단자일 수 있다. 또한, 휴대용 원심분리 장치에 내장된 모터(102)로서 시판 중인 소형 모터를 사용할 수 있는 바, 이러한 모터만으로도 전술한 회전 속도를 구현할 수 있다.Referring to the above drawings, the portable centrifugal separation device 100 is provided with a motor portion A on the upper side of the upright support portion 101. The motor unit may be driven by power supplied from a mobile device, and may include a motor 102 enclosed in a case and a rotating wing 103 that rotates together according to driving of the motor. In this regard, the motor 103 may use a small motor known in the art, but as low as possible to be suitable for portable power, specifically a range of power supplied from a mobile device or an auxiliary power supply (for example, about 1 To 5 V, specifically about 3 V). In this regard, a connector terminal (or a power input terminal) is provided at a lower end (opposite end of the motor unit) of the support 101 to be electrically connected to a power input/output terminal of a mobile device or an auxiliary power supply. In this case, the power input/output terminal may be a power input/output terminal of a USB port. In addition, a commercially available small motor can be used as the motor 102 embedded in the portable centrifugal separation device, and the above-described rotational speed can be realized by using only such a motor.
도시된 구체예에 따르면, 회전 윙(103)은 모터의 케이스와 일체형으로 형성된 것이거나, 또는 모터의 케이스와 회전 윙 각각이 개별적으로 제작된 후에 기계적으로 상호 결합된 것일 수도 있다. 이때, 직립형 지지부(101)를 중심으로, 모터부(A)에서 상호 대향하면서 배치된 한 쌍의 회전 윙(103)이 모터(102)에 체결되어 있는 바, 이는 예시적인 것으로 1개 이상의 회전 윙이 더 형성될 수도 있다(예를 들면, 2 쌍의 회전 윙이 형성될 수 있음).According to the illustrated embodiment, the rotary wing 103 may be formed integrally with the case of the motor, or may be mechanically coupled to each other after each of the case and the rotary wing of the motor are individually manufactured. At this time, centered on the upright support 101, a pair of rotating wings 103 arranged while facing each other in the motor portion A are fastened to the motor 102, which is exemplary, and includes one or more rotating wings This may be further formed (for example, two pairs of rotating wings may be formed).
택일적 구체예에 있어서, 모터(102)와 후술하는 고리 부재(104)를 직접 연결하여 고리 부재(104)를 회전시킬 수 있다면 회전 윙(103)을 생략할 수 있다. In an alternative embodiment, if the motor 102 and the ring member 104 described later can be directly connected to rotate the ring member 104, the rotary wing 103 can be omitted.
예시적 구체예에 있어서, 각각의 회전 윙(103)에는 고리 부재(104)가 기계적으로 연결(또는 체결)되어 있다. 이를 위하여, 회전 윙(103)에 체결 홈이 형성되어 고리 부재(104)의 적어도 일부를 이에 삽입하는 방식으로 결합 또는 체결될 수 있으며, 필요에 따라서는 체결 홈에 턱(도시되지 않음)이 형성되어 원심 분리 과정에서 고리 부재가 이탈되는 것을 방지할 수도 있다. 이처럼, 고리 부재(104)는 모터(102)와 직접적으로 또는 간접적으로 체결되어 있기 때문에 모터(102) 구동에 따라 회전하는 바, 전형적으로는 수평 방향으로 회전하게 된다. In the exemplary embodiment, the ring member 104 is mechanically connected (or fastened) to each rotary wing 103. To this end, a fastening groove is formed in the rotating wing 103 so that at least a portion of the ring member 104 can be coupled or fastened by inserting it, and if necessary, a jaw (not shown) is formed in the fastening groove. It is also possible to prevent the ring member from being separated during the centrifugation process. As such, since the ring member 104 is directly or indirectly engaged with the motor 102, it rotates according to the driving of the motor 102, and typically rotates in the horizontal direction.
한편, 도시된 구체예에 따르면, 원심 분리 장치(100)의 경우, 제1 캐비티(106) 및 제2 캐비티(108)가 형성된 제1 튜브 홀더(B)가 제공된다. 이때, 제1 캐비티(106)는 고리 부재(104)와 고리 연결 또는 인터로킹(interlocking)됨으로써, 움직임의 자유도를 가지면서도 고리 부재(104)로부터 이탈되지 않도록 구성된다. Meanwhile, according to the illustrated embodiment, in the case of the centrifugal separation device 100, a first tube holder B in which the first cavity 106 and the second cavity 108 are formed is provided. At this time, the first cavity 106 is configured to be free from movement of the ring member 104 while having a degree of freedom of movement, by connecting or interlocking the ring member 104 with the ring member 104.
도 5b를 참조하면, 제1 튜브 홀더(B)는 제1 면(105) 및 제2 면(107)이 "L"자 형상으로 연결되어 있으며, 제1 면(105)에 제1 캐비티(106), 그리고 제2 면(107)에 제2 캐비티(108)가 각각 형성되어 있다. 예시적으로, 제1 튜브 홀더(B)는 금속 재질의 단일 프레임이 벤딩되어 형성된 것일 수도 있고, 또한 고분자 재료를 성형(예를 들면, 사출 성형) 또는 3D 프리팅에 의하여 제작한 것일 수도 있다. 예시적 구체예에 있어서, 제1 튜브 홀더(B)를 구성하는 고분자 재료는 열가소성 고분자로서, 폴리에스테르, 폴리올레핀(예를 들면, 폴리에틸렌, 폴리프로필렌 등), 폴리아미드, 폴리비닐알코올, 폴리우레탄, 폴리스티렌, 폴리염화비닐 또는 이의 조합일 수 있다.Referring to FIG. 5B, the first tube holder B is connected to the first surface 105 and the second surface 107 in an “L” shape, and the first cavity 106 is connected to the first surface 105. ), and a second cavity 108 is formed on the second surface 107, respectively. For example, the first tube holder B may be formed by bending a single metal frame, or may be formed by molding (eg, injection molding) or 3D printing a polymer material. In an exemplary embodiment, the polymer material constituting the first tube holder (B) is a thermoplastic polymer, polyester, polyolefin (for example, polyethylene, polypropylene, etc.), polyamide, polyvinyl alcohol, polyurethane, Polystyrene, polyvinyl chloride, or a combination thereof.
도시된 예에서, 제1 튜브 홀더(B)는 제1 면에 형성된 제1 캐비티(106)에 의하여 고리 부재(104)와 고리 연결 또는 인터로킹되어 있는 바, 제1 캐비티(106)는 걸림부로서 기능하게 된다. 한편, 제1 튜브 홀더(B)의 제2 면에 형성된 제2 캐비티(108)는 튜브 수용부로 기능하는 바, 이에 튜브 시험관(C)을 삽입한다. 이때, 도 6을 참조하면, 삽입된 튜브 시험관(C)은 모터(103)가 구동되지 않은 경우에는 수직 위치(예를 들면, 중력 방향)에 있게 되며, 모터부(A)의 구동 시, 고리 부재(104)의 회전에 따라 원심력에 의하여 수평 위치(horizontal position)로 전환되어 회전한다.In the illustrated example, the first tube holder B is hooked or interlocked with the ring member 104 by the first cavity 106 formed on the first surface, and the first cavity 106 is the locking portion Will function as On the other hand, the second cavity 108 formed on the second surface of the first tube holder (B) functions as a tube receiving portion, into which the tube test tube (C) is inserted. At this time, referring to Figure 6, the inserted tube test tube (C) is in the vertical position (for example, in the direction of gravity) when the motor 103 is not driven, when driving the motor unit (A), the ring According to the rotation of the member 104, it is converted to a horizontal position by a centrifugal force and rotates.
도 7a 및 도 7b 각각은 예시적 구체예에 있어서 휴대용 원심 분리 장치 내 제1 튜브 홀더의 변형 예를 도시한다.7A and 7B each show a variant of the first tube holder in a portable centrifugal device in an exemplary embodiment.
상기 도면에 도시된 제1 튜브 홀더의 구조는 원심력을 균일하게 가해주기 위하여 튜브 홀더의 무게 중심이 중앙에 정렬할 수 있도록 설계된 것이다.The structure of the first tube holder shown in the figure is designed to align the center of gravity of the tube holder in the center to uniformly apply centrifugal force.
도 7a를 참조하면, 제1 튜브 홀더(B)의 상측 부위에 상당하는 제1 면(115) 내에 걸림부로서 제1 캐비티(116)가 형성되어 있다. 한편, 제1 튜브 홀더(B)의 하측 부위에는 제1 면(115)의 면 방향과 수직의 면 방향을 갖는 바닥면으로서, 예를 들면 사각형(구체적으로 정사각형)의 제2 면(117)에 튜브 수용부로서 제2 캐비티(118)가 형성되어 있다. 이때, 제1 면(115)과 같은 폭을 갖는 한 쌍의 제1 연결 면(121) 각각이 제1 면(115)의 하측 모서리와 소정 각도를 유지하며 연결(연장)되어 있고, 또한 제2 면(117)으로부터 점차적으로 폭이 감소되는 사다리꼴 형상을 갖도록 형성된 한 쌍의 제2 연결 면(122) 각각의 상측 모서리가 제1 연결 면(121)의 하측 모서리와 만나도록 구성된다. 도시된 예에 있어서, 제1 연결 면 및 제2 연결 면은 단일 부재가 벤딩되거나, 성형 또는 3D 프린팅에 의하여 일체적으로 형성된 것일 수도 있다. Referring to FIG. 7A, the first cavity 116 is formed as a locking portion in the first surface 115 corresponding to the upper portion of the first tube holder B. On the other hand, the lower portion of the first tube holder (B) is a bottom surface having a surface direction perpendicular to the surface direction of the first surface 115, for example, to the second surface 117 of a square (specifically square) The second cavity 118 is formed as a tube receiving portion. At this time, each of the pair of first connection surfaces 121 having the same width as the first surface 115 is connected (extended) to the lower edge of the first surface 115 while maintaining a predetermined angle, and also a second The upper edge of each of the pair of second connecting surfaces 122 formed to have a trapezoidal shape that gradually decreases in width from the surface 117 is configured to meet the lower edge of the first connecting surface 121. In the illustrated example, the first connecting surface and the second connecting surface may be a single member bent or integrally formed by molding or 3D printing.
한편, 상기 도면에서는 제2 면(117)이 사각형의 평면 부재로 구성되어 있으나, 다각형의 평면 부재로 변형 가능하다. 이 경우, 제2 연결 면은 사각 형상(예를 들면, 제2 연결 면의 폭이 제1 연결 면의 폭과 동일한 경우) 또는 역 사다리꼴 형상의 면(예를 들면, 제2 연결 면의 폭이 제1 연결 면의 폭보다 작은 경우)을 갖도록 구성될 수도 있다. On the other hand, in the drawing, the second surface 117 is composed of a rectangular planar member, but can be transformed into a polygonal planar member. In this case, the second connection surface has a square shape (for example, when the width of the second connection surface is equal to the width of the first connection surface) or an inverted trapezoidal surface (for example, the width of the second connection surface) It may be configured to have a) less than the width of the first connection surface.
