Field of the Invention The present invention relates to labelled nanoparticles, and electrochemical methods for their modification and detection.

Background to the Invention Labelled or tagged nanoparticles (NPs) have become increasingly used in sensing and biosensing applications. In one class of applications, modified silver or gold NPs are used to detect molecules binding to the modifier via surface-sensitive spectroscopies such as SPR and SERS [see references 1-6 at the end of the description, which are incorporated herein by reference in their entirety]. Similarly, fluorescent and colour- coded tags have also been used to enable rapid optical detection of target molecules [see references 7-10, which are incorporated herein by reference in their entirety]. Of particular interest has been the use of electrochemical methods to detect the tagged NPs: typically square wave or cyclic voltammetry [see references 11-15, which are incorporated herein by reference in their entirety]. In these applications, the tagged NPs have been immobilised onto the electrode before the voltammetric or electrochemical measurement.

Other methods for detecting tagged NPs include ICP-MS [see reference 16, which is incorporated herein by reference in its entirety], and electrochemiluminescence [see reference 17, which is incorporated herein by reference in its entirety].

It would be desirable to provide a method that is an alternative to or an improvement upon at least one method of the prior art. In particular, it would be desirable to provide a method that allows modification of a label of a labelled nanoparticle while in suspension. Additionally, it would be desirable to provide a method that allows detection and analysis of labelled nanoparticles while they are in suspension. Summary of the Invention

In a first aspect, there is provided a method of electrochemically modifying labelled nanoparticles, the method comprising:

(i) providing a working electrode, a counter electrode and the labelled nanoparticles suspended in a liquid carrier medium, wherein the liquid carrier medium is in contact with the working electrode;

(ii) applying a potential between the working electrode and the counter electrode, such that, at the working electrode, the label of a labelled nanoparticle is reduced or oxidised during a collision of a labelled nanoparticle in suspension in the liquid carrier medium with the working electrode. The method may further comprise detecting the current during a collision, preferably during a plurality of collisions, of a labelled nanoparticle, or the labelled nanoparticles, with the working electrode. The reduced or oxidised form of the label may or may not enable further reaction or modification of the label or other species in a desired manner.

In a second aspect, there is provided an apparatus for carrying out the method according to the first aspect, the apparatus comprising:

a working electrode, a counter electrode and labelled nanoparticles suspended in a liquid carrier medium, wherein the liquid carrier medium is in contact with the working electrode;

wherein a potential can be applied between the working electrode and the counter electrode, such that, at the working electrode, the label of a labelled nanoparticle is reduced or oxidised during a collision of a labelled nanoparticle in suspension in the liquid carrier medium with the working electrode.

In a third aspect, the present invention provides a method of electrochemically detecting labelled nanoparticles, the method comprising:

(i) providing a working electrode, a counter electrode and the labelled nanoparticles suspended in a liquid carrier medium, wherein the liquid carrier medium is in contact with the working electrode,

(ii) applying a potential between the working electrode and the counter electrode, such that, at the working electrode, the label of a labelled nanoparticle is oxidised or reduced during a collision of the labelled nanoparticle with the working electrode, and detecting the current during the collision. In a fourth aspect, the present invention provides an apparatus for detecting labelled nanopartieles, the apparatus comprising:

a working electrode, a counter electrode and the labelled nanopartieles suspended in a liquid carrier medium, wherein the liquid carrier medium is in contact with the working electrode;

wherein a potential can be applied between the working electrode and the counter electrode, such that, at the working electrode, the label of a labelled nanoparticle is oxidised or reduced during a collision of the labelled nanoparticle with the working electrode, and the current can be detected during the collision.

The present inventors have found that they can modify the labels of labelled nanopartieles, and, if desired, monitor the progress of the modification of the labels. They have also found that they can detect the presence of, and analyse, labelled nanopartieles by monitoring the charge transfer during collisions of the particles with the working electrode. Moreover, the detection can be selective in that it is possible to detect the presence of labelled nanopartieles over the presence of non-labelled nanopartieles and free labels that may have detached themselves from a nanoparticle and which are present in the liquid medium and/or on the working electrode. Monitoring the collisions and the charge passed during the collisions can provide valuable information about the labelled nanopartieles, such as an estimation of the average number of labels per nanoparticle, and the concentration of the nanopartieles in the liquid medium.

