WO2018008898A1 - Kit de détection électrochimique d'une cellule isolée - Google Patents

Kit de détection électrochimique d'une cellule isolée Download PDF

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Publication number
WO2018008898A1
WO2018008898A1 PCT/KR2017/006941 KR2017006941W WO2018008898A1 WO 2018008898 A1 WO2018008898 A1 WO 2018008898A1 KR 2017006941 W KR2017006941 W KR 2017006941W WO 2018008898 A1 WO2018008898 A1 WO 2018008898A1
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WIPO (PCT)
Prior art keywords
single cell
electrode
detection kit
cell detection
active electrode
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PCT/KR2017/006941
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English (en)
Korean (ko)
Inventor
김병권
박준희
이지영
강미정
Original Assignee
숙명여자대학교산학협력단
전북대학교산학협력단
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Publication of WO2018008898A1 publication Critical patent/WO2018008898A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/42—Measuring deposition or liberation of materials from an electrolyte; Coulometry, i.e. measuring coulomb-equivalent of material in an electrolyte
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/42—Measuring deposition or liberation of materials from an electrolyte; Coulometry, i.e. measuring coulomb-equivalent of material in an electrolyte
    • G01N27/423—Coulometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48735—Investigating suspensions of cells, e.g. measuring microbe concentration