도 7b에 도시된 제1 튜브 홀더(B)의 경우, 바닥면(127)으로부터 복수의 빔 부재(125) 각각이 바닥면(127)의 중앙 상측 공간을 향하도록 소정 각도로 연장 형성되어 서로 접합한다. 도시된 예에 따르면, 사각형(구체적으로 정사각형)의 바닥면(127) 중 4개의 꼭짓점 또는 이의 근처 부위로부터 4개의 빔 부재(125) 각각이 연장 형성되어 있다. 이때, 바닥면(127)과 빔 부재(125)을 용이하게 상호 부착할 수 있도록 바닥면의 모서리(도면에서는 대향하는 한 쌍의 모서리)에 턱이 형성되어 빔 부재의 부착면을 제공할 수 있다. 상기 도면에서는 한 쌍의 턱이 형성되어 있으나, 필요 시 바닥면의 모서리 전부에 부착용 턱이 형성될 수도 있다. In the case of the first tube holder B shown in FIG. 7B, each of the plurality of beam members 125 from the bottom surface 127 is formed to extend at a predetermined angle so as to face the central upper space of the bottom surface 127 and is joined to each other do. According to the illustrated example, each of the four beam members 125 extends from four vertices or a nearby portion of the bottom surface 127 of a square (specifically a square). At this time, the chin is formed at the edge of the bottom surface (a pair of opposite edges in the drawing) so that the bottom surface 127 and the beam member 125 can be easily attached to each other, thereby providing the attachment surface of the beam member. . In the drawing, a pair of jaws are formed, but if necessary, attachment jaws may be formed at all corners of the bottom surface.
한편, 도시된 예에서 4개의 빔 부재(125) 각각은 바닥면(127)으로부터 연장되는 방향으로 점차적으로 감소된 폭을 가질 수 있다. 이와 같이 4개의 빔 부재(125)가 서로 만나는 접합부(126)는 전술한 고리 부재(104)와 고리 결합 또는 인터로킹되는 걸림부를 형성하게 된다. 상기 도면의 경우, 바닥면은 사각형 면으로 이루어져 있으나, 경우에 따라서는 원형 또는 사각형 이외의 다각형 면으로 이루어질 수 있다. 원심 분리 시 안정적인 회전이 가능하도록 균형을 제공할 수 있는 한, 빔 부재의 개수 역시 변경될 수 있다. 또한, 바닥면(127)의 하측 방향으로 실린더 형상의 캐비티(128)가 형성되어 튜브 수용부로서 기능할 수 있다. 이와 관련하여, 캐비티(128)의 하단은 폐쇄되거나(즉, 포켓 형태) 또는 개방될 수 있다. 이외에도, 도 7a 및 도 7b에 도시된 제1 튜브 홀더는 성형 또는 3D 프린팅 방식으로 제작될 수 있다.Meanwhile, in the illustrated example, each of the four beam members 125 may have a gradually reduced width in a direction extending from the bottom surface 127. In this way, the junction 126 where the four beam members 125 meet each other forms a locking portion that is interlocked or interlocked with the aforementioned ring member 104. In the case of the drawing, the bottom surface is made of a rectangular surface, but in some cases, it may be formed of a polygonal surface other than a circular or rectangular surface. The number of beam members can also be changed so long as the balance can be provided for stable rotation during centrifugation. In addition, a cylinder-shaped cavity 128 is formed in the downward direction of the bottom surface 127 to function as a tube receiving portion. In this regard, the bottom of the cavity 128 may be closed (ie in the form of a pocket) or opened. In addition, the first tube holder shown in FIGS. 7A and 7B may be manufactured by molding or 3D printing.
튜브 홀더는 원심분리 동안 시험 튜브를 안정적으로 잡아두어야 하며, 튜브 홀더의 튜브 수용부의 직경은 사용되는 튜브 시험관의 직경과 실질적으로 동일하도록 제작할 수 있다. The tube holder should hold the test tube stably during centrifugation, and the diameter of the tube receiving portion of the tube holder can be made to be substantially the same as the diameter of the tube test tube used.
한편, 예시적 구체예에 있어서, 튜브 시험관(C)은 전체적으로 공지된 형상의 형상을 갖는 바, 크게 시험관 본체(109') 및 내용물을 수용하거나 배출하기 위한 개폐용 뚜껑(109")을 포함할 수 있다. 이와 관련하여, 튜브 시험관 본체(109')는 제1 튜브 홀더(B)에 구비된 튜브 수용부로 삽입하는데 적합한 치수를 갖는 한, 특별히 한정되는 것은 아니다. 다만, 튜브 시험관(C) 내에 분석 플랫폼, 즉 복수의 이동상 입자, 복수의 고정상 입자, 그리고 시료의 증폭 생성물(구체적으로 증폭 유전자)을 수용하고, 이를 외부에서 시각적으로 또는 조도 센서에 의하여 확인 가능해야 하는 만큼, 튜브 시험관 본체(109')는 투명 재질로 구성하는 것이 바람직하다. 예시적으로, 튜브 시험관 본체(109')는, 다양한 무기물 재질(예를 들면, 글라스) 또는 고분자 재질을 사용할 수 있다. On the other hand, in the exemplary embodiment, the tube test tube (C) has a shape of a generally known shape, and largely includes a test tube body 109' and a lid 109" for opening and closing for receiving or discharging the contents. In this connection, the tube test tube body 109' is not particularly limited as long as it has dimensions suitable for insertion into the tube receiving portion provided in the first tube holder B. However, it is not limited to the tube test tube C. As an analysis platform, i.e., a plurality of mobile phase particles, a plurality of stationary phase particles, and an amplification product (specifically, amplification gene) of a sample, it is necessary to visually check it externally or by an illuminance sensor. ') is preferably made of a transparent material, for example, the tube test tube body 109' may use various inorganic materials (for example, glass) or polymer materials.
개폐용 뚜껑(109")의 경우, 제1 튜브 홀더(B)의 튜브 수용부 내로 튜브 시험관이 삽입된 후에는 모터부(102)의 구동 시 가해지는 원심력 하에서 튜브 홀더(B)로부터 이탈되지 않고 고정된 상태로 유지되는 것으로 바람직할 수 있다. 이를 위하여, 예시적 구체예에 따르면, 개폐용 뚜껑(109")의 둘레를 따라 걸림 턱 또는 돌출부가 형성되어 있다.In the case of the lid 109" for opening and closing, after the tube test tube is inserted into the tube accommodating part of the first tube holder B, it does not deviate from the tube holder B under the centrifugal force exerted when the motor part 102 is driven. It may be desirable to remain fixed, for this purpose, according to an exemplary embodiment, a locking jaw or protrusion is formed along the perimeter of the lid 109" for opening and closing.
예시적 구체예에 따르면, 튜브 시험관(C)의 수용 체적 또는 용량은, 예를 들면 약 200 내지 2000 ㎕, 구체적으로 약 300 내지 1000 ㎕, 보다 구체적으로 약 350 내지 600 ㎕의 범위일 수 있으나, 이는 예시적인 의미로 이해될 수 있다. 다만, 소형 모터를 이용하는 만큼, 지나치게 수용 체적이 큰 튜브 시험관은 이동상(구체적으로 자성 입자)의 원심 분리에 적합하지 않을 수 있다. 예시적으로, 튜브 시험관(C)의 직경은, 예를 들면 약 5 내지 15 mm, 구체적으로 약 6 내지 12 mm 범위 내에서 정하여질 수 있으며, 이는 예시적인 의미로 이해될 수 있다.According to an exemplary embodiment, the volume or capacity of the tube in vitro (C) may be, for example, in the range of about 200 to 2000 μl, specifically about 300 to 1000 μl, more specifically about 350 to 600 μl, This can be understood in an exemplary sense. However, as a small motor is used, a tube test tube with an excessively large receiving volume may not be suitable for centrifugation of a mobile phase (specifically, magnetic particles). Illustratively, the diameter of the tube test tube (C) can be defined, for example, in the range of about 5 to 15 mm, specifically about 6 to 12 mm, which can be understood in an exemplary sense.
모바일 기기의 조도 센서를 이용한 센싱부Sensing unit using the illuminance sensor of a mobile device
일 구체예에 따르면, 전술한 분석 플랫폼에 있어서 물리적 분리 수단(구체적으로 원심 분리 수단)에 의하여 고정 상과 바인딩되지 않은 이동 상 입자를 육안에 의하여 시각적으로 확인 가능할 뿐만 아니라, 조도 센서를 이용하여 정성적 및 정량적으로 보다 정확하게 판독할 수 있다. According to one embodiment, in the above-described analysis platform, the mobile phase particles that are not bound to the stationary phase by physical separation means (specifically, centrifugation means) can be visually confirmed by the naked eye, and can be determined using an illuminance sensor. It can be read more accurately in grades and quantity.
도 8은 예시적 구체예에 따라 조도 센서에 의하여 분석 플랫폼으로부터 양성 및 음성을 판정하는 원리를 개략적으로 도시한다.8 schematically illustrates the principle of determining positive and negative from an analytical platform by an illuminance sensor according to an exemplary embodiment.
상기 도면을 참조하면, 광원과 조도 센서 사이에 물리적 분리, 구체적으로 원심 분리를 거친 테스트 튜브(분석 플랫폼을 수용한 튜브 시험관)를 위치시키고, 테스트 튜브 내 이동상의 분리 정도에 따라 다른 값을 나타내는 투과도를 조도 센서에 의하여 측정함으로써 판독을 수행할 수 있다. Referring to the drawings, a physical separation between a light source and an illuminance sensor, specifically, a test tube (tube test tube containing an analytical platform) that has undergone centrifugal separation is placed, and the transmittance exhibits different values depending on the degree of separation of the mobile phase in the test tube. The reading can be performed by measuring with an illuminance sensor.
이때, 광원으로부터 조사된 광은 테스트 튜브(구체적으로 테스트 튜브의 하측 영역)를 투과하도록 하고, 이를 투과한 광이 조도 센서에 의하여 감지되도록 배열하는 것이 바람직하다. At this time, it is preferable to arrange the light irradiated from the light source to pass through the test tube (specifically, the lower region of the test tube), and the transmitted light is sensed by the illuminance sensor.
양성인 경우, 원리 분리에 의하여 이동상 입자가 테스트 튜브의 하측에 농축되고, 농축된 이동상 입자가 광 투과도를 저감시켜 조도 센서에 도달하는 광의 조도를 감소시킨다. 반면, 음성인 경우에는 이동상 입자가 고정상 입자에 결합되어 있으므로 테스트 튜브의 하측 부위에서 이동상 입자의 농축은 상대적으로 낮은 수준이다. 따라서, 조사된 광의 투과도에 미치는 정도가 작기 때문에 조도 센서로 도달하는 광의 조도는 양성인 경우에 비하여 높다. If positive, the mobile phase particles are concentrated to the lower side of the test tube by principle separation, and the concentrated mobile phase particles reduce light transmittance to reduce the light intensity of the light reaching the illuminance sensor. On the other hand, in the negative case, since the mobile phase particles are bound to the stationary phase particles, the concentration of the mobile phase particles in the lower part of the test tube is relatively low. Therefore, since the degree of influence on the transmittance of the irradiated light is small, the illuminance of the light reaching the illuminance sensor is higher than that of the positive case.
