WO2018008898A1 - Kit for electrochemically detecting single cell - Google Patents

Kit for electrochemically detecting single cell Download PDF

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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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single cell
electrode
detection kit
cell detection
active electrode
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PCT/KR2017/006941
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French (fr)
Korean (ko)
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김병권
박준희
이지영
강미정
Original Assignee
숙명여자대학교산학협력단
전북대학교산학협력단
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Publication of WO2018008898A1 publication Critical patent/WO2018008898A1/en

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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

The present invention relates to a kit for electrochemically detecting the concentration of live single cells in a reaction solution through a single-particle collision method on an ultramicroelectrode (UME). Specifically, provided is the device, which is a single cell detection kit, comprising: a reactor including a relative electrode, a reference electrode, and an active electrode; and a reaction solution, which includes an oxidation-reduction species, and measuring the concentration of single cells through the intensity change of a current which occurs when the single cells in the reactor collide with or are absorbed onto the surface of the active electrode.

Description

단일 세포의 전기화학적 검출 키트Single Cell Electrochemical Detection Kit
본 발명은 초미세전극(ultramicroelectrode, UME) 표면에 대한 단일입자 충돌법을 이용하여 용액 중의 살아있는 단일 세포의 농도를 전기화학적으로 검출하기 위한 키트에 관한 것이다.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.
단일입자 충돌법(single-particle collision)은 단일입자의 특성, 예컨대, 크기, 농도, 확산계수, 표면 전하 및 수명에 대한 중요한 정보를 제공하기 때문에 상당한 주목을 받아왔다. 처음에는, 이 방법의 작용 원리를 설명하기 위하여, 비전도성 경질 물질로 구성된 마이크로 또는 나노 스케일의 비드를 사용하였고, 다양한 경질 입자의 흥미 있는 특성이 이 독특한 분석 플랫폼을 이용하여 관찰되어 왔으며, 최근 연구 결과에 따르면, 단일입자 충돌 기반 기술을 통한 연구 범위가 연질 입자의 범위까지 확장되었음을 확인할 수 있다.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. Initially, in order to explain the working principle of this method, 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.
환경은 핵산, 단백질과 같은 생체 분자부터 박테리아와 같은 생균에 이르기까지 작지만 중요한 다양한 연질 입자로 구성되어 있기 때문에, 이러한 단일입자 충돌에 기반한 연구 범위의 확장은 상당한 진보를 나타낸다. 이러한 소형 연질 입자는 많은 연구 분야에서 중요하나, 그들의 특성은 연구되기 어려운 실정이었다. 그러나, 최근 에멀젼 입자 충돌 기술의 도입으로 인하여, 바이러스, 단백질 및 DNA를 조사하는 생체 분자 검출에 관한 연구들이 보고되어 왔으며, 본 발명자들이 이러한 연질 물질로 구성된 개별 입자를 특성 짓는 것을 가능하게 하였다.Because the environment consists of a variety of small but important soft particles, ranging from biomolecules such as nucleic acids and proteins to live bacteria such as bacteria, the expansion of research based on such single particle collisions represents a significant advance. These small soft particles are important in many fields of study, but their properties have been difficult to study. However, with the recent introduction of emulsion particle collision techniques, studies have been reported on the detection of biomolecules that examine viruses, proteins and DNA, and have enabled the inventors to characterize individual particles composed of such soft materials.
이와 같이, 다양한 단일 생체 분자의 특징이 관찰되어 왔으나, 단일 미생물에 대한 연구는 지금까지 보고된 적이 없다. 박테리아와 같이 살아있는 물질은 시간에 따라 증식할 수 있고 궁극적으로 질병 등을 발생시킬 수 있기 때문에, 단일입자 충돌법에 기반한 분석을 통한 미생물의 신속한 실시간 검출은 살아있는 미생물의 특성에 대한 정보 또는 살아있는 미생물을 제어에 필요한 정보를 제공할 수 있다.As such, the characteristics of various single biomolecules have been observed, but studies of single microorganisms have not been reported to date. Since living materials such as bacteria can multiply over time and ultimately cause disease, rapid real-time detection of microorganisms through analysis based on single particle collisions can provide information on the characteristics of living microorganisms or living microorganisms. Information necessary for control can be provided.
박테리아를 비롯한 미생물의 검출 및 정량은 임상병리학, 식품과학 및 생물학 분야에서 그 필요성이 강조되고 있다. 그러나, 이러한 미생물은 그 크기가 작고 수영(swimming), 운집(swarming), 활공(gliding) 및 경련(twitching)에 의하여 이동하는 특성을 가지고 있어, 살아있는 세포를 관찰하거나 포획하기 어렵다. 또한, 살아있는 세포의 빠른 자가증식은 그들의 정확한 농도를 예상하는 것을 더욱 어렵게 만든다. 현재, 광학 밀도, 현미경 기술 및 세포 배양 기술을 활용하는 다양한 접근은 시료 중의 박테리아 세포 등을 측정할 수 있게 한다. 각 기술은 각각의 장점을 가지나, 그들의 다양한 적용을 방해하는 몇몇 제한사항이 존재한다. 예컨대, 세포 배양법은 박테리아 농도를 측정하기 위해 최대 5일까지 소요되므로, 시간을 많이 소비하게 되며, 분광광도계로 광학 밀도(즉, 박테리아에 의하여 유발된 시료 중의 혼탁도)를 측정하는 것은 신속하고 간단하나, 조사된 박테리아 및 배지에 대한 특정 반응 요소가 우선 계산되어야 하는 번거로움이 존재하며, 결과값의 정확도 및 민감도가 충분히 높지 않다. 현미경상에서의 계수(counting)는 박테리아를 정량 하는 간단한 방법이나, 이 방법은 (2㎛ 미만의) 작은 박테리아에서는 사용될 수 없다. 따라서, 특정 박테리아 세포를 검출하고 이들의 농도를 측정할 수 있는 신속하고 안정적인 기술이 시급히 요구되고 있다.The detection and quantification of microorganisms, including bacteria, has highlighted the need for clinical pathology, food science and biology. However, 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. In addition, rapid self-proliferation of living cells makes it more difficult to predict their exact concentration. Currently, 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. For example, 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. However, there is the hassle that certain reaction components for the bacteria and medium investigated have to be calculated first, and the accuracy and sensitivity of the results are not high enough. Counting on the microscope is a simple way to quantify bacteria, but this method cannot be used on small bacteria (<2 μm). Therefore, there is an urgent need for a fast and stable technique for detecting specific bacterial cells and measuring their concentration.
