WO2014188166A1

WO2014188166A1 - Electrochemically determining the concentration of a halide or a pseudohalide

Info

Publication number: WO2014188166A1
Application number: PCT/GB2014/051530
Authority: WO
Inventors: Her Shuang TOH; Christopher BATCHELOR-MCAULEY; Kristina TSCHULIK; Richard Guy Compton
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2013-05-20
Filing date: 2014-05-19
Publication date: 2014-11-27
Anticipated expiration: 2015-11-20
Also published as: GB201309055D0

Abstract

The present invention relates to a method of determining the concentration of an anion selected from a halide ion and a pseudohalide ion in a sample of interest, the method involving: providing a working electrode having a substance in a first oxidation state on a surface thereof, wherein the electrode is in contact with the sample of interest that contains the anion; carrying out a voltammetry or chronoamperometry experiment that involves increasing the potential applied to the working electrode to a first potential to oxidise the substance to a second oxidation state, wherein, at the first potential, the electrochemical response of the substance is different when the anion is present in the sample compared to when the anion is absent in the sample, and obtaining electrochemical information during the voltammetry or chronoamperometry experiment; using a determined relationship between the electrochemical information and the concentration of the anion in a reference sample to calculate the concentration of the anion in the sample of interest. An apparatus for carrying out the method and use of an electrode having silver on a surface thereof are also disclosed herein.

Description

Electrochemically determining the concentration of a halide or a pseudohalide

Field of the Invention The present invention relates to the determination of the concentration of a halide ion or pseudohalide ion in a sample, e.g. a biological sample. It can be used, for example, to determine the concentration of chloride in a sweat sample, as a preliminary screening for cystic fibrosis. Other possible uses are described herein. Background

The concentration of halides, such, as chlorides, and pseudohalides, such as cyanides, in a biological sample can give valuable information on the health of an individual. As just one example, cystic fibrosis is a common disease which has an associated characteristic symptom of high sweat chloride content. Cystic fibrosis (CF) is a common genetic disease caused by an autosomal recessive gene known as the cystic fibrosis transmembrane conductance regulator (CFTR) gene.¹ It affects multiple organs including the lungs and the intestines. The defective gene leads to an expression of a non-functional channel protein, leading to irregular transport of sodium and chloride ions across the epithelial cells.² " In the bronchial tissues, chloride ions are unable to move across the epithelial cells, leading to a high chloride concentration in the mucus in the lungs. CF patients suffer from chronic bacterial lung infection as the salt-sensitive antibodies are inactivated.-' - It is a life-long incurable disease with only symptom alleviation available.⁶ A person is diagnosed as a cystic fibrosis patient when one has a high sweat chloride content and two genetic mutations of CFTR gene.^{5, z}. Since one of the main symptoms for CF is a high level of chloride in sweat, a borderline sweat test is used in hospitals as a primary screening and diagnosis method for CF, along with genetic analysis of FTIR gene as a diagnosis test.^2, - For the borderline sweat test, the sweat chloride level is measured and matched against a guideline. The references for sweat chloride levels for adults are categorised into: less than 39 mM (negative), 40 - 59 mM (borderline) and greater than 60 mM (positive). For infants, sweat chloride levels are categorised as: less than 29mM (negative), 40 - 59 mM (borderline) and greater than 60 mM (positive). '-^'1" The Cystic Fibrosis Foundation from the US recommends a sweat chloride sensor range to be 10 - 160 mM.— The Association of Clinical Biochemistry from the UK states that the maximum level of sweat chloride in human is 160 mM. ^{"6 "7} The current methods for determination of chloride levels include colorimetry, coulometry, indirect ion selective electrodes, direct ion selective electrodes and manual titration using Schales procedure.^{12 1}" The Association of Clinical Biochemistry based in United Kingdom recommends coulometry out of the various methods available.— This method uses argentometry where a standard silver ion solution (e.g silver nitrate) is used to titrate the chloride ions through the spontaneous formation of silver chloride. The conductivity of excess silver ions is used to determine the end point and hence the chloride level in sweat. Nonetheless, the coulometry equipment faces laboratory constraints. Trained personnel are required to operate the equipment and special buffers are required for selectivity and sensitivity.¹⁸