이처럼, 조도 센서에 도달하는 조도를 측정하면 시료 내 타겟 유전자를 정성적 및 정량적으로 판독(또는 진단)할 수 있는 것이다. As described above, by measuring the illuminance reaching the illuminance sensor, the target gene in the sample can be qualitatively and quantitatively read (or diagnosed).
더욱이, 조도 센서로서 모바일 기기, 구체적으로 스마트폰 또는 태블릿 기기에 내장된 조도 센서를 이용하고, 모바일 기기에 조도 측정용 어플리케이션을 설치함으로써 조도 센서에 도달하는 광의 량을 정확하면서도 용이하게 확인할 수 있다.Moreover, as an illuminance sensor, it is possible to accurately and easily check the amount of light reaching the illuminance sensor by using an illuminance sensor embedded in a mobile device, specifically a smart phone or tablet device, and installing an illuminance measurement application on the mobile device.
이처럼, 본 구체예에서는 모바일 기기에 내장된 조도 센서를 이용하여 전술한 판독 원리를 구현할 수 있는 센싱부를 제공한다. As such, the present embodiment provides a sensing unit capable of implementing the above-described reading principle using the illuminance sensor embedded in the mobile device.
센싱부는 판독이 수행되는 센서 구조물 및 조도 센서를 포함하는 개념으로서, 센서 구조물(200)에 조도 센서에 필요한 광을 제공하는 광원, 그리고 판독 대상인 테스트 튜브를 도입 또는 삽입하고, 센서 구조물(200)이 조도 센서가 노출되는 면을 커버하도록 모바일 기기에 부착함으로써 전술한 판독 원리를 구현할 수 있다. The sensing unit is a concept including a sensor structure and an illuminance sensor on which reading is performed. A sensor structure 200 introduces or inserts a light source that provides light required for the illuminance sensor and a test tube to be read, and the sensor structure 200 The above-described reading principle can be implemented by attaching the illumination sensor to the mobile device to cover the exposed surface.
이때, 광원은 이동상 입자, 특히 자성 입자의 분리 정도를 광학 분석하기 위하여 사용되는 것으로, 예를 들면 백색 광원을 적용할 수 있다. 다만, 백색 광원에 한정되는 것은 아니며, 자성 입자 및 스마트 기기 내 조도 센서의 특성 상 가시광 범위 내 다양한 파장의 광을 방출하거나 조사할 수 있는 다양한 광원을 사용할 수 있다. 특정 구체예에 따르면, 광원으로서 통상적으로 알려진 발광 다이오드(LED) 소자를 사용할 수 있으며, 조사되는 광의 세기는, 예를 들면 약 100000 Lux 이하, 구체적으로 약 100 내지 30000 Lux 범위 내에서 선정될 수 있고, 또한 파장 대역은, 예를 들면 약 400 내지 1100 nm, 구체적으로 약 500 내지 800 nm 범위일 수 있다.In this case, the light source is used to optically analyze the degree of separation of mobile phase particles, particularly magnetic particles, for example, a white light source can be applied. However, it is not limited to a white light source, and various light sources capable of emitting or irradiating light of various wavelengths within a visible light range may be used due to the characteristics of the magnetic particles and the illuminance sensor in the smart device. According to a specific embodiment, a light emitting diode (LED) device commonly known as a light source may be used, and the intensity of the irradiated light may be selected, for example, within a range of about 100000 Lux or less, specifically in a range of about 100 to 30000 Lux, , Also, the wavelength band may be, for example, in the range of about 400 to 1100 nm, specifically about 500 to 800 nm.
한편, 예시적 구체예에서 센싱부를 구성하는데 적용 가능한 센서 구조물은 도 9에 도시된 바와 같다.Meanwhile, the sensor structure applicable to constructing the sensing unit in the exemplary embodiment is as illustrated in FIG. 9.
상기 도면을 참조하면, 센서 구조물(200)은 크게 조도 센서가 내장된 모바일 기기에 부착하기 위한 모바일 기기 홀더(201), 테스트 시험관을 삽입하기 위한 제2 튜브 홀더(202) 및 광원을 장착하기 위한 광원 홀더(203)를 포함한다. 도시된 예에서 센서 구조물(200)은 전체적으로 6면체 형상을 갖고 있다. Referring to the drawings, the sensor structure 200 is largely for mounting a mobile device holder 201 for attaching to a mobile device with a built-in illuminance sensor, a second tube holder 202 for inserting a test tube, and a light source. It includes a light source holder 203. In the illustrated example, the sensor structure 200 has a hexagonal shape as a whole.
도시된 구체예에 따르면, 통상적으로 모바일 기기, 특히 스마트 폰의 상측 부위에 조도 센서가 내장되어 조도 센서의 노출면이 형성되어 있다. 이점을 고려하여, 모바일 기기의 상측 부위를 삽입할 수 있도록 센서 구조물(200)의 하측 부위 또는 하단 면에 연장된 관통 홈 형태로 모바일 기기 홀더(201)가 제공된다. 도시된 예의 경우, 관통 홈은 센서 구조물 하면의 중앙보다는 광원 홀더(203)가 형성된 면에 대향하는 면 측으로 치우친 지점 또는 위치를 가로질러 형성되는 바, 전체 폭을 기준으로 관통 홈의 중앙이 대략 광원 홀더(203)가 형성된 면으로부터 약 0.55 내지 0.9, 구체적으로 약 0.6 내지 0.8에 상당하는 위치에 형성될 수 있다. 이는 제2 튜브 홀더(202)의 형성 공간 및 광원 홀더(203)를 통한 광원의 장착 공간을 제공하기 위함이다. 이때, 관통 홈의 폭은 삽입에 의하여 모바일 기기 내 조도 센서의 노출면을 커버할 수 있는 한, 특정 수치로 한정되는 것은 아니며, 삽입되는 모바일 기기의 폭, 삽입 용이성 등을 고려하여 정하여질 수 있다. According to the illustrated embodiment, an illumination sensor is usually built in an upper portion of a mobile device, particularly a smart phone, to form an exposed surface of the illumination sensor. In consideration of this, the mobile device holder 201 is provided in the form of a through groove extending in a lower portion or a lower surface of the sensor structure 200 so as to insert the upper portion of the mobile device. In the illustrated example, the through groove is formed across a point or position biased toward the side opposite to the surface where the light source holder 203 is formed, rather than the center of the bottom surface of the sensor structure. The holder 203 may be formed at a position corresponding to about 0.55 to 0.9, specifically about 0.6 to 0.8, from the formed surface. This is to provide a space for forming the second tube holder 202 and a space for mounting the light source through the light source holder 203. At this time, the width of the through groove is not limited to a specific value, as long as it can cover the exposed surface of the illuminance sensor in the mobile device by insertion, and may be determined in consideration of the width of the inserted mobile device, ease of insertion, and the like. .
한편, 센서 구조물(200)의 상측 면에는 테스트 시험관(즉, 분석 플랫폼 및 증폭 유전자가 도입된 튜브 시험관)을 삽입하기 위한 제2 튜브 홀더(202)가 형성되어 있다. 도시된 예에서 제2 튜브 홀더(202)는 전형적으로 원형 단면의 홀(hole) 형태로 형성될 수 있는 바, 이러한 제2 튜브 홀더(202)를 통하여 테스트 시험관이 삽입될 수 있다. 도시된 예에서는 홀의 하측은 뚫려 있으면서 바닥은 폐쇄되어 있기 때문에 삽입된 테스트 시험관의 수용 공간을 형성하면서 센서 구조물 아래로 빠져 나가지 않는다. 택일적으로, 홀의 하측 공간의 단부는 개방될 수 있는 바, 이러한 태양에서는 전술한 바와 같이 튜브 시험관의 기폐용 뚜껑(109")에 형성된 걸림 턱에 의하여 고정시킬 수 있다. 제2 튜브 홀더(202)의 위치 및 치수는 테스트 시험관의 치수, 센서 구조물 내 모바일 기기 홀더 및 광원 홀더의 위치 및 간격 등을 고려하여 적절히 조절할 수 있다.On the other hand, a second tube holder 202 for inserting a test tube (ie, an analysis platform and a tube tube into which amplification genes are introduced) is formed on the upper surface of the sensor structure 200. In the illustrated example, the second tube holder 202 may be formed in a hole shape, which is typically a circular cross-section, so that a test tube can be inserted through the second tube holder 202. In the illustrated example, the lower side of the hole is open and the bottom is closed, so that it does not escape under the sensor structure while forming the receiving space of the inserted test tube. Alternatively, the end of the lower space of the hole can be opened, in this aspect it can be secured by a locking jaw formed in the lid 109" for the tube's airflow as described above. Second Tube Holder 202 ) The position and dimensions of the test tube can be appropriately adjusted in consideration of the dimensions of the test tube, the position and spacing of the mobile device holder and the light source holder in the sensor structure.
또한, 센서 구조물(202)에 있어서 제2 튜브 홀더(202)를 기준으로 모바일 기기 홀더(201) 측 방향의 대향 면에 광원 홀더(203)가 사각 형상의 홀로 형성되어 있다. 도시된 예에서 광원 홀더(203)는 사각 형상의 홀이 센서 구조물(200)의 중앙 쪽으로 연장 형성되어 제2 튜브 홀더(202)의 하측 방향으로 뚫려 있는 공간, 그리고 모바일 기기 홀더(201)의 연장된 관통 홈의 측면과 연통될 수 있다. 이러한 연통 구조는 광원으로부터 조사된 광이 테스트 튜브를 투과한 후에 모바일 기기의 조도 센서에 전달될 수 있는 경로를 제공한다. 도시된 예에서는 광원 홀더의 형상이 사각 형상을 갖고 있으나, 이는 예시적인 것으로서 광원을 장착할 수 있는 한, 다양한 형상의 단면이 가능하다. In addition, in the sensor structure 202, the light source holder 203 is formed as a square-shaped hole on the opposite surface of the mobile device holder 201 side with respect to the second tube holder 202. In the illustrated example, the light source holder 203 is a space in which a square-shaped hole extends toward the center of the sensor structure 200 and is drilled in a downward direction of the second tube holder 202, and an extension of the mobile device holder 201 It can be communicated with the side of the through groove. This communication structure provides a path through which light emitted from the light source passes through the test tube and can be transmitted to the illumination sensor of the mobile device. In the illustrated example, the shape of the light source holder has a square shape, but as an example, as long as the light source can be mounted, various cross-sections are possible.