본 발명은 초미세전극 표면에 대한 단일입자 충돌법을 활용하여 살아있는 단일 세포의 농도를 전기화학적으로 검출하기 위한 키트를 제공하기 위한 것이다.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.
본 발명의 일 양상은 상대전극, 기준전극 및 활성전극을 포함하는 반응기; 및 산화환원종을 포함하는 반응 용액을 포함하는 단일 세포 검출 키트로서, 상기 반응기 내의 단일 세포가 상기 활성전극의 표면에 충돌 또는 흡착함으로써 발생하는 전류의 세기 변화를 통하여 단일 세포의 농도를 측정하는 단일 세포 검출 키트를 제공한다.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. Provide a cell detection kit.
이하, 본 발명의 단일 세포 검출 키트의 구체적인 실시예를 설명하기로 한다. 그러나 이는 예시적 실시예에 불과하며 본 발명은 이에 제한되지 않는다.Hereinafter, specific examples of the single cell detection kit of the present invention will be described. However, this is only an exemplary embodiment and the present invention is not limited thereto.
도 1은 본 발명의 일 실시예에 따른 단일 세포 검출 키트를 나타낸 도면이다. 1 is a diagram showing a single cell detection kit according to an embodiment of the present invention.
도 1을 참조하면, 단일 세포 검출 키트(10)는 반응기(20), 활성전극(12), 기준전극(14), 상대전극(16) 및 반응 용액(18)을 포함할 수 있다.Referring to FIG. 1, 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.
반응기(20)는 본 발명에서 활용하는 사건인 활성전극과 시료 중의 단일입자 간의 충돌이 발생하는 곳으로, 검출대상인 단일 세포가 일정 기간 동안 반응기 내의 반응 용액 중에 저장되면서 활성전극과 충돌을 일으키는 공간을 제공한다. 상기 반응기의 형상은 원통, 플레이트, 정육면체, 직육면체, 정다면체, 다각기둥, 구일 수 있으나, 이에 한정되는 것은 아니며, 원통, 다각기둥인 것이 바람직하다. 상기 반응기의 크기에는 제한이 없으나, 한 면의 길이가 0.5cm 이상인 것이 바람직하다.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. to provide. 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. Preferably, 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.
활성전극(12)은 전극 반응에 직접 참가하여 반응을 일으키는 전극을 의미하며, 본 발명에서 사용되는 활성전극은 반응 용액 중의 산화환원종이 산화 반응을 일으키는 전극을 의미한다. 본 발명의 일 실시예에 따르면, 상기 초미세전극은 탄소 섬유, 인듐 산화주석, 불소도핑 산화주석, 보론도핑 다이아몬드, 금, 은, 백금, 구리, 니켈 등 금속 및 비금속 전도성 물질로 구성될 수 있다. 본 발명의 일 실시예에 따르면, 상기 활성전극 표면의 형상은 원형, 타원형, 삼각형, 사각형, 오각형 등 다각형, 또는 비정형일 수 있으나, 이에 한정되는 것은 아니며, 원형인 것이 바람직하다. 이는 원형이 전극 중심으로부터 전극 가장자리까지의 최대거리가 일정한 것에 기인한다. 본 발명의 일 실시예에 따르면, 상기 활성전극은 초미세전극으로 이루어질 수 있으며, 전극 표면의 최대대각선 길이가 1 내지 500㎛인 것이 바람직하다. 본 발명의 일 실시예에 따르면, 활성전극의 크기는 미생물의 충돌에 따른 전류 변화, 구체적으로는 계단 전류를 명백히 생산하기 위하여 최적화되어야 한다. 바람직하게는, 활성전극의 표면의 최대대각선 길이 대 분석물의 최대길이의 비는 대략 20보다 작아야 한다. 즉, 활성전극 및 분석물이 원형인 경우, 활성전극의 직경 대 분석물의 직경의 비는 대략 20보다 작아야 한다. 예를 들어, 대략 500 ㎚ 이하의 수치를 갖는 Escherichia coli(E.coli)를 검출하기 위하여는, 활성전극의 직경은 10 내지 20㎛, 바람직하게는 5㎛일 수 있다.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. According to an embodiment of the present invention, 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. . According to an embodiment of the present invention, the shape of the surface of the active electrode may be circular, elliptical, triangular, rectangular, pentagonal, polygonal, or atypical, but is not limited thereto. This is due to the fact that the circular shape has a constant maximum distance from the electrode center to the electrode edge. According to one embodiment of the invention, the active electrode may be made of an ultra-fine electrode, the maximum diagonal length of the electrode surface is preferably 1 to 500㎛. According to one embodiment of the present invention, 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. Preferably, 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. For example, having a numerical value of about 500 nm or lessEsherichia coli(E.coli), The diameter of the active electrode may be 10 to 20 μm, preferably 5 μm.
본 발명의 일 실시예에 따르면, 반응기에 장착되는 활성전극은 1개, 또는 활성전극의 표면의 최대대각선 길이가 상이한 2개 이상일 수 있으며, 활성전극의 최대대각선 길이를 달리 하는 2개 이상의 활성전극을 장착함으로써 상이한 크기를 갖는 미생물의 존재 여부 또는 그의 농도를 동시에 검출할 수 있다.According to one embodiment of the present invention, 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.
기준전극(14)은 전위 측정 시 단극전위가 일정하여 기준이 될 수 있는 전극을 의미한다. 본 발명의 일 실시예에 따르면, 상기 기준전극은 은(Ag)으로 이루어질 수 있으며, Ag/AgCl(3M KCl)일 수 있다.The reference electrode 14 refers to an electrode which may be a reference because the monopole potential is constant during electric potential measurement. According to an embodiment of the present invention, the reference electrode may be made of silver (Ag), and may be Ag / AgCl (3M KCl).
상대전극(16)은 활성전극 또는 기준전극과 짝지어 전극반응을 일으키는 전극을 의미한다. 본 발명의 일 실시예에 따르면, 상기 상대전극은 백금(Pt), 금(Au), 산화이리듐(IrO2) 등으로 이루어질 수 있으며, 바람직하게는 활성전극의 면적보다 5배 이상 큰 면적을 가지는 다양한 형태의 전극일 수 있다.The counter electrode 16 refers to an electrode that pairs with an active electrode or a reference electrode to cause an electrode reaction. According to an embodiment of the present invention, 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.
