WO2017116267A1

WO2017116267A1 - A nanoelectrode for detecting cu(ii) ions and a method of producing and using thereof

Info

Publication number: WO2017116267A1
Application number: PCT/RU2015/000958
Authority: WO
Inventors: Alexander Sergeyevich EROFEEV; Petr Vladimirovich GORELKIN; Alexander Georgiyevich MAJOUGA; Artem Radiyevich USMANOV; Igor Vladimirovich YAMINSKY; Yuri Evgenyevich KORCHEV
Original assignee: Autonomous Non-Profit Organization For Higher Education "Skolkovo Institute Of Science And Technology"
Priority date: 2015-12-30
Filing date: 2015-12-30
Publication date: 2017-07-06
Also published as: EA037290B1; EA201891540A1

Abstract

The present disclosure relates to a sensitive, reusable, selective Cu(II) nanoelectrode. The disclosed nanoelectrode provides for high resolution quantitative and label-free copper ions detection inside or close to micro objects such as living cells of tissue.

Description

A NANOELECTRODE FOR DETECTING CU(II) IONS AND A METHOD OF

PRODUCING AND USING THEREOF

FIELD

The present invention generally relates to devices and methods for metal ions detection. In particular, it relates to a nanoelectrode for detecting Cu(ll) ions and a method of producing and using thereof for detection copper ions in a sample, as well as to a system comprising the nanoelectrode. BACKGROUND

Copper is an essential element for life, but alterations in its cellular homeostasis can lead to serious neurodegenerative diseases, including Menkes and Wilson diseases, familial amyotropic lateral sclerosis, Alzheimer's disease, and prion diseases. Furthermore, it has been suggested that altered cellular copper concentration is also associated with development of cancer. Therefore, it is important to develop devices and methods for precise monitoring of cellular copper concentration.

Electrochemical methods can be more suitable than other methods for cupper ions detection due to improved durability, sensitivity, rapid response, and integration with other device components. US3821 100 discloses one of the first electrochemical methods for ion copper detection using a cupric ion selective sensor. CN103941 185 discloses a copper metal vapor concentration detection system that is based on a micrometer scale sensor. CN103940882 discloses a sensor for detecting trace copper ions in a water sample, and a construction method thereof. CN103913502 discloses a copper rapid determination method based on a square-wave stripping voltammetry and a three-electrode sensor. CN102507698 discloses a novel sensor for synchronously detecting copper ions and lead ions.

The existing devices for copper ions detection, however, have macro or micro size and are not affordable for local and intracellular detection. These devices are usually large enough to provide testing of typical mammalian cells (5 to 10 μηι), and procedures are often limited to measurements in oocytes and embryos, which are at least ten times larger.

Miniaturization of existing devices for detecting Cu(ll) ions is a complicated technical problem. It is difficult to create an inexpensive nanosized Cu(ll) electrode having high sensitivity with respect to Cu(ll) ions, in particular, because the measurement of low intensity signals is a severe problem. Furthermore, the area of contact between a cell and the electrode has to be separated from the environment. Accordingly, there is a continuous need for inexpensive analytical tools for measuring concentration of copper ions with a nanoscale resolution, for example, inside a single cell. In particular, there is a continuous need to provide a high selectivity and high sensitivity Cu(ll) ions electrodes having nanoscale spatial resolution. This and other needs are addressed in the present disclosure as described in more details below.

SUMMARY

Some embodiments disclosed herein relate to a nanoelectrode for detecting Cu(ll) ions, comprising:

a nanopipette having a tip defining an opening;

a carbon plug occupying at least part of an interior of the tip and having a first surface facing the opening;

a metal coating covering at least part of the first surf ace; and at least one Cu(ll) ions chelating compound bound to the metal coating. In some embodiments, the metal in the metal coating is selected gold, silver, platinum, and alloys thereof. In some embodiments, the Cu(ll) ions chelating compound has a thiol group in its structure. In some embodiments, the Cu(ll) ions chelating compound is selected from: natural peptides; noncanonical peptides; nitrogen, oxygen, sulfur containing heterocycles; nitrogen, oxygen, sulfur crown ethers. In some embodiments, a diameter of the opening is in the range from 1 to 1000 nm, from 5 to 100 nm, from 10 to 70 nm, or from 20 to 50 nm. In some embodiments, an outer diameter of an end of the tip is in the range from 10 to 10000 nm, from 100 to 800 nm, or from 200 to 500 nm. In some embodiments, at least part of the first surface is concave, forming a cavity.