그 결과, 센서 구조물(200)의 모바일 기기 홀더(201)를 모바일 기기의 상측 부위(즉, 내장된 조도 센서의 노출면이 위치하는 부위)에 삽입할 경우, 광원 홀더(203)에 장착된 광원으로부터 조사된 광은 테스트 시험관(구체적으로, 원심 분리에 의하여 고정상과 결합되지 않은 이동상이 농축되는 테스트 시험관의 하측 부위)를 투과하여 조도 센서의 노출면으로 전달될 수 있다. 이와 같이 전달된 광은 조도 센서에 의하여 측정되며, 측정된 값은 특히 모바일 기기에 설치된 어플리케이션에 의하여 처리되어 모바일 기기의 화면에 측정된 조도를 디스플레이할 수 있다. 따라서, 화면에 표시된 조도가 낮으면 양성으로 판단할 수 있는 것이다. As a result, when the mobile device holder 201 of the sensor structure 200 is inserted into the upper part of the mobile device (ie, the part where the exposed surface of the built-in illuminance sensor is located), the light source mounted on the light source holder 203 The light irradiated from can pass through the test tube (specifically, the lower portion of the test tube where the mobile phase not bound to the stationary phase is concentrated by centrifugation) and can be transmitted to the exposed surface of the illuminance sensor. The transmitted light is measured by the illuminance sensor, and the measured value can be processed by an application installed in the mobile device, in particular, to display the measured illuminance on the screen of the mobile device. Therefore, if the illuminance displayed on the screen is low, it can be judged as positive.
한편, 예시적 구체예에 따르면, 센서 구조물(200)은 일체형 구조물일 수 있는 바, 이를 위하여 몰드(또는 성형) 또는 3D 프린팅 방식으로 제작할 수 있으나, 작은 사이즈의 입체 구조 내에 복수의 홀 및 이의 연통 구조를 형성하는 것이 요구되는 만큼, 3D 프린팅 방식을 이용하여 간편하게 제작할 수 있다. 예시적 구체예에 따르면, 3D 프린팅 방식으로 제작 가능한 센서 구조물(200)의 재질은 공지되어 있는 바, 전형적으로는 합성 고분자 또는 플라스틱을 사용할 수 있으며, 보다 전형적으로 폴리우레탄, 탄소섬유, 열가소성 수지, 광경화성 수지 또는 이의 조합을 예시할 수 있다. On the other hand, according to an exemplary embodiment, the sensor structure 200 may be an integral structure, and for this purpose, a mold (or molding) or 3D printing method may be used, but a plurality of holes and communication therebetween in a small-sized three-dimensional structure As it is required to form a structure, it can be easily produced using a 3D printing method. According to an exemplary embodiment, the material of the sensor structure 200 that can be manufactured by 3D printing is known, and typically a synthetic polymer or plastic can be used, more typically polyurethane, carbon fiber, thermoplastic resin, And photo-curable resins or combinations thereof.
이와 같이 센서 구조물이 제작되면, 이에 광원(예를 들면 LED 소자), 및 광원의 구동에 필요한 전기를 제공하기 위한 배터리를 센서 구조물(200)에 설치하고, 모바일 기기 홀더(201)를 이용하여 모바일 기기에 삽입한다. 이후, 판독 대상인 테스트 튜브를 제2 튜브 홀더(202) 내로 삽입하고 광원을 조사하면 설치된 어플리케이션이 실행되어 모바일 기기의 화면 상에 조도가 표시된다. 이때, 모바일 기기에 설치되는 조도 측정용 어플리케이션은 특별히 한정되는 것은 아니며, 해당 분야에서 공개된 다양한 종류를 특별한 제한없이 적용 가능하다. 또한, 전술한 절차는 예시적인 것으로서 일부 순서는 변경 가능하다.When the sensor structure is manufactured as described above, a light source (for example, an LED element) and a battery for providing electricity required for driving the light source are installed in the sensor structure 200, and the mobile device holder 201 is used for mobile. Insert it into the device. Thereafter, when a test tube to be read is inserted into the second tube holder 202 and irradiated with a light source, the installed application is executed, and illuminance is displayed on the screen of the mobile device. At this time, the application for measuring the illuminance installed on the mobile device is not particularly limited, and various types disclosed in the field can be applied without particular limitation. In addition, the above-described procedure is exemplary, and some of the procedures may be changed.
이하, 본 발명의 이해를 돕기 위해 바람직한 실시예를 제시하지만, 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐 본 발명이 이에 한정되는 것은 아니다.Hereinafter, preferred examples are provided to help the understanding of the present invention, but the following examples are provided only to more easily understand the present invention, and the present invention is not limited thereto.
실시예Example
실시예에서 사용된 물질 및 장치는 하기와 같다:The materials and devices used in the examples are as follows:
- The N-Hydroxysuccinimidyl sepharoseㄾ 4 Fast Flow (4B)는 Sigma Aldrich로부터 구입하였다(Cat # H 8280).-The N-Hydroxysuccinimidyl sepharoseㄾ 4 Fast Flow (4B) was purchased from Sigma Aldrich (Cat # H 8280).
- 자성입자(Dynabeadsㄾ Myone™ Streptavidin C1)는 미국 Life Science사로부터 구입하였다(Cat #65001). -Magnetic particles (Dynabeads ㄾ Myone™ Streptavidin C1) were purchased from Life Science, USA (Cat #65001).
- 테스트 튜브로서 Axygen, Cat # AXY-MCT-060를 사용하였다.-Axygen, Cat # AXY-MCT-060 was used as a test tube.
- PCR 혼합물로서 Qiagen HotStartTaqㄾ Plus Master Mix Kit를 사용하였다(Valencia, USA,Cat # 203643). -Qiagen HotStartTaq® Plus Master Mix Kit was used as PCR mixture (Valencia, USA, Cat # 203643).
- 수식된 프라이머 합성물은 Bioneer(Daejeon, Korea)로부터 구입하였다.-Modified primer composites were purchased from Bioneer (Daejeon, Korea).
- GelRed™ Nucleic Acid Gel stain(10,000X)은 Biotium으로부터 구입하였다(Cat # 41002)-GelRed™ Nucleic Acid Gel stain (10,000X) was purchased from Biotium (Cat # 41002)
- 원심분리기는 Hanil사(Combi-514R)로부터 구입하여 사용하였다.-Centrifuge was purchased from Hanil (Combi-514R) and used.
- DLS은 Malvern로부터 구입하여 사용하였다(Zetasizer nano zs).-DLS was purchased from Malvern and used (Zetasizer nano zs).
- NMR 또는 IR, Quick Exrtact™ DNA 추출 용액 1.0은 상품명 Epicentriㄾ (Cat # QE09050, USA)를 사용하였다. -NMR or IR, Quick Exrtact™ DNA extraction solution 1.0 was used under the trade name Epicentriㄾ (Cat # QE09050, USA).
- PBS 완충액은 Gibco사 제품(Cat #70013-032)을 4℃에서 저장하여 사용하였다.-PBS buffer was used by storing Gibco (Cat #70013-032) at 4°C.
- PCR 장치로서 C1000 Touch™ thermal cycler PCR System (Bio-rad, CA, USA)을 사용하였다.-As a PCR device, a C1000 Touch™ thermal cycler PCR System (Bio-rad, CA, USA) was used.
실시예 1Example 1
A. 실험 절차A. Experimental procedure
E.coli O157:H7E.coli O157:H7 제조 및 DNA 추출 Manufacturing and DNA extraction
E.coli는 Luria-Bertani (LB) 배지 내에 37℃에서 16 시간 동안 배양하였다. 그 다음, 콜로니 계수 기법(colony counting method)을 이용하여 E.coli의 CFU(colony forming unit)를 확인하였다. E.coli는 원심분리기(13000 rpm)을 이용하여 테스트 튜브 내에서 수집하였고, Quick Exrtact™ DNA 추출 용액 1.0을 이용하여 98℃에서 15분에 걸쳐 E.coli 펠렛으로부터 DNA를 추출하였다. E.coli was cultured in Luria-Bertani (LB) medium at 37° C. for 16 hours. Next, the colony forming unit (CFU) of E.coli was identified using a colony counting method. E.coli was collected in a test tube using a centrifuge (13000 rpm), and DNA was extracted from E.coli pellets at 98° C. over 15 minutes using Quick Exrtact™ DNA Extraction Solution 1.0.
E.coli O157:H7E.coli O157:H7 에 대한 PCR 반응PCR reaction for
타겟 DNA 앰플리콘을 신속하게 시각적으로 확인하는 개념에 대한 증명을 위하여, E.coli에 대한 PCR을 수행하였다. 프라이머는 stx 2 gene DNA (ref)에 기반하여 설계하였다. 역방향 프라이머는 염기서열의 5' 말단에서 Cy3으로 라벨링하였다. 바이오틴은 정방향 프라이머의 5' 말단에서 라벨링되어 스트렙트아비딘-라벨링된 자성 비드에 의하여 포획될 수 있도록 하였다. 정방향 프라이머 및 역방향 프라이머의 염기서열 각각은 하기 표 1과 같다.To demonstrate the concept of quickly and visually identifying the target DNA amplicon, PCR for E.coli was performed. Primers were designed based on stx 2 gene DNA (ref). The reverse primer was labeled with Cy3 at the 5'end of the base sequence. Biotin was labeled at the 5'end of the forward primer so that it could be captured by streptavidin-labeled magnetic beads. The base sequences of the forward primer and the reverse primer are shown in Table 1 below.
프라이머 primer 염기서열Sequence
정방향Forward 5′-GGG CAG TTA TTT TGC TGT GGA-3′ 5′-GGG CAG TTA TTT TGC TGT GGA-3′
역방향Reverse 5′-TGT TGC CGT ATT AAC GAA CCC-3′5′-TGT TGC CGT ATT AAC GAA CCC-3′
E.col의 증폭을 위하여, HotStartTaqㄾ Plus Master Mix Kit를 이용하여 수행하였다. 반응 혼합물은 2X 마스터 믹스, 0.08 μM의 정방향 프라이머, 0.08 μM의의 역방향 프라이머, 1 μL의 추출된 DNA 및 탈이온수를 함유하였다. DNA 증폭을 위하여, C1000 Touch™ thermal cycler PCR System을 사용하였다. 열적 사이클 조건은 95℃에서 5분, 30초 동안 95℃, 30초 동안 60℃ 및 30초 동안 72℃의 열적 사이클을 35회 반복한 다음, 5분 동안 72℃에서 최종 연장 반응(final extension)을 수행하여 유전자 증폭 반응을 완료하였다. 타겟 DNA는 바이오틴으로 라벨링된 정방향 프라이머 및 역방향 프라이머를 이용하여 120-bp 사이즈의 앰플리콘에 대하여 증폭되었다. For amplification of E.col , it was performed using HotStartTaq aq Plus Master Mix Kit. The reaction mixture contained 2X master mix, 0.08 μM forward primer, 0.08 μM reverse primer, 1 μL extracted DNA and deionized water. For DNA amplification, a C1000 Touch ™ thermal cycler PCR System was used. The thermal cycle conditions were repeated 35 times for 5 minutes at 95°C, 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by a final extension at 72°C for 5 minutes. Was performed to complete the gene amplification reaction. The target DNA was amplified against a 120-bp size amplicon using biotin labeled forward and reverse primers.