본 발명의 일 실시예에 따르면, 상대전극 및 기준전극은 활성전극의 형상 및 크기와 동일하거나 다르게 형성될 수 있다.According to one embodiment of the present invention, the counter electrode and the reference electrode may be formed the same or different from the shape and size of the active electrode.
본 발명의 일 실시예에 따르면, 활성전극, 상대전극 및/또는 기준전극이 반응기 내에 장착되는 위치에는 제한이 없으나, 반응기 벽에 부착되는 것보다는 반응기 벽과 이격되어 장착된 것이 바람직하다. 또한, 기준전극과 활성전극 간의 거리에는 제한이 없으나, 1㎝ 이내에 배치되는 것이 바람직하다.According to one embodiment of the present invention, 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. In addition, the distance between the reference electrode and the active electrode is not limited, but is preferably disposed within 1 cm.
세 전극은 연속적인 전해질 수용액 속에 담겨있어 전기적으로 연결되어 있어야 하며, 각 전극이 오염되는 것을 방지하기 위하여 분리막을 통해 전해질로 연결되어 사용하는 것도 가능하다.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.
반응 용액(18)은 전극 반응을 수행하기 위한 각종 이온 등 물질을 제공하는 역할을 하며, 배양 배지 및 산화환원종을 포함할 수 있다. 상기 배양 배지는 검출대상인 미생물이 본 발명에 따른 검출 키트의 반응기에서 활성전극과의 충돌과정을 일으키는 동안 일정 시간 저장됨에 기인하여, 상기 미생물의 생존 및 증식을 위하여 포함된다. 용액 중의 배양 배지는 검출 대상인 미생물 각각에 대하여, 당 분야의 통상의 기술자의 상식에 따라 적절한 구성 및 농도로 포함될 수 있다. 나아가, 본 발명의 반응기 내부의 조건은 검출대상인 미생물의 생존 및 증식에 알맞은 조건, 예컨대 온도(예컨대, 박테리아의 경우 37℃에서 배양) 또는 반응 시간을 결정할 수 있다.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. Furthermore, 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.
본 발명의 일 실시예에 따르면, 상기 반응 용액은 산화환원종으로서 페로시안화(ferrocyanide) 이온, 페리시안화(ferricyanide) 이온, 육아민화 루테늄(ruthenium, Ru) 이온, 하이드로퀴논(hydronquinone), 아스코르브산(ascorbic acid), 도파민(dopamine), 페로센메탄올(ferrocenemethanol), 페로센(ferrocene), 페로센다이메탄올(ferrocenedimethanol), α-메틸페로센메탄올, 페로센카복시산(ferrocene carboxylic acid), 페로센다이카복시산(ferrocene dicarboxylic acid), 페로센알데하이드(ferrocene aldehyde) 등으로 구성되는 군으로부터 선택되는 하나 이상을 포함할 수 있다. 본 발명의 일 실시예에 따르면, 상기 산화환원종이 페로시안화 이온, 페리시안화 이온, 육아민화 루테늄 이온, 하이드로퀴논, 아스코르브산 및 도파민으로 구성되는 군으로부터 선택되는 하나 이상인 경우, 상기 반응 용액 중 산화환원종의 농도는 1 내지 400mM, 바람직하게는 1 내지 200mM, 보다 바람직하게는 2 내지 100mM의 값을 가질 수 있으며, 상기 산화환원종이 페로센메탄올, 페로센, 페로센다이메탄올, α-메틸페로센메탄올, 페로센카복시산, 페로센다이카복시산 및 페로센알데하이드로 구성되는 군으로부터 선택되는 하나 이상인 경우, 상기 반응 용액 중 산화환원종의 농도는 100㎛ 내지 5mM, 바람직하게는 1 내지 5mM, 보다 바람직하게는 2 내지 5mM의 값을 가질 수 있다. 상기 산화환원종의 농도는 활성전극에서 검출하고자 하는 대상 물질의 종류 및 크기에 따라 적절히 선택될 수 있다. 일반적으로, 개별 분석 물질 신호는 산화환원종의 농도에 비례하여 증가하는 경향을 가지며, 개별 신호가 커질 경우 기기의 잡음 전류로부터 검출 신호를 구분하기 수월해진다. 그러나, 산화환원종의 농도는 산화환원종의 용해도에 의하여 최대 농도에 제한을 받고, 바이오 분자의 경우 삼투현상 등에 따라 분석 물질이 변형될 수 있으므로, 허용 최대농도가 제한된다. 상기 산화환원종이 이온성일 경우, 산화환원종의 농도가 증가하면 이온 세기가 증가하게 되므로, 높은 이온세기에서는 분석 물질의 뭉침, 침전현상이 일어날 수 있으므로 지양해야 한다. 상기 산화환원종이 중성 전하를 가지는 경우, 이온 세기를 적정한 수준으로 조절하기 위하여 안정한 염, 구체적으로 염화나트륨, 질산칼륨, 염화칼륨 등을 10mM 이하의 농도로 첨가할 수 있다. 적절한 이온 세기는 안정적인 대기 전류를 보여준다. 위의 변수들을 고려하여 분석 물질의 종류에 따라 적절한 산화환원종의 농도를 선택하여야 하며, 바람직하게는 2 내지 100mM의 값이 적절하다.According to one embodiment of the present invention, 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, α-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. According to an embodiment of the present invention, 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 The 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, α-methyl ferrocene methanol, ferrocene carboxy. In the case of at least one selected from the group consisting of acid, ferrocenedicarboxylic acid and ferrocenealdehyde, the concentration of redox species in the reaction solution is 100 μm 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. However, 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. If 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. When the redox species have a neutral charge, 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. In consideration of the above variables, 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.
본 명세서에서 사용되는 용어 "세포"는 생명체를 구성하는 구조적인 기본단위로, 본 발명에서 농도 측정의 대상이 되는 대상을 의미하며, 미생물, 혈구, 효소, 항체, 항원 등을 포함하나 이에 한정되는 것은 아니다. 상기 세포는 단리된 세포 또는 후생동물의 단리된 조직 단편으로 이루어질 수 있다. 본 발명의 일 실시예에 따르면, 상기 세포는 포유동물 세포, 특히 종양 세포, 예컨대 림프구, 말초 혈액 단핵구 세포 등의 혈액 세포와 같은 인간 세포일 수 있다.As used herein, 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. According to one embodiment of the invention, 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.