In some embodiments, the nanoelectrode further comprises an inner electrode, arranged to be in contact with a second surface of the carbon plug, the second surface facing an interior of the nanopipette.

Some embodiments disclosed herein relate to a method of producing a nanoelectrode for detecting Cu(ll) ions, comprising the steps of:

(a) providing a nanopipette having a tip defining an opening;

(b) at least partially filling an interior of the tip with carbon to form a carbon plug having a first surface facing the opening;

(c) at least partially coating the first surface with a metal to form a metal coating; and

(d) contacting the metal coating with at least one copper chelating compound to bind the copper chelating compound to the metal coating.

In some embodiments, the method after step (b) and before step (c) further comprises the step of:

(e) creating a cavity on the first surface.

In some embodiments, the cavity is created by electrochemical treatment, in particular, by electrochemical etching. In some embodiments, the first surface is coated with a metal by electrochemical deposition. In some embodiments, the metal is selected from a group comprising gold, silver, platinum, and alloys thereof. In some embodiments, the Cu(ll) ions chelating compound has a thiol group in its structure. In some embodiments, the Cu(ll) ions chelating compound has been selected from: natural peptides; noncanonical peptides; nitrogen, oxygen, sulfur containing heterocycles; nitrogen, oxygen, sulfur crown ethers. In some embodiments, the method further comprises the step of

(f) arranging an electrode to be in contact with a second surface of the carbon plug, the second surface facing an interior of the nanopipette.

Some embodiments disclosed herein relate to a system for detecting Cu(ll) ions in a sample, comprising the nanoelectrode disclosed herein as a working electrode. In some embodiments, the system further comprises a counter electrode, a reference electrode, a voltage supply to said working and counter-electrodes and a current meter for determining the current between said working and counter-electrodes. In some embodiments, the counter electrode is a platinum wire electrode and the reference electrode is Ag/AgCI. In some embodiments, the nanoelectrode, the counter electrode, the reference electrode, the voltage supply, and the current meter are electrically connected.

Some embodiments disclosed herein relate to a method of detecting Cu(ll) ions in a sample, comprising the steps of:

(a) contacting the sample with an electrode assembly comprising the nanoelectrode disclosed herein as a working electrode;

(b) determining a response of the working electrode in a voltammogram by varying a potential applied to the nanoelectrode;

(c) measuring the height of the peak in the voltammogram corresponding to the reduction of Cu(ll) ions to Cu(l) ions at the working electrode in order to determine the concentration of Cu(ll) ions.

In some embodiments, the method further comprises the steps of :

preparing calibration curves for the height of the voltammogram peaks for known concentrations of Cu(ll) ions; and

extrapolating the measured height of the sample's voltammogram peaks against the calibration curve to determine the concentration of Cu(ll) ions in the sample.

In some embodiments, the electrode assembly further comprises a counter electrode, a reference electrode, a voltage supply to said working and counter-electrodes and a current meter for determining the current between said working and counter-electrodes. In some embodiments, the counter electrode is a platinum wire electrode and the reference electrode is Ag/AgCI. In some embodiments, the voltammogram is selected from the group consisting of a cyclic voltammogram, a square-wave voltammogram, and a square-wave adsorptive stripping voltammogram. In some embodiments, the Cu(ll) to Cu(l) transition displays characteristic peaks at reduction potentials in the range from +200.0 mV to +700 mV, in particular, between +400.0 mV to +600 mV. In some embodiments, the applied potential is varied in a range that covers the potential at which the Cu(ll) to Cu(l) transition occurs, in particular, in the range from -500 mV to 800 mV. In some embodiments, varying the potential applied to the nanoelectrode involves applying a negative accumulation potential. In some embodiments, the accumulation potential is varied from -500 mV to +0.0 mV, in particular from -500 mV to -100.0 mV. In some embodiments, the negative accumulation potential is applied for a period of time at least 10ms, at least 30ms, or at least 50ms. In some embodiments, varying the potential applied to the nanoelectrode involves applying a positive potential with a scan rate in the range from 8 V/s to 2-10³ V/s. In some embodiments, the sensitivity of detection of Cu(ll) ions concentration increases with the scan rate and/or value (more negative) of the accumulation potential and/or duration of applying the accumulation potential.

