WO2018054320A1 - 检测试剂盒及其制备方法、包含检测试剂盒的分析系统、及它们的用途 - Google Patents

检测试剂盒及其制备方法、包含检测试剂盒的分析系统、及它们的用途 Download PDF

Info

Publication number
WO2018054320A1
WO2018054320A1 PCT/CN2017/102648 CN2017102648W WO2018054320A1 WO 2018054320 A1 WO2018054320 A1 WO 2018054320A1 CN 2017102648 W CN2017102648 W CN 2017102648W WO 2018054320 A1 WO2018054320 A1 WO 2018054320A1
Authority
WO
WIPO (PCT)
Prior art keywords
capillary
electrode
conductive
composite
wire
Prior art date
Application number
PCT/CN2017/102648
Other languages
English (en)
French (fr)
Inventor
江德臣
潘荣容
陈洪渊
Original Assignee
南京大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京大学 filed Critical 南京大学
Publication of WO2018054320A1 publication Critical patent/WO2018054320A1/zh

Links

Images

Classifications

    • 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
    • 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
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements

Definitions

  • the invention belongs to the field of biochemical detection.
  • the present invention relates to a detection kit for detecting an analyte in a single cell, a method of preparing the same, an analysis system comprising the detection kit, and uses thereof.
  • kit-based analytical techniques for measuring small molecules and proteins have been greatly developed.
  • the components of the kit enable the conversion of substrates or small molecules of the target protein into products and by-products for direct detection.
  • the kits typically use highly specific, commercially available enzymes for detecting the activity of small molecules and proteins compared to the fluorescent assays described above, thereby avoiding the structural design of fluorescent probes for individual molecules. Therefore, kit-based assays can provide specific detection of substances in a single cell, thereby facilitating analysis of individual cells.
  • nano-sized devices/devices For the detection of the contents contained in a single cell, due to the size limitations of the cells themselves, it is often necessary to employ nano-sized devices/devices for detection purposes.
  • Orane GG et al, Cell, 2016, 166: 506-516 discloses a nanosyringe for extracting contents from a single living cell, however the contents obtained by the nanoinjector do not reflect the material within a single cell. Real-time status.
  • a chemical field effect transistor-based nanobiosensor is disclosed in US Patent Application No. US 20080044911 A1, wherein the nanobiosensor comprises a nanowire and a nanowire array comprising functionalized or non-functionalized nanowires.
  • One or more cellular components and the like are detected by, for example, thermally lysing the cells.
  • Chinese patent application CN 103616361A discloses a fluorescent glucose nanobiosensor for detecting intracellular cells.
  • a glucose concentration wherein the nanobiosensor comprises fluorescent oxygen nanoparticles and glucose oxidase coupled to the nanoparticles.
  • glucose oxidase in the nanobiosensor requires coupling of fluorescent oxygen nanoparticles, it is possible to affect the reactivity and activity of glucose oxidase in some environments.
  • WO 2016138116 A1 discloses a nanosensor for measuring pH in a single cell, wherein the nanosensor comprises a nanopipette structure operatively connected to a micromanipulator and Sensing device, and including working electrode and polymer coating), amplifier circuit and logic circuit, and the like.
  • Alimujiang F et al, Sensors and Actuators B, 2010, 150: 673-680 discloses an intracellular glucose biosensor based on ZnO nanosheets produced on a borosilicate glass capillary tube head for measuring human fat cells and frog eggs The concentration of glucose in the mother cell, wherein the biosensor comprises a separate aluminum working electrode covered with ZnO nanosheets (the electrode needs to be extremely sharp to be inserted into living cells) and a separate Ag/AgCl reference electrode (by chrome and Silver is deposited on the outer surface of the borosilicate glass capillary tube head and formed by forming an AgCl coating on the tube head.
  • Nascimento RA et al, Nano Lett., 2016, 16(2): 1194-200 discloses a glucose nanosensor for measuring glucose levels in a single cancer cell, wherein the nanosensor comprises nanopipetting a tube and a custom scanning ion conductivity microscopy (SICM) device, the nanopipette is a quartz nanotube and its inner surface is coated with polylysine, and an Ag/AgCl working electrode needs to be placed in the nanopipette, and Another separate Ag/AgCl electrode was immersed in the solution to be tested as a reference electrode.
  • SAM custom scanning ion conductivity microscopy
  • a Pt (platinum) layer can be directly deposited on a non-conductive capillary as a first electrode, and a conductive wire is placed in the capillary as a second electrode and a detection reagent is placed, so that the second electrode can be utilized.
  • the detection reagent is discharged to the nozzle of the non-conductive capillary when necessary, and the Pt ring electrode formed on the end face of one end of the capillary of the Pt layer is used to make the measuring device simpler and more convenient (no fluorescent labeling detection reagent is required) There is no need for a separate reference electrode or the like, and it is also possible to achieve an effect of accurately detecting the level of a target small molecule or the activity of a protein in a single cell.
  • the invention provides a test kit, characterized in that the test kit comprises:
  • the composite capillary comprises a non-conductive capillary having a cavity, a Pt layer covering the outside of the non-conductive capillary, and an insulating layer covering the outside of the Pt layer;
  • the composite capillary has an end face having one end of the formed Pt ring electrode.
  • the present invention provides an analysis system comprising the above detection kit, characterized in that the analysis system comprises: the detection kit; a Pt layer connected to a composite capillary in the detection kit a conductive line and a fuel gauge connected to the other side of the conductive line; and a power source connected to the fuel gauge and the second electrode of the composite capillary by two additional wire wires, respectively.
  • the present invention provides a method of preparing the above test kit, characterized in that the method comprises the following steps:
  • the detection kit further comprises instructions.
  • the invention provides the use of the above detection kit and assay system for preparing a test for electrochemical analysis of a single cell.
  • the invention relates to a method of performing single cell electrochemical analysis using the above described detection kit and analysis system.
  • a detection kit characterized in that the detection kit comprises:
  • the composite capillary comprises a non-conductive capillary having a cavity, a Pt layer covering the outside of the non-conductive capillary, and an insulating layer covering the outside of the Pt layer;
  • the composite capillary has an end face having one end of the formed Pt ring electrode.
  • non-conductive capillary is selected from the group consisting of a glass capillary, a plastic capillary, and/or a photoresist capillary.
  • the conductive wire is selected from the group consisting of a Pt wire, an Ag wire, an Au wire, a Cu wire, an Al wire, and/or Graphene wire.
  • the detection reagent is selected from the group consisting of glucose oxidase; sphingomyelin, alkaline phosphatase, and choline oxidase a mixture; cholesterol oxidase; or choline oxidase.
  • test kit of any of paragraphs [1] to [15], characterized in that the detection reagent is added in an amount of more than 0 femtoliter to less than or equal to 10 ⁇ l, preferably 1 ⁇ l, before use.
  • the detection reagent is added in an amount of more than 0 femtoliter to less than or equal to 10 ⁇ l, preferably 1 ⁇ l, before use.
  • the detection reagent is added in an amount of more than 0 femtoliter to less than or equal to 10 ⁇ l, preferably 1 ⁇ l, before use.
  • test kit of any of paragraphs [1] to [17] wherein a voltage is applied to the second electrode for 2-30 seconds, preferably 2-15 seconds, most preferably 2 seconds. .
  • An analysis system comprising the detection kit of any of paragraphs [1] to [18], wherein the analysis system comprises:
  • the detection kit ;
  • a conductive line connected to a Pt layer of the composite capillary in the detection kit and a fuel gauge connected to the other side of the conductive line;
  • Power is supplied to the fuel gauge and the second electrode of the composite capillary by two additional conductive lines, respectively.
  • the conductive wire is selected from the group consisting of a Pt wire, an Ag wire, an Au wire, a Cu wire, an Al wire, and/or a graphene wire.
  • the detection kit further comprises instructions.
  • the non-conductive capillary is selected from the group consisting of a glass capillary, a plastic capillary, and/or a photoresist Capillary.
  • the platinum layer has a thickness of 20 to 100 nm, preferably 70 nm.
  • the insulating layer is a wax layer or a cathodic electrophoretic paint layer.
  • the insulating layer has a thickness of 20 to 50 nm, preferably about 30 nm.
  • an outer diameter of the opening of the annular electrode end of the composite capillary It is from 290 nm to 315 nm, preferably 300 nm.
  • the length of the composite capillary is from 1 mm to 5 cm, preferably from 1 mm to 10 mm. Most preferably about 5 mm.
  • an inner diameter of the opening of the composite capillary that is away from the annular electrode end It is from 0.4 mm to 0.9 mm, preferably 0.5 mm.
  • the conductive wire is selected from the group consisting of a Pt wire, an Ag wire, an Au wire, and a Cu. Wire, Al wire and/or graphene wire.
  • the detection reagent is selected from the group consisting of glucose oxidase; sphingomyelin, alkaline phosphate a mixture of enzyme and choline oxidase; cholesterol oxidase; or choline oxidase.
  • test kit of any of paragraphs [1] to [18] and the analysis system of paragraph [19] or [20] for the preparation of a test for single cell electrochemical analysis.
  • the detection kit of the present invention and the analysis system including the detection kit are used in comparison with the detection device/technology disclosed in the prior art that utilizes a more complicated structure or requires surface functionalization of the detection reagent to identify the analyte.
  • Pt ring on the end face at one end The hollow composite capillary of the electrode detects the level of small molecules in a single cell and the activity of proteins and the like, thereby providing a simpler means of detecting intracellular molecules.
  • FIG. 1 is a schematic diagram of a composite capillary in an exemplary detection kit of the present invention and an analysis system including the composite capillary.
  • FIG. 2A-2F are respectively a flow chart for preparing an exemplary composite capillary of the present invention (Fig. 2A); a side view of a non-conductive capillary coated with a Pt layer and a scanning electron microscope image of a front view (Fig. 2B and Figure 2C, scanning rate of 100 mV/s); side view of a non-conductive capillary with a Pt layer further coated with a wax insulating layer and a scanning electron microscope image of the front view (Fig. 2D and Fig. 2E, scanning rate 100 mV, respectively) /s); and voltammetric test results of composite capillaries with Pt ring electrodes in PBS buffer (Fig. 2F).
  • Figures 3A-3F show detection reagents exposed to 10 mM PBS (pH 7.4) (curve a) or 10 mM PBS (pH 7.4) with 0.2 mM (curve b), 1 mM (curve c) or 5 mM (curve d) glucose, respectively.
  • the charge of the cartridge (Fig. 3A); the difference in charge collected in 0.2 mM (curve a), 1 mM (curve b) and 5 mM (curve c) glucose after subtraction of the illegal primal charge collected in PBS (Fig. 3B) Difference in charge of the test kit after exposure to 0.2 mM glucose and different voltages for 2 seconds (Fig.
  • Figure 5D shows a statistical analysis of cells in different states.
  • the term "detection reagent” has the meaning commonly understood in the art and encompasses any biochemical reagent known to those skilled in the art that can be used to detect a target analyte, such as, but not limited to, glucose oxidase (preferably used) Concentration is 0.1 mg/ml - 10 mg/ml, most preferably 0.2 mg/ml); sphingomyelin (preferably used at a concentration of 0.1 mM to 10 mM, most preferably 1 mM), alkaline phosphatase (preferably at a concentration of 1-20 U/mL) Most preferably 5 U/mL) and a mixture of choline oxidase (preferably at a concentration of 1-20 U/mL, most preferably 5 U/mL); cholesterol oxidase; choline oxidase and the like.
  • glucose oxidase preferably used
  • Concentration is 0.1 mg/ml - 10 mg/ml, most preferably 0.2 mg/
  • target analyte and “analyte” are used interchangeably and refer to various target substances to be detected in a cell, such as, but not limited to, small molecules in cells such as glucose, cholesterol, and the like. And proteins (such as various enzymes, such as sphingomyelinase, transaminase) and the like.
  • the term "about” means that the parameter modified by the term has a measurement error as generally understood in the art, such as, but not limited to, covering ⁇ 5% of the parameter, such as ⁇ 2%, ⁇ 1%. Any value.