본 명세서에서 사용되는 용어 "미생물"은 단세포 또는 다세포 원핵(prokaryotic) 또는 진핵(eukaryotic) 유기체를 지칭한다. 상기 미생물은 발달 또는 번식 단계에 따라 단세포 형태의 다세포 유기체일 수 있다. 바람직하게는, 상기 미생물은 박테리아, 곰팡이, 효모, 해조, 원생동물, 및 후생동물의 단리된 세포로 이루어진 군으로부터 선택되는 단세포 미생물일 수 있으며, 바람직하게는 박테리아, 보다 바람직하게는 스트랩토코커스(Streptococcus) 또는 대장균(Escherichia) 속의 박테리아이다.As used herein, the term “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. Preferably, 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 .
본 명세서에서 사용되는 용어 "시료"는 미생물을 함유할 수 있는 것이라면 어떠한 유형도 가능하다. 예를 들어, 상기 시료는 생물학적 시료, 식품 시료, 물 시료, 예컨대 폐수, 담수 또는 해수 시료, 토양 시료, 슬러지 시료 또는 공기 시료일 수 있으나, 이에 한정되는 것은 아니다. 또한, 상기 시료는 본 발명에 따른 반응기에 투입되기 전에 정제 또는 농축되지 않은 상태로 사용할 수 있으며, 시료에 따라 입자가 지나치게 큰 경우, 예를 들어 수 십 혹은 수 백 ㎛ 이상의 크기를 가지는 입자가 포함되어 있는 경우, 적절한 필터를 여과함으로써 큰 입자를 제거할 수 있다. The term "sample" as used herein may be of any type as long as it can contain microorganisms. For example, 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. In addition, 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.
본 발명에서, 단일입자 충돌법을 이용한 미생물의 검출은 1) 전기영동적 이동 및 2) 전기활성화 영역의 차단이라는 두 순차적 전략에 따라 수행된다. 용액 중의 산화환원종인 페로시안화 이온이 활성전극 표면에서 산화되는 경우, 정상 상태 전류 흐름에 의하여 활성전극 표면 근처의 양의 전기장이 발생하므로, 음으로 하전된 미생물 개체는 전기영동적 이동을 통하여 활성전극 표면으로 끌려간다. 전류 수준은 충돌 사건이 일어날 때까지 방사상 확산에 의하여 유지된다. 미생물 개체가 활성전극 표면에 충돌하여 부착되면, 산화환원종의 흐름이 부착된 미생물에 의하여 차단되기 때문에, 정상 상태 전류의 수준은 즉시 감소된다.In the present invention, 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. When 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. When a microbial entity strikes and attaches to the active electrode surface, the level of steady-state current is immediately reduced because the flow of redox species is blocked by the attached microorganism.
본 발명의 일 실시예에 따르면, 상기 단일 세포 검출 키트는 상기 반응기의 일측 상부에 형성되고 시료가 유입되는 유입유로(30); 및 상기 반응기의 타측 하부에 형성되고 시료가 배출되는 배출유로(40)를 추가로 포함할 수 있다.According to one embodiment of the invention, 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.
본 발명의 일 실시예에 따르면, 상기 단일 세포 검출 키트는 상기 활성전극에서의 시간의 흐름에 따른 전류의 세기 변화를 표시하는 표시부(60)를 추가로 포함할 수 있다.According to one embodiment of the invention, 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.
본 발명에 따른 단일 세포 검출 키트는 살아있는 상태의 단일 세포, 특히 미생물의 존재여부를 검출 및 용액 중 단일 세포의 농도를 동정하는 것을 기존 방법에 비하여 보다 신속하고 단순하게 수행할 수 있도록 한다.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.
또한, 활성전극의 크기에 따라 검출 가능한 미생물의 크기가 상이하므로, 용액 중의 특정 크기의 미생물의 농도를 선택적으로 측정할 수 있다.In addition, since the detectable microorganisms vary in size depending on the size of the active electrode, the concentration of the microorganisms having a specific size in the solution may be selectively measured.
도 1은 본 발명에 따른 단일 세포 검출 키트의 일 구현예를 도식적으로 나타낸다.1 diagrammatically shows one embodiment of a single cell detection kit according to the invention.
도 2는 초미세전극 표면에서의 산화환원종(페로시안화 이온)의 산화 모식도, 및 미생물 충돌에 의한 산화환원종의 산화 중단 및 그에 의한 전류의 변화 개요를 나타낸다.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.
도 3은 탄소섬유-초미세전극(C-UME)에서의 단일 E. coli 충돌의 전류-시간(i-t) 곡선을 나타낸다. 이때, E. coli의 농도는 53fM이며, 페로시안화 칼륨의 농도는 각각 20mM(A), 50mM(B), 100mM(C) 및 200mM(D)이다.3 shows a current-time (it) curve of a single E. coli collision at a carbon fiber-ultrafine electrode (C-UME). At this time, the concentration of E. coli is 53fM, the concentration of potassium ferrocyanide is 20mM (A), 50mM (B), 100mM (C) and 200mM (D), respectively.
도 4는 대조군 실험에서의 전류-시간(i-t) 곡선을 나타낸다.4 shows current-time (i-t) curves in control experiments.
도 5는 충돌 후의 초미세전극 상의 E. coli의 형광 현미경 이미지(A) 및 SEM 이미지(B)를 나타낸다.5 shows fluorescence microscopy images (A) and SEM images (B) of E. coli on the ultrafine electrodes after collision.
도 6은 탄소섬유-초미세전극(C-UME) 상에서의 E. coli 충돌의 전류-시간(i-t) 곡선과 형광 현미경 이미지의 관계를 나타낸다.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).
도 7은 3-D 시뮬레이션 도메인에서 원통형으로 표현된 E. coli(A), 탄소섬유-초미세전극에 부탁된 E. coli의 SEM 이미지(B), 및 E.coli와 초미세전극 중심과의 거리에 따른 E. coli 부착 후의 전극 전류 변화의 시뮬레이션된 상대적 크기(Δi/ilim)(C)를 나타낸다. 이때, 적색 원은 초미세전극 표면을, Δr은 초미세전극의 중심으로부터 E.coli의 중심까지의 거리를 나타낸다.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 (Δi / ilim) (C) of electrode current change after E. coli adhesion over distance is shown. In this case, the red circle represents the surface of the ultrafine electrode, and Δr represents the distance from the center of the ultrafine electrode to the center of E. coli .