Similarly, the excess or deficiency of iodide in the human body can be indicative of a range of health problems, which are often associated with the thyroid. The current method used for determination of iodine in a human body involves a urine sample collection. Patients receive and ingest an iodine tablet and the iodine level in the urine is measured over 24 hours (preferred) or on a single morning. Sandell-Kolthoff reaction is performed on the urine sample before the iodine levels are measured spectrophotometrically or colorimetrically. Sandell-Kolthoff involves a pretreatment of chloric acid digestion or oxidation with ammonium persulphate to remove interference. The main reaction involves reduction of yellow eerie ions by arsenic in presence of iodide to form colourless cerous ions. The disappearance of yellow eerie ions determines the iodine concentration by cross referencing with a determined relationship between known iodine concentration and absorbance. Alternatively, ICP- MS can also be used to determine iodine concentration.

It would be desirable to provide an alternative to, and, ideally, an improvement upon the methods of the prior art that allow the efficient determination of halide ion or pseudohalide ion concentration. Brief Description of the Figures

Figure 1 shows a schematic diagram for the preparation and use of a silver nanoparticle modified screen printed electrode and the anodic stripping voltammogram of silver nanoparticles, as described in the Examples below. Step 1 : Drop casting of silver nanoparticle suspension onto a screen printed electrode with micropipette. Step 2: Dry electrode in a 50°C oven for 12 minutes. Steps 1 and 2 are repeated five times. The modified electrodes are used for anodic stripping voltammetry in two conditions: in the absence of chloride ions and in the presence of chloride ions. In the presence of different concentration of chloride ions, the peak height of the voltammetric signal at the lower potential increases with increasing chloride concentration.

Figure 2 shows nanoparticle tracking analysis for silver nanoparticles used in the Examples below. Mean diameter = 35 nm.

Figure 3 shows scanning electron microscopy image for silver nanoparticles used in the Examples below.

Figure 4 shows the oxidative stripping of silver nanoparticles from a glassy carbon electrode in 0.1 M sodium nitrate spiked with different amount of potassium chloride (KCI) at a scan rate of 0.05 V s^' The experiment is described in more detail in the Examples below. Grey dashed line: No KCI; Black solid line: 2mM KCI; Black dashed Line: 8mM KCI; Black dotted line: 16mM KCI; Grey solid line: 32mM KCI. Reference electrode: Ag/AgN0₃ (10 mM) & NaN0₃ (90 mM) Counter electrode: Platinum mesh; Inlay: Calibration plot of peak height of peak A against the concentration of chloride ions. Each data point consists of a minimum of 3 repeats.

Figure 5 shows the oxidative stripping of silver nanoparticles from a screen printed electrode in 0.1 M sodium nitrate spiked with different amount of potassium chloride at a scan rate of 0.05 V s^' The peak height of peak A (see Figure 4) is plotted against concentration of chloride. Each data point consists of a minimum of 3 repeats. The experiment is described in more detail in the Examples below.

Figure 6 shows the oxidative stripping of silver nanoparticles from a screen printed electrode in 0.1 M sodium nitrate with different synthetic sweat samples (diluted by a factor of 4) at a scan rate of 0.05 V s^' Black solid line: Synthetic sweat sample 1 ; Black dash line: Synthetic sweat sample 2. Inlay: Black line: Calibration plot of peak height of peak A against concentration of chloride from screen printed electrode data; Triangle: Diluted synthetic sweat sample 1 ; Star: Diluted synthetic sweat sample 2. Each data point consists of three repeats.

Summary of the Invention

In a first aspect, the present invention provides a method of determining the concentration of an anion selected from a halide ion and a pseudohalide ion in a sample of interest, the method involving:

providing a working electrode having a substance in a first oxidation state on a surface thereof, wherein the electrode is in contact with the sample of interest that contains the anion;