Definitions

  • the present invention relates to a kit for electrochemically detecting the concentration of live single cells in a solution using a single particle collision method on an ultramicroelectrode (UME) surface.
  • UAE ultramicroelectrode
  • Single-particle collisions have received considerable attention because they provide important information about the properties of single particles, such as size, concentration, diffusion coefficient, surface charge and lifetime.
  • micro or nano scale beads composed of non-conductive hard materials were used, and interesting properties of various hard particles have been observed using this unique analysis platform, a recent study. According to the results, it can be seen that the scope of research through the single particle collision based technology has been extended to the range of soft particles.
  • microorganisms including bacteria
  • these microorganisms are small in size and have the property of moving by swimming, swarming, gliding and twitching, making it difficult to observe or capture living cells.
  • rapid self-proliferation of living cells makes it more difficult to predict their exact concentration.
  • various approaches utilizing optical density, microscopy, and cell culture techniques enable the determination of bacterial cells and the like in samples. Each technique has its own advantages, but there are some limitations that prevent their various applications.
  • cell culture methods can take up to 5 days to measure bacterial concentrations, which can be time consuming and measuring optical density (ie, turbidity in samples caused by bacteria) with a spectrophotometer is quick and easy.
  • optical density ie, turbidity in samples caused by bacteria
  • spectrophotometer ie, turbidity in samples caused by bacteria
  • Counting on the microscope is a simple way to quantify bacteria, but this method cannot be used on small bacteria ( ⁇ 2 ⁇ m). Therefore, there is an urgent need for a fast and stable technique for detecting specific bacterial cells and measuring their concentration.
  • the present invention is to provide a kit for the electrochemical detection of the concentration of a single live cell using a single particle collision method on the surface of the ultra-fine electrode.
  • One aspect of the invention is a reactor comprising a counter electrode, a reference electrode and an active electrode; And a reaction solution comprising a redox species, wherein the single cell detection kit comprises a single cell measuring the concentration of a single cell through a change in the intensity of a current generated by impinging or adsorbing a single cell on the surface of the active electrode.
  • the single cell detection kit comprises a single cell measuring the concentration of a single cell through a change in the intensity of a current generated by impinging or adsorbing a single cell on the surface of the active electrode.
  • FIG. 1 is a diagram showing a single cell detection kit according to an embodiment of the present invention.
  • the single cell detection kit 10 may include a reactor 20, an active electrode 12, a reference electrode 14, a counter electrode 16, and a reaction solution 18.
  • the reactor 20 is a place where collision between the active electrode, which is an event utilized in the present invention, and a single particle in a sample occurs, and a space in which a single cell to be detected collides with the active electrode while being stored in the reaction solution in the reactor for a predetermined time.
  • the shape of the reactor may be a cylinder, a plate, a cube, a cube, a cube, a polygonal pillar, or a sphere, but is not limited thereto.
  • the reactor is a cylinder or a polygonal pillar.
  • the size of the reactor is not limited, but the length of one side is preferably 0.5 cm or more.
  • the active electrode 12 refers to an electrode that directly participates in an electrode reaction to cause a reaction.
  • the active electrode used in the present invention refers to an electrode in which a redox species in a reaction solution causes an oxidation reaction.
  • the ultrafine electrode may be made of metal and nonmetal conductive materials such as carbon fiber, indium tin oxide, fluorine-doped tin oxide, boron-doped diamond, gold, silver, platinum, copper, nickel, and the like.
  • the shape of the surface of the active electrode may be circular, elliptical, triangular, rectangular, pentagonal, polygonal, or atypical, but is not limited thereto.
  • the active electrode may be made of an ultra-fine electrode, the maximum diagonal length of the electrode surface is preferably 1 to 500 ⁇ m.
  • the size of the active electrode should be optimized to clearly produce a change in current caused by the impact of the microorganism, specifically, a stepped current.
  • the ratio of the maximum diagonal length of the surface of the active electrode to the maximum length of the analyte should be less than approximately 20. That is, when the active electrode and the analyte are circular, the ratio of the diameter of the active electrode to the diameter of the analyte should be less than approximately 20.
  • the diameter of the active electrode may be 10 to 20 ⁇ m, preferably 5 ⁇ m.
  • the active electrode mounted on the reactor may be one, or two or more different in the maximum diagonal length of the surface of the active electrode, two or more active electrodes different in the maximum diagonal length of the active electrode By attaching the presence or absence of microorganisms having different sizes can be detected at the same time.
  • the reference electrode 14 refers to an electrode which may be a reference because the monopole potential is constant during electric potential measurement.
  • the reference electrode may be made of silver (Ag), and may be Ag / AgCl (3M KCl).
  • the counter electrode 16 refers to an electrode that pairs with an active electrode or a reference electrode to cause an electrode reaction.