Brief Description of the Figures

Figure 1 shows cyclic voltammograms for (a) a NTP-modified Ag macroelectrode, (b) a NTP-modified GC macroelectrode and (c) NTP-tagged AgNPs modified GC macroelectrode. All scans were performed in a solution of 0.1 M NaCI0 ₄ and 10mM HCI0 ₄ at a scan rate of 50 mV s ^' NTP indicates 1 ,4-nitrothiophenol. The scans have been offset vertically for clarity. The tests carried out to produce these results, and those shown in the other Figures, are described in the Examples below.

Figure 2 shows chronoamperometric profiles for (a) reduction spikes for NTP-tagged AgNPs with zoom of circled transient (inset) and (b) no spikes for untagged AgNPs potentiostatted at -0.17V. Figure 3 shows the variation of the average charge per impact transient, Q, with the time allowed for NTP to adsorb onto AgNPs.

Figure 4 shows the distribution of charge passed per spike during NTP-tagged AgNPs collision with the substrate GC electrode.

Detailed Description

The present invention provides the first to fourth aspects described above. Optional and preferred features of the various aspects are described below. Unless otherwise stated, any optional or preferred feature may be combined with any other optional or preferred feature, and with any of the aspects of the invention mentioned herein.

Nanoparticles

In the present context, a labelled nanoparticle comprises a nanoparticle core having a label bound to the core.

The nanoparticle core may have a diameter of from 1 to 500 nm, optionally from 1 to 200 nm, optionally from 10 to 90 nm, optionally from 20 to 80 nm, optionally from 30 to 70 nm. The liquid carrier medium may comprise labelled nanoparticles having a nanoparticle core mean diameter of from 1 to 500 nm, optionally from 1 to 200 nm, optionally from 10 to 90 nm, optionally from 20 to 80 nm, optionally from 30 to 70 nm. The diameter of the nanoparticle core can be determined by scanning electron microscope (SEM) imaging, as would be appreciated by the skilled person. The nanoparticle core can be any suitable regular or irregular shape, including spherical, and elongated, for example a rod-shaped nanoparticle core. If a nanoparticle core is non-spherical, the diameter of the nanoparticle core as measured herein will be the smallest diameter across the core. The mean diameter of nanoparticle cores in the liquid carrier medium can be measured using SEM imaging of a sample of the nanoparticles, e.g. before labelling, and calculating the number average (i.e. mean) diameter for 100 nanoparticles or more, optionally 200 nanoparticles or more, optionally 300 nanoparticles or more. The nanoparticle core may comprise any suitable material. Preferably, the material of the nanoparticle core is not itself oxidised or reduced at the potential at which the label is oxidised or reduced during the method of the first or second aspect. The nanoparticle core may comprise a metal, a semi-metal or a non-metal. Preferably, the nanoparticle core is or comprises an electrically-conducting material. Optionally, the nanoparticle core comprises a semi-conductor material. Preferably, the nanoparticle core comprises a metal, preferably in elemental form. The metal may be any suitable metal, including, but not limited to, a metal selected from any of groups 3 to 14 of the periodic table. The metal may be selected from a transition metal of any of groups 3 to 12 of the periodic table. Optionally, the nanoparticle core comprises a metal selected from a group 11 of the periodic table. Optionally, the nanoparticle core comprises a metal in elemental form selected from copper, silver, gold, ruthenium, osmium, rhodium, iridium, palladium and platinum. Preferably, the nanoparticle core comprises a metal in elemental form selected from silver and gold.

In an embodiment, the nanoparticle core comprises a semiconductor. The nanoparticle core may comprise a semi-conductor material selected from one or more of a group IV elemental semiconductor, such as silicon (Si) and germanium (Ge); group IV compound semiconductors, such as silicon germanide (SiGe); and group lll-V semiconductors, such as aluminium antimonide (AlSb) and indium phosphide (InP). Semiconductor nanoparticles, sometimes termed semiconductor nanocrystals, are described, for example, in WO2007/013877, which is incorporated herein by reference in its entirety.