carrying out a voltammetry or chronoamperometry experiment that involves increasing the potential applied to the working electrode to a first potential to oxidise the substance to a second oxidation state, wherein, at the first potential, the electrochemical response of the substance is different when the anion is present in the sample compared to when the anion is absent in the sample, and obtaining electrochemical information during the voltammetry or chronoamperometry experiment; using a determined relationship between the electrochemical information and the concentration of the anion in a reference sample to calculate the concentration of the anion in the sample of interest. Electrochemical information may include, but is not limited to, peak current, peak charge and peak potential. In a second aspect, the present invention provides an apparatus for determining the concentration of an anion selected from a halide ion and a pseudohalide ion in a sample of interest, the apparatus comprising a working electrode having a substance in a first oxidation state on a surface thereof, the apparatus adapted to carry out a method involving:

contacting the working electrode with the sample of interest;

carrying out a voltammetry or chronoamperometry experiment that involves increasing the potential applied to the working electrode to a first potential to oxidise the substance to a second oxidation state, wherein, at the first potential, the electrochemical response of the substance is different when the anion is present in the sample compared to when the anion is absent in the sample, and obtaining electrochemical information during the voltammetry or chronoamperometry experiment; using a determined relationship between the electrochemical information and the concentration of the anion in a reference sample to calculate the concentration of the anion in the sample of interest. In a third aspect, the present invention provides a use of an electrode having silver on a surface thereof in an electrochemical test method to determine the concentration of chloride ions in a sweat sample, wherein the electrochemical test method involves oxidation of the silver from Ag (0) to Ag(l) on the electrode in the sweat sample. As explained below, preferably a finite amount of silver is present on the working electrode, for example in the form of silver nanoparticles, such that peaks of current of a desired height are observed.

In a fourth aspect, the present invention provides use of an electrode having silver on a surface thereof in an electrochemical test method to determine the concentration of cyanide ions in a sample, wherein the electrochemical test method involves oxidation of the silver from Ag (0) to Ag(l) on the electrode in the sample.

The present inventors have developed an electrochemical method for determining the concentration of a halide or a pseudohalide of interest in a sample, e.g. a biological sample. The method can be cost effective and convenient. They have found that certain substances, e.g. certain metals, give a different electrochemical response in a sample, depending on whether or not a halide ion or pseudohalide ion is present in the sample, and that the response has a relationship with the concentration of the halide or pseudohalide in the sample. In one embodiment, and as shown in the Examples below, the electrochemical oxidation of silver nanoparticles in the absence of chloride ions gives one voltammetric signal related to the oxidation of silver to silver ions (in solution). The presence of chloride ions in the solution causes the appearance of an additional signal at a lower potential which is related to the oxidation of silver to silver chloride (an insoluble salt). This signal has a peak height which correlates linearly with the concentration of chloride ions, e.g. from 2 mM to 40 mM, when the electrochemical experiments are performed on silver nanoparticle modified screen printed electrodes or glassy carbon electrodes. Accordingly, reliable quantification was found to be achievable. Furthermore, chloride ion levels of diluted synthetic sweat samples are measured accurately with the modified electrodes. Thus, the method and apparatus described herein may be used in a cost-efficient, fast and reliable point-of-care system for the preliminary screening of cystic fibrosis. Detailed Description

The present invention provides the first to the fourth aspects described herein. Preferable and optional features are described below. Any of the preferable and optional features are applicable to any aspect of the invention, unless otherwise stated. Any preferable or optional feature may be combined with any other preferable or optional feature. In the method, typically the substance in its first oxidation state contacts the sample of interest. Preferably, at the first potential, when the anion is present in the sample, the substance in its second oxidation state forms a species with the anion in which the substance is in a different local environment compared to if it were oxidised to its second oxidation state in the sample in the absence of the anion in question. Preferably, at the first potential, the substance in its second oxidation state forms a solid compound with the anion on the electrode or forms a complex with the anion; and such a complex may or may not be dissolved in the sample. Preferably, in the absence of the anion in the sample of interest, if the potential is raised to oxidise the substance from the first oxidation state to the second oxidation state, the substance in its second oxidation state is soluble in the sample. If the complex is formed at the first potential, raising the potential above the first potential to a second potential may involve the dissociation of the substance and the anion. If the solid compound is formed on the electrode at the first potential, raising the potential above the first potential to a second potential may involve the dissolution of the compound into the sample in its second oxidation state. As an illustration, the substance may be silver, and the anion may be chloride. This is demonstrated in the Examples. At the start of the method in the Examples, the silver is in an elemental form (i.e. its first oxidation state is 0). When the silver on the working electrode is oxidised in the aqueous sample in the absence of chloride ions, a single peak in current is observed; this relates to the formation of silver (I) ions in solution. However, when chloride ions are present in the sample, two peaks are observed in the voltammetry experiment: a first peak that relates to the formation of solid silver chloride compound on the electrode, and, at a higher potential, a second peak that relates to the formation of silver (I) ions in solution. The method involves a voltammetry or a chronoamperometry experiment. The voltammetry will typically involve monitoring of the current at the working electrode as the potential between the working electrode and a further electrode is changed; the further electrode may be selected from a reference electrode and a counter electrode; in some examples a counter electrode and a reference electrode are both used in the voltammetry, and a potential may be applied between the working electrode and the reference electrode. The voltammetry may involve a voltammetry technique selected from cyclic voltammetry, square wave voltammetry, linear sweep voltammetry, pulse voltammetry, such as normal pulse or differential pulse voltammetry; or an amperometry technique such as chronoamperometry may be used. The voltammetry may be used to obtain electrochemical information, such as the potential, peak current and/or peak charge at which the oxidation of the substance on the working electrode occurs.