  • the counter electrode may be made of platinum (Pt), gold (Au), iridium oxide (IrO 2 ), or the like, and preferably has an area of at least 5 times larger than the area of the active electrode. It may be various types of electrodes.
  • the counter electrode and the reference electrode may be formed the same or different from the shape and size of the active electrode.
  • the active electrode, the counter electrode and / or the reference electrode is not limited in the position to be mounted in the reactor, but is preferably mounted to be spaced apart from the reactor wall rather than attached to the reactor wall.
  • the distance between the reference electrode and the active electrode is not limited, but is preferably disposed within 1 cm.
  • the three electrodes are contained in a continuous aqueous electrolyte solution and must be electrically connected. In order to prevent contamination of the electrodes, the three electrodes can be connected to the electrolyte through a separator.
  • the reaction solution 18 serves to provide materials such as various ions for performing the electrode reaction, and may include a culture medium and a redox species.
  • the culture medium is included for the survival and proliferation of the microorganism due to being stored for a predetermined time during the collision process with the active electrode in the reactor of the detection kit according to the present invention.
  • Culture medium in solution may be included in the appropriate configuration and concentration for each microorganism to be detected, according to the common knowledge of those skilled in the art.
  • the conditions inside the reactor of the present invention can determine conditions suitable for the survival and proliferation of the microorganism to be detected, such as temperature (eg, culture at 37 ° C. for bacteria) or reaction time.
  • the reaction solution is a redox species, ferrocyanide (ferrocyanide) ions, ferricyanide ions, ruthenium (ruthenium, Ru) ions, hydroquinone (hydronquinone), ascorbic acid ( ascorbic acid, dopamine, dorocamine, ferroceneemethanol, ferrocene, ferrocene, ferrocenedimethanol, ⁇ -methyl ferrocenemethanol, ferrocene carboxylic acid, ferrocene dicarboxylic acid ), Ferrocene aldehyde (ferrocene aldehyde) and the like may include one or more selected from the group consisting of.
  • the redox species when the redox species is at least one selected from the group consisting of ferrocyanide ions, ferricyanide ions, hexahedral ruthenium ions, hydroquinone, ascorbic acid and dopamine, redox in the reaction solution
  • concentration of the species may have a value of 1 to 400 mM, preferably 1 to 200 mM, more preferably 2 to 100 mM, and the redox species are ferrocene methanol, ferrocene, ferrocene dimethanol, ⁇ -methyl ferrocene methanol, ferrocene carboxy.
  • the concentration of redox species in the reaction solution is 100 ⁇ m to 5 mM, preferably 1 to 5 mM, more preferably 2 to 5 mM. It can have a value.
  • the concentration of the redox species may be appropriately selected according to the type and size of the target material to be detected in the active electrode. In general, individual analyte signals tend to increase in proportion to the concentration of redox species, and when the individual signals become larger, it becomes easier to distinguish the detection signal from the noise current of the instrument.
  • the concentration of the redox species is limited to the maximum concentration by the solubility of the redox species, and in the case of biomolecules, the analyte may be modified according to osmosis, etc., so the maximum allowable concentration is limited.
  • the redox species is ionic, the ionic strength increases as the concentration of the redox species increases, so it should be avoided at high ionic strength because aggregation of the analyte may occur and precipitation may occur.
  • stable salts specifically sodium chloride, potassium nitrate, potassium chloride, and the like may be added at a concentration of 10 mM or less in order to adjust the ionic strength to an appropriate level. Appropriate ionic strength shows stable quiescent current.
  • an appropriate concentration of redox species should be selected according to the type of analyte, and preferably, a value of 2 to 100 mM is appropriate.
  • the term "cell” is a structural basic unit constituting a living organism, and means a subject to be measured in the present invention, and includes, but is not limited to, microorganisms, blood cells, enzymes, antibodies, antigens, and the like. It is not.
  • the cells may consist of isolated cells or isolated tissue fragments of epigenetics.
  • the cell may be a mammalian cell, in particular a human cell such as a tumor cell such as blood cells such as lymphocytes, peripheral blood mononuclear cells and the like.
  • microorganism refers to unicellular or multicellular prokaryotic or eukaryotic organisms.
  • the microorganism may be a multicellular organism in unicellular form depending on the stage of development or reproduction.
  • the microorganism may be a single cell microorganism selected from the group consisting of isolated cells of bacteria, fungi, yeasts, seaweeds, protozoa, and epidermis, preferably bacteria, more preferably Straptococcus ( Streptococcus ) or bacteria of the genus Escherichia .
  • sample as used herein may be of any type as long as it can contain microorganisms.
  • the sample may be, but is not limited to, biological samples, food samples, water samples, such as wastewater, freshwater or seawater samples, soil samples, sludge samples, or air samples.
  • the sample may be used without being purified or concentrated before being introduced into the reactor according to the present invention. If the particles are too large depending on the sample, for example, the sample may include particles having a size of tens or hundreds of micrometers or more. If so, large particles can be removed by filtering the appropriate filter.