  • the invention relates to a detection kit, characterized in that the detection kit comprises:
  • the composite capillary comprises a non-conductive capillary having a cavity, a Pt layer covering the outside of the non-conductive capillary, and an insulating layer covering the outside of the Pt layer;
  • a detection reagent wherein the detection reagent is added to the cavity of the non-conductive capillary before use, and is passed during the detection process Applying a voltage to the second electrode discharges the detection reagent onto an end surface of the composite capillary having one end of the formed Pt ring electrode.
  • the composite capillary of the present invention can achieve the objects of the present invention in any shape.
  • the end of the composite capillary having the Pt ring electrode is preferably in the form of a cone, which can further contribute to the ease of insertion of the composite capillary into the cell and further damage to the cells.
  • the composite capillary can have any size that is smaller than the size of the cells to be detected, and thus is suitable for insertion into a single cell without affecting cell viability.
  • an outer diameter of the opening of the composite capillary to be inserted into one end of the second electrode is 0.8 mm to 1.5 mm, preferably 1 mm; further preferably, the inner diameter of the opening is 0.4 mm to 0.9 mm, preferably 0.5 mm.
  • the outer diameter of the opening of the composite capillary having one end of the Pt ring electrode is 290 nm to 315 nm, preferably 300 nm; further preferably, the inner diameter of the opening is 120- 145 nm, preferably 130 nm.
  • the composite capillary has a length of from 1 mm to 5 cm, preferably from 1 mm to 10 mm, most preferably about 5 mm.
  • non-conductive material capillary can be used for the purposes of the present invention to prepare a composite capillary.
  • the non-conductive capillary of the present invention may exemplify a glass capillary (for example, a borate glass capillary), a plastic capillary (for example, a PDMS plastic capillary), and/or a photoresist capillary (for example, SU). -8 photoresist capillaries and the like, but materials suitable for the preparation of the non-conductive capillary of the present invention are not limited thereto.
  • the insulating layer can be prepared using any insulating material known in the art that is biologically compatible with the cells to be detected.
  • wax and cathodic electrocoating are preferably employed to obtain a wax layer (e.g., Apiezon wax layer) and a cathodic electrocoat layer.
  • the conductive wires of the present invention can be made of any material that is electrically conductive.
  • Exemplary conductive lines that can satisfy the requirements of the present invention include, but are not limited to, Pt wires, Ag wires, Au wires, Cu wires, Al wires, and/or graphene wires, and the like.
  • the volume of the detection reagent to be added to the cavity of the composite capillary of the present invention is not particular limitation.
  • the volume of the detection reagent added to the lumen of the composite capillary is greater than 0 femtoliter (fl) to less than or equal to 10 ⁇ l, preferably 1 ⁇ l.
  • accurate detection of analytes (small molecules and proteins) in a single cell can be achieved by applying a voltage to the end face of the ring-shaped electrode end of the composite capillary tube to discharge less than 10 femtoliters of the detection reagent.
  • the voltage applied to the second electrode is 1-10V simply to minimize the volume of test reagent entering the cell, or to analyze a smaller area or to produce as low a disturbance as possible to cell viability.
  • 1-5 V, most preferably 1 V, more preferably a voltage is applied to the second electrode for 2-30 seconds, preferably 2-15 seconds, and most preferably 2 seconds.
  • the present invention relates to an analysis system comprising the above detection kit, characterized in that the analysis system comprises: the detection kit; and a Pt layer connected to a composite capillary in the detection kit a conductive line and a fuel gauge coupled to the other side of the conductive line; and a power source coupled to the fuel gauge and the second electrode of the composite capillary by two additional conductive lines, respectively.
  • the fuel gauge can be any existing charge measuring device capable of measuring the amount of charge released by the analyte to be detected.
  • the invention relates to a method of preparing the above test kit, characterized in that the method comprises the following steps:
  • the detection kit further comprises instructions.
  • one end of the non-conductive capillary to be formed on the end surface of the annular electrode is drawn into a cone shape.
  • the current of the coater is controlled to be 5-30 mA, preferably 15 mA, to spray the platinum layer.
  • the time for spraying the platinum layer is 200 to 1000 seconds, preferably 700 seconds.
  • the platinum layer has a thickness of from 20 to 100 nm, preferably about 70 nm.
  • an insulating layer is applied to the outside of the platinum layer in a drop coating manner.
  • the insulating layer has a thickness of 20 to 50 nm, preferably about 30 nm.
  • the outer diameter of the opening of the annular electrode end of the composite capillary is 290 nm to 315 nm, preferably 300 nm; further preferably The inner diameter of the opening is 120-145 nm, preferably 130 nm. Further preferably, the outer diameter of the opening of the composite capillary away from the annular electrode end (ie, the end to be inserted into the second electrode) is 0.8 mm - 1.5 mm, preferably 1 mm; further preferably the inner diameter of the opening It is from 0.4 mm to 0.9 mm, preferably 0.5 mm.
  • the detection reagent is individually packaged in a volume of from greater than 0 femtoliter to less than or equal to 10 ⁇ l, preferably 1 ⁇ l, for ease of use only.
  • the invention relates to the use of the above detection kit and assay system for the preparation of a test for electrochemical analysis of a single cell.
  • the single cell electrochemical assay comprises detecting the level of small molecules and/or the reactivity or activity of the protein within a single cell.
  • the detection kit of the present invention can detect various small molecules in a single cell and using a suitable detection reagent.
  • protein
  • the small molecules within a single cell of the invention may be glucose, cholesterol, PO 4 3- ion, reactive oxygen species, and/or H 2 O 2 .
  • the protein within a single cell of the invention may be various enzymes within a single cell, such as, but not limited to, sphingomyelinase, transaminase, and the like.
  • a detection reagent when a conductive wire is inserted into a non-conductive capillary and a detection reagent is added to a cavity of a non-conductive capillary, when an appropriate voltage is applied to the conductive wire, The detection reagent is enabled to flow out of the composite capillary and react with the target molecule or protein in a single cell at the orifice.
  • the obtained by-product hydrogen peroxide was electrochemically oxidized on the Pt ring electrode to collect the generated charges.
  • the composite capillary is pretreated in a solution containing no target analyte to determine the amount of illicit charge, and the illicit charge is excluded from the collected charge, thereby quantifying the activity and concentration of the target analyte in the cell. .
  • the detection kit of the present invention and an analysis system comprising the same use a hollow composite capillary having a Pt ring electrode on an end surface of one end thereof to detect the level of small molecules in a single cell and the activity of a protein or the like, This provides a simpler and more accurate means of detecting intracellular analytes.
  • hollow composite capillaries 1-5 were prepared according to the following procedure:
  • a non-conductive capillary having a cavity having a size as described in Table 1 below wherein a borate glass capillary, a PDMS plastic capillary, a borate glass capillary, a SU-8 photoresist capillary, and a borate glass capillary as a non-conductive capillary to prepare a composite capillary 1-5), wherein one end of the non-conductive capillary to be formed on the end surface thereof to form a ring-shaped electrode is drawn into a cone;
  • O.D. indicates the outer diameter
  • I.D. indicates the inner diameter
  • the prepared composite capillary 1-5 was placed in 100 mM phosphate buffer (PBS, pH 7.4) having 5 mM ferrocyanide for voltammetry test as follows: before and after coating the insulating layer
  • the non-conductive capillary covered with the Pt layer was characterized by SEM (Hitachi S-4800 Instrument, Japan).
  • a 10 kV accelerating voltage was applied to the Au-coated sample, and the composite capillary 1-5 was exposed to 100 mM phosphate buffer (PBS, pH 7.4) having 5 mM ferrocyanide to characterize exposure to the capillary.
  • the area of the Pt layer at one end. Ag/AgCl electrodes and Pt electrodes were used as the reference electrode and the counter electrode, respectively.
  • the voltage cycle ranged from -0.1 volt to 0.6 volt and a scan rate of 100 mv/s was applied to collect current using an electrochemical workstation (CHI 630E, CH Instruments). The result is shown in Figure 2F.
  • a conductive wire is inserted into a cavity of the composite capillary 1-5 prepared in Embodiment 1 away from the end of the annular electrode end (this end is referred to as "away from the annular electrode end") (wherein the composite capillary 1 -5 into the Pt wire, Ag wire, Au wire, Cu wire and Pt wire) as the second electrode, and then with the following 5 kinds of detection in 10 ⁇ L of 10 mM PBS buffer (pH 7.4) placed in an ampoule
  • the reagent mixtures are assembled together to obtain a test kit 1'-5': (1) a mixture of 1 mM sphingomyelin, 5 U/ml alkaline phosphatase, and 5 U/ml choline oxidase (hereinafter referred to as "mixture 1"); (2) a mixture of 0.1 mM sphingomyelin, 1 U/ml alkaline phosphatase, and 1 U/ml choline oxidase (herein
  • the composite capillary 1-5 in the detection kits 1-5 and 1'-5' prepared in Example 2 was connected to a fuel gauge through a Cu wire connected to its Pt layer, and the other two Cu wires were used to The fuel gauge and the second electrode in the capillary are respectively connected to a power source, thereby respectively preparing a corresponding analysis system 1-5 and an analysis system 1'-5' (wherein the analysis system is used for detecting a single cell)
  • the detection reagent or detection reagent mixture placed in the ampoule in the test kit was previously added to the capillary 1-5 as described in Example 2, respectively.
  • the present inventors measured the glucose level in a single cell using the analysis system 1-5 (in which glucose oxidase was used as a detection reagent) prepared in Example 3, wherein glucose oxidase will be as shown in Reaction Scheme 1 below.
  • the ⁇ -D-glucose is oxidized to D-glucono-1,5-lactone and hydrogen peroxide (the amount of hydrogen peroxide is related to the amount of glucose in the cells).
  • a voltage of 1 V was applied to the second electrode inserted into the composite capillary 1-5 by a power source for 30 seconds, a voltage of 1 V for 2 seconds, a voltage of 2 V for 15 seconds, a voltage of 5 V for 10 seconds, and a voltage of 10 V for 2 seconds.
  • Glucose oxidase added to the cavity of the composite capillary 1-5 is discharged onto the end face of the composite capillary 1-5 having a ring-shaped Pt electrode.
  • Fig. 4E The results of the detection are shown in Fig. 4E, in which the glucose concentrations in the individual cells corresponding to the 29 samples tested are shown in Table 2 below (since one glucose molecule will release two electrons, while one cell is known)
  • the volume is 1 pL, whereby the amount of glucose measured from the fuel gauge can be used to calculate the amount of glucose and further calculate the glucose concentration in a single cell).
  • Sphingomyelinase is a hydrolase involved in sphingolipid metabolism.
  • Existing studies eg, Hannun YA et al, J. Biol. Chem., 2002, 277(29): 25847-25850
  • SMase Activation is an important pathway for ceramide production in response to cellular stress. Analysis of the activity of SMase in a single cell has not been achieved so far in the prior art.
  • the present inventors measured the activity of SMase in cells using the analysis system 1'-5' prepared in Example 3 (in which a mixture of sphingomyelin, alkaline phosphatase (ALP) and choline oxidase was used as a detection reagent). Among them, as shown in Reaction Scheme 1 below, sphingomyelin is sequentially subjected to the action of SMase, ALP, and choline oxidase to finally produce hydrogen peroxide (the amount of hydrogen peroxide is related to the activity of SMase in the cells).
  • a voltage of 1 V is applied to the second electrode inserted into the composite capillary 1-5 by a power source for 30 seconds, a voltage of 10 V for 2 seconds, a voltage of 1 V for 5 seconds, a voltage of 5 V for 15 seconds, and a voltage of 8 V for 20 seconds.
  • the detection reagent mixture 1-5 added to the cavity of the composite capillary 1-5 is discharged onto the end face of the composite capillary 1-5 having a ring-shaped Pt electrode.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Electrochemistry (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