The voltammetry may involve increasing the potential at the working electrode until a peak in current is observed at a certain potential, and continued until the current has dropped from this peak and then started to rise again. The potential may be raised at a constant rate, for example at a rate of 0.01 to 0.1 Vs^"1 , optionally at a rate of from 0.03 Vs^"1 to 0.07 Vs^{"1 ,} optionally at a rate of 0.04 to 0.06 Vs^"1 , optionally about 0.05 Vs^"1. Preferably, when the anion is present in the sample of interest, two peaks in current can be seen in the voltammetry experiment: a first peak that may, for example, represent formation on the working electrode of a solid compound of the substance in its second oxidation state with the anion or a complex of the substance in its second oxidation state with the anion, and the second peak that represents formation of ions of the anion and the substance in its second oxidation state, both of which are in solution in the sample and may be dissociated from one another, e.g. not forming a complex together; optionally the height or area of the first peak may be used as the electrochemical information. The first potential may be a potential in the range of -0.3 V to -0.1 V, optionally in the range of -0.3 to -0.2 V, optionally about -0.25V (vs Ag/AgN0₃ (10 mM) as a reference electrode), for example when using a glassy carbon working electrode having silver nanoparticles thereon, and optionally using other conditions given in the Examples below.

The electrochemical information may be the height or area of a peak of current at the first potential, and preferably when the substance in its second oxidation state forms a solid compound on the electrode with the anion or forms a complex with the anion. In an embodiment, a first peak of current is observed at the first potential, and the method further involves raising the potential above the first potential, so that a second peak in current is observed at a second potential. In an embodiment, the difference between the first and second potentials at which there is a peak in current is used to identify the anion and/or used as the electrochemical information to calculate the concentration of the anion in the sample of interest. In an embodiment a ratio of (i) the area under the peak of current at the first potential and (ii) the area under the peak of current at the second potential is used as the electrochemical information to calculate the concentration of the anion in the sample of interest.

The method involves using a determined relationship between the electrochemical information and the concentration of the anion in a reference sample to calculate the concentration of the anion in the sample of interest.

The determined relationship may have been determined in a reference experiment in which is measured the variation of the electrochemical information, e.g. the height or area or a peak of current at the first potential, over a range of concentrations of the anion in the reference sample.

The reference sample can be a sample suitable for calibrating the concentration of the anion in the sample of interest. The reference sample and the sample of interest may be similar to one another, for example in that they contain the same liquid medium, e.g. water, in approximately the same amount, and any other components, aside from the anion, in the same amounts. In an embodiment, the sample of interest may be sweat containing a certain concentration of chloride ions, and the reference sample is a biological or synthetic sweat sample that contains chloride ions; and optionally the reference experiment involves measuring the variation of the electrochemical information, e.g. the height or area of a peak of current at the first potential, over a range of concentrations of the chloride in the reference sample. In an embodiment, the electrochemical information is the height of a peak of current at the first potential, and the determined relationship may be represented by formula (a)

I = C + n[anion] formula (a) wherein I is the height of a peak of current at the first potential, C is a constant, and n is a coefficient, and [anion] is the concentration of the anion in the sample of interest (or in the reference experiment, the concentration of the anion in the reference sample). C and n may be determined in the reference experiment. From determining I in the sample of interest, and using the formula above, i.e. (l-C)/n, the concentration of anion in the sample of interest, [anion], can be determined. The present inventors have found that the variation of peak height at the first potential can vary linearly with anion concentration within useful ranges.