The proposed nanoelectrode can be used for local copper ions detection in liquids, inside or near micro-objects of various natures and sizes, for example, cells, bacterial cells, tissues and animals, liposomes, microdroplets, etc. It is applicable to prokaryotes and eukaryotes. Suitable microobjects are sufficiently soft to allow penetration of a nanocapillary inside them. The proposed nanoelectrode has high sensitivity and selectivity to copper ions.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows current-voltage characteristic of a carbon nanoelectrode fabricated by pyrolytic deposition of carbon inside a pulled nanopipette. The characteristic was recorded in a 1 mM ferrocene methanol (FeMeOH) solution.

Figure 2 shows current-voltage characteristic recorded during the process of etching the carbon nanoelectrode in a 0.1 M NaOH, 10 mM KCI solution during 40 cycles for 10 seconds each to create a cavity on the nanoelectrode surface. The curve for each subsequent cycle is above that for the preceding cycle. Figure 3 shows current-voltage characteristic of the carbon nanoelectrode after etching a cavity. The characteristic was recorded in a 1 mM FeMeOH solution.

Figure 4 shows current-voltage characteristic recorded during the process of gold deposition on the carbon nanoelectrode in a 10 mM HAuCI₄ ^■ H₂0 solution. The curve for each subsequent cycle is below that for the preceding cycle.

Figure 5 shows current-voltage characteristic of the nanoelectrode after deposition a layer of gold on its surface. The characteristic was recorded in a 1 mM FeMeOH solution.

Figure 6 shows current-voltage characteristic of the nanoelectrode after deposition a layer of gold and prior to deposition of a copper chelating compound on its surface. The characteristic was recorded at various concentrations of copper ions in PBS (phosphate buffered saline solution having pH of 7.4 and prepared from 7.2 mM Na₂HP0₄, 2.8 mM KH₂P0₄, and 150 mM NaCI) solution. Circles - PBS, squares - 3-10^"7 M copper, triangles - 10^"6 M copper.

Figure 7 shows current-voltage characteristic of an embodiment of the proposed copper nanoelectrode recorded at various concentrations of copper ions in PBS (phosphate buffered saline solution having pH of 7.4 and prepared from 7.2 mM Na₂HP0₄, 2.8 mM KH₂P0₄, and 150 mM NaCI) solution. Circles - PBS, squares - 3-10^~7 M copper, triangles - 10^"6 M copper.

DETAILED DESCRIPTION

The proposed copper nanoelectrode comprises a nanopipette having a tip. The term "nanopipette" as used herein means a tube, or a capillary, with a narrow tip at one end. The tip has an opening with an internal diameter in the nanoscale range, for example, 1 to 1000 nm, from 5 to 100 nm, from 10 to 70 nm, or from 20 to 50 nm. The outer diameter of the end of the tip is adjusted so as not to exceed the size of the microscopic object, and can be, for example, in the range 10 to 10000 nm, from 100 to 800 nm, or from 200 to 500 nm. The internal and the external diameters of the end of the tip depend on the application and are adjusted by the parameters of the fabrication process in a known manner. The other end of the nanopipette is sized to allow insertion of a wire inside the nanoelectrode. The equipment and the procedures for fabrication of nanopipettes are known, see, for example, Actis, P. et al. // Biosensors and Bioelectronics 26, pp. 333-337. In some embodiments the nanopipettes can be fabricated by laser pulling of capillary tubes.

The length of the nanopipette tip vary depending on the application. The length in the range from 5 to 100 micrometers (microns) is convenient for using with microobjects, such as mammalian cells. The nanopipette can be manufactured from any suitable nonconductive material with high melting temperature, for example, quartz, glass, borosilicate. The nanopipette can have two or more parallel bores. The bores can be radially spaced or concentric. Such nanopipettes can be fabricated from multibore capillary tubes, which are commercially available. The multibore nanopipettes allow using different copper chelating compounds in different compartments or combining copper nanoelectrode with other analytical microdevices.