도 8은 E. coli의 농도에 따른 충돌빈도의 함수를 나타낸다.8 shows the function of the collision frequency with the concentration of E. coli .
이하 하나 이상의 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 실시예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, one or more embodiments will be described in more detail. However, these examples are intended to illustrate one or more embodiments by way of example, but the scope of the present invention is not limited to these examples.
본 발명자들은 초미세전극 상에서의 살아있는 단일 박테리아 세포의 검출을 보고하였다. 살아있는 박테리아의 농도는 초미세전극 표면에서의 포획된 세포의 수(충돌빈도로 표현됨)에 기초하여 측정할 수 있고, 전기화학적 신호는 박테리아 크기의 추정에 사용될 수 있었다. 본 발명에서의 모델 박테리아 세포로서, 대략 2㎛ 길이의 막대 모양을 갖는 그람-음성 박테리아인, E. coli를 이용하였다.We have reported detection of live single bacterial cells on ultrafine electrodes. The concentration of live bacteria can be measured based on the number of captured cells (expressed as collision frequency) on the ultramicroelectrode surface, and electrochemical signals could be used to estimate bacterial size. As model bacterial cells in the present invention, E. coli , a Gram-negative bacterium having a rod shape of approximately 2 μm in length, was used.
제조예Production Example 1. 활성전극의 제조 1. Preparation of active electrode
C-UME를 본 연구실에서 발전시킨 하기의 일반적인 절차로 제조하였다. 간단히, 헥세인, 톨루엔, IPA, 에탄올 및 물(초순수)로 세척한 후, 직경 10㎛의 탄소 섬유를 붕규산염 유리 튜브(borosilicate glass tubing, 외경 1.5㎜×내경 0.75㎜)에 밀봉하였다. 그 후, 상기 전극을 경면(mirror finish)과 같이 되도록 알루미나 분말 수 현탁액으로 연마하였다. 표면 영역을 페로센메탄올 용액 중의 표준 산화환원 전기화학으로 체크하였다. 모든 실험 전에, 모든 전극은 사용하기 전에 알루미나 페이스트(0.05㎛)로 연마하였다.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 탆 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 μm) before use.
활성전극 직경 대 분석물 직경의 비는 대략 20보다 작아야 하므로, E. coli를 검출하기 위한 활성전극으로서 직경 10㎛의 것을 선택하였다.Since 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 μm was selected as the active electrode for detecting E. coli .
실시예Example 1. 초미세전극 표면에서의 단일 박테리아 세포의 검출 1. Detection of Single Bacteria Cells on the Surface of Ultrafine Electrodes
E. coli 충돌 사건을 관찰하기 위하여 산화환원종으로 사용된 페로시안화 칼륨을 초미세전극 표면에서 연속적으로 산화시킨 결과, 계단 전류 반응을 확인할 수 있었다(도 1 참조).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).
실시예Example 2. 용액 중의  2. in solution 산화환원종의Redox species 농도 선택 Select concentration
초미세전극 표면 차단에 기초한 살아있는 단일 미생물의 충돌 사건과 미생물 이외의 다른 물질의 충돌을 구별하기 위하여, 용액 조성 및 전극 반경 등의 실험 조건은 신중하게 선택하여야 한다. 이를 위하여, 단일입자 차단 실험을 고농도의 산화환원종의 존재 하(즉, 200mM 또는 400mM 페로시안화 칼륨)에 전형적으로 수행하여, 높은 전류 강도를 수득하였다. 그러나 이러한 고농도의 산화환원종은 살아있는 E. coli에 예상치 못한 피해를 야기할 수 있다.Experimental conditions such as solution composition and electrode radius should be carefully chosen to distinguish between single live microbial collisions based on ultra-microelectrode surface blocking and other microbial collisions. To this end, single particle blocking experiments were typically performed in the presence of high concentrations of redox species (ie, 200 mM or 400 mM potassium ferrocyanide) to obtain high current strengths. However, these high levels of redox species can cause unexpected damage to live E. coli .
따라서, 관찰 가능한 충돌 반응을 일으킬 수 있는, 상대적으로 낮은 페로시안화 이온 농도에 관하여 조사하였다. 4종의 상이한 농도의 산화환원종에서 단일 E. coli 세포의 충돌에 대하여 시험하였다. 모든 농도에서 단일 E. coli 세포 충돌 상에서의 계단 전류 반응을 관찰하였고, 전류의 높이 차는 산화환원종의 농도에 비례하였다(도 2 참조). 20mM 페로시안화 칼륨의 존재 하에서, E. coli 세포의 충돌은 수십 피코암페어의 변화를 야기했고, 이는 자릿수로 기계의 하한(몇 피코암페어)을 넘어선 것이었다. 이 농도(20mM)의 페로시안화는 삼투성 세포 사멸을 방지할 정도로 충분히 낮으나, 관찰 가능한 신호 강도를 야기할 정도로 충분히 높은 것이었다.Thus, the investigation was made with respect to relatively low ferrocyanide ion concentrations that could lead to observable collision reactions. Four different concentrations of redox species were tested for collisions of single E. coli cells. Stepped current responses on single E. coli cell collisions were observed at all concentrations, and the height difference of the currents was proportional to the concentration of redox species (see FIG. 2). In the presence of 20 mM potassium ferrocyanide, collisions of E. coli cells resulted in a change of tens of picoamps, which exceeded the lower limit of the machine (a few picoamps) by orders of magnitude. Perocyanation at this concentration (20 mM) was low enough to prevent osmotic cell death, but high enough to cause observable signal intensity.