In an alternative embodiment, if desired, higher order polynomials may be used for the determined relationship between the peak of current at the first potential and the concentration of anion in the reference sample. For example, the relationship may be expressed by a second degree polynomial of formula (b)

I = C + n[anion] + m[anion]² formula (b) wherein I, C, n and [anion] are as defined above and m is a further coefficient. Again, C, n and m may be determined in a reference experiment by measuring I over a range of known anion concentrations in the reference sample. Higher degree polynomials relating I and [anion] can also be used, such as third degree polynomials, fourth degree polynomials, and so on. Other functional forms can be used and the most appropriate can be determined by the skilled person. However, in many circumstances, the relationship between I and [anion] has been found to be sufficiently linear that formula (a) can be used and is adequate for determination of the concentration in a sample of interest.

The voltammetry or chronoamperometry experiment may be carried out using a suitable electrochemical analytical device, for example a potentiostat.

The method typically involves use of a counter electrode. The counter electrode may be made of any suitable material, for example a metal or carbon. The counter electrode may be selected such that it is inert, i.e. not oxidised or reduced under the conditions at which the electrochemical analysis is carried out. The counter electrode may comprise a metal selected from gold, silver, copper 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. The method may involve use of a reference electrode. A reference electrode, for example a Ag/Ag⁺ (e.g. 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. In an embodiment, a pseudo reference electrode may be used. A pseudo reference electrode may be defined as electrode through which no appreciable current is allowed to flow and is used to observe or control the potential at a working electrode. A pseudo-reference electrode, such as a Ag or Pt wire, may be used. A means for applying a potential, e.g. a potentiostat, is electrically connected to the working, counter and, if present, (pseudo)reference electrodes.

Optionally, the working electrode forms part of a screen printed electrode, which further comprises one or both of a counter electrode and a reference electrode, which may be as described herein. The working electrode, before carrying out the voltammetry experiment, may comprise an electrically conductive or semi-conductive material having on a surface thereof the substance in the first oxidation state; the substance in the first oxidation state may be different from the electrically conductive or semi-conductive material. For example, if the substance on the working electrode comprises silver (and the first oxidation state is 0), then the electrically conductive or semi-conductive substance is not silver and is inert during the oxidation; for example the electrically conductive or semi-conductive substance is or comprises carbon. During the voltammetry experiment, the electrically conductive or semi-conductive material is preferably inert. The electrically conductive material may comprise a metal or carbon. The electrically conductive material may comprise a metal selected from gold, silver and platinum. In an embodiment, the electrically conductive material comprises indium tin oxide. In an embodiment, the electrically conductive material comprises a conductive polymer. The electrically conductive material may comprise a carbon-containing material, which may be selected from glassy carbon, edge plane pyrolytic graphite, basal plane pyrolytic graphite, boron doped diamond, highly ordered pyrolytic graphite, carbon powder and carbon nanotubes. In a preferred embodiment, the electrically conductive comprises a glassy carbon material. In an embodiment, the working electrode may form part of a screen printed electrode, and the working electrode may be or comprise a screen printed carbon electrode having on a surface thereof the substance in the first oxidation state (before the voltammetry experiment). In an embodiment, the working electrode may be a carbon electrode and form part of a screen printed electrode that further comprises a reference electrode that may comprise, for example, silver.