提供了一种检测试剂盒及其制备方法、包含检测试剂盒的分析系统、及它们的用途。该检测试剂盒包含：含有非导电性毛细管、Pt层和绝缘层的复合毛细管，第二电极以及检测试剂。该分析系统包含通过导电线相互连接的检测试剂盒、电量计和电源。该分析系统构造更加简单，同时实现了对单个细胞内的分析物（包括小分子和蛋白质）的精确测量。

Description

检测试剂盒及其制备方法、包含检测试剂盒的分析系统、及它们的用途 技术领域
本发明属于生化检测领域。具体而言，本发明涉及一种用于检测单个细胞内的分析物的检测试剂盒及其制备方法、包含检测试剂盒的分析系统、及它们的用途。
背景技术
目前的研究(例如，Schubert C，Nature，2011，480(7375):133-137)表明，分析单个细胞内的小分子和蛋白质等对于理解与细胞异质性和疾病状态相关的通路来说是很关键的。为了实现这一目的，现有技术中开发出了大量的荧光探针，该荧光探针能够在结合至靶分子时发出特定波长的荧光，这促进了我们对细胞内信号传导的了解。然而，现有的荧光检测技术在细胞毒性和结构化需求方面存在缺陷。对于荧光标记的蛋白质来说，关于该蛋白改变的化学结构对细胞过程的影响的问题使得本领域技术人员开始开发用于细胞分析的新的分析技术。由于蛋白的局部环境能够显著改变其活性，有必要研究蛋白与其生理伴侣的相互作用以了解该蛋白在细胞通路中的作用。毛细管微米/纳米制造技术的出现使得能够研究靶标物质的化学反应性。例如，Lee CL等，Nat.Biotechnol.，1999，17(8):759-762和Meredith GD等，Nat.Biotechnol.，2000，18(3):309-312等中公开了利用毛细管电泳分析胞内激酶活性的方法。开发出更小且更精细的装置来表征细胞化合物的特性(例如浓度、活性、反应性等)将有助于我们更好地理解细胞异质性和信号传导等。
在生物领域，用于测量小分子和蛋白质等的基于试剂盒的分析技术已得到了长足的发展。试剂盒的组分使得能够将靶蛋白的底物或小分子转化为产物和副产物，以直接进行检测。并且，与上述的荧光检测相比，试剂盒通常使用高度特异性的可商购的酶用于对小分子和蛋白质的活性进行检测，从而避免了用于单个分子的荧光探针的结构设计。因此，基于试剂盒的分析能够对单个细胞中的物质提供特异性检测，从而促进了单个细胞的分析。
对于单个细胞包含的内容物的检测而言，由于细胞本身的尺寸限制，往往需要采用纳米尺寸的设备/装置以实现检测目的。例如，Orane GG等，Cell，2016，166:506-516公开了一种纳米注射器用于提取单个活细胞中的内容物，然而通过该纳米注射器得到的内容物并不能反映出单个细胞内的物质的实时状态。类似地，美国专利申请US 20080044911A1中公开了一种基于化学场效晶体管的纳米生物传感器，其中，该纳米生物传感器包含纳米导线以及含有官能化和或非官能化的纳米导线的纳米导线阵列，可通过例如热裂解细胞来检测一种或多种细胞组分等。
对于其它类型的用于检测细胞内的物质的纳米设备/装置，本领域技术人员也进行了诸多尝试，例如，中国专利申请CN 103616361A中公开了一种荧光葡萄糖纳米生物传感器用于检测细胞内的葡萄糖浓度，其中，该纳米生物传感器包括荧光氧气纳米粒子及偶联至该纳米粒子的葡萄糖氧化酶。正如上文所述，由于该纳米生物传感器中的葡萄糖氧化酶需要偶联荧光氧气纳米粒子，使得有可能在一些环境中影响葡萄糖氧化酶的反应性和活性。国际专利申请WO 2016138116A1公开了一种用于测量单个细胞内的pH的纳米传感器，其中，该纳米传感器包含纳米移液管结构(该纳米移液管结构可操作地连接至显微操作器和传感装置，并且其中包含工作电极和聚合物涂层)、放大器电路以及逻辑电路等。Alimujiang F等，Sensors and Actuators B，2010，150:673-680公开了一种基于硼硅酸玻璃毛细管管头上生成的ZnO纳米片的胞内葡萄糖生物传感器，用于测量人脂肪细胞和蛙卵母细胞中的葡萄糖浓度，其中，该生物传感器包含单独的覆盖有ZnO纳米片的铝工作电极(该电极需要极其锋利以插入活细胞中)和单独的Ag/AgCl参比电极(通过将铬和银先后沉积在硼硅酸玻璃毛细管管头的外表面，并在管头形成AgCl涂层而制得)。类似地，Nascimento RA等，Nano Lett.，2016，16(2):1194-200公开了一种用于测量单个癌细胞中的葡萄糖水平的葡萄糖纳米传感器，其中，所述纳米传感器包含纳米移液管以及定制的扫描离子电导显微镜(SICM)装置，所述纳米移液管为石英纳米管并且其内表面涂覆聚赖氨酸，且需要向纳米移液管中放置Ag/AgCl工作电极，并将另一单独的Ag/AgCl电极浸入待测试溶液中作为参比电极。
如何使得用于检测单个细胞内的物质(包括小分子、蛋白质等)的设备本身结构更加简单且同时满足精确检测的效果，成为本领域技术人员关注的热点。
发明内容
本发明人通过研究发现，通过直接在非导电性毛细管上喷镀Pt(铂)层作为第一电极，同时在该毛细管内放置导电线作为第二电极并放置检测试剂，从而能够利用第二电极使检测试剂在需要时被排出到非导电性毛细管的管口处，并利用Pt层在毛细管的一端的端面上形成的Pt环状电极来使测量装置更加简单便捷化(无需荧光标记检测试剂且无需独立的参比电极等)，同时还能够实现精确地检测单个细胞内的靶标小分子的水平或蛋白质的活性的效果。
在一个方面，本发明提供了一种检测试剂盒，其特征在于，所述检测试剂盒包含：
中空的复合毛细管，其中，所述复合毛细管包含具有空腔的非导电性毛细管、覆盖在所述非导电性毛细管外侧的Pt层以及覆盖在所述Pt层外侧的绝缘层；
插入所述非导电性毛细管的空腔中的导电线，作为第二电极；其中，所述复合毛细管的远离所插入的第二电极的一端的端面未用所述绝缘层包覆，从而在所述端面上形成Pt环状电极；以及
检测试剂，其中，在临用前将所述检测试剂加入到所述非导电性毛细管的空腔中，并在检测过程中通过向所述第二电极施加电压将所述检测试剂排出至所述复合毛细管的具有所形成的Pt环状电极的一端的端面上。
在另一方面，本发明提供了一种包含上述检测试剂盒的分析系统，其特征在于，所述分析系统包含：所述检测试剂盒；连接至所述检测试剂盒中的复合毛细管的Pt层的导电线和与所述导电线的另一侧连接的电量计；以及通过两条另外的导线电线分别连接至电量计和所述复合毛细管的第二电极的电源。
在另一方面，本发明提供了一种制备上述检测试剂盒的方法，其特征在于，所述方法包括如下步骤：
(1)提供包含空腔的非导电性毛细管；
(2)向所述非导电性毛细管的外侧喷镀铂层，从而制备得到半成品毛细管；以及
(3)向所述铂层的外侧涂覆绝缘层，其中，保留在所述半成品毛细管的一端的端面上的铂层免于用所述绝缘层进行涂覆，由此在所述端面上形成Pt环状电极，将该端称为环状电极端，从而得到复合毛细管；
(4)向所述复合毛细管的远离所述环状电极端的一端的空腔中插入导电线作为第二电极，其中，将所述复合毛细管的远离所述环状电极端的一端称为远离环状电极端；
(5)将所述步骤(4)中插入导电线的复合毛细管与检测试剂分别单独包装，并组装在一起；
其中，任选所述检测试剂盒进一步包含说明书。
在又一方面，本发明提供了上述检测试剂盒和分析系统在制备用于单个细胞电化学分析的检测物中的用途。或者，本发明涉及使用上述检测试剂盒和分析系统来进行单个细胞电化学分析的方法。
本发明的示例性的技术方案通过如下编号的段落的记载进行描述：
[1].一种检测试剂盒，其特征在于，所述检测试剂盒包含：
中空的复合毛细管，其中，所述复合毛细管包含具有空腔的非导电性毛细管、覆盖在所述非导电性毛细管外侧的Pt层以及覆盖在所述Pt层外侧的绝缘层；
插入所述非导电性毛细管的空腔中的导电线，作为第二电极；其中，所述复合毛细管的远离所插入的第二电极的一端的端面未用所述绝缘层包覆，从而在所述端面上形成Pt环状电极；以及
检测试剂，其中，在临用前将所述检测试剂加入到所述非导电性毛细管的空腔中，并在检测过程中通过向所述第二电极施加电压将所述检测试剂排出至所述复合毛细管的具有所形成的Pt环状电极的一端的端面上。
[2].如段落[1]所述的检测试剂盒，其特征在于，所述复合毛细管的具有所述Pt环状电极的一端处于圆锥体形式。
[3].如段落[1]或[2]所述的检测试剂盒，其特征在于，所述复合毛细管的待插入所述第二电极的一端的开口的外径为0.8mm-1.5mm、优选1mm。
[4].如段落[1]-[3]中任一段所述的检测试剂盒，其特征在于，所述复合毛细管的待插入所述第二电极的一端的开口的内径为0.4mm-0.9mm、优选0.5mm。
[5].如段落[1]-[4]中任一段所述的检测试剂盒，其特征在于，所述复合毛细管的具有所述Pt环状电极的一端的开口的外径为290nm-315nm、优选300nm。
[6].如段落[1]-[5]中任一段所述的检测试剂盒，其特征在于，所述复合毛细管的具有所述Pt环状电极的一端的开口的内径为120-145nm、优选130nm。