실시예Example 3. 실험 결과에 대한 용액 중 불순물의 영향 확인 3. Check the effect of impurities in the solution on the test results
최적의 조건을 이용하여, 많은 불순물(즉, Luria-Bertani(LB) 배지)을 함유하는 실제 E. coli 시료를 시험하였다. 계단 전류 감소는 주로 전극 표면 상에의 분석물의 부착으로부터 유래하나, 불순물의 흡착은 또한 계단 전류 감소를 야기할 수 있다. 따라서, 분석물이 없는(E. coli가 없는) 용액을 이용하여 대조군 실험을 수행하였다(도 3 참조). 그 결과, E. coli 세포 배양에 사용된 LB 배지의 존재 하에서 500초가 지나도록 의미 있는 계단 신호를 관찰할 수 없었다. LB 배지는 340nm 미만의 다양한 작은 분자(예컨대, 효모 추출물)를 함유하지만, 이들 작은 입자들이 상기 실시예들의 조건 하에서 검출 가능한 계단 전류 감소를 야기하지 않음을 확인할 수 있었다.Using optimal conditions, real E. coli samples containing many impurities (ie, Luria-Bertani (LB) medium) were tested. Step current reduction results primarily from the attachment of the analyte on the electrode surface, but adsorption of impurities can also result in step current reduction. Therefore, control experiments were performed using a solution without analyte (without E. coli ) (see FIG. 3). As a result, meaningful step signals could not be observed over 500 seconds in the presence of LB medium used for E. coli cell culture. The LB medium contained various small molecules of less than 340 nm (eg yeast extract), but it was confirmed that these small particles did not cause a detectable step current reduction under the conditions of the above examples.
따라서, 본 발명의 시스템은 불순물인 LB 배지를 제거하지 않고 E. coli를 검출하는 데에 사용될 수 있는 것이다.Therefore, the system of the present invention can be used to detect E. coli without removing the impurity LB medium.
실시예Example 4. 형광현미경을 통한 단일 박테리아 세포의 검출 검증 4. Verification of single bacterial cell detection by fluorescence microscope
살아있는 E. coli 세포가 전기장이 소멸된 후에도 초미세전극 표면 상에 남아있을지 여부를 검출하기 위하여, 전기영동적 힘을 제거한 후에 초미세전극 표면 상에 부착된 형광 E. coli 세포를 형광현미경을 이용하여 관찰하였다. 이 과정을 통하여, 발명자들은 E. coli 세포의 부착을 확인하고, 충돌빈도를 초미세전극 상에 부착된 E.coli 세포의 실제 수와 연관지었다.In order to detect whether live E. coli cells remain on the surface of the microelectrode even after the electric field is extinguished, the fluorescent E. coli cells attached to the surface of the microelectrode after removal of the electrophoretic force using a fluorescence microscope Observed. Through this process, 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.
초미세전극 표면 상에 부착된 E. coli 세포를 시각적으로 확인하기 위하여, 강화된 녹색 형광 단백질(enhanced green fluorescent protein, EGFP)을 발현시킨 E.coli 세포를 사용하였다. 전기화학적 측정을 마친 후, 전극을 증류수로 부드럽게 세정하여 전해질 염을 제거하였다. 다양한 현미경 기술을 통해 초미세전극 표면을 조심히 관찰하여, 세정 단계 후에 초미세전극 표면에 남아있는 E. coli 세포의 존재를 동정하였다.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. After the electrochemical measurement, 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.
형광 현미경을 이용하여 초미세전극 표면 상의 녹색 표지 된 E. coli 세포를 명확하게 관찰할 수 있었으며(도 4의 (A) 참조), 상기 형광 현미경의 이용하여 동정된 부위와 동일한 부위에 대한 전자 주사 현미경(SEM) 이미지도 E. coli 세포의 흡착을 뒷받침하였다(도 4의 (B) 참조). 이들 현미경 이미지를 통하여, 전극 표면 상에 흡착된 입자가 살아있는 E. coli 세포라는 것 및 상기 세포가 초미세전극 표면으로부터 쉽게 분리될 수 없다는 것을 확인하였다.Using a fluorescence microscope, the green labeled E. coli cells on the surface of the ultrafine electrode could be clearly observed (see FIG. 4 (A)), and electron scanning of the same region as the region identified using the fluorescence microscope was performed. Microscopic (SEM) images also supported the adsorption of E. coli cells (see FIG. 4B). These microscopic images confirmed that the particles adsorbed on the electrode surface were living E. coli cells and that the cells could not be easily separated from the ultramicroelectrode surface.
따라서, 본 발명의 검출 모듈을 이용한 검출 방법은 초미세전극 표면에 부착된 단일 박테리아를 조사하기 위하여 사용될 수 있다.Therefore, 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.
전극 표면 상에의 E. coli 세포의 안정한 흡착은 세포를 안정화시킬 대안 방법을 추가적으로 발전시켜야 할 요구를 제거하였으며, 발명자들이 전기화학적 계단 전류 응답의 수와 전극에 충돌된 EGFP-발현된 E. coli의 실제수 사이의 관계를 편리하게 해명할 수 있도록 하였다. 53fM EGFP-발현 E. coli 세포의 존재 하에서, 하나의 충돌 신호를 90초 동안 검출하고(도 5의 (A) 참조), 전기화학적 충돌 신호를 수집한 후, 형광 현미경으로 상기 전극 표면을 관찰하였으며(도 5의 (B) 참조), 다중 충돌 결과를 보여주는 전극도 조사하였다. 실험 결과, 53fM EGFP-발현 E. coli의 존재 하에서, 500초에 걸쳐 7번의 충돌 응답을 관찰하였고(도 5의 (C) 참조), 7개의 형광 E. coli가 초미세전극 표면 상에 존재하는 것을 확인하였으며(도 5의 (D) 참조), 흡착된 E. coli 세포의 수는 전기화학적 충돌 응답의 수와 일치하였다. 따라서, 이러한 접근은 현재의 일시(transient)에 기초하여 초미세전극 상에 흡착된 E.coli의 수를 예측하는 데에 사용될 수 있다.Stable adsorption of E. coli cells on the electrode surface eliminated the need to further develop alternative methods to stabilize the cells, and the inventors found the number of electrochemical step current responses and the EGFP-expressed E. coli impinged on the electrode. The relationship between the actual numbers of can be conveniently explained. In the presence of 53fM EGFP-expressing E. coli cells, one collision signal was detected for 90 seconds (see FIG. 5 (A)), and after collecting the electrochemical collision signals, the electrode surface was observed by fluorescence microscopy. (See FIG. 5B), the electrodes showing multiple collision results were also investigated. As a result, we observed seven collision responses over 500 seconds in the presence of 53fM EGFP-expressing E. coli (see FIG. 5 (C)), and seven fluorescent E. coli were present on the surface of the ultrafine electrode. (See FIG. 5D), the number of adsorbed E. coli cells was consistent with the number of electrochemical collision responses. Thus, this approach can be used to predict the number of E. coli adsorbed on ultrafine electrodes based on current transients.