The substance on the working electrode may be selected from silver, gold, platinum, palladium, iron, copper, zinc, mercury, tin, lead, bismuth, gallium and indium, and the first oxidation state is 0. In an embodiment, the substance is selected from silver, lead, mercury, copper, and thallium, and the first oxidation state is 0. In an embodiment, the substance is silver, the first oxidation state is 0 and the second oxidation state is 1. The substance may be in elemental form. If the substance comprises a metal, e.g. silver, the substance may be present, in the first oxidation state, as a pure metal or an alloy of this metal. Silver, for example, can be present as an alloy with a metal selected from copper, gold, platinum, bismuth, and combinations thereof. Preferably, the substance, in its second oxidation state, can form a compound with the anion that is substantially insoluble in water at 25 °C or can form a complex with the anion, which may or not be soluble in water at 25 °C. Preferably, the substance, in its second oxidation state, can form a compound with the anion that has a solubility product constant, K_sp,(at 25 °C in water) of 1 x 10^"4 or less, optionally 1 x 10^"5 or less, optionally 1 x 10^"6 or less, optionally 1 x 10^"7 or less.

The substance may be in any suitable form on the working electrode. In an embodiment, the substance is in a form selected from nanoparticles and a film. Preferably, the entirety of the substance is oxidised during the method, and preferably substantially none of the material of the working electrode on which it was disposed is oxidised. Preferably, the substance is in the form of nanoparticles. The present inventors have found that having a finite amount of oxidisable material on the electrode (i.e. the substance as described herein), such that the entirety of which is oxidised during the method, produces peaks of potential of a desired size. When the substance is in the form of nanoparticles, the amount of such substance deposited onto a working electrode (before use in the method) can be carefully controlled, such that it is entirely oxidised during the method.

If the substance is in the form of nanoparticles on the electrode, the nanoparticles may be or comprise particles having a diameter of from 1 to 500 nm, optionally from 1 to 200 nm, optionally from 10 to 100 nm. The diameter of a nanoparticle, and/or particle size distributions of nanoparticles in a sample, can be determined by a technique selected from scanning electron microscope (SEM) imaging, atomic force microscopy, dynamic light scattering techniques, UV-visible spectroscopy, and other suitable techniques, as would be appreciated by the skilled person. The nanoparticle can be any suitable shape, including spherical, and elongated, for example a rod-shaped nanoparticle. If a nanoparticle is non-spherical, the diameter of the nanoparticle as measured herein will be the smallest diameter across the particle.

The nanoparticles may comprise, consist essentially of, or consist of, a type of substance, as described herein. If a nanoparticle consists essentially of a material, this indicates that preferably the particle comprises at least 95 % by weight of this material, preferably 98 % by weight, preferably 99 % by weight, of this material.

The anion may be a halide and may be selected from chloride, bromide and iodide.

Optionally, the substance is silver, the first oxidation state is 0 and the halide is chloride, and optionally the second oxidation state is 1.

A pseudohalide may be defined as the anion of a pseudohalogen. A pseudohalogen may be defined as a monovalent, inorganic group, consisting of two or more electronegative elements, including, but not limited to, C, N, O, S, Se and Te. Pseudohalogens can have similar chemical and physical properties to halogens. The pseudohalides may be selected from CN^", N₃ ^", NCO^", SCN^", NCS^", SeCN^", NCSe^", CS₂N₃ ^" and TeCN^". Pseudohalogens are described, for example, in New Pathways in Inorganic Chemistry, edited by E.A.V. Ebsworth et al, and published by Cambridge University Press (1968), which is incorporated herein by reference in its entirety. In an embodiment, the substance is silver, the first oxidation state is 0, the second oxidation state is 1 , and the anion is CN^". The sample of interest and/or the reference sample may be a liquid sample. Optionally, the sample of interest and/or the reference sample is a biological sample, which may be selected from a sweat sample, a blood sample, saliva and a urine sample. The blood sample may be selected from a whole blood sample, a plasma sample and a serum sample. If the sample of interest is a sweat sample from a human patient, the reference sample may be a biological or synthetic sweat sample. The synthetic sweat sample may be prepared according to a known standard, including, but not limited to, the German industrial standard (DIN-53160-2) or the British Standard (BS EN181 1-1999), adapted as required to alter the concentration of the anion, e.g. chloride, therein, and by the addition of one or more electrolytes, e.g. sodium nitrate, to a desired concentration.

The sample of interest and/or the reference sample may be selected from sea water, river water, drinking water, waste water and effluent.