증폭된 DNA의 시각화Visualization of amplified DNA
도 4에 도시된 바와 같이, PCR 증폭 반응 후, 4 μL의 바이오틴화 증폭 DNA, 4 μL의 자성 비드 서스펜션(10 mg/mL, Dynabeadsㄾ Myone™ Streptavidin C1) 및 1 μL의 GelRed (20x, Biotium, Hayward, CA, USA)의 혼합물을 개별 겔 컬럼(Ortho BioVue system polycassettes) 내로 피펫팅하였고, 37℃에서 5분 동안 인큐베이션하였다. 인큐베이션 후, 겔 카드를 1000 rpm에서 2분 동안 1회 원심분리하였다. 시각적 확인은 30초, 60초, 120초 및 300초에서 수행되었다. 겔 매트릭스에서 플랫 라인(flat line)에 대응하는 로딩 부위는 유입 현상이 일어나지 않았음을 지시하며, 음성 결과를 보여준다. 양성 결과는 앰플리콘-운반 자성 비드(민감화-처리된 자성 비드)가 겔 매트릭스를 통과하며 겔 컬럼의 바닥까지 이동하였음을 지시한다. 4, after the PCR amplification reaction, 4 μL of biotinylated amplification DNA, 4 μL of magnetic bead suspension (10 mg/mL, Dynabeads yna Myone™ Streptavidin C1) and 1 μL of GelRed (20x, Biotium, The mixture of Hayward, CA, USA) was pipetted into individual gel columns (Ortho BioVue system polycassettes) and incubated at 37° C. for 5 minutes. After incubation, the gel card was centrifuged once at 1000 rpm for 2 minutes. Visual confirmation was performed at 30 seconds, 60 seconds, 120 seconds and 300 seconds. The loading site corresponding to the flat line in the gel matrix indicates that no inflow phenomenon occurred, and shows a negative result. A positive result indicates that the amplicon-carrying magnetic beads (sensitized-treated magnetic beads) passed through the gel matrix and moved to the bottom of the gel column.
B. 결과 분석B. Results Analysis
기능화된 세파로오스 기반의 바이오 분석 플랫폼의 원리Principle of functionalized Sepharose-based bio-analysis platform
특정 분석 장치 없이 PCR 생성물을 분석하기 위하여, 육안 식별 가능한 센싱 원리를 설계하였고, 리간드 친화성 크로마토그래피-기반의 광학 센싱 플랫폼을 개발하였다. 스트렙트아비딘-접합된(conjugated) 자성 입자(MP)를 이동상으로 사용하였고, 또한 이의 고유한 붉은색을 띄는 갈색에 기반하는 분자 시그널을 위하여 사용하였다. PCR 방법에 의한 유전자 증폭에 있어서, MP 및 PCR 생성물과 직접 바인딩하도록 바이오틴화 프라이머를 합성하였다. NHS-기능화된 세파로오스를 고정상으로 사용하였다. 육안 식별 가능한 광학 센싱 시스템을 확립하기 위하여, 통상의 PCR 장치를 이용하여 먼저 병원체로부터의 유전자를 증폭시켰고 제조된 자성 입자와 혼합하였는 바, MP 표면의 모이티가 PCR로부터의 증폭된 유전자를 바인딩함에 따라 변화하였다. 그 다음, MP 혼합물과 PCR의 혼합물을 NHS-수식된 세파로오스 컬럼에 적용하였다. 음성 테스트에 있어서, 자성 입자 표면 상의 스트렙트아비딘이 노출되었고, 스트렙트아비딘 상의 아민기가 세파로오스의 NHS기와 반응할 수 있었다. 그러나, 양성 테스트에 있어서는 스트렙트아비딘이 증폭된 유전자에 의하여 피복되어 있고, 이후 자성 입자가 세파로오스 네트워크 구조로부터부터 분리되었다. 이러한 특성에 기반하여, 세파로오스 겔 내의 자성 입자의 이동(migration) 범위는 원심 분리 과정에서 점차적으로 변화하였고, 자성 입자의 붉은색을 띄는 갈색으로 인하여 자성 입자의 움직임을 육안 관찰할 수 있었다.In order to analyze PCR products without a specific analysis device, a visually identifiable sensing principle was designed, and a ligand affinity chromatography-based optical sensing platform was developed. Streptavidin-conjugated magnetic particles (MP) were used as the mobile phase and were also used for molecular signals based on their unique reddish brown color. In gene amplification by PCR method, biotinylated primers were synthesized to directly bind MP and PCR products. NHS-functionalized sepharose was used as the stationary phase. In order to establish a visually identifiable optical sensing system, a gene from a pathogen was first amplified using a conventional PCR device and mixed with the prepared magnetic particles, so that the moiety on the MP surface binds the amplified gene from PCR. It changed accordingly. The mixture of MP mixture and PCR was then applied to an NHS-modified Sepharose column. In the negative test, streptavidin on the magnetic particle surface was exposed, and the amine group on streptavidin was able to react with the NHS group of sepharose. However, in the positive test, streptavidin was coated by the amplified gene, and then the magnetic particles were separated from the Sepharose network structure. Based on these properties, the migration range of the magnetic particles in the Sepharose gel gradually changed during the centrifugation process, and the movement of the magnetic particles could be visually observed due to the reddish-brown color of the magnetic particles.
결과적으로, 타겟 유전자는 육안 관찰에 의하여 간단히 진단할 수 있었다.As a result, the target gene could be easily diagnosed by visual observation.
육안 관찰에 의한 PCR 생성물의 분석Analysis of PCR products by visual observation
본 실시예에서는 식품 유래의 병원체에 대한 바이오마커로서 E.coli를 선택하였다. 1.0×105 CFU의 E.coli를 제조하였고, 이의 유전자 DNA(gDNA)를 추출하여 정제하였다. 탈이온수를 음의 대조군 테스트로서 사용하였고, 상기 용액을 30분 동안 자성 입자와 혼합하였다. 그 다음, 10배로 희석된 GelRed를 10분 동안 반응시켰다. 제조된 200 μL NHS-수식된 세파로오스를 테스트 튜브 내에서 조심스럽게 팩킹하였다. 용액 내에서 부유된 세파로오스로 인하여, 테스트 튜브를 30초 동안 원심분리시켰다. 이후, 각각의 제조된 테스트 시료를 세파로오스 컬럼에 적용하였고, 상기 컬럼을 2분 동안 1000 rpm에서 원심분리시켰다. In this example, E.coli was selected as a biomarker for food-derived pathogens. E.coli of 1.0×10 5 CFU was prepared, and its genetic DNA (gDNA) was extracted and purified. Deionized water was used as a negative control test, and the solution was mixed with magnetic particles for 30 minutes. Then, the GelRed diluted 10-fold was reacted for 10 minutes. The prepared 200 μL NHS-modified Sepharose was carefully packed in test tubes. Due to the suspended sepharose in solution, the test tube was centrifuged for 30 seconds. Then, each prepared test sample was applied to a Sepharose column, and the column was centrifuged at 1000 rpm for 2 minutes.
그 결과, 양성 테스트에서 자성 입자는 도 10a에서와 같이 자성 입자는 명확하게 세파로오스 컬럼을 통과하여 이동하였고, 붉은색을 띄는 갈색 입자가 육안으로도 관찰되었다. 반면, 음성 테스트에서는 도 10b에서와 같이 자성 입자의 움직임은 관찰되지 않았는 바, 이는 자성 입자가 세파로오스와 견고하게 바인딩되어 있음을 지시한다.As a result, in the positive test, the magnetic particles were clearly moved through the Sepharose column, as shown in FIG. 10A, and reddish brown particles were also observed with the naked eye. On the other hand, in the negative test, the movement of the magnetic particles was not observed as in FIG. 10B, indicating that the magnetic particles were tightly bound to the sepharose.
상기 결과는 실시예에 따른 분석 플랫폼이 의도된 바와 같이 성공적으로 작동하고, 타겟 병원체로부터 증폭된 유전자가 육안으로 용이하게 검출 또는 확인될 수 있음을 보여준다. The above results show that the analysis platform according to the example operates successfully as intended, and the gene amplified from the target pathogen can be easily detected or identified with the naked eye.
분석 플랫폼의 Analysis platform E.coliE.coli 에 대한 검출 한계(LOD) 평가Limit of detection (LOD)
음식물 내 극히 소량의 E.coli를 포함하는 박테리아가 식중독을 유발하기 때문에 높은 감도 특성을 나타낼 것이 요구된다. E.coli 분석 시 검출 한계를 확인하기 위하여 E.coli를 1.0×106 CFU에서부터 1.0×101 CFU까지 순차적으로 희석시켰고, 전술한 절차와 같이 DNA를 추출하였다. 각각의 시료로부터 증폭된 PCR 생성물에 대하여 동일 조건 하에서 본 실시예에 따른 분석 시스템을 적용하였으며, 그 결과를 전기영동 테스트 결과와 함께 도 11에 나타내었다. 상기 도면에 따르면, 음성 대조군 테스트를 제외하고는 자성 입자의 이동이 관찰되었는 바, 이는 본 실시예에 따른 검출 시스템을 이용할 경우, 육안 관찰에 의하여 E.coli를 진단할 수 있음을 지시한다. 또한, 1.0×101 CFU 미만의 E.coli 농도에서는 자성 입자는 약간 이동하였다. 이와 같이 낮은 E.coli 농도에서도 MP 레벨이 변하였으며, 육안으로 평가하기에 음성 시료 테스트(대조군) 결과에 비하여 뚜렷하게 구별된다. 따라서, 검출 한계(LOD)는 1.0×101 CFU로 판단하였다. Bacteria containing a very small amount of E. coli in foods cause food poisoning, so it is required to exhibit high sensitivity properties. In order to confirm the detection limit during E.coli analysis, E.coli was sequentially diluted from 1.0×10 6 CFU to 1.0×10 1 CFU, and DNA was extracted as described above. For the PCR product amplified from each sample, the analysis system according to this example was applied under the same conditions, and the results are shown in FIG. 11 together with the electrophoresis test results. According to the figure, the movement of magnetic particles was observed except for the negative control test, which indicates that when using the detection system according to the present embodiment, E. coli can be diagnosed by visual observation. In addition, the magnetic particles slightly shifted at an E. coli concentration of less than 1.0×10 1 CFU. Even at such low concentrations of E. coli , the MP level was changed, and it was clearly distinguished from the negative sample test (control) result for visual evaluation. Therefore, the detection limit (LOD) was determined to be 1.0×10 1 CFU.
실제 시료에 기반한 육안 관찰 분석Visual observation analysis based on real samples
E.coli가 식중독의 주된 요인인 만큼, 실제 시료에 기반하는 E.coli 분석 실험을 수행하여 본 실시예에 따른 분석 플랫폼의 실제 적용 가능성을 평가하였다. 본 실시예에서는 10 μL의 우유 내로 1.0×106 CFU에서부터 1.0×101 CFU까지의 E.coli를 주입하여 인위적으로 감염된 실제 시료를 제조하였다. 박테리아-주입된 우유 시료를 용해시키고, 용해된 용액을 통상의 PCR에 적용하였다. PCR 생성물을 본 실시예에 따른 시스템에 적용하였는 바, 재현성 확보를 위하여 동일 조건 하에서 적어도 3회에 걸쳐 분석 실험을 수행하였다. 그 결과를 도 12에 나타내었다. Since E.coli is the main factor of food poisoning, the possibility of practical application of the analysis platform according to the present embodiment was evaluated by performing an E.coli analysis experiment based on an actual sample. In this example, an actual sample artificially infected was prepared by injecting E.coli from 1.0×10 6 CFU to 1.0×10 1 CFU into 10 μL of milk. The bacterial-infused milk sample was dissolved and the dissolved solution was subjected to conventional PCR. When the PCR product was applied to the system according to this example, analytical experiments were performed at least three times under the same conditions to ensure reproducibility. The results are shown in FIG. 12.