[7].如段落[1]-[6]中任一段所述的检测试剂盒，其特征在于，所述复合毛细管的长度为1mm-5cm、优选1mm-10mm、最优选约5mm。
[8].如段落[1]-[7]中任一段所述的检测试剂盒，其特征在于，所述非导电性毛细管选自玻璃毛细管、塑料毛细管和/或光刻胶毛细管。
[9].如段落[8]所述的检测试剂盒，其特征在于，所述非导电性毛细管为硼酸盐玻璃毛细管。
[10].如段落[8]所述的检测试剂盒，其特征在于，所述非导电性毛细管为PDMS塑料毛细管。
[11].如段落[8]所述的检测试剂盒，其特征在于，所述非导电性毛细管为SU-8光刻胶毛细管。
[12].如段落[1]-[11]中任一段所述的检测试剂盒，其特征在于，所述绝缘层为蜡层或阴极电泳漆层。
[13].如段落[12]所述的检测试剂盒，其特征在于，所述蜡层为Apiezon蜡层。
[14].如段落[1]-[13]中任一段所述的检测试剂盒，其特征在于，所述导电线选自Pt导线、Ag导线、Au导线、Cu导线、Al导线和/或石墨烯导线。
[15].如段落[1]-[14]中任一段所述的检测试剂盒，其特征在于，所述检测试剂选自葡萄糖氧化酶；鞘磷脂、碱性磷酸酶和胆碱氧化酶的混合物；胆固醇氧化酶；或者胆碱氧化酶。
[16].如段落[1]-[15]中任一段所述的检测试剂盒，其特征在于，在临用前将大于0毫微微升至小于等于10μl、优选1μl的所述检测试剂加入到所述非导电性毛细管的空腔中。
[17].如段落[1]-[16]中任一段所述的检测试剂盒，其特征在于，向所述第二电极施加的电压为1-10V、优选1-5V、最优选1V。
[18].如段落[1]-[17]中任一段所述的检测试剂盒，其特征在于，向所述第二电极施加电压2-30秒、优选2-15秒、最优选2秒。
[19].一种包含段落[1]-[18]中任一段所述的检测试剂盒的分析系统，其特征在于，所述分析系统包含：
所述检测试剂盒；
连接至所述检测试剂盒中的复合毛细管的Pt层的导电线和与所述导电线的另一侧连接的电量计；以及
通过两条另外的导电线分别连接至电量计和所述复合毛细管的第二电极的电源。
[20].如段落[19]所述的分析系统，其特征在于，所述导电线选自Pt导线、Ag导线、Au导线、Cu导线、Al导线和/或石墨烯导线。
[21].一种制备段落[1]-[18]中任一段所述的检测试剂盒的方法，其中，所述方法包括如下步骤：
(1)提供包含空腔的非导电性毛细管；
(2)向所述非导电性毛细管的外侧喷镀铂层，从而制备得到半成品毛细管；以及
(3)向所述铂层的外侧涂覆绝缘层，其中，保留在所述半成品毛细管的一端的端面上的铂层免于用所述绝缘层进行涂覆，由此在所述端面上形成Pt环状电极，将该端称为环状电极端，从而得到复合毛细管；
(4)向所述复合毛细管的远离所述环状电极端的一端的空腔中插入Pt导线作为第二电极，其中，将所述复合毛细管的远离所述环状电极端的一端称为远离环状电极端；
(5)将所述步骤(4)中插入导电线的复合毛细管与检测试剂分别单独包装，并组装在一起；
其中，任选所述检测试剂盒进一步包含说明书。
[22].如段落[21]所述的方法，其特征在于，在所述步骤(1)中，将所述非导电性毛细管的待在其端面上形成环状电极的一端拉制成圆锥体形状。
[23].如段落[21]或[22]所述的方法，其特征在于，在所述步骤(1)中，所述非导电性毛细管选自玻璃毛细管、塑料毛细管和/或光刻胶毛细管。
[24].如段落[23]所述的方法，其特征在于，在所述步骤(1)中，所述非导电性毛细管为硼酸盐玻璃毛细管。
[25].如段落[23]所述的方法，其特征在于，在所述步骤(1)中，所述非导电性毛细管为PDMS塑料 毛细管。
[26].如段落[23]所述的方法，其特征在于，在所述步骤(1)中，所述非导电性毛细管为SU-8光刻胶毛细管。
[27].如段落[21]-[26]中任一段所述的方法，其特征在于，在所述步骤(2)中，控制镀膜机的电流为5-30mA、优选15mA，从而喷镀所述铂层。
[28].如段落[21]-[27]中任一段所述的方法，其特征在于，在所述步骤(2)中，喷镀所述铂层的时间为200-1000秒、优选700秒。
[29].如段落[21]-[28]中任一段所述的方法，其特征在于，在所述步骤(2)中，所述铂层的厚度为20-100nm、优选70nm。
[30].如段落[21]-[29]中任一段所述的方法，其特征在于，在所述步骤(3)中，所述绝缘层为蜡层或阴极电泳漆层。
[31].如段落[30]所述的方法，其特征在于，所述蜡层为Apiezon蜡层。
[32].如段落[21]-[31]中任一段所述的方法，其特征在于，在所述步骤(3)中，以滴涂的方式向所述铂层的外侧涂覆绝缘层。
[33].如段落[21]-[32]中任一段所述的方法，其特征在于，在所述步骤(3)中，所述绝缘层的厚度为20-50nm、优选约30nm。
[34].如段落[21]-[33]中任一段所述的方法，其特征在于，在所述步骤(3)中，所述复合毛细管的所述环状电极端的开口的外径为290nm-315nm、优选300nm。
[35].如段落[21]-[34]中任一段所述的方法，其特征在于，在所述步骤(3)中，所述复合毛细管的所述环状电极端的开口的内径为120-145nm、优选130nm。
[36].如段落[21]-[35]中任一段所述的方法，其特征在于，在所述步骤(3)中，所述复合毛细管的长度为1mm-5cm、优选1mm-10mm、最优选约5mm。
[37].如段落[21]-[36]中任一段所述的方法，其特征在于，在所述步骤(4)中，所述复合毛细管的所述远离环状电极端的开口的外径为0.8mm-1.5mm、优选1mm。
[38].如段落[21]-[37]中任一段所述的方法，其特征在于，在所述步骤(4)中，所述复合毛细管的所述远离环状电极端的开口的内径为0.4mm-0.9mm、优选0.5mm。
[39].如段落[21]-[38]中任一段所述的方法，其特征在于，在所述步骤(4)中，所述导电线选自Pt导线、Ag导线、Au导线、Cu导线、Al导线和/或石墨烯导线。
[40].如段落[21]-[39]中任一段所述的方法，其特征在于，在所述步骤(5)中，所述检测试剂选自葡萄糖氧化酶；鞘磷脂、碱性磷酸酶和胆碱氧化酶的混合物；胆固醇氧化酶；或者胆碱氧化酶。
[41].如段落[21]-[40]中任一段所述的方法，其特征在于，在所述步骤(5)中，将所述检测试剂以大于0毫微微升至小于等于10μl、优选1μl的体积进行单独包装。
[42].段落[1]-[18]中任一段所述的检测试剂盒和段落[19]或[20]所述的分析系统在制备用于单个细胞电化学分析的检测物中的用途。
[43].如段落[42]所述的用途，其特征在于，所述单个细胞电化学分析包括检测单个细胞内的小分子的水平和/或蛋白质的反应性或活性。
[44].如段落[42]或[43]所述的用途，其特征在于，所述单个细胞内的小分子选自葡萄糖、胆固醇、PO4 3-离子、活性氧和/或H2O2。
[45].如段落[42]-[44]中任一段所述的用途，其特征在于，所述蛋白质为所述单个细胞内的酶。
[46].如段落[45]所述的用途，其特征在于，所述酶选自鞘磷脂酶、转氨酶。
相比起现有技术公开的利用更加复杂的结构或者需要对检测试剂进行表面官能化来识别分析物的检测装置/技术，本发明所述的检测试剂盒及包含该检测试剂盒的分析系统采用在其一端的端面上具有Pt环 状电极的中空的复合毛细管来检测单个细胞内的小分子的水平以及蛋白等的活性，从而提供更加简单的胞内分子检测手段。
附图说明
图1为本发明的示例性的检测试剂盒中的复合毛细管及包含所述复合毛细管的分析系统的示意图。
图2A-图2F分别为本发明的示例性的复合毛细管的制备流程图(图2A)；包覆有Pt层的非导电性毛细管的侧视图和主视图的扫描电镜图片(分别为图2B和图2C，扫描速率为100mV/s)；进一步包覆有蜡绝缘层的具有Pt层的非导电性毛细管的侧视图和主视图的扫描电镜图片(分别为图2D和图2E，扫描速率为100mV/s)；以及具有Pt环状电极的复合毛细管在PBS缓冲液中的伏安法测试结果(图2F)。
图3A-图3F分别表示暴露至10mM PBS(pH 7.4)(曲线a)或者具有0.2mM(曲线b)、1mM(曲线c)或5mM(曲线d)葡萄糖的10mM PBS(pH 7.4)的检测试剂盒的电荷(图3A)；扣除在PBS中收集的非法拉第电荷之后，在0.2mM(曲线a)、1mM(曲线b)和5mM(曲线c)葡萄糖中收集的电荷方面的差异(图3B)；曝露至0.2mM葡萄糖和不同的电压2秒后的检测试剂盒的电荷方面的差异(图3C)；曝露至0.2mM葡萄糖和1V的电压不同时间后的检测试剂盒的电荷方面的差异(图3D)；葡萄糖浓度和电荷差异之间的相关性(图3E)；以及SMase活性和电荷差异之间的相关性(图3F)；其中，所述图中的误差棒表示三重复独立实验的标准差。