Nanopipette has a carbon plug occupying at least part of the interior of the tip. The term "carbon plug" as used herein means a piece of carbonaceous material that completely fills or blocks the interior of the tip in the transverse direction and at least partially occupies the interior of the tip in the longitudinal direction (the direction parallel to the axis of the nanopipette).

A part of the surface of the carbon plug contacts the nanopipette walls. Another part of the surface faces the opening. This part of the surface is referred to as "a first surface" of the carbon plug. Still another part of the surface faces the interior of the nanopipette, i.e. the broader part of the nanopipette, the part opposite to the tip. This part of the surface is referred to as "a second surface" of the carbon plug. Methods and apparatuses for filling nanopipettes with carbon are known in the art. The method of pyrolytic decomposition of hydrocarbons described in Takahashi Y. et al, Angewandte Chemie 50, pp. 9638-9642 is an example. In some embodiments, the exterior surface of the carbon plug is flat and flush with the end of the tip.

In other embodiments, at least part of the exterior surface of the carbon plug is concave, i.e. is hollowed inward. This configuration is also referred to as a "nanocavity". The presence of a nanocavity provides for better adhesion of the subsequent metal layer to the carbon plug.

Methods and apparatuses for creating cavities in carbon bodies are known in the art. The electrochemical etching method described in Clausmeyer J. et al. // Electrochemistry Communications 40, pp. 28-30 is an example.

At least part of the first surface of the carbon plug is covered with a metal coating. The metal is suitable for forming a bond with the copper chelating compound, in particular, a covalent bond. Examples of such metals are gold, silver, and platinum. Usage of metal alloys is also covered by the term "metal coating". Deposition of metal on the carbon surface can be done, for example, electrochemically from suitable salts or acids. Deposition of metal in the cavity provides stronger binding of the copper chelating compound to the nanoelectrode. The nanoelectrode comprises at least one copper chelating compound bound to the metal coating. The molecules of the copper chelating compound act as specific collectors of copper ions and increase sensitivity and selectivity of the nanoelectrode to copper ions. The copper chelating compound reversibly and selectively binds Cu²⁺ ions, thus increasing local concentration of the ions in the vicinity of the nanoelectrode. Upon application of positive potential to the nanoelectrode, Cu ions are reduced to Cu⁺ ions and are releases from the nanoelectrode.

Another characteristic of a suitable copper chelating compound is its ability to bind to the metal coating.

In some embodiments this ability is provided by the presence of a thiol group in the structure of the copper chelating compound. The thiol group is able to form strong covalent bond with the metal coating. The binding is further strengthened when the coating metal is gold because the sulfur - gold bond has particularly high binding energy (44 kcal / mol).

In one particular embodiment, the structure of a copper chelating compound comprises peptides capable of coordinating copper ions. Such peptides can be isolated from natural sources or be chemically synthesized.

In particular embodiment, peptides capable of coordinating copper ions comprise hystidine peptide fragments in their structure. Particular example of such peptide is a glycyl-L-histidyl-L-lysine peptide (GHK) having a structure shown below

Because it has three amino acids it is called a tripeptide. The GHK tripeptide has strong affinity for copper(ll) and was first isolated from human plasma. The synthesis of the peptides for using in the nanoelectrode can be performed by classical peptide synthesis methodology, in particular, in liquid phase synthesis or in solid phase peptide synthesis. In still another embodiment, the peptide capable of coordinating copper ions is modified by introducing a linker peptide for enhancing binding of the compound to the surface of the metal coating.

In a particular embodiment, the linker peptide comprises at least one sulfur group.

An example of the linker peptide comprising a sulfur group is lipoic acid. Introduction of lipoic acid moiety into the peptide capable of coordinating copper ions provides a strong binding to the metal surface, without affecting the coordination properties of the peptide. Nonlimiting list of copper chelating agents that can be used in the present nanoelectrode include natural peptides, noncanonical peptides, nitrogen, oxygen, sulfur containing heterocycles, nitrogen, oxygen, sulfur crown ethers.