상기 도면을 참조하면, 전체 사진에서 MP의 이동이 관찰되었는 바, 이는 본 실시예에 따른 시스템에 실제 음식물 시료에 기반한 E.coli가 효과적으로 검출 가능함을 뒷받침한다. 구체적으로, MP는 1.0×101 CFU를 이상의 E.coli 농도에서는 명확하게 이동한 반면, 음성 대조군에서는 MP 변화가 관찰되지 않았다. 1.0×101 CFU 농도의 경우, 자성 입자는 약간 이동하였으나, 음성 대조군에 비하여 뚜렷한 이동이 관찰되었다. 따라서, 실제 시료에 있어서, 검출 한계는 1.0×101 CFU E.coli로 결정할 수 있다.Referring to the above figure, the movement of the MP was observed in the whole picture, which supports that the E.coli based on the actual food sample can be effectively detected in the system according to the present embodiment. Specifically, MP clearly shifted 1.0×10 1 CFU at an E.coli concentration of greater than or equal to, whereas no MP change was observed in the negative control. In the case of 1.0×10 1 CFU concentration, the magnetic particles slightly shifted, but a clear shift was observed compared to the negative control. Therefore, in an actual sample, the detection limit can be determined as 1.0×10 1 CFU E.coli .
DLS(dynamic light scattering)을 이용한 DNA 유연성 평가DNA flexibility assessment using DLS (dynamic light scattering)
본 실시예에서 제시된 바이오센싱 원리에 따르면, 양성 테스트에서 자성 입자 상에 부착된 증폭된 PCR 생성물이 스트렙트아비딘의 아민기와 세파로오스 상의 NHS기의 바인딩이 입체 장애에 의하여 간섭받는다. 따라서, 이중 스트랜드 DNA(dsDNA)의 구조를 강성 형태로 유지하는 것이 반응 효율을 증가시키는데 중요하다. 그러나, DNA의 이중 스트랜드는 수계 상에서 높은 유연성을 갖고 있어 정확한 분석이 방해받는다. dsDNA-특정 삽입 물질(분자)의 결합 시, DNA 유연성이 변화하는 바, 본 실시예에서는 GelRed를 사용하여 평가하였다.According to the biosensing principle presented in this example, the binding of the amine group of streptavidin and the NHS group on sepharose in the amplified PCR product attached to the magnetic particles in the positive test is interfered by steric hindrance. Therefore, maintaining the structure of the double stranded DNA (dsDNA) in a rigid form is important to increase the reaction efficiency. However, the double strand of DNA has high flexibility in water system, which prevents accurate analysis. When the dsDNA-specific insertion material (molecule) was bound, DNA flexibility was changed. In this example, GelRed was used for evaluation.
GelRed가 염기 쌍 사이에 스태킹될 수 있기 때문에, DNA 움직임은 수계 상 내에서 구속될 수 있다. GelRed 반응에 따른 DNA 유연성을 평가하기 위하여, DLS 방법을 통하여 DNA 사이즈 변화를 분석하였는 바, 상기 방법은 바이오분자의 역학(dynamics)을 측정하여 사이즈 변화를 관찰한다. 약 100개의 염기 쌍으로서 증폭된 DNA 주형의 경우, 작은 사이즈의 길이 변화를 명확하게 검출하기 위하여 100 nm의 스트렙트아비딘-접합된 자성입자를 사용하였다. 1.0×105 CFU E.coli는 추출 및 정제되었고, 제조된 유전자는 바이오틴화 프라이머를 함유하는 PCR 혼합물과 혼합되었다. PCR 수행 후, 증폭된 유전자는 100 nm 자성 입자와 반응하였고, 이후 GelRed를 상기 용액과 혼합하였으며, DLS(dynamic light scattering) 사이즈를 측정하여 그 결과를 도 13에 나타내었다. Since GelRed can be stacked between base pairs, DNA movement can be confined within the aqueous phase. In order to evaluate the DNA flexibility according to the GelRed reaction, the DNA size change was analyzed through the DLS method, and the method observes the size change by measuring the dynamics of biomolecules. In the case of a DNA template amplified with about 100 base pairs, streptavidin-conjugated magnetic particles of 100 nm were used to clearly detect small size length changes. 1.0×10 5 CFU E.coli was extracted and purified, and the prepared gene was mixed with a PCR mixture containing biotinylated primers. After PCR was performed, the amplified gene reacted with 100 nm magnetic particles, and then GelRed was mixed with the solution, and the dynamic light scattering (DLS) size was measured and the results are shown in FIG. 13.
상기 도면에 나타낸 바와 같이, 초기 자성 입자의 사이는 158 nm이었다. 자성 입자 표면 상의 전체 스트렙트아비딘 사이즈(약 20 nm) 및 입자의 수력학적 직경을 고려하면, 상기 사이즈는 신뢰할 수 있다. 또한, DNA가 바인딩된 MP는 191 nm로 측정되었다. 증폭된 유전자는 평균 100 bs이었고, 이론 사이즈는 약 34 nm이었다. 증폭된 DNA가 MP 표면과 균일하게 바인딩된다는 것을 가정하면, 전체 사이즈는 68 nm 증가하였다. 그러나, 전술한 바와 같이, DNA 길이는 수용액 내에서 변화가능한 만큼, 얻어진 결과는 DNA가 유연성을 나타냄을 뒷받침한다. GelRed, DNA 및 MP 테스트 결과에 있어서 관찰된 사이즈는 약 224 nm이었다. 상기 결과는 dsDNA의 염기 쌍 사이에 GelRed가 성공적으로 삽입되었고, GelRed가 증폭된 DNA의 유연성을 효과적으로 감소시켰음을 보여준다. As shown in the figure, the distance between the initial magnetic particles was 158 nm. Considering the overall streptavidin size (about 20 nm) on the magnetic particle surface and the hydrodynamic diameter of the particle, the size is reliable. In addition, the DNA-bound MP was measured to be 191 nm. The amplified gene averaged 100 bs, and the theoretical size was about 34 nm. Assuming that the amplified DNA binds uniformly to the MP surface, the overall size is increased by 68 nm. However, as described above, the DNA length is variable in aqueous solution, and the results obtained support that the DNA exhibits flexibility. The observed size for GelRed, DNA and MP test results was about 224 nm. The results show that GelRed was successfully inserted between the base pairs of dsDNA, and GelRed effectively reduced the flexibility of the amplified DNA.
GelRed 농도 및 반응 시간의 최적화Optimization of GelRed concentration and reaction time
GelRed의 기능성을 이중 스트랜드 DNA의 유연성 변화로 고려할 때, 유전자 분석에서 세파로오스와의 MP 바인딩 친화성은 GelRed의 농도와 밀접하게 관련되어 있다. GelRed 농도를 최적화하기 위하여, 세파로오스-충진(팩킹)된 테스트 튜브 및 1.0×105 CFU E.coli는 앞선 테스트와 동일한 방법으로 제조하였다. PCR에 의하여 증폭된 유전자는 동일 농도의 MP와 반응시켰다. 그 다음, 5배, 10배, 20배, 50배 및 100배 희석된 GelRed를 순차적으로 10분 동안 적용하였고, 이와 같이 제조된 테스트 튜브는 2분 동안 1000 rpm에서 원심분리되었다. 도 14a에 나타낸 바와 같이, 5배 희석된 GelRed 테스트에서는 MP의 응집 현상이 관찰되었다. 이처럼, 지나치게 높은 농도의 GelRed는 세파로오스 상에서 응집되어 MP 상의 아민과 세파로오스 상의 NHS의 반응에 관계없이 MP의 이동이 교란되는 것으로 판단된다. 그러나, 10배 희석된 GelRed 테스트에 있어서, MP는 양성 테스트에서 명확하게 이동한 반면, 음성 컨트롤 테스트에서는 MP의 움직임 변화는 관찰되지 않았다. Considering the functionality of GelRed as a change in flexibility of double stranded DNA, the MP binding affinity with Sepharose in genetic analysis is closely related to the concentration of GelRed. To optimize the GelRed concentration, a sepharose-filled (packed) test tube and 1.0×10 5 CFU E.coli were prepared in the same manner as in the previous test. Genes amplified by PCR were reacted with the same concentration of MP. Then, 5, 10, 20, 50, and 100-fold diluted GelRed were sequentially applied for 10 minutes, and the test tubes thus prepared were centrifuged at 1000 rpm for 2 minutes. As shown in FIG. 14A, in the 5-fold diluted GelRed test, aggregation of MP was observed. As such, an excessively high concentration of GelRed is aggregated on the sepharose, and it is determined that the movement of the MP is disturbed regardless of the reaction of the amine on MP and NHS on sepharose. However, in the 10-fold diluted GelRed test, MP clearly shifted in the positive test, whereas in the negative control test, no change in the movement of the MP was observed.
20배, 50배 및 100배 희석된 GelRed 테스트에 있어서, 음성 컨트롤에서의 MP 레벨은 효과적으로 변화하지 않았고, 양성 테스트에서의 MP는 초기 상태와 유사하였는 바, 이는 사용된 GelRed가 증폭된 타겟 유전자에 따른 시그널 변화를 차별화하는데 불충분함을 지시한다. 상술한 테스트 결과, 약 10배 희석된 GelRed를 사용하는 것이 적합하다는 결론을 내릴 수 있다.In the 20, 50 and 100-fold diluted GelRed tests, the MP level in the negative control did not change effectively, and the MP in the positive test was similar to the initial state, indicating that the GelRed used was targeted to the amplified target gene. Insufficient in differentiating the signal changes that follow. As a result of the above-mentioned test, it can be concluded that it is suitable to use GelRed diluted about 10 times.
한편, 최적화된 원심 분리 조건을 도출하기 위한 실험을 수행하였으며, 그 결과를 도 14b에 나타내었다.Meanwhile, experiments were conducted to derive optimized centrifugal separation conditions, and the results are shown in FIG. 14B.
상기 도면에 나타낸 바와 같이, 모든 테스트 결과 중 양성 테스트에서 자성 비드의 이동 레벨은 순차적으로 변화한 반면, 음성 테스트에서 자성 비드 움직임의 현저한 변화는 검출되지 않았는 바, 이는 본 개시 내용에서 제안된 바이오 분석 플랫폼의 작동 원리가 적절하다는 것을 지시한다. 이는 PCR 생성물과 자성 비드의 접합에 따른 표면 기능기의 차이때문으로 판단된다. 양성 테스트 및 음성 테스트에 따른 자성 비드의 이동 변화는 30초에서 60초까지의 반응 시간에서 관찰되었음에도 불구하고, 자성 비드 이동 변화가 적기 때문에 질병 감염을 결정하는데 불충분하였다. 반면, 120초 동안 반응시킨 경우, 양성 테스트 및 음성 테스트 모두에서의 비드 변화가 명확히 확인되었는 바, 이는 육안으로도 E.coli PCR 생성물을 신뢰성 있게 진단할 수 있도록 한다. 300초의 반응 시간에서는 충분한 진단 결과를 확보할 수 있었으나, 이러한 원심 분리 조건은 현장 테스트에 적합하지 않다. E.coli의 경우, 약 2분의 반응 시간이 가장 이상적임을 알 수 있다.As shown in the figure, the movement level of the magnetic beads was sequentially changed in the positive test among all the test results, while a significant change in the magnetic bead movement was not detected in the negative test, which is a bio-analysis proposed in the present disclosure. It indicates that the working principle of the platform is appropriate. This is due to the difference in surface functional groups according to the conjugation of the PCR product and the magnetic beads. Although the movement change of the magnetic beads according to the positive test and the negative test was observed in the reaction time from 30 seconds to 60 seconds, it was insufficient to determine disease infection because the change of the magnetic bead movement was small. On the other hand, when reacted for 120 seconds, the bead change in both the positive test and the negative test was clearly confirmed, which makes it possible to reliably diagnose the E.coli PCR product even with the naked eye. Sufficient diagnostic results could be obtained at a reaction time of 300 seconds, but these centrifugation conditions are not suitable for field testing. In the case of E.coli , it can be seen that a reaction time of about 2 minutes is most ideal.