In an embodiment, the nanoparticle core may comprise a polymer. The polymer may be crosslinked. The nanoparticle core may comprise a natural polymer and/or a synthetic polymer. The natural polymer may be selected from, for example, sugars, starches, dextrans, polysaccharides, proteins, alginates, and cellulose. The synthetic polymer may, for example, be a polymer of a monomer selected from an acrylate monomer, an ethylenic monomer and a vinylaromatic monomer. Polymeric nanoparticles and their methods of production are described, for example, in European Patent Application No. EP 1447074, which is incorporated herein by reference in its entirety. The nanoparticle core, may comprise, consist essentially of, or consist of, any of the materials mentioned above. If a nanoparticle core consists essentially of a material, this indicates that preferably the core comprises at least 95 % by weight of the material, preferably 98 % by weight, preferably 99 % by weight, of the material.

Optionally, the nanoparticle core has a core-shell structure. Label As mentioned above, a labelled nanoparticle comprises a nanoparticle core having a label bound to the core. The label may comprise a plurality of label molecules bound to the nanoparticle core. The label molecules may form a partial or complete covering of the nanoparticle core. Molecules, in this context, includes species covalently or ionically bound to the nanoparticles core. Optionally, the label molecules form a partial or complete monolayer, for example a self-assembling monolayer, on the nanoparticle core.

The label may be any label capable of being oxidised or reduced within the electrochemical window of the liquid carrier medium and attached to a nanoparticle core. The label may be or comprise an organic or inorganic species. In an embodiment, the label is or comprises a species of the formula— L— F, where L is a linker group, optionally an organic linker group, that binds to the nanoparticle core, and F is a functional group that is oxidised or reduced during step (ii) of the method according to the first or third aspects. F may extend away from the nanoparticle core.

In an embodiment, -L- is of the formula -X-R -, wherein X is a species that binds to the core nanoparticle and R is a linker species, optionally an organic species, that covalently binds to F. In an embodiment, X is selected from S and C0 ₂ . In an embodiment, R comprises or is an organic linker group, optionally selected from an aliphatic group, an aromatic group, a polymeric moiety formed from the polymerisation of one or more monomers, and combinations thereof. In an embodiment, F may be selected from a nitro group, a carboxy group, an ester group, an aldeyhyde group, a ketone group, a nitrile group, and a hydroxy group. The carboxy group may be in free or acidic form. The reduction of a label molecule or the functional group F of a label molecule, during step (ii) of the method of the first or third aspect, may be at least a one-electron reduction, optionally a two-electron reduction, optionally a three-electron reduction, optionally a four-electron reduction. The oxidation of a label molecule or the functional group F of a label molecule, for example a label molecule, during step (ii) of the method of the first or third aspect, may be at least a one-electron oxidation, optionally a two- electron oxidation, optionally a three-electron oxidation, optionally a four-electron oxidation. The number or electrons involved in the oxidation or reduction will depend on the nature of the label being oxidised or reduced and the potential applied to the working electrode, as would be appreciated by the skilled person.

Aliphatic, when mentioned herein, includes, but it not limited to, linear, cyclic or branched optionally substituted alkyl, alkylene, alkene, alkenylene, alkyne and alkynylene groups, preferably alkyl or alkylene, optionally containing from 1 to 20 carbon atoms, optionally preferably from 2 to 10 carbon atoms, not including any substituents that may be present.

Alkylene, when mentioned herein, includes, but it not limited to, linear, cyclic or branched optionally substituted alkylene, optionally containing from 1 to 20 carbon atoms, optionally from 2 to 10 carbon atoms, not including any substituents that may be present. Alkylene includes, but is not limited to, a species of formula -(CH2)n-, where n is 1 to 20, preferably from 2 to 10, more preferably from 4 to 8, optionally 5, 6 or 7.

Aromatic, when mentioned herein, includes, but is not limited to, optionally substituted phenyl and naphthyl.

Optional substituents include, but are not limited to, -N02, optionally substituted phenyl, aryl, heteroaryl, arylalkyl, alkylaryl, heteroarylalkyl, alkylheteroaryl, alkoxy, aryloxy, arylalkoxy, acyl, aroyl, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, arylalkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl.

Alkyl, when mentioned herein, includes, but is not limited to, linear, cyclic or branched optionally substituted alkyl group, preferably containing from 1 to 20 carbon atoms, most preferably from 2 to 10 carbon atoms, not including any substituents that may be present on the alkyl group. Aryl, where mentioned herein, includes an aromatic group, including, but not limited to, optionally substituted phenyl and naphthyl.