The nanoelectrode can be used as a working electrode in a system for detecting Cu(ll) ions in a sample. Such system can comprise a counter electrode, a reference electrode, a voltage supply to said working and counter electrodes and a current meter for determining the current between said working and counter electrodes, the nanoelectrode, the counter electrode, the reference electrode, the voltage supply, and the current meter being electrically connected to each other. The proposed nanoelectrode, however, is not limited for using in the particular system described above. Other configurations of the electrodes and measuring and controlling devices are possible as is well known to those skilled in the art.

For performing measurement, a metal wiring or another conductor can be connected to the second surface of the carbon plug of the nanoelectrode. This wiring is used to apply electrical potential to the nanoelectrode during measurement and to electrically connect the nanoelectrode to the other components of the measuring set up. The proposed nanoelectrode can be used for local copper ions detection in liquids, inside or near micro-objects of various natures and sizes, for example, cells, bacterial cells, tissues and animals, liposomes, microdroplets, etc. It is applicable to prokaryotes and eukaryotes. Suitable microobjects are sufficiently soft to allow penetration of a nanocapillary inside them for copper ions detection. The proposed nanoelectrode has high sensitivity and selectivity with respect to Cu(ll) ions.

The navigation of the nanoelectrode in or near the microobject can be done mechanically "manually" under the control of a relevant optical systems. It is also possible to carry out navigation of the nanoelectrode in micro-object by using automated tools. For example, a scanning ion-conductance microscope can be used to determine the position of the object, and then a positioning system can be used to carry out the three-dimensional positioning of a controlled navigation of the nanoelectrode in or close to the microobjects.

The proposed nanoelectrode can be used in an electroanalytical method of detecting Cu(ll) ions in a sample, the method is based upon voltammetric analysis. The common characteristic of all voltammetric techniques is that they involve the application of a potential (E) to a working electrode and the monitoring of the resulting current (I) flowing through the electrochemical cell. In many cases the applied potential is varied or the current is monitored over a period of time (t) as a function of the applied potential (E). Thus, all voltammetric techniques can be described as some function of E, I, and t. The electrochemical cell where the voltammetric experiment is carried out consists of a working (indicator) electrode, a reference electrode, and usually a counter (auxiliary) electrode. The reduction or oxidation of a substance at the surface of a working electrode at the appropriate applied potential results in the mass transport of new material to the electrode surface and the generation of a current (I).

In accordance with the present method of detecting Cu(ll) ions, cyclic voltammetry is used to perform voltammetric measurements where the potential from an initial potential (E1 ) to a final potential (E2) is varied over time through a complete cycle. Cyclic voltammetry is based on varying the applied potential at a working electrode in both forward and reverse directions (at some scan rate) while monitoring the current.

Typically, resulting voltammogramm displays one or more peaks each corresponding to particular electrochemical transformation occurring at the working electrode. In the present method the electrochemical transformation is the Cu(ll) to Cu(l) transition that displays characteristic peaks at reduction potentials in the range from +200.0 mV to +700 mV, in particular, between +400.0 mV to +600 mV. The peak height is directly proportional to the concentration of the electroactive species, Cu(ll) ions. Therefore, by preparing appropriate calibrating curve it is possible to measure Cu(ll) concentration in a sample.

For electrochemical measurements standard electronic circuits and devices that include a controllable amplifier and a sensitive voltage and current detector can be used. They may comprise any sensitive device for detecting changes in current on the order of 1-10 picoamperes, based on a baseline current of 10-1000 picoamperes. Such electronic circuits and devices are time responsive and relatively temperature independent or allow for changes in temperature to be compensated for. It has an input in a circuit where a known voltage is supplied. Sensitive detecting circuits are known, including voltage clamp amplifiers and transimpedance amplifiers. The output current follows changes in the input voltage and small changes in current can be detected.