실시예 2Example 2
스마트 폰을 전원공급원으로 하는 휴대용 원심 분리 장치를 이용한 병원체의 진단Diagnosis of pathogens using a portable centrifugal device using a smart phone as a power supply
본 실시예에서는 실시예 1에서와 동일한 절차에 따라 E.coli 분석 플랫폼을 제조하였는 바, 구체적으로 시험 튜브에 NHS-수식된 세파로오스를 충진하였고, 증폭된 유전자와 반응된 스트렙트아비딘-접합된 자성 입자(MP)를 이에 적하하였다. 이때, 실제 시료로서 브로스(broth) 및 우유(milk)를 각각 사용하였다(1.0×101-1.0×106 CFU, E. coli O157:H7)In this example, the E.coli analysis platform was prepared according to the same procedure as in Example 1, specifically, the test tube was filled with NHS-modified Sepharose, and streptavidin-conjugated reacted with the amplified gene. The magnetic particles (MP) were added dropwise thereto. At this time, broth and milk were used as actual samples (1.0×10 1 -1.0×10 6 CFU, E. coli O157:H7 ).
후속적으로, 도 15a에서와 같이 3D 프린팅으로 제작된 제1 튜브 홀더를 이용하여 도 15b와 같이 휴대용 원심 분리 장치에 앞서 제조된 분석 플랫폼을 함유하는 시험 튜브를 삽입하였다. 그 다음, 도 16에 나타낸 바와 같이 휴대용 원심 분리 장치의 지지부의 하단에 설치된 전원 커넥터를 스마트 폰(LG사 제품명 G4)의 USB 단자에 연결시켜 2분에 걸쳐 원심분리를 수행하였다. 이때, 시험 튜브의 체적은 400 μL이었고, 회전 속도는 2000 rpm이었다.Subsequently, a test tube containing the analytical platform prepared prior to the portable centrifugal separation apparatus as shown in FIG. 15B was inserted using a first tube holder made of 3D printing as in FIG. 15A. Then, as shown in Figure 16, the power connector installed at the bottom of the support of the portable centrifugal separator was connected to a USB terminal of a smart phone (LG product name G4) to perform centrifugation over 2 minutes. At this time, the volume of the test tube was 400 μL, and the rotation speed was 2000 rpm.
그 결과를 도 17a 및 도 17b에 각각 나타내었다.The results are shown in FIGS. 17A and 17B, respectively.
상기 도면에서 확인되는 바와 같이, 기능화된 입자 기반의 분석 플랫폼과 스마트 폰으로 구동되는 휴대용 원심분리 장치를 조합하여 물리적 분리를 수행함으로써 양성 테스트 및 음성 테스트 결과를 육안으로 관찰할 수 있었으며, 이는 2가지의 실제 시료(브로스 및 우유)에서 유효하게 적용되었다. 특히, 실시예 1에서 확인되는 검출 한계와 동등한 수준의 진단 정확성을 확보할 수 있었다.As can be seen from the figure, a physical separation was performed by combining a functionalized particle-based analysis platform and a smart phone-driven portable centrifugal separation device, whereby positive and negative test results could be visually observed. Effectively applied in the actual samples (broth and milk). In particular, it was possible to ensure a diagnostic accuracy equivalent to the detection limit identified in Example 1.
실시예 3Example 3
스마트 폰에 내장된 조도 센서를 이용한 병원체의 진단Diagnosis of pathogens using the illumination sensor built into the smartphone
실시예 2에서 원심 분리된 테스트 튜브를 육안으로 관찰하는 대신에 스마트 폰에 내장된 조도 센서를 이용하여 양성 및 음성 판독을 수행하였다.In Example 2, instead of visually observing the centrifuged test tube, positive and negative readings were performed using an illuminance sensor built into the smart phone.
본 분석 플랫폼이 이동 상(자성 입자; MP)과 고정 상(세파로오스) 간의 흡광도 및 투과도 차이를 이용하여 진단을 수행하는 만큼, 사전 실험으로서 스마트 폰에 내장된 조도 센서의 노출 면 상에 세파로오스 및 자성 입자(MP) 각각이 충진된 투명한 기판을 위치시킨 후 LED를 조사하여 광량의 차이를 분석하였다. 그 결과를 도 18에 나타내었다.As the analysis platform performs diagnosis using the difference in absorbance and transmittance between the mobile phase (magnetic particle; MP) and the stationary phase (sepharose), as a pre-experiment, a separation wave on the exposed surface of the illumination sensor embedded in the smartphone After placing a transparent substrate filled with each of rose and magnetic particles (MP), LEDs were irradiated to analyze the difference in the amount of light. The results are shown in FIG. 18.
상기 도면에서 확인되듯이, 자성 입자는 조사된 광의 투과도가 낮기 때문에 스마트 폰 화면에서 표시된 조도는 낮은 수준인 반면, 세파로오스는 조사된 광의 투과도가 상대적으로 높기 때문에 높은 조도 값이 표시되었다. 이처럼, 본 분석 플랫폼에 기반하고, 스마트 폰에 내장된 조도 센서를 이용하여 진단할 수 있을 것으로 판단된다.As can be seen from the figure, since the magnetic particles have low transmittance of irradiated light, the illuminance displayed on the smartphone screen is low, while Sepharose has a relatively high transmittance of irradiated light, so a high illuminance value is displayed. As such, it is judged that it is based on the present analysis platform and can be diagnosed using the illumination sensor built into the smart phone.
상술한 사전 실험 결과를 고려하여, 도 19a에 나타낸 바와 같이 3D 프린팅을 이용하여 센서 구조물(몰드)를 제작하였다. 이와 같이 제작된 센서 구조물의 광원 홀더에 광원으로서 LED 소자를 부착하였고, 광원은 측면에 부착된 3V 배터리에 의하여 작동하도록 하였다. 또한, 실시예 2에서 2가지 시료((101 내지 106 CFU 범위의 E. coli O157:H7)에 대하여 분석 플랫폼을 적용하고 원심 분리를 거친 시험 튜브를 센서 구조물의 제2 튜브 홀더에 삽입하였다. In consideration of the above-mentioned pre-experiment results, a sensor structure (mold) was fabricated using 3D printing as shown in FIG. 19A. The LED element was attached as a light source to the light source holder of the sensor structure thus manufactured, and the light source was operated by a 3V battery attached to the side. In addition, in Example 2, an analysis platform was applied to two samples (( E. coli O157:H7 in the range of 10 1 to 10 6 CFU)) and a test tube subjected to centrifugation was inserted into the second tube holder of the sensor structure. .
이후, 도 19b에서와 같이 테스트 튜브가 삽입된 센서 구조물에 의하여 스마트 폰의 조도 센서 노출 면을 커버하도록 하였으며, 조도 센서를 이용하여 테스트 튜브를 투과한 LED 광의 조도를 측정하고, 설치된 어플리케이션을 구동함으로써 스마트 폰의 화면에 표시된 조도 값을 확인하였다. 그 결과를 도 20a 및 도 20b에 나타내었다.Subsequently, as shown in FIG. 19B, a sensor structure in which a test tube is inserted is configured to cover the exposed surface of the illumination sensor of the smart phone, and the illuminance sensor is used to measure the illuminance of the LED light passing through the test tube and drive the installed application. The illuminance value displayed on the screen of the smart phone was confirmed. The results are shown in FIGS. 20A and 20B.
상기 도면에 따르면, 스마트 폰에 내장된 조도 센서를 이용하여 측정한 결과, 양성의 경우에는 모두 자성입자가 침전되어 낮은 광량을 보였으나, 음성의 경우 광원이 세파로즈를 투과하여 높은 광량을 보였다. 즉, 음성군과 테스트군의 광량 차이가 뚜렷하여 재현성 및 민감도를 확보할 수 있음을 확인하였다. According to the figure, as a result of measurement using an illuminance sensor embedded in a smart phone, in the positive case, magnetic particles precipitated and showed a low amount of light, but in the case of negative light, the light source transmitted through Sepharose and showed a high amount of light. That is, it was confirmed that the difference in the amount of light between the negative group and the test group was clear, and thus reproducibility and sensitivity could be secured.
이처럼, 모바일 기기를 이용하여 간편하게 현장에서 분석 가능할 수 있으며, 이에 사용된 센서 구조물은 간단한 구조를 갖고 있어 저렴하게 제작할 수 있는 등, 상용화 가능성이 높은 것으로 판단된다.As described above, it can be easily analyzed in the field using a mobile device, and the sensor structure used for this has a simple structure, so it can be manufactured inexpensively, and thus, it is considered that there is a high possibility of commercialization.
본 발명의 단순한 변형 내지 변경은 이 분야의 통상의 지식을 가진 자에 의하여 용이하게 이용될 수 있으며, 이러한 변형이나 변경은 모두 본 발명의 영역에 포함되는 것으로 볼 수 있다.Simple modifications or changes of the present invention can be easily used by those skilled in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (15)

  1. (A) 시각적 확인(identification)이 가능하고, 제1 기능기-함유 성분으로 수식된 복수의 이동상(mobile phase) 입자, 및 상기 제1 기능기-함유 성분과 결합능을 갖는 화학적 기능기에 의하여 활성화된 복수의 고정상(stationary phase) 입자를 포함하고, 상기 제1 기능기-함유 성분이 타겟 병원체의 유전자에 특이적인 프라이머와 접합된 제2 기능기-함유 성분과 바인딩 특성을 갖되, 상기 제2 기능기-함유 성분이 프라이머를 통하여 증폭된 타겟 병원체의 유전자와 접합되어 있는 경우에는 입체 장애로 인하여 상기 이동상 입자와 상기 고정상 입자 간의 바인딩이 억제되며, 그리고 상기 고정상 입자에 바인딩되지 않은 이동상 입자를 분리함으로써, 시각적으로 또는 조도 센서에 의하여 확인 가능하도록 구성된 병원체의 분석 플랫폼; 그리고(A) A plurality of mobile phase particles capable of visual identification and modified with a first functional group-containing component, and activated by a chemical functional group having binding ability with the first functional group-containing component A plurality of stationary phase particles, wherein the first functional group-containing component has binding properties with a second functional group-containing component conjugated with a primer specific to the gene of the target pathogen, but the second functional group -When the containing component is conjugated to the gene of the target pathogen amplified through the primer, binding between the mobile phase particle and the stationary phase particle is suppressed due to steric hindrance, and by separating the mobile phase particle that is not bound to the stationary phase particle, An analysis platform for pathogens configured to be identifiable visually or by an illuminance sensor; And
    (B) (b1) 모바일 기기의 전원 입출력 단자와 연결되고 이로부터 공급된 전원에 의하여 상기 고정상 입자에 바인딩되지 않은 이동상 입자를 분리하는 휴대용 원심 분리 장치, 및 (b2) 모바일 기기에 내장된 조도 센서를 이용하여 상기 분석 플랫폼에 의하여 타겟 병원체를 분석하기 위한 센싱부 중 적어도 하나를 포함하는 병원체의 진단 시스템.(B) (b1) a portable centrifugal separation device that is connected to a power input/output terminal of a mobile device and separates mobile phase particles that are not bound to the stationary phase particles by power supplied therefrom, and (b2) an illuminance sensor embedded in the mobile device A diagnostic system of a pathogen comprising at least one of a sensing unit for analyzing a target pathogen by the analysis platform using the.