In an embodiment, the nanoparticle core comprises or consists of a metal, optionally selected from silver and gold, and the label is or comprises the species of the formula - -L— F, wherein -L- is of the formula -X-R -, wherein X is a species that binds to the nanoparticle core and R is a linker species that covalently binds to F, X being optionally selected from S and C0 ₂ and R comprising or being optionally selected from an aromatic or aliphatic group, and F is optionally selected from a nitro group, a carboxy group, an ester group, an aldeyhyde group, a ketone group, a nitrile group, and a hydroxy group. The carboxy group may be in free or acidic form.

In an embodiment, the nanoparticle core comprises or consists of a metal, selected from silver and gold, and the label is or comprises the species of the formula— L— F, wherein -L- is of the formula -X-R ¹ -, wherein X is a species that binds to the nanoparticle core and R is an linker species that covalently binds to F, X being selected from S and C0 ₂ and R comprising or being selected from an aromatic or aliphatic group, and F is selected from a nitro group, a carboxy group, an ester group, an aldeyhyde group, a ketone group, a nitrile group, and a hydroxy group. The carboxy group may be in free or acidic form.

In an embodiment, the label comprises or is 1 ,4-nitrothiophenol.

In an embodiment, the label may comprise one or more species selected from amino acids, purines, pyrimidines, nucleosides, nucleotides, proteins, DNA, RNA, antibodies, dye molecules, and fluorescent species.

Liquid carrier medium The liquid carrier medium may be any suitable medium. Preferably, the liquid carrier medium comprises, consists essentially of, or consists of (excluding any dissolved solutes and suspended nanoparticles), water. If the carrier medium consists essentially of water, preferably the liquid carrier medium (excluding any dissolved solutes and labelled nanoparticles) comprises at least 98% by weight water, preferably at least 99% by weight water, preferably at least 99.5% by weight water. The water, before the addition of the nanoparticles, may have a resistivity of at least 18 MQ.cm, preferably 18.2 MQ.cm. Optionally, the liquid carrier medium is degassed before the addition of the labelled nanoparticles. The liquid carrier medium may be degassed using any suitable technique, for example degassing using N ₂ under an atmosphere of N ₂ .

The labelled nanoparticles are suspended in the liquid carrier medium. Preferably, the labelled nanoparticles are dispersed in the liquid carried medium. The labelled nanoparticles may be dispersed in the liquid carrier medium using any suitable technique. In an embodiment, the nanoparticles are added to the liquid carrier medium and then dispersed in the liquid carrier medium using ultrasound, optionally stirring or otherwise agitating the liquid carrier medium as required.

Electrodes The working electrode may comprise any suitable material. Preferably, the surface of the working electrode comprises an inert material, such that it is not itself oxidised or reduced under the conditions at which the method is carried out. Suitable electrodes are available to the skilled person. The working electrode may have a surface comprising a metal or carbon. Optionally, the whole of the electrode comprises a metal or carbon. The working electrode may comprise a metal selected from gold, silver and platinum. The working electrode may comprise a carbon-containing material, which may be selected from edge plane pyrolytic graphite, basal plane pyrolytic graphite, glassy carbon, boron doped diamond, highly ordered pyrolytic graphite, carbon powder and carbon nanotubes. In a preferred embodiment, the working electrode is a glassy carbon electrode.

The present inventors have found that using a carbon-containing working electrode, the potential at which a label is reduced or oxidised when bound to the nanoparticle is of smaller magnitude than the potential at which the label is reduced or oxidised when bound to the electrode in the absence of nanoparticles. The difference between the potential at which the label is oxidised or reduced when bound to the nanoparticle compared to when it is bound to the electrode has been found to be sufficiently high that it is possible to selectively detect the labelled nanoparticles over dissociated or free labels that may be present in the liquid medium or on the surface of the electrode. The counter electrode may be made of any suitable material, for example a metal or carbon. If the counter electrode is in contact with the liquid carrier medium, preferably the material of the counter electrode is selected such that it is not itself oxidised or reduced under the conditions at which the method is carried out. The counter electrode may comprise a metal selected from gold, silver and platinum. The counter electrode may comprise a carbon-containing material, which may be selected from edge plane pyrolytic graphite, basal plane pyrolytic graphite, a glassy carbon, boron doped diamond, highly ordered pyrolytic graphite, carbon powder and carbon nanotubes. In a preferred embodiment, the counter electrode is a graphite electrode.