  • detection of microorganisms using the single particle collision method is performed according to two sequential strategies: 1) electrophoretic migration and 2) blocking of the electroactivation region.
  • the redox species the redox species
  • the redox species are oxidized at the surface of the active electrode, a positive electric field is generated near the surface of the active electrode due to the steady-state current flow. Is drawn to. The current level is maintained by radial diffusion until a collision event occurs.
  • the level of steady-state current is immediately reduced because the flow of redox species is blocked by the attached microorganism.
  • the single cell detection kit is formed on one side of the reactor and the inlet flow path 30, the sample is introduced; And a discharge passage 40 formed at the other lower portion of the reactor and discharged from the sample.
  • the single cell detection kit may further include a display unit 60 for displaying the change in the intensity of the current over time in the active electrode.
  • the single cell detection kit according to the present invention enables the detection of the presence of single cells, in particular microorganisms in a live state, and the identification of the concentration of single cells in solution, more quickly and simply compared to conventional methods.
  • the concentration of the microorganisms having a specific size in the solution may be selectively measured.
  • FIG. 1 diagrammatically shows one embodiment of a single cell detection kit according to the invention.
  • FIG. 2 shows a schematic diagram of the oxidation of redox species (ferrocyanide ions) on the surface of the ultrafine electrode, and the cessation of oxidation and reduction of the redox species due to microbial collision and a change in current.
  • redox species ferrocyanide ions
  • C-UME 3 shows a current-time (it) curve of a single E. coli collision at a carbon fiber-ultrafine electrode (C-UME).
  • concentration of E. coli is 53fM
  • concentration of potassium ferrocyanide is 20mM (A), 50mM (B), 100mM (C) and 200mM (D), respectively.
  • FIG. 5 shows fluorescence microscopy images (A) and SEM images (B) of E. coli on the ultrafine electrodes after collision.
  • FIG. 6 shows the relationship between current-time (it) curves of fluorescence microscopy images of E. coli collisions on carbon fiber-ultrafine electrodes (C-UME).
  • FIG. 7 shows E. coli (A) expressed in a cylindrical shape in the 3-D simulation domain, SEM image (E) of E. coli attached to a carbon fiber ultrafine electrode, and E. coli with the ultrafine electrode center.
  • the simulated relative magnitude ( ⁇ i / ilim) (C) of electrode current change after E. coli adhesion over distance is shown.
  • the red circle represents the surface of the ultrafine electrode
  • ⁇ r represents the distance from the center of the ultrafine electrode to the center of E. coli .
  • E. coli a Gram-negative bacterium having a rod shape of approximately 2 ⁇ m in length, was used.
  • C-UME was prepared by the following general procedure developed in our laboratory. Briefly, after washing with hexane, toluene, IPA, ethanol and water (ultra pure water), a carbon fiber having a diameter of 10 ⁇ was sealed in a borosilicate glass tubing (outer diameter 1.5 mm x inner diameter 0.75 mm). The electrode was then polished with an alumina powder water suspension to give a mirror finish. The surface area was checked by standard redox electrochemistry in ferrocenemethanol solution. Before all experiments, all electrodes were polished with alumina paste (0.05 ⁇ m) before use.
  • the ratio of the diameter of the active electrode to the diameter of the analyte should be less than approximately 20, a diameter of 10 ⁇ m was selected as the active electrode for detecting E. coli .
  • Potassium ferrocyanide used as a redox species, was continuously oxidized on the surface of the ultrafine electrode to observe the E. coli collision event. As a result, the step current reaction was confirmed (see FIG. 1).
  • the system of the present invention can be used to detect E. coli without removing the impurity LB medium.
  • the fluorescent E. coli cells attached to the surface of the microelectrode after removal of the electrophoretic force using a fluorescence microscope Observed.
  • the inventors have built check the adhesion of E. coli cells, and associates the second collision frequency and the actual number of E.coli cells attached to the microelectrode.
  • Ultrafine electrodes in order to visually identify the E. coli cells attached to the surface, which was used in which the expression of the enhanced green fluorescent protein (enhanced green fluorescent protein, EGFP) E.coli cells.
  • the electrode was gently washed with distilled water to remove the electrolyte salt.
  • the microscopic surface of the microelectrode was carefully observed through various microscopic techniques to identify the presence of E. coli cells remaining on the surface of the microelectrode after the cleaning step.
  • the detection method using the detection module of the present invention can be used to examine a single bacteria attached to the surface of the ultrafine electrode.
  • the magnitude of the step current reduction resulting from a single E. coli collision is predicted through 3D Comsol Multiphysics simulation (FIG. 6).
  • similar simulations with 2D axis symmetry were used to predict the change in current resulting from spherical particle collisions.
  • the 2D simulation can be used because of the symmetrical structure of the particles to be observed, but 3D simulation was performed because the E. coli cells used in this experiment were assumed to be cylindrical rather than symmetrical.
  • the orientation of the cylinder i.e., E. coli cells, should also be taken into account (see FIG.
  • the radius of the E. coli cells is 0.4 ⁇ m and the cylindrical length is 2 ⁇ m.
  • the height of the stepped current (ie, 83pA) observed experimentally matches well with the predicted signal height (75pA) based on the simulation results (see FIG. 6 (C)). It was.