图4A-图4F分别表示插入细胞中的复合毛细管的明视场图像(图4A)；在复合毛细管插入并施加电压排出检测试剂之前和之后，在细胞(n＝10)中观察到的荧光强度(细胞内钙浓度)(图4B)；在将复合毛细管插入细胞之前和之后，复合毛细管的电荷(图4C)；使用包含葡萄糖氧化酶(轨迹a)和仅PBS(轨迹b)的复合毛细管在扣除于细胞外部收集的非法拉第电荷之后，电荷方面的差异(图4D)；在29个HeLa细胞中收集的法拉第电荷(图4E)；以及在27个饥饿细胞中收集的法拉第电荷(图4F)。
图5A-图5C分别表示在扣除于细胞外部收集的非法拉第电荷之后，在如下细胞中收集的电极的法拉第电荷：Zn刺激的J774细胞(n＝26)(图5A)；未刺激的J774细胞(n＝20)(图5B)；以及Zn刺激的HeLa细胞(图5C)。图5D表示在不同状态下的细胞的统计学分析。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
在本发明中，术语“检测试剂”具有本领域所通常理解的含义，涵盖了本领域技术人员已知的可用于检测靶分析物的任何生化试剂，例如但不限于：葡萄糖氧化酶(优选使用浓度为0.1mg/ml-10mg/ml、最优选0.2mg/ml)；鞘磷脂(优选使用浓度为0.1mM-10mM、最优选1mM)、碱性磷酸酶(优选使用浓度为1-20U/mL、最优选5U/mL)和胆碱氧化酶(优选使用浓度为1-20U/mL、最优选5U/mL)的混合物；胆固醇氧化酶；胆碱氧化酶等。
在本发明中，术语“靶分析物”和“分析物”可互换使用，是指细胞内的待检测的各种靶标物质，例如但不限于，细胞内的小分子(诸如葡萄糖、胆固醇等)和蛋白质(例如各种酶，如鞘磷脂酶、转氨酶)等。
在本发明中，术语“约”表示该术语所修饰的参数具有本领域所通常理解的测量误差，例如但不限于涵盖了该参数的±5％、如±2％、±1％范围内的任意值。
在一个实施方式中，本发明涉及一种检测试剂盒，其特征在于，所述检测试剂盒包含：
中空的复合毛细管，其中，所述复合毛细管包含具有空腔的非导电性毛细管、覆盖在所述非导电性毛细管外侧的Pt层以及覆盖在所述Pt层外侧的绝缘层；
插入所述非导电性毛细管的空腔中的导电线，作为第二电极；其中，所述复合毛细管的远离所插入的第二电极的一端的端面未用所述绝缘层包覆，从而在所述端面上形成Pt环状电极(在本发明中，复合毛细管的这一端也简称为“环状电极端”；所述复合毛细管的插入第二电极的一端简称为“远离环状电极端”)；以及
检测试剂，其中，在临用前将所述检测试剂加入到所述非导电性毛细管的空腔中，并在检测过程中通 过向所述第二电极施加电压将所述检测试剂排出至所述复合毛细管的具有所形成的Pt环状电极的一端的端面上。
对本领域技术人员来说可以理解的是，本发明所述的复合毛细管具有任意形状均可实现本发明的目的。在优选的实施方式中，所述复合毛细管的具有所述Pt环状电极的一端优选处于圆锥体形式，从而能够更加有助于该复合毛细管易于插入细胞中且对细胞带来的伤害进一步减少。
本领域技术人员能够理解的是，所述复合毛细管可具有小于待检测细胞的尺寸的任意尺寸，从而适合用于插入单个细胞中而不影响细胞活力。优选的是，所述复合毛细管的待插入所述第二电极的一端(即，远离环状电极端)的开口的外径为0.8mm-1.5mm、优选1mm；进一步优选所述开口的内径为0.4mm-0.9mm、优选0.5mm。另外优选的是，所述复合毛细管的具有所述Pt环状电极的一端(即，环状电极端)的开口的外径为290nm-315nm、优选300nm；进一步优选所述开口的内径为120-145nm、优选130nm。
此外，优选所述复合毛细管的长度为1mm-5cm、优选1mm-10mm、最优选约5mm。
本领域技术人员能够理解的是，可将任意的非导电性材质的毛细管用于本发明的目的，从而制备复合毛细管。仅作为优选的示例性方式，本发明的非导电性毛细管可列举出玻璃毛细管(例如，硼酸盐玻璃毛细管)、塑料毛细管(例如，PDMS塑料毛细管)和/或光刻胶毛细管(例如，SU-8光刻胶毛细管)等，但适用于制备本发明的非导电性毛细管的材质并不仅限于此。
在本发明中，所述绝缘层可采用本领域已知的能够与待检测的细胞在生物学上兼容的任何绝缘材料制备得到。优选的是，作为形成本发明所述的绝缘层的优选绝缘材料，优选采用蜡和阴极电泳漆，从而得到蜡层(例如Apiezon蜡层)和阴极电泳漆层。
本发明所述的导电线可以通过能够导电的任意材料制成。能够满足本发明要求的示例性的导电线包括但不限于：Pt导线、Ag导线、Au导线、Cu导线、Al导线和/或石墨烯导线等。
对加入本发明所述的复合毛细管的空腔中的检测试剂的体积并无特定限制，只要能够得出本发明期望的测试结果即可。仅作为优选的实例，加入复合毛细管的管腔中的检测试剂的体积为大于0毫微微升(fl)至小于等于10μl、优选1μl。其中，通过施加电压向复合毛细管的环形电极端的端面排出小于10毫微微升的检测试剂时即可实现对单个细胞内的分析物(小分子和蛋白质)的准确检测。
在优选的实施方式中，仅为了使进入细胞的测试试剂的体积最小化、或者为了分析更小的区域或者为了对细胞活力产生尽可能低的干扰，向第二电极施加的电压为1-10V、优选1-5V、最优选1V，进一步优选向所述第二电极施加电压2-30秒、优选2-15秒、最优选2秒。
在另一实施方式中，本发明涉及包含上述检测试剂盒的分析系统，其特征在于，所述分析系统包含：所述检测试剂盒；连接至所述检测试剂盒中的复合毛细管的Pt层的导电线和与所述导电线的另一侧连接的电量计；以及通过两条另外的导电线分别连接至电量计和所述复合毛细管的第二电极的电源。
其中，所述电量计可为能够对待检测的分析物释放出的电荷量进行测量的任何现有的电荷测量装置。
在另一实施方式中，本发明涉及制备上述检测试剂盒的方法，其特征在于，所述方法包括如下步骤：
(1)提供包含空腔的非导电性毛细管；
(2)向所述非导电性毛细管的外侧喷镀铂层，从而制备得到半成品毛细管；以及
(3)向所述铂层的外侧涂覆绝缘层，其中，保留在所述半成品毛细管的一端的端面上的铂层免于用所述绝缘层进行涂覆，由此在所述端面上形成Pt环状电极，将该端称为环状电极端，从而得到复合毛细管；
(4)向所述复合毛细管的远离所述环状电极端的一端的空腔中插入导电线作为第二电极，其中，将所述复合毛细管的远离所述环状电极端的一端称为远离环状电极端；
(5)将所述步骤(4)中插入导电线的复合毛细管与检测试剂分别单独包装，并组装在一起；
其中，任选所述检测试剂盒进一步包含说明书。
在优选的实施方式中，在步骤(1)中，将所述非导电性毛细管的待在其端面上形成环状电极的一端拉制成圆锥体形状。
在优选的实施方式中，在步骤(2)中，控制镀膜机的电流为5-30mA、优选15mA，从而喷镀所述铂层。进一步优选的是，在步骤(2)中，喷镀所述铂层的时间为200-1000秒、优选700秒。在更进一步优 选的实施方式中，在步骤(2)中，所述铂层的厚度为20-100nm、优选约70nm。
在优选的实施方式中，在步骤(3)中，以滴涂的方式向所述铂层的外侧涂覆绝缘层。在进一步优选的实施方式中，所述绝缘层的厚度为20-50nm、优选约30nm。
在优选的实施方式中，所述复合毛细管的环状电极端(即，在其端面上具有所形成的Pt环状电极的一端)的开口的外径为290nm-315nm、优选300nm；进一步优选所述开口的内径为120-145nm、优选130nm。另外优选的是，所述复合毛细管的远离环状电极端(即，待插入所述第二电极的一端)的开口的外径为0.8mm-1.5mm、优选1mm；进一步优选所述开口的内径为0.4mm-0.9mm、优选0.5mm。
在优选的实施方式中，仅为了更加便于使用，将所述检测试剂以大于0毫微微升至小于等于10μl、优选1μl的体积进行单独包装。
在又一实施方式中，本发明涉及上述检测试剂盒和分析系统在制备用于单个细胞电化学分析的检测物中的用途。
在优选的实施方式中，所述单个细胞电化学分析包括检测单个细胞内的小分子的水平和/或蛋白质的反应性或活性。
由于本领域已知能够用于测定细胞内的各种小分子和蛋白质的检测试剂，因此，在采用合适的检测试剂的情况下，本发明的检测试剂盒可检测单个细胞内各种小分子和蛋白质。仅作为示例，本发明所述的单个细胞内的小分子可以为葡萄糖、胆固醇、PO4 3-离子、活性氧和/或H2O2。作为示例，本发明所述的单个细胞内的所述蛋白质可以为单个细胞内的各种酶，例如但不限于，鞘磷脂酶、转氨酶等。
在本发明的示例性的检测试剂盒中，通过将导电线插入到非导电性毛细管中并将检测试剂加入到非导电性毛细管的空腔中，当向所述导电线施加适当的电压时，使得检测试剂能够流出复合毛细管并在管口处与单个细胞中的靶分子或蛋白反应。所得到的副产物过氧化氢在Pt环状电极上进行电化学氧化，收集所产生的电荷。将复合毛细管在不含靶分析物的溶液中进行预处理，确定非法拉第电荷的量，并从所收集的电荷中排除非法拉第电荷，由此对细胞内的靶分析物的活性和浓度进行定量。
本发明所述的检测试剂盒及包含该检测试剂盒的分析系统采用在其一端的端面上具有Pt环状电极的中空的复合毛细管来检测单个细胞内的小分子的水平以及蛋白等的活性，从而提供更加简单且准确的胞内分析物的检测手段。