In a preferred embodiment, both the working electrode and the counter electrode are carbon-containing electrodes.

A reference electrode, for example a Ag/AgCI reference electrode or a saturated calomel reference electrode, may be connected to the working and/or counter electrodes as is known in the art. The working electrode, the counter electrode and the reference electrode may be controlled by a potentiostat, as is known by the skilled person. Any appropriate number of electrodes may be used. For example, just two electrodes may be used, i.e. a single working electrode and a counter electrode, or optionally three electrodes may be present, e.g. a single working electrode, a single counter electrode and a single reference electrode. In embodiments, a second working electrode may be present with a reference electrode as part of a four-electrode system.

A means for applying a potential is electrically connected to the working and counter electrodes. The means for applying a potential can be any suitable means, for example a potentiostat, as mentioned above. In an embodiment, if the label is reduced in the method, the potential applied to the working electrode is of sufficient magnitude to effect the reduction of the label on the nanoparticle, but at a potential more positive than the potential required to effect reduction of the label when bound to the working electrode in the absence of the nanoparticles, but all other conditions being the same. In an embodiment, if the label is reduced in the method, the potential applied to the working electrode is of sufficient magnitude to effect the reduction of the label on the nanoparticle, but at a potential of at least 0.1 V, optionally at least 0.2 V, optionally 0.3 V, optionally at least 0.4 V, optionally at least 0.5V more positive than the potential required to effect reduction of the label when bound to the working electrode in the absence of the nanoparticles, but all other conditions being the same. For example, in the Examples, the present inventors describe the electrochemical detection of silver nanoparticles tagged with 1 ,4-nitrothiophenol (NTP). Here, they found the potential at which the NTP is reduced when on the silver nanoparticles is -0.17 V - this was the potential at which a peak in current was shown in the cyclic voltametry experiment. However, when the NTP was bound to the same electrode (but not to any silver nanoparticles), the NTP was not reduced until the potential was more negative at about -0.31 V. Accordingly, when detecting the NTP-tagged silver nanoparticles at a potential of -0.17 V, the inventors could be certain that the reduction spikes they observed were only those of NTP reduction during a collision of NTP-tagged nanoparticles, not reduction of dissociated or free NTP. In an embodiment, if the label is oxidised in the method, the potential applied to the working electrode is of sufficient magnitude to effect the oxidation of the label on the nanoparticle, but is at a potential more negative than the potential required to effect oxidation of the label when bound to the working electrode in the absence of the nanoparticles, but all other conditions being the same. In an embodiment, if the label is oxidised in the method, the potential applied to the working electrode is of sufficient magnitude to effect the oxidation of the label on the nanoparticle, but at a potential of at least 0.1 V, optionally at least 0.2 V, optionally 0.3 V, optionally at least 0.4 V, optionally at least 0.5V more negative than the potential required to effect oxidation of the label when bound to the working electrode in the absence of the nanoparticles, but all other conditions being the same.

The method of the first aspect may be used to modify labelled nanoparticles, while detecting the current during collisions of the labelled nanoparticles with the working electrode. Optionally, in the method of the first or second aspects, the frequency of collision of the labelled nanoparticles with the working electrode is determined. This can be determined by, for example, monitoring the current generated by each collision. With each collision, a spike in current is observed, and the number of spikes N, within a given time period T, can be used to determine the frequency of collision (N/T). The time period T may be, for example a period of at least 0.5 s, optionally at least 1 s, optionally at least 5 s. The time period T may be, for example, a period of from 0.5 s to 30 s, optionally from 1 s to 15 s. The present inventors have found that the rate of collision correlates with the concentration of the labelled nanoparticles in the liquid medium. Optionally, the rate of collision is used to determine the concentration of the labelled nanoparticles in the liquid carrier medium. The rate of collision found during the method can be compared, for example, to reference values for rates of collision determined in a calibration method in which the concentration of labelled nanoparticles was known; this allow the rate of collision to be used to calculate a concentration. Optionally, the method of the first or third aspect includes a step before step (ii), and optionally before step (i), of carrying out a calibration to determine how the rate of collision varies with the concentration of the labelled nanoparticles in the liquid carrier medium.