단일 E. coli 충돌로부터 야기된 계단 전류 감소의 크기는 3D Comsol Multiphysics simulation을 통하여 예측된다(도 6). 이전에는, 구형 입자 충돌로부터 야기되는 전류 변화를 예측하기 위하여 2D 축 대칭을 갖는 유사한 시뮬레이션을 사용하였다. 상기 과정에서, 2D 시뮬레이션은 관찰대상인 입자의 대칭적 구조 때문에 사용될 수 있으나, 본 실험에 사용된 E. coli 세포가 대칭이 아닌 원통형이라고 추정하였기 때문에 3D 시뮬레이션을 수행하였다. E. coli 세포의 배향에 따라, E.coli 충돌에 따른 상이한 전류 응답이 예상될 수 있다. 따라서, 원통, 즉, E.coli 세포의 배향 또한 고려되어야 한다(도 6의 (A) 참조): 1) 원통의 장축이 전극의 중심을 향함(파선), 2) 원통의 장축이 전극에 대한 직교선에 평행함(점선), 및 3) 원통의 장축이 전극의 중심에 수직함(실선). 상기 시뮬레이션 결과는 전류 단계의 상대적 크기(Δi/ilim, 이때, ilim은 초미세전극으로부터의 제한 전류를 나타냄)가, E. coli의 배향과 무관하게, 그 착지점(landing position)이 초미세전극의 가장자리에 가까울수록 증가하는 경향이 있음을 시사하였다(도 6의 (C) 참조). 이에 기초하여, 특정 크기의 E. coli 세포가 초미세전극과 충돌하는 경우의 최대 및 최소 계단 전류 높이는 예측할 수 있고, 본 실시예에서, E. coli 세포의 반경은 0.4μm이고 길이가 2μm인 원통형이라고 추정할 수 있었다. 실험적으로 관찰되는 계단 전류(즉, 83pA)의 높이(도 5의 (A) 참조)는 시뮬레이션 결과(도 6의 (C) 참조)에 기초하여 예측된 신호 높이(75pA)와 잘 들어맞음을 확인하였다.The magnitude of the step current reduction resulting from a single E. coli collision is predicted through 3D Comsol Multiphysics simulation (FIG. 6). Previously, similar simulations with 2D axis symmetry were used to predict the change in current resulting from spherical particle collisions. In the above process, 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. Depending on the orientation of E. coli cells, different current responses due to E. coli collisions can be expected. Therefore, the orientation of the cylinder, i.e., E. coli cells, should also be taken into account (see FIG. 6 (A)): 1) the long axis of the cylinder faces the center of the electrode (dashed line), 2) the long axis of the cylinder with respect to the electrode. Parallel to the orthogonal line (dotted line), and 3) the long axis of the cylinder is perpendicular to the center of the electrode (solid line). The simulation results show that the relative magnitude of the current stage (Δi / ilim, where ilim represents the limiting current from the ultrafine electrode), is independent of the orientation of E. coli , and its landing position is It was suggested that there is a tendency to increase closer to the edge (see Fig. 6 (C)). Based on this, the maximum and minimum step current heights when the specific size of E. coli cells collide with the ultrafine electrode can be predicted. In this embodiment, the radius of the E. coli cells is 0.4 μm and the cylindrical length is 2 μm. Could be estimated. The height of the stepped current (ie, 83pA) observed experimentally (see FIG. 5 (A)) matches well with the predicted signal height (75pA) based on the simulation results (see FIG. 6 (C)). It was.
실시예Example 5. 미생물의 충돌빈도와 용액 중 농도와의 상관관계 분석 5. Correlation analysis between collision frequency of microorganisms and concentration in solution
E. coli 세포의 실험적 및 이론적 충돌빈도는 세포의 농도와 관련되므로, 대칭적 초미세전극의 flux를 고려한 2D 시뮬레이션을 수행하여, 초미세전극에서 정상 상태 전류(즉, 평균 실험적 전류)를 이용하여 이주적 운동(migrational transport) 하에서의 이론적 충돌빈도 결과를 수득하였다. 본 시뮬레이션에 기초하여, 예상되는 빈도(주파수)는 (초미세전극 상에서 관찰된 E. coli의 총 flux에 기초하여 계산된) 약 0.47pM-1s-1이었다. 이는 실험적 (0.26 pM-1s-1) 및 예상된 빈도값과 같은 자리수의 수치로서, 이 결과는 E. coli의 복잡한 운동은 거의 전체 충돌빈도에 영향을 미치지 않음을 시사하였다. 다양한 E. coli 농도를 동일한 방식으로 측정하였으며(도 7 참조), 상기 결과는 10 내지 100fM의 농도 범위에서의 우수한 선형성을 보여주었다. 212fM의 E.coli 세포를 사용하여 수득한 결과는, 완전히 E. coli로 뒤덮인(감소된 전기활성 영역을 갖는) 초미세전극은 그의 표면에 대하여 감소된 이주적 E.coli flux를 나타내기 때문에, 포화된 빈도를 야기하였다. 따라서, 시료 중의 세포의 농도를 이론적 단일 입자 충돌빈도를 사용하여 예측할 수 있다. i-t 곡선에서 관찰된 충돌 응답의 수는 형광 현미경을 통하여 관찰되고 이론적 계산에 의해 예측된 E.coli의 수와 관계됨을 확인하였다. Since the experimental and theoretical collision frequency of E. coli cells is related to the concentration of the cells, a 2D simulation considering the flux of the symmetrical ultrafine electrodes is performed, using a steady state current (ie, average experimental current) at the ultrafine electrode. Theoretical frequency results were obtained under migration transport. Based on this simulation, the expected frequency (frequency) was about 0.47 pM-1 s-1 (calculated based on the total flux of E. coli observed on the ultrafine electrode). This is the same number of digits as experimental (0.26 pM-1s-1) and expected frequency values, suggesting that the complex motion of E. coli hardly affects the overall collision frequency. Various E. coli concentrations were measured in the same manner (see FIG. 7) and the results showed good linearity in the concentration range of 10-100 fM. The results obtained using 212 fM of E. coli cells indicate that the ultrafine electrode completely covered with E. coli (with reduced electroactive region) exhibits a reduced migratory E. coli flux to its surface. It caused a saturated frequency. Thus, the concentration of cells in a sample can be predicted using theoretical single particle collision frequency. The number of collision responses observed in the it curve was confirmed to be related to the number of E. coli observed through fluorescence microscopy and predicted by theoretical calculations.