实施例
实施例1制备中空的复合毛细管
按照下表1中示出的制备条件，根据如下步骤制备得到中空的复合毛细管1-5：
(1)提供具有如下表1中所述尺寸的包含空腔的非导电性毛细管(其中，分别采用硼酸盐玻璃毛细管、PDMS塑料毛细管、硼酸盐玻璃毛细管、SU-8光刻胶毛细管和硼酸盐玻璃毛细管作为非导电性毛细管来制备复合毛细管1-5)，其中，将所述非导电性毛细管的待在其端面上形成环状电极的一端拉制成圆锥体；
(2)向所述非导电性毛细管的外侧喷镀铂层，从而制备得到半成品毛细管；以及
(3)向所述半成品毛细管的铂层的外侧涂覆绝缘层，其中，保留在所述半成品毛细管的一端的端面上的铂层免于用绝缘层进行涂覆，由此在所述端面上形成Pt环状电极(该端称为“环状电极端”)，从而得到中空的复合毛细管1-5。
表1复合毛细管1-5的制备条件
Figure PCTCN2017102648-appb-000001
Figure PCTCN2017102648-appb-000002
注：O.D.表示外径；I.D.表示内径。
按照如下方法，将所制备的复合毛细管1-5放置入具有5mM亚铁氰化物的100mM的磷酸盐缓冲液中(PBS，pH 7.4)中进行伏安法测试：在涂覆绝缘层之前和以后，将覆盖有Pt层的非导电性毛细管用SEM(Hitachi S-4800Instrument，日本)进行表征。将10kv加速电压施加在Au-包覆样品上，将复合毛细管1-5暴露至具有5mM亚铁氰化物的100mM的磷酸盐缓冲液中(PBS，pH 7.4)中，以表征暴露在所述毛细管的一端的Pt层的区域。分别将Ag/AgCl电极和Pt电极用作参比电极和对电极。电压循环的范围为-0.1v至0.6v，并施加100mv/s的扫描速率以使用电化学工作站(CHI 630E，CH Instruments)收集电流。结果在图2F中示出。
由于实验电流大于理论值，从而确认了所得到的毛细管1-5在它们的一端的端面均具有Pt环状电极。实施例2制备检测试剂盒
向实施例1中制备的复合毛细管1-5的远离所述环状电极端的一端(该端称为“远离环状电极端”)的空腔中插入导电线(其中，复合毛细管1-5中分别插入Pt导线、Ag导线、Au导线、Cu导线和Pt导线)作为第二电极，并随后分别与放置在安瓿瓶中的10μL处于10mM的磷酸盐缓冲液(PBS，pH 7.4)中的0.1mM、0.2mM、1mM、5mM、10mM的葡萄糖氧化酶组装在一起，得到检测试剂盒1-5。在临用前，将所述放置在安瓿瓶中的检测试剂分别加入所述复合毛细管1-5中。
同时，向实施例1中制备的复合毛细管1-5的远离所述环状电极端的一端(该端称为“远离环状电极端”)的空腔中插入导电线(其中，复合毛细管1-5中分别插入Pt导线、Ag导线、Au导线、Cu导线和Pt导线)作为第二电极，并随后与放置在安瓿瓶中的10μL处于10mM PBS缓冲液(pH 7.4)中的如下5种检测试剂混合物组装在一起，得到检测试剂盒1’-5’：(1)1mM鞘磷脂、5U/ml碱性磷酸酶和5U/ml胆碱氧化酶的混合物(下文称为“混合物1”)；(2)0.1mM鞘磷脂、1U/ml碱性磷酸酶和1U/ml胆碱氧化酶的混合物(下文称为“混合物2”)；(3)5mM鞘磷脂、10U/ml碱性磷酸酶和10U/ml胆碱氧化酶的混合物(下文称为“混合物3”)；(4)10mM鞘磷脂、20U/ml碱性磷酸酶和20U/ml胆碱氧化酶的混合物(下文称为“混合物4”)；以及(5)8mM鞘磷脂、15U/ml碱性磷酸酶和15U/ml胆碱氧化酶的混合物(下文称为“混合物5”)。在临用前，将所述放置在安瓿瓶中的检测试剂混合物1-5分别加入所述复合毛细管1-5中。
实施例3制备分析系统
将实施例2中制备的检测试剂盒1-5和1’-5’中的复合毛细管1-5通过与其Pt层连接的Cu导线连接至电量计，再通过另外的两条Cu导线将所述电量计以及所述毛细管中的第二电极分别连接至电源，从而分别制备得到相应的分析系统1-5和分析系统1’-5’(其中，在将分析系统临用于对单个细胞进行检测前，将所述检测试剂盒中的放置在安瓿瓶中的检测试剂或检测试剂混合物按照实施例2中所述分别加入毛细管1-5中)。
效果例
效果例1细胞内的葡萄糖水平的测量
准确分析细胞内的葡萄糖水平对于了解细胞内的生化过程以及对于一些相关疾病(例如癌症)的辅助诊断具有重要意义。本发明人采用实施例3中制备的分析系统1-5(其中，使用葡萄糖氧化酶作为检测试剂)来测量单个细胞内的葡萄糖水平，其中，如下述的反应方案1所示，葡萄糖氧化酶将β-D-葡萄糖氧化为D-葡萄糖酸-1,5-内酯和过氧化氢(过氧化氢的量与细胞内的葡萄糖的量相关)。
反应方案1
Figure PCTCN2017102648-appb-000003
具体而言，向所述中空的复合毛细管1-5的空腔中分别加入1μL的处于10mM的磷酸盐缓冲液(PBS，pH 7.4)中的0.1mM、0.2mM、1mM、5mM、10mM的葡萄糖氧化酶。使所述复合毛细管1-5的Pt环状电极分别接触待检测的单个HeLa细胞样品(n＝29，其中，所述复合毛细管1-5的Pt环状电极分别接触编号为1-6、7-12、13-18、19-24、25-29的样品)。通过电源向插入到所述复合毛细管1-5中的第二电极分别施加1V的电压30秒、1V的电压2秒、2V的电压15秒、5V的电压10秒和10V的电压2秒，从而使加入所述复合毛细管1-5的空腔中的葡萄糖氧化酶排出至所述复合毛细管1-5的具有环状Pt电极的端面上。
检测结果在图4E中示出，其中，所检测的29个样品对应的单个细胞中的葡萄糖浓度分别在下表2中示出(由于1个葡萄糖分子将释放2个电子，同时已知一个细胞的体积为1pL，由此根据电量计测得的电荷量可以推算出葡萄糖的量，并进一步计算得出单个细胞中的葡萄糖浓度)。
表2 HeLa细胞中的葡萄糖浓度检测结果
单个HeLa细胞样品编号 浓度(mM)
1 2.17
2 1.48
3 3.26
4 1.58
5 3.52
6 5.67
7 0.82
8 4.03
9 1.17
10 1.21
11 1.51
12 1.76
13 5.15
14 1.03
15 0.43
16 2.07
17 2.02
18 1.65
19 1.12
20 3.25
21 2.70
22 1.28
23 0.61
24 0.92
25 1.20
26 1.27
27 1.89
28 1.30
29 1.35
效果例2细胞内的鞘磷脂酶的活性的测量
鞘磷脂酶(SMase)是参与鞘脂代谢反应的一种水解酶，现有的研究(例如，Hannun YA等，J.Biol.Chem.，2002，277(29):25847-25850)表明SMase的活化是应答细胞应激而产生神经酰胺的重要途径。现有技术中迄今并未实现对单个细胞中的SMase的活性进行分析。
本发明人使用实施例3中制备的分析系统1’-5’(其中，使用鞘磷脂、碱性磷酸酶(ALP)和胆碱氧化酶的混合物作为检测试剂)来测量细胞内的SMase的活性，其中，如下述的反应方案1所示，鞘磷脂依次经过SMase、ALP和胆碱氧化酶的作用，最终产生过氧化氢(过氧化氢的量与细胞内的SMase的活性相关)。
反应方案2
Figure PCTCN2017102648-appb-000004
具体而言，向所述中空的复合毛细管1-5的空腔中分别加入1μL的处于10mM PBS缓冲液中的上述实施例2中的混合物1-5。使所述复合毛细管1-5的Pt环状电极分别接触待检测的单个J774细胞样品(n＝26，其中，所述复合毛细管1-5的Pt环状电极分别接触编号为1-6、7-11、12-16、17-21、22-26的样品)(在进行检测前，预先用0.1mM Zn(II)离子对J774细胞进行刺激，使J774细胞的SMase活性上调)。通过电源向插入到所述复合毛细管1-5中的第二电极分别施加1V的电压30秒、10V的电压2秒、1V的电压5秒、5V的电压15秒和8V的电压20秒，从而使加入所述复合毛细管1-5的空腔中的所述检测试剂混合物1-5排出至所述复合毛细管1-5的具有环状Pt电极的端面上。
检测结果在图5A中示出，其中，所检测的26个样品对应的单个细胞中的SMase的活性分别在下表3中示出(由于1个鞘磷脂分子在单位时间内SMase转化将释放2个电子，同时已知一个细胞的体积为1pL，由此根据电量计测得的电荷量可以计算出SMase的活度)。
表3 J774细胞中的SMase活性检测结果
单个J774细胞样品编号 活度(nU)
1 3.46
2 1.35
3 1.04
4 0.64
5 0.66
6 1.26
7 0.68
8 0.85
9 0.81
10 1.52
11 1.16
12 1.41
13 4.58
14 0.14
15 0.56
16 2.68
17 0.54
18 2.26
19 1.53
20 0.46
21 1.11
22 0.76
23 0.28
24 1.2
25 0.73
26 0.76