Optionally, in the method of the first or second aspects, the charge passed per collision of a labelled nanoparticle with the working electrode is monitored. The charge passed per collision may be monitored by integrating the current over a time period of a collision, which may be by integrating the current over a spike of current produced by the collision. The spike in current corresponds to a collision. Optionally, the average charge passed per collision is determined. The average charge passed per collision may be determined by dividing the sum of the total charge passed for a given number M of collisions by M. M may be any suitable amount, e.g. 10 or more, optionally 20 or more, optionally 50 or more, optionally 10 to 100, optionally 10 to 300. Optionally, the average charge passed per collision is determined and used to estimate the average number of label molecules on a nanoparticle. The average charge passed Q (in Coulombs) is related to the average number of label molecules N on a nanoparticle and the number of electrons M required to oxidise or reduce a single label molecule by the equation: Q = MeN, where e is the charge on an electron in Coulombs. Accordingly, where Q and M are known, N can be calculated. Average in the present context is the number mean. Similarly, the surface area S for a spherical nanoparticle is given by the equation S=4TTr ² , wherein r is the radius of the nanoparticle. This equation can also be used for non-spherical nanoparticles to give an approximation of their surface area. Where nanoparticles have full surface coverage, and S is known and Q is known (e.g. calculated from the equation above), it is possible to calculate or estimate the average area on the surface of a nanoparticle covered by each label molecule. Additionally, using the equations above, where the average surface coverage of labels on a nanoparticle is known, the average charge passed per collision may by used to calculate the radius of a nanoparticle. The calculations or estimates mentioned herein may be carried out on an automated apparatus, for example using a computer.

The method may be carried out at any suitable temperature and pressure. Optionally, the method is carried out at a temperature of from 0 °C to 50 °C, optionally from 10 °C to 30 °C. Optionally, the method is carried out at a pressure of from 90 kPa to 1 10 kPa, optionally from 95 KPa to 105 kPa, optionally from 98 kPa to 102 kPa.

Embodiments of the present invention will now be described with reference to the following non-limiting Examples and the accompanying drawings.

EXAMPLES

Citrate-capped silver nanoparticles (AgNPs) of mean radius 45 ± 5 nm were synthesised according to the method according to Pyatenko et al [27, 28]. Sodium perchlorate (≥ 98%, NaCI0 ₄ ) and NTP (80%) were supplied by Sigma-Aldrich®. Perchloric acid (70%, HCI0 ₄ ) was purchased from Fisher Chemicals. All solutions were made using ultrapure water of resistivity ≥18.2 ΜΩ.αη at 298K (Millipore). AgNPs were modified with NTP by mixing AgNPs colloid with 0.5mM NTP aqueous solution with a volume ratio of 1 : 1 for 2 to 40 hours. The excess NTP molecules were removed by centrifuging and washing the tagged NPs three times. The electrochemistry experiments were conducted within a faraday cage using a μΑυίο^ III (Metrohm®-Autolab BV, Utrecht, Netherlands) and a three electrode arrangement. Working electrodes used were: a bare silver macro electrode (0.3mm in diameter), or a bare glassy carbon (GC, 3mm in diameter) electrode, or a AgNP-modified GC electrode, or a 1 1 μηι diameter GC microelectrode. Reference and counter electrodes were a Ag/AgCI (Radiometer®, Copenhagen), and a graphite rod respectively. To modify the GC electrode, 2.5 μΙ_ of the suspension of AgNPs was drop-cast on the GC electrode and left to dry under a N ₂ atmosphere. Figure 1 shows cyclic voltammograms for (a) a NTP-modified Ag macroelectrode, (b) a NTP-modified GC macroelectrode and (c) NTP-tagged AgNPs modified GC macroelectrode. All scans were performed in a solution of 0.1 M NaCI0 ₄ and 10mM HCI0 ₄ at a scan rate of 50 mV s ^' The scans have been offset vertically for clarity.

Example 1

Control experiments were conducted to confirm the voltammetry of NTP adsorbed on silver and GC electrode surfaces. The Ag macroelectrode was first dipped into a 0.1 mM solution of NTP for 1 hour, then sonicated in water to remove the excess molecules from the electrode surface, and then transferred to a solution of 100mM NaCI0 ₄ and 10mM HCI0 ₄ . Cyclic voltammograms (CV) were recorded as shown in Figure 1a, which shows the first reduction peak at ca -0.17 V on the silver surfaces (both bulk and NP-modified) and at ca -0.31 V on naked GC: in agreement with the literature [29-31]. The first reduction peak was observed to occur at -0.167 V, corresponding to the four-electron, four-proton reduction to the hydroxylamine (see Scheme 1). No peaks were observed for the second scan showing that a self- assembled NTP monolayer was formed on AgNPs.