The proposed method of detecting Cu(ll) ions comprises the step of contacting the sample with an electrode assembly comprising the proposed nanoelectrode as a working electrode. The counter electrode and the reference electrode can be located outside the sample, but are in electrical contact with the working electrode, for example, though an electrolyte solution. Then voltammogram is recorded by varying a potential applied to the nanoelectrode. The height of the peak corresponding to Cu(ll) to Cu(l) transition is measured and compared with a calibration curve to determine concentration of Cu(ll) ions in the sample. In some embodiments, an increasing positive potential is applied to the working electrode during recording of the voltammogram. The potential is varied in a range that covers the potential at which the Cu(ll) to C(l) transition occurs. In some embodiments, a positive potential is applied with a scan rate in the range from 8 V/s to 2-10³ V/s. Such rapid application of positive potential provides simultaneous reduction of all copper ions collected at the electrode and, as a result, high current that increases sensitivity of the measurement. In case of slow voltage ramp, the current caused by the transition of the copper ions is distributed in time that can lead to a poor response of electrochemical signal.

In some embodiments, a negative accumulation potential is applied to the working electrode before application a positive potential. The negative accumulation potential enhances collection of copper ions at the electrode via migration, thus further increasing their local concentration. Application of positive potential after application of accumulation potential provides even higher currents and better sensitivity.

In some embodiments, the accumulation potential is in the range from -500 mV to +0.0 mV, in particular from -500 mV to -100.0 mV.

In some embodiments, the negative accumulation potential is applied for a period of time at least 10 ms, at least 30 ms, or at least 50 ms. The rate at which the accumulation potential is applied is not particularly important. It can be just set at some particular value and held for certain period of time.

The sensitivity of detection of Cu(ll) ions concentration increases with an increase in value of the scan rate and/or value (more negative) of the accumulation potential and/or duration of applying the accumulation potential. In some embodiments, the potential during recording of a voltammogram is varied in the range from -500 mV to 800 mV.

EXAMPLE

A number of nanopipettes was fabricated using a P-2000 laser puller (Sutter Instrument) from quartz capillaries with an outer diameter of 1.2 mm and an inner diameter of 0.90 mm (Q120-90-7.5; Intracel). The parameters used for the fabrication were: Heat 790, Filament 3, Velocity 45, Delay 130, and Pull 90. Each nanopipette was filled with butane gas via a tygon tubing. A quartz tubing, which was connected to an argon tank to provide constant flow of argon, was slit to cover the nanopipette tip. The nanopipette tip was then heated by a butane burner to cause pyrolytic decomposition of the butane gas, thus forming a carbon plug inside the nanopipette tip.

After the deposition of carbon, the internal surface of the carbon plug was contacted with a Ag/AgCI wire, thus forming a working electrode. The working electrode and another Ag/AgCI electrode, acting as auxiliary/reference electrode, were immersed into 2 ml of a 1 mM FeMeOH solution in PBS (phosphate buffered saline solution having pH of 7.4 and prepared from 7.2 mM Na₂HP0₄, 2.8 mM KH₂P0₄, and 150 mM NaCI). Both electrodes were connected to an Axopatch 700B amplifier with the DigiData 1322A digitizer (Molecular Devices), and a PC equipped with pClamp 10 software (Molecular Devices).

The potential of the working electrode was linearly ramped in a cyclic manner. The time of one cycle was 10 seconds. Figure 1 shows the resulting current-voltage characteristic of the working electrode. The value of the plateau at the positive potential indicates the steady-state current, giving the apparent radius of the carbon plug to be about 20 nm (for details of the calculation procedure see Actis P. et al. // ACS Nano 8, pp. 875-884). After the carbon deposition, a cavity in the carbon plug was etched by immersing the electrode in a 0.1 M NaOH, 10 mM KCI solution and applying 40 cycles of positive voltage of up to 2.1 mV during 10 seconds each. Figure 2 shows the current-voltage characteristic during the process of etching of the carbon plug. Figure 3 shows the current-voltage characteristic of the nanoelectrode with a cavity. After the etching, the current decreased in the positive part of the potential and increased in the negative part of the potential showing that the nanoelectrode changed its shape from a disk to a tube.