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Abstract

La présente invention concerne un kit pour la détection électrochimique de la concentration de cellules isolées vivantes dans une solution de réaction par l'intermédiaire d'un procédé de collision de particules isolées sur une ultramicroélectrode (UME). Plus particulièrement, l'invention concerne un dispositif, qui est un kit de détection de cellules isolées, comprenant : un réacteur comprenant une électrode relative, une électrode de référence et une électrode active ; et une solution de réaction, qui comprend une espèce d'oxydo-réduction, et mesurant la concentration de cellules isolées par l'intermédiaire d'une variation d'intensité de courant qui se produit lorsque des cellules isolées dans le réacteur entrent en collision avec la surface de l'électrode active, ou sont absorbées sur celle-ci.
PCT/KR2017/006941 2016-07-04 2017-06-30 Kit de détection électrochimique d'une cellule isolée WO2018008898A1 (fr)

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KR10-2016-0083937 2016-07-04
KR1020160083937A KR101768382B1 (ko) 2016-07-04 2016-07-04 단일 세포의 전기화학적 검출 키트

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KR102024995B1 (ko) * 2017-12-20 2019-09-24 숙명여자대학교산학협력단 혈액 분석 장치 및 이를 이용한 혈액 분석 방법
KR102102174B1 (ko) * 2019-09-17 2020-04-21 숙명여자대학교산학협력단 혈액 분석 장치 및 이를 이용한 혈액 분석 방법
KR102327269B1 (ko) * 2019-12-03 2021-11-17 한국과학기술원 고분자 분자량 측정 장치 및 그 측정 방법
KR102314023B1 (ko) * 2019-12-30 2021-10-18 충북대학교 산학협력단 유기 용매 내 물방울 검출을 위한 조성물, 물방울 검출 장치 및 방법

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JP2000121590A (ja) * 1998-10-15 2000-04-28 Nippon Telegr & Teleph Corp <Ntt> 電気化学検出器
KR20090002778A (ko) * 2007-07-04 2009-01-09 (주)엔바이오닉스 미생물 검출 및 판별장치와 그 방법
KR20110129528A (ko) * 2010-05-26 2011-12-02 고려대학교 산학협력단 전기화학적 바이오센서 및 그 제조방법
JP2013511354A (ja) * 2009-11-20 2013-04-04 メドトロニック ミニメド インコーポレイテッド 医療装置システムに有用な多導体リードの構造並びにその製造方法及び使用方法
KR20150041146A (ko) * 2012-08-17 2015-04-15 오사카 유니버시티 시료의 분석 방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000121590A (ja) * 1998-10-15 2000-04-28 Nippon Telegr & Teleph Corp <Ntt> 電気化学検出器
KR20090002778A (ko) * 2007-07-04 2009-01-09 (주)엔바이오닉스 미생물 검출 및 판별장치와 그 방법
JP2013511354A (ja) * 2009-11-20 2013-04-04 メドトロニック ミニメド インコーポレイテッド 医療装置システムに有用な多導体リードの構造並びにその製造方法及び使用方法
KR20110129528A (ko) * 2010-05-26 2011-12-02 고려대학교 산학협력단 전기화학적 바이오센서 및 그 제조방법
KR20150041146A (ko) * 2012-08-17 2015-04-15 오사카 유니버시티 시료의 분석 방법

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