  2. 제1항에 있어서, 상기 전원 입출력 단자는 USB 포트의 전원 입출력 단자인 것을 특징으로 하는 병원체의 진단 시스템.The diagnostic system for a pathogen according to claim 1, wherein the power input/output terminal is a power input/output terminal of a USB port.
  3. 제1항에 있어서, 상기 휴대용 원심 분리 장치는,According to claim 1, The portable centrifugal separation device,
    직립형 지지부의 상측에 설치되며, 모바일 기기로부터 공급된 전원에 의하여 구동되는 모터부;A motor unit installed on an upper side of the upright support unit and driven by power supplied from a mobile device;
    상기 직립형 지지부의 하측에 구비되어 상기 모바일 기기의 전원 입출력 단자에 전기적으로 연결 가능한 커넥터 단자;A connector terminal provided at a lower side of the upright support portion and electrically connectable to a power input/output terminal of the mobile device;
    상기 모터부와 기계적으로 연결되어 모터부의 회전 시 수평 방향으로 회전하도록 구성된 적어도 하나의 고리 부재; 및At least one ring member mechanically connected to the motor part and configured to rotate in a horizontal direction when the motor part rotates; And
    상기 적어도 하나의 고리 부재와 고리 결합 또는 인터로킹(interlocking)되는 걸림부 및 튜브 시험관을 삽입하도록 형성된 튜브 수용부를 구비하는 제1 튜브 홀더;A first tube holder having a locking portion interlocking or interlocking with the at least one ring member and a tube receiving portion formed to insert a tube test tube;
    를 포함하며,It includes,
    상기 모터부의 회전 시 제1 튜브 홀더에 삽입된 튜브 시험관이 원심력에 의하여 수직 위치에서 수평 위치로 전환되면서 회전하는 것을 특징으로 하는 병원체의 진단 시스템.When the motor portion is rotated, the tube test tube inserted into the first tube holder is rotated while being converted from a vertical position to a horizontal position by centrifugal force.
  4. 제3항에 있어서, 상기 제1 튜브 홀더는 걸립부로서 제1 캐비티가 구비된 제1 면, 및 튜브 수용부로서 제2 캐비티가 구비된 제2 면이 "L"자 형상을 이루도록 금속 재질의 단일 프레임이 벤딩되어 형성되거나, 또는 고분자 재료가 성형 또는 3D 프린팅에 의하여 형성된 것을 특징으로 하는 병원체의 진단 시스템.The method according to claim 3, wherein the first tube holder is made of a metal material such that a first surface provided with a first cavity as a hanging part and a second surface provided with a second cavity as a tube receiving part have an “L” shape. A diagnostic system for a pathogen, characterized in that a single frame is formed by bending, or a polymer material is formed by molding or 3D printing.
  5. 제3항에 있어서, 상기 제1 튜브 홀더의 재질은 열가소성 고분자로서, 폴리에스테르, 폴리올레핀, 폴리아미드, 폴리비닐알코올, 폴리우레탄, 폴리스티렌, 폴리염화비닐 또는 이의 조합인 것을 특징으로 하는 병원체의 진단 시스템.The diagnostic system for pathogens according to claim 3, wherein the material of the first tube holder is a thermoplastic polymer, and is polyester, polyolefin, polyamide, polyvinyl alcohol, polyurethane, polystyrene, polyvinyl chloride, or a combination thereof. .
  6. 제3항에 있어서, 상기 제1 튜브 홀더는,The method of claim 3, wherein the first tube holder,
    상측 부위에는 걸림부로서 제1 면에 형성된 제1 캐비티; 및The upper portion has a first cavity formed on a first surface as a locking portion; And
    하측 부위에는 제1 면의 면 방향과 수직의 면 방향을 갖는 바닥면으로서 제2 면에 형성된 제2 캐비티;The lower portion includes a second cavity formed on the second surface as a bottom surface having a surface direction perpendicular to the surface direction of the first surface;
    를 포함하고,Including,
    여기서, 제1 면과 같은 폭을 갖는 한 쌍의 제1 연결 면 각각이 제1 면의 하측 모서리와 소정 각도를 유지하며 연결되어 있고, 또한 제2 면으로부터 점차적으로 폭이 감소되는 단면을 갖도록 형성된 한 쌍의 제2 연결 면 각각의 상측 모서리가 제1 연결 면의 하측 모서리와 만나도록 구성된 것을 특징으로 하는 병원체의 진단 시스템.Here, each of the pair of first connecting surfaces having the same width as the first surface is connected to the lower edge of the first surface while maintaining a predetermined angle, and is formed to have a cross section that gradually decreases in width from the second surface. A diagnostic system for a pathogen, characterized in that the upper edge of each of the pair of second connection surfaces is configured to meet the lower edge of the first connection surface.
  7. 제3항에 있어서, 상기 제1 튜브 홀더는,The method of claim 3, wherein the first tube holder,
    바닥면; 및Bottom surface; And
    상기 바닥면으로부터 소정 각도로 각각 연장 형성되어 바닥면의 중앙 상측 공간에서 서로 접합하는 복수의 빔 부재;A plurality of beam members formed to extend at a predetermined angle from the bottom surface and joined to each other in a central upper space of the bottom surface;
    를 포함하며,It includes,
    여기서, 상기 빔 부재가 서로 만나는 접합부가 걸림부를 형성하며, 또한 바닥면의 하측 방향으로 튜브 수용부로서 캐비티가 형성되는 것을 특징으로 하는 병원체의 진단 시스템.Here, the diagnostic system of the pathogen, characterized in that the joint where the beam member meets each other forms a locking portion, and a cavity is formed as a tube receiving portion in a downward direction of the bottom surface.
  8. 제7항에 있어서, 상기 빔 부재 각각은 바닥면으로부터 연장되는 방향으로 점차적으로 감소된 폭을 갖는 것을 특징으로 하는 병원체의 진단 시스템.The diagnostic system of a pathogen according to claim 7, wherein each of the beam members has a gradually reduced width in a direction extending from the bottom surface.
  9. 제7항에 있어서, 상기 캐비티의 하단은 폐쇄되거나, 또는 개방되어 있는 것을 특징으로 하는 병원체의 진단 시스템.The diagnostic system of a pathogen according to claim 7, wherein the lower end of the cavity is closed or opened.
  10. 제2항에 있어서, 상기 튜브 시험관은,According to claim 2, The tube test tube,
    상단부에 개구가 형성되고 하단부는 폐쇄되어 있는 광 투과성 재질의 시험관 본체; 및A test tube body made of a light transmissive material having an opening formed at an upper portion and closed at an lower portion; And
    상기 시험관 본체의 개구에 체결되도록 구성되며, 상기 제1 튜브 홀더의 튜브 수용부 내로 튜브 시험관이 삽입된 후에는 모터부의 구동 시 가해지는 원심력 하에서 고정 상태에 있도록 둘레를 따라 돌출되어 있는 걸림 턱이 구비된 개폐용 뚜껑;It is configured to be fastened to the opening of the test tube body, and after the tube test tube is inserted into the tube accommodating part of the first tube holder, a locking jaw protruding along the circumference is provided to be in a fixed state under centrifugal force applied when driving the motor part. Closed lid;
    을 포함하는 것을 특징으로 하는 병원체의 진단 시스템.Diagnosis system of a pathogen comprising a.
  11. 제10항에 있어서, 상기 튜브 시험관의 수용 체적은 200 내지 2000 ㎕ 범위인 것을 특징으로 하는 병원체의 진단 시스템.The diagnostic system of a pathogen according to claim 10, wherein the volume of the test tube is 200 to 2000 μl.
  12. 제2항에 있어서, 상기 센싱부는, According to claim 2, The sensing unit,
    삽입 방식으로 모바일 기기와 체결되면서 상기 모바일 기기 내 조도 센서의 노출면을 수용하도록 하측에 연장된 관통 홈이 형성된 모바일 기기 홀더;A mobile device holder having a through groove extending at a lower side to accommodate an exposed surface of the illuminance sensor in the mobile device while being fastened with the mobile device by an insertion method;
    튜브 시험관 내에 분석 플랫폼이 수용되어 있는 테스트 시험관을 삽입하기 위한 제2 튜브 홀더; 및A second tube holder for inserting a test tube in which the analysis platform is housed in a tube test tube; And
    상기 삽입된 테스트 시험관으로 광을 조사하기 위한 광원이 장착되는 광원 홀더;A light source holder equipped with a light source for irradiating light to the inserted test tube;
    를 구비하는 센서 구조물을 포함하고, It includes a sensor structure having a,
    상기 센서 구조물 내에서 모바일 기기 홀더, 제2 튜브 홀더 및 광원 홀더는 광원으로부터 조사된 광이 테스트 시험관을 투과하여 조도 센서의 노출면으로 전달되도록 배열되는 것을 특징으로 하는 병원체의 진단 시스템.A mobile device holder, a second tube holder, and a light source holder in the sensor structure are arranged such that light irradiated from the light source passes through the test tube and is transmitted to the exposed surface of the illuminance sensor.
  13. 제12항에 있어서, 상기 센서 구조물은 일체형 구조물인 것을 특징으로 하는 병원체의 진단 시스템.The diagnostic system of a pathogen according to claim 12, wherein the sensor structure is an integral structure.
  14. 제13항에 있어서, 상기 센서 구조물은 3D 프린팅 방식으로 제작된 것을 특징으로 하는 병원체의 진단 시스템.The diagnostic system of a pathogen according to claim 13, wherein the sensor structure is manufactured by 3D printing.
  15. 제12항에 있어서, 상기 광원은 발광 다이오드(LED) 소자로서 이의 파장 대역은 400 내지 1100 nm 범위인 것을 특징으로 하는 병원체의 진단 시스템.The diagnostic system of a pathogen according to claim 12, wherein the light source is a light emitting diode (LED) element and its wavelength band is in the range of 400 to 1100 nm.
PCT/KR2019/018446 2019-01-31 2019-12-26 Pathogen diagnostic system integrated with mobile device WO2020159084A1 (en)

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