Claims (10)

  1. 一种检测试剂盒，其特征在于，所述检测试剂盒包含：
    中空的复合毛细管，其中，所述复合毛细管包含具有空腔的非导电性毛细管、覆盖在所述非导电性毛细管外侧的Pt层以及覆盖在所述Pt层外侧的绝缘层；
    插入所述非导电性毛细管的空腔中的导电线，作为第二电极；其中，所述复合毛细管的远离所插入的第二电极的一端的端面未用所述绝缘层包覆，从而在所述端面上形成Pt环状电极；以及
    检测试剂，其中，在临用前将所述检测试剂加入到所述非导电性毛细管的空腔中，并在检测过程中通过向所述第二电极施加电压将所述检测试剂排出至所述复合毛细管的具有所形成的Pt环状电极的一端的端面上。
  2. 如权利要求1所述的检测试剂盒，其特征在于，所述复合毛细管的具有所述Pt环状电极的一端处于圆锥体形式。
  3. 如权利要求1或2所述的检测试剂盒，其特征在于，所述复合毛细管的具有所述Pt环状电极的一端的开口的外径为290nm-315nm、优选300nm；另外优选所述开口的内径为120-145nm、优选130nm。
  4. 如权利要求1-3中任一项所述的检测试剂盒，其特征在于，所述非导电性毛细管选自玻璃毛细管、塑料毛细管和/或光刻胶毛细管。
  5. 如权利要求1-4中任一项所述的检测试剂盒，其特征在于，所述绝缘层为蜡层或阴极电泳漆层。
  6. 如权利要求1-5中任一项所述的检测试剂盒，其特征在于，所述导电线选自Pt导线、Ag导线、Au导线、Cu导线、Al导线和/或石墨烯导线。
  7. 一种包含权利要求1-6中任一项所述的检测试剂盒的分析系统，其特征在于，所述分析系统包含：
    所述检测试剂盒；
    连接至所述检测试剂盒中的复合毛细管的Pt层的导电线和与所述导电线的另一侧连接的电量计；以及通过两条另外的导电线分别连接至电量计和所述复合毛细管的第二电极的电源。
  8. 一种制备权利要求1-6中任一项所述的检测试剂盒的方法，其中，所述方法包括如下步骤：
    (1)提供包含空腔的非导电性毛细管；
    (2)向所述非导电性毛细管的外侧喷镀铂层，从而制备得到半成品毛细管；以及
    (3)向所述铂层的外侧涂覆绝缘层，其中，保留在所述半成品毛细管的一端的端面上的铂层免于用所述绝缘层进行涂覆，由此在所述端面上形成Pt环状电极，将该端称为环状电极端，从而得到复合毛细管；
    (4)向所述复合毛细管的远离所述环状电极端的一端的空腔中插入Pt导线作为第二电极，其中，将所述复合毛细管的远离所述环状电极端的一端称为远离环状电极端；
    (5)将所述步骤(4)中插入导电线的复合毛细管与检测试剂分别单独包装，并组装在一起；
    其中，任选所述检测试剂盒进一步包含说明书。
  9. 如权利要求8所述的方法，其特征在于，在所述步骤(1)中，将所述非导电性毛细管的待在其端面上形成环状电极的一端拉制成圆锥体形状。
  10. 权利要求1-6中任一项所述的检测试剂盒和权利要求7所述的分析系统在制备用于单个细胞电化学分析的检测物中的用途。
PCT/CN2017/102648 2016-09-23 2017-09-21 检测试剂盒及其制备方法、包含检测试剂盒的分析系统、及它们的用途 WO2018054320A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610847435.9 2016-09-23
CN201610847435.9A CN107870188B (zh) 2016-09-23 2016-09-23 检测试剂盒及其制备方法、包含检测试剂盒的分析系统、及它们的用途