단일 E. coli 충돌의 이론적 전류 응답은 Comsol Multiphysics simulations에 의하여 수득되었다. 이들 시뮬레이션 결과는 또한 실험적 결과와 잘 일치하였다.Theoretical current responses of single E. coli collisions were obtained by Comsol Multiphysics simulations. These simulation results were also in good agreement with the experimental results.
이제까지 본 발명에 대하여 그 바람직한 실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로, 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 상세한 설명보다는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown by the following claims rather than the foregoing description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention. Should be.
[부호의 설명][Description of the code]
10 : 단일 세포 검출 키트10: single cell detection kit
12 : 활성전극12: active electrode
14 : 기준전극14: reference electrode
16 : 상대전극16: counter electrode
18 : 반응 용액 18: reaction solution
20 : 반응기20: reactor
30 : 유입유로30: inflow channel
40 : 배출유로40: discharge flow path
50 : 교반기50: stirrer
60 : 표시부60 display unit

Claims (13)

  1. 상대전극, 기준전극 및 활성전극을 포함하는 반응기; 및A reactor including a counter electrode, a reference electrode, and an active electrode; And
    산화환원종을 포함하는 반응 용액을 포함하는 단일 세포 검출 키트로서,A single cell detection kit comprising a reaction solution comprising a redox species,
    상기 반응기 내의 단일 세포가 상기 활성전극의 표면에 충돌 또는 흡착함으로써 발생하는 전류의 세기 변화를 통하여 단일 세포의 농도를 측정하는 단일 세포 검출 키트.Single cell detection kit for measuring the concentration of a single cell through the change in the intensity of the current generated by the single cell in the reactor impinge or adsorb on the surface of the active electrode.
  2. 제1항에 있어서,The method of claim 1,
    상기 단일 세포는 미생물인 것인 단일 세포 검출 키트.Single cell detection kit is the single cell is a microorganism.
  3. 제2항에 있어서,The method of claim 2,
    상기 미생물은 박테리아인 것인 단일 세포 검출 키트.The microorganism is a single cell detection kit.
  4. 제1항에 있어서,The method of claim 1,
    상기 활성전극은 탄소 섬유, 인듐 산화주석, 불소도핑 산화주석, 보론도핑 다이아몬드, 금, 은, 백금, 구리 및 니켈로 구성되는 군으로부터 선택되는 어느 하나인 것인 단일 세포 검출 키트.The active electrode is any one selected from the group consisting of carbon fiber, indium tin oxide, fluorine-doped tin oxide, boron-doped diamond, gold, silver, platinum, copper and nickel.
  5. 제1항에 있어서,The method of claim 1,
    상기 활성전극은 전극 표면의 최대대각선 길이가 1 내지 500μm인 것인 단일 세포 검출 키트.The active electrode is a single cell detection kit of the maximum diagonal length of the electrode surface of 1 to 500μm.
  6. 제1항에 있어서,The method of claim 1,
    상기 활성전극은 상기 반응기 중에 1개, 또는 전극 표면의 최대대각선 길이가 상이한 2개 이상이 포함된 것인 단일 세포 검출 키트.Wherein the active electrode is one, or a single cell detection kit containing two or more different in the maximum diagonal length of the electrode surface.
  7. 제1항에 있어서,The method of claim 1,
    상기 활성전극은 원형인 것인 단일 세포 검출 키트.The active electrode is a circular single cell detection kit.
  8. 제1항에 있어서,The method of claim 1,
    상기 반응 용액은 산화환원종으로서 페로시안화(ferrocyanide) 이온, 페리시안화(ferricyanide) 이온, 육아민화 루테늄(Ru) 이온, 하이드로퀴논(hydronquinone), 아스코르브산(ascorbic acid) 및 도파민(dopamine)으로 구성되는 군으로부터 선택되는 하나 이상을 포함하는 것인 단일 세포 검출 키트.The reaction solution is a redox species that is composed of ferrocyanide ions, ferricyanide ions, ruthenium arsenide (Ru) ions, hydronquinone, ascorbic acid and dopamine. Single cell detection kit comprising one or more selected from the group.
  9. 제8항에 있어서,The method of claim 8,
    상기 반응 용액 중 산화환원종의 농도는 1 내지 400mM인 것인 단일 세포 검출 키트.Single cell detection kit is the concentration of the redox species in the reaction solution is 1 to 400mM.
  10. 제1항에 있어서,The method of claim 1,
    상기 반응 용액은 산화환원종으로서 페로센메탄올(ferrocenemethanol), 페로센(ferrocene), 페로센다이메탄올(ferrocenedimethanol), α-메틸페로센메탄올, 페로센카복시산(ferrocene carboxylic acid), 페로센다이카복시산(ferrocene dicarboxylic acid) 및 페로센알데하이드(ferrocene aldehyde)로 구성되는 군으로부터 선택되는 하나 이상을 포함하는 것인 단일 세포 검출 키트.The reaction solution is a redox species ferroceneemethanol, ferrocene (ferrocene), ferroceneimethanol (ferrocenedimethanol), α-methyl ferrocenemethanol, ferrocene carboxylic acid (ferrocene carboxylic acid), ferrocene dicarboxylic acid (ferrocene dicarboxylic acid) And one or more selected from the group consisting of ferrocene aldehyde.
  11. 제10항에 있어서,The method of claim 10,
    상기 반응 용액 중 산화환원종의 농도는 100μM 내지 5mM인 것인 단일 세포 검출 키트.The concentration of redox species in the reaction solution is a single cell detection kit of 100μM to 5mM.
  12. 제1항에 있어서,The method of claim 1,
    상기 반응기의 일측 상부에 형성되고 시료가 유입되는 유입유로; 및An inflow passage formed at one side of the reactor and into which a sample is introduced; And
    상기 반응기의 타측 하부에 형성되고 시료가 배출되는 배출유로를 추가로 포함하는 것인 단일 세포 검출 키트.Single cell detection kit that is formed in the other lower portion of the reactor and further comprises a discharge passage through which the sample is discharged.
  13. 제1항에 있어서,The method of claim 1,
    상기 활성전극에서의 시간의 흐름에 따른 전류의 세기 변화를 표시하는 표시부를 포함하는 단일세포 검출 키트.Single cell detection kit comprising a display unit for displaying the change in the intensity of the current over time in the active electrode.
PCT/KR2017/006941 2016-07-04 2017-06-30 Kit for electrochemically detecting single cell WO2018008898A1 (en)

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