Scheme 1 - the 4-electron reduction of adsorbed NTP Next an analogous experiment was conducted using a clean GC macroelectrode

(see Figure 1 b), where the reduction wave was now observed at -0.308 V.

Finally, the clean GC macroelectrode was modified with the NTP-tagged AgNPs by drop-casting and a CV recorded (shown in Figure 1 c). This shows the first reduction peak of NTP occurring at -0.158 V: in the context of the following impact experiments it is important to note that there is no reduction signal observable on the bare GC macroelectrode at this potential.

Figure 2 shows chronoamperometric profiles for (a) reduction spikes for NTP- tagged AgNPs with zoom of circled transient (inset) and (b) no spikes for untagged AgNPs potentiostatted at -0.17V.

Example 2 To measure the particle impacts, the cell was assembled with the GC microelectrode, reference and counter electrodes, and with 20ml_ of an aqueous solution of 100mM NaCI0 ₄ and 10mM HCI0 ₄ which was thoroughly degassed with nitrogen prior to the experiment. Chronoamperograms were recorded at the potential of -0.17 V in the absence of NPs to confirm that no impact spikes could be observed. Next, a known amount of NTP-tagged AgNPs were added, the solution briefly bubbled with nitrogen to evenly disperse NPs and the potential held first at 0V and then at -0.17V. No spikes were observed at the former potential whilst they were clearly observed at the latter shown in Fig 2 (a) due to the reduction of nitro group while NTP-tagged AgNPs were in contact of the substrate GC microelectrode. The collision frequency was found to be in the range of 0.1 s ^" pM ^" for all NP concentrations studied, which is in good agreement with our previous result for the oxidation of AgNPs [24,32]. Comparative Example 3

Another control experiment was done at the potential of -0.17V with untagged AgNPs in the solution and no spikes were shown (Fig 2(b)), confirming again that the reduction of NTP on the surface of NPs is the source of the transients.

Example 4 - Results and Analysis

The variation in the average charge passed per transient was found to vary according to the length of time that the AgNPs were immersed in NTP solution during the modification process: the charge passed per impact spikes increases up to 8 hrs and almost levels off when the immersion time is over 8 hrs (see figure 3, which shows the variation of the average charge per impact transient, Q, with the time allowed for NTP to adsorb onto AgNPs).

For a modification time of 30 hours, the analysis of the obtained several hundred spikes gave a mean charge (Q) of 2.72 x 10 ^"13 C passed per impact transient. Q, on the other hand, is related to the number of NTP molecules reduced per collision, N, via the electronic charge according to

Q = 4eN (1)

The present inventors consider that all NTP molecules on a nanoparticle would be reduced in a collision. This allows us to calculate an average number of NTP molecules attached to the surface of single AgNPs of 4.3 x 10 ⁵ . Given the surface area of a single AgNP is

S=4TTI- ² (2) and the size of a NTP molecule, and assuming NTP molecules are vertically aligned on the NPs surface, a number of 5.5 x 10 ⁵ NTP molecules can be calculated in terms of a full monolayer coverage on the surface of a single AgNP. Therefore, we can conclude that an approximate monolayer of NTP was formed on the surface of AgNPs in our experiment and can be detected during the tagged NPs colliding with the electrode. Figure 4 shows the distribution of charge passed per spike during NTP-tagged AgNPs collision with the substrate GC electrode. We note that the majority of the charges are centred in the range of 1.0 to 4.0 x 10 ^"13 C. The variation in the charges passed will reflect variations in (i) the NTP coverage of the NPs, (ii) the size and degree of aggregation of the NPs, (iii) the contact times between NP and electrode. In addition the contact time required to effect full reduction of the complete NTP coverage of the impacting NP will depend on NP size [26] as well as the kinetics of the NTP reduction: note there are two possible routes for the electron transfer in this scenario, namely direct electron conduction 'through' the metal NP, or a hopping mechanism around the NTP-covered surface [33,34].

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