Publications (1)

Publication Number Publication Date
WO2018054320A1 true WO2018054320A1 (zh) 2018-03-29

Family

ID=61690721

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/102648 WO2018054320A1 (zh) 2016-09-23 2017-09-21 检测试剂盒及其制备方法、包含检测试剂盒的分析系统、及它们的用途

Country Status (2)

Country Link
CN (1) CN107870188B (zh)
WO (1) WO2018054320A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114441264A (zh) * 2022-01-20 2022-05-06 复旦大学 一种皮升级体积单细胞样品裂解酶解反应器

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113355387B (zh) * 2020-09-01 2024-04-30 南京大学 一种检测组织中单细胞的酶活性的检测系统及其检测方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040262636A1 (en) * 2002-12-09 2004-12-30 The Regents Of The University Of California Fluidic nanotubes and devices
CN101241097A (zh) * 2007-09-18 2008-08-13 中国科学院上海应用物理研究所 一种采用茎环结构检测探针的电化学dna检测方法及其试剂盒
CN101421616A (zh) * 2006-04-10 2009-04-29 迪埃诺斯维斯股份有限公司 安培计检测优化的小型化生物传感器
CN102590302A (zh) * 2011-01-13 2012-07-18 同济大学 一种制造金纳米阵列超微电极的方法
CN103675060A (zh) * 2013-12-05 2014-03-26 南京大学 一种微电极阵列及其在检测单细胞表面活化胆固醇上的应用
CN104379261A (zh) * 2012-04-16 2015-02-25 昆南诺股份有限公司 用于生物分子检测的纳米毛细管装置、流体网络结构和其制造方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2609420B1 (en) * 2010-08-24 2021-03-03 Bioptic, Inc. Disposible bio-analysis cartridge and instrument for conducting bio-analysis using same

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040262636A1 (en) * 2002-12-09 2004-12-30 The Regents Of The University Of California Fluidic nanotubes and devices
CN101421616A (zh) * 2006-04-10 2009-04-29 迪埃诺斯维斯股份有限公司 安培计检测优化的小型化生物传感器
CN101241097A (zh) * 2007-09-18 2008-08-13 中国科学院上海应用物理研究所 一种采用茎环结构检测探针的电化学dna检测方法及其试剂盒
CN102590302A (zh) * 2011-01-13 2012-07-18 同济大学 一种制造金纳米阵列超微电极的方法
CN104379261A (zh) * 2012-04-16 2015-02-25 昆南诺股份有限公司 用于生物分子检测的纳米毛细管装置、流体网络结构和其制造方法
CN103675060A (zh) * 2013-12-05 2014-03-26 南京大学 一种微电极阵列及其在检测单细胞表面活化胆固醇上的应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114441264A (zh) * 2022-01-20 2022-05-06 复旦大学 一种皮升级体积单细胞样品裂解酶解反应器
CN114441264B (zh) * 2022-01-20 2023-05-30 复旦大学 一种皮升级体积单细胞样品裂解酶解反应器

Also Published As

Publication number Publication date
CN107870188B (zh) 2020-07-14
CN107870188A (zh) 2018-04-03

Similar Documents

Publication Publication Date Title
CN1230679C (zh) 具有改善加样性能的生物传感器
Perry et al. Review of recent advances in analytical techniques for the determination of neurotransmitters
Weltin et al. Polymer-based, flexible glutamate and lactate microsensors for in vivo applications
Zhang et al. New frontiers and challenges for single-cell electrochemical analysis
Lin et al. Chemical analysis of single cells
JP5456885B2 (ja) バイオセンサー及びその使用方法
Hu et al. Nanoelectrodes for intracellular measurements of reactive oxygen and nitrogen species in single living cells
He et al. Hollow nanoneedle-electroporation system to extract intracellular protein repetitively and nondestructively
US20050269214A1 (en) Biosensors having improved sample application and uses thereof
JP2005530179A (ja) 多成分の微量分析のための微細加工で作られたセンサーアレイ
JP2016529515A (ja) 高アンモニア血症の検出のためのデバイス及びデバイスを使用する方法
Collins et al. Ion-transfer voltammetric determination of the β-blocker propranolol in a physiological matrix at silicon membrane-based liquid| liquid microinterface arrays
Ino et al. Intracellular electrochemical sensing
US20180338712A1 (en) Mutli-probe microstructured arrays
Wu et al. Analytical and quantitative in vivo monitoring of brain neurochemistry by electrochemical and imaging approaches
WO2018054320A1 (zh) 检测试剂盒及其制备方法、包含检测试剂盒的分析系统、及它们的用途
Zhu et al. A gold nanoparticle-modified indium tin oxide microelectrode for in-channel amperometric detection in dual-channel microchip electrophoresis
Marinesco Micro-and nano-electrodes for neurotransmitter monitoring
Han et al. Development of multi-well-based electrochemical dissolved oxygen sensor array
Budai Carbon fiber-based microelectrodes and microbiosensors
Ly Implementation of a biocircuit implants for neurotransmitter release during neuro-stimulation
Goyal et al. Voltammetric quantification of adenine and guanine at C60 modified glassy carbon electrodes
CN114487055A (zh) 一种多路闭合式双极电化学发光芯片及其在检测传感中的应用
Majeed et al. Electrochemical biosensors for determination of anticancer medicine etoposide in human blood by glassy carbon modified electrode based on film of poly (L-lysine) with MWCNTS
Pan et al. Nanokits for the electrochemical quantification of enzyme activity in single living cells

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17852388

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17852388

Country of ref document: EP

Kind code of ref document: A1