In the next step, a film of gold was electrochemically deposited on the surface of the carbon nanoelectrode by immersing the nanoelectrode in a 10 mM solution of HAuCI_r H₂0 and applying voltage. Figure 4 shows current-voltage characteristic of the deposition process. The initial deposition of gold occurred within the cavity as indicated by the slow change of the current because the region of interaction of the gold surface with the solution remains the same. Then, the avalanche current started to rise indicating that the cavity was completely filled and gold is being deposited outside the cavity. At this point, the electrochemical deposition was stopped to prevent formation of a microsized electrode.

Figure 5 shows the current-voltage characteristic of the nanoelectrode after deposition of gold. As can be seen, the current at the positive potentials significantly increased indicating substantial increase in the size of the electrode because of a solid gold deposition in the etched cavity. This conclusion is also confirmed by the increased oxygen current at the negative potentials.

Figure 6 shows the current-voltage characteristic of the nanoelectrode after deposition of gold, recorded at various concentrations of copper ions in PBS. The regime of applying voltage was the same as in Figure 7 as discussed below.

In the next step, a copper chelating compound was immobilized on the surface of gold by keeping the nanoelectrode obtained in the previous step in a 10^"3 M solution of glycyl- L-histidyl-L-lysine peptide in ethanol during one night. The structural formula of glycyl-L- histidyl-L-lysine peptide is shown below.

Figure 7 shows voltammograms of the resulting copper nanoelectrode recorded at different concentrations of Cu ⁺ ions. Each cycle shown in Figure 7, as well as in Figure 6, consisted of several stages. At the first stage, slowly decreasing negative potential was applied to the nanoelectrode until the potential reached -500 mV relative to the reference electrode. During this stage, the copper chelating agent collected Cu²⁺ ions. At a second stage, the potential was rapidly increased until it reached 800 mV. During the second stage, the increase in the potential from 0 to 800 mV that took 4 ms (scan rate 200 V/s) caused reduction of Cu²⁺ ions to Cu⁺ ions. This transition corresponds to a maximum on the voltammograms at around 500 mV. At a third stage, the potential was rapidly decreased from 800 mV to 0. The duration of the complete cycle was 60 ms.

Comparison of Figure 7 to Figure 6 (nanoelectrode without a copper chelating agent) shows that application of the copper chelating compound to the metal surface significantly increases sensitivity of the nanoelectrode with respect to Cu²⁺ ions at low concentrations. Further, Figure 7 shows that the current associated with reduction of copper ions can be reliably measured in the presence of other ions in the solution, thus demonstrating selectivity of the copper nanoelectrode. The prepared copper nanoelectrode was used to measure concentration of copper ions inside MCF-7 cells (human breast adenocarcinoma). Micromanipulator was used for positioning the nanoelectrode. Concentration of copper ions was detected before and after exposure cells with binuclear Cu complexes described in J. Med. Chem. 57, pp. 6252-6258. Initial concentration of copper ions inside the cells was 6±2 μΜ. After incubation of cells with 9 μΜ binuclear Cu complexes concentration inside the cells increased to 220±30 μΜ.

Claims

1. A nanoelectrode for detecting Cu(ll) ions, comprising:

a nanopipette having a tip defining an opening;

a metal coating covering at least part of the first surface; and

at least one Cu(ll) ions chelating compound bound to the metal coating.

2. The nanoelectrode of claim 1 , wherein the metal is selected from gold, silver, platinum, and alloys thereof.

3. The nanoelectrode of any one of claims 1 -2, wherein the Cu(ll) ions chelating compound has a thiol group in its structure.

4. The nanoelectrode of any one of claims 1 -3, wherein the Cu(ll) ions chelating compound is selected from: natural peptides; noncanonical peptides; nitrogen, oxygen, sulfur containing heterocycles; nitrogen, oxygen, sulfur crown ethers.

5. The nanoelectrode of any one of claims 1 -4, wherein a diameter of the opening is in the range from 1 to 1000 nm, from 5 to 100 nm, from 10 to 70 nm, or from 20 to 50 nm.

6. The nanoelectrode of any one of claims 1-5, wherein an outer diameter of an end of the tip is in the range from 10 to 10000 nm, from 100 to 800 nm, or from 200 to 500 nm.

7. The nanoelectrode of any one of claims 1 -6, wherein at least part of the first surface is concave, forming a cavity.

8. The nanoelectrode of at least one of claims 1 -7 further comprising an inner electrode, arranged to be in contact with a second surface of the carbon plug, the second surface facing an interior of the nanopipette.

9. A method of producing a nanoelectrode for detecting Cu(ll) ions, comprising the steps of:

(a) providing a nanopipette having a tip defining an opening;

10. The method of claim 9, wherein the method after step (b) and before step (c) further comprises the step of:

(e) creating a cavity on the first surface.

1 1. The method of claim 10, wherein the cavity is created by electrochemical treatment, in particular, by electrochemical etching.

12. The method of any one of claims 9-1 1 , wherein the first surface is coated with a metal by electrochemical deposition.

13. The method of any one of claims 9-12, wherein the metal has been selected from gold, silver, platinum, and alloys thereof.

14. The method of any one of claims 9-13, wherein the Cu(ll) ions chelating compound has a thiol group in its structure.

15. The method of any one of claims 9-14, wherein the Cu(ll) ions chelating compound has been selected from: natural peptides, noncanonical peptides; nitrogen, oxygen, sulfur containing heterocycles; nitrogen, oxygen, sulfur crown ethers.

16. The method of any one of claims 9-15, further comprising the step of

17. A system for detecting Cu(ll) ions in a sample, comprising the nanoelectrode according to any one of claims 1-8 as a working electrode.

18. The system according to claim 17, further comprising a counter electrode, a reference electrode, a voltage supply to said working and counter-electrodes and a current meter for determining the current between said working and counter-electrodes.

19. The system according to claim 18, wherein the counter electrode is a platinum wire electrode and the reference electrode is Ag/AgCI.

20. The system according to any one of claims 18-19, wherein the nanoelectrode, the counter electrode, the reference electrode, the voltage supply , and the current meter are electrically connected.

21 . A method of detecting Cu(ll) ions in a sample, comprising the steps of:

(a) contacting the sample with an electrode assembly comprising the nanoelectrode according to any one of claims 1 -8 as a working electrode;

(b) determining a response of the working electrode in a voltammogram by varying a potential applied to the nanoelectrode; and

22. The method of claim 21 , further comprising the steps of: preparing calibration curves for the height of the voltammogram peaks for known concentrations of Cu(ll) ions; and

23. The method according to any one of claims 21-22, wherein said electrode assembly further comprises a counter electrode, a reference electrode, a voltage supply to said working and counter-electrodes and a current meter for determining the current between said working and counter-electrodes.

24. The method according to claim 23, wherein the counter electrode is a platinum wire electrode and the reference electrode is Ag/AgCI.

25. The method according to any one of claims 21-24, wherein said voltammogram is selected from the group consisting of a cyclic voltammogram, a square-wave voltammogram, and a square-wave adsorptive stripping voltammogram.

26. The method according to any one of claims 21 -25, wherein the Cu(ll) to Cu(l) transition displays characteristic peaks at reduction potentials in the range from +200.0 mV to +700 mV, in particular, between +400.0 mV to +600 mV.

27. The method according to any one of claims 21 -26, wherein the applied potential is varied in a range that covers the potential at which the Cu(ll) to Cu(l) transition occurs, in particular, in the range from -500 mV to 800 mV.

28. The method according to any one of claims 21 -27, wherein varying the potential applied to the carbon plug of the nanoelectrode involves applying a negative accumulation potential.

29. The method according to any one of claims 21 -28, wherein the accumulation potential is varied from -500 mV to +0.0 mV, in particular from -500 mV to -100.0 mV.

30. The method according to any one of claims 21 -29, wherein the negative accumulation potential is applied for a period of time at least 10ms, at least 30ms, or at least 50ms.

31. The method according to any one of claims 21 -30, wherein varying the potential applied to the nanoelectrode involves applying a positive potential with a scan rate in the range from 8 V/s to 2-10³ V/s.

32. The method according to any one of claims 21-31 , wherein the sensitivity of detection of Cu(ll) ions concentration increases with the scan rate and/or value (more negative) of the accumulation potential and/or duration of applying the accumulation potential.