Optionally, before contacting with the working electrode, the sample may be diluted, e.g. with a liquid medium such as water, the diluted sample used in the method, and the dilution is taken into account when calculating the concentration of the anion in the undiluted sample. If diluted with a liquid medium, such as water, the liquid medium may comprise an electrolyte, including, but not limited to sodium nitrate. The sample of interest, which may be a biological sample, may be diluted so that its concentration is 1/N of its original concentration (e.g. biological concentration), e.g. by the addition of water (that optionally contains an electrolyte), and N may be 2 or more, optionally 3 or more, optionally 4 or more, optionally 2 to 5, optionally about 4.

Optionally, before contacting with the working electrode, the sample may be concentrated, e.g. by allowing liquid, e.g. water, to evaporate from the sample, the concentrated sample used in the method, and the pre-concentration is taken into account when calculating the concentration of the anion in the unconcentrated sample.

Optionally, the anion, e.g. chloride, concentration in the sample of interest, as tested in the voltammetry experiement, and after any dilution or concentration of the sample of interest, is within the range of 1 mM to 160 mM, optionally in the range of 10 mM to 160 rtiM. Optionally, the anion, e.g. chloride, concentration in the sample of interest, as tested in the voltammetry experiement, and after any dilution or concentration, is within the range of 2 mM to 40 mM.

In an embodiment the anion is chloride, the sample of interest is a sweat sample from a human subject, and optionally the method gives an indication of whether the concentration of the chloride ion in the sweat sample is above a threshold value, and, optionally, if above a threshold value, further tests are carried out to determine if the subject has cystic fibrosis. The further tests may involve, for example, genetic tests, e.g. CFTR-related genetic tests. The threshold value may be, for example, a value of 40 mM or more, optionally a value of 50 mM or more, optionally a value of 60 mM or more. If the sample of interest has been previously diluted or concentrated before contacting with the working electrode, the threshold value may be the value in the undiluted or non-concentrated sample of interest.

The method may be used to determine if a subject, e.g. a human subject, is sufficiently hydrated. Accordingly, in an embodiment, the sample may be a sample from a human subject, e.g. a sweat sample, the anion is chloride, and if the sample contains 40 mM or more chloride ion, optionally 60 mM or more chloride ion, this may indicate dehydration in the subject, particularly if the subject is not found to (e.g. in a subsequent test) or known not to have cystic fibrosis.

The present inventors have found that the method can be used to determine the concentration of a halide ion or pseudohalide ion in a very small amount of sample. In some embodiments, the amount of the sample of interest in contact with the working electrode is 1 ml or less, optionally 500 μΙ_ or less, optionally 200 μΙ_ or less, optionally 100 μΙ_ or less, optionally 75 μΙ_ or less, optionally 60 μΙ_ or less. In some embodiments, the amount of sample of interest in contact with the working electrode is at least 1 μΙ_, optionally at least 5 μΙ_, optionally at least 10 μΙ_, optionally at least 20 μΙ_.

In an alternative embodiment, the method may be used to determine the amount of iodide in a sample, including, but not limited to, a urine sample from a human subject. An excess or deficiency of iodide in a human subject can indicate and/or cause certain health problems. In a human adult or child, a urinary iodine concentration of 100 - 199 μg/L is adequate. In a pregnant women, a urinary iodine concentration of 150-249 μg/L is adequate. In a human subject, a urinary iodine concentration of less than 20 μg/L can be insufficient and indicate a severe iodine deficiency. In a human subject, a urinary iodine concentration of 20 μg/L to 49 μg/L can be insufficient and indicate a moderate iodine deficiency. In a human subject, a urinary iodine concentration of 50 μg/L to 99 μg/L can be insufficient and indicate a moderate iodine deficiency. In a human subject, a urinary iodine concentration of 200 μg/L to 299 μg/L can be more than adequate and indicate a risk of iodine-induced hyperthyroidism within 5-10 years. In a human subject, a urinary iodine concentration of 300 μg/L or more can indicate an excessive risk of adverse health consequences (iodine induced hyperthyroidism, autoimmune thyroid diseases). More information on iodine levels in a human subject can be found in a publication by M.Andersson, entitled Iodine Deficiency in Europe: A continuing public health problem, World Health Organisation, 2007.

The method may be used to detect whether the iodide/iodine level in a biological sample, e.g. urine, is above or below a threshold value, e.g. the values given above.

As mentioned, the present invention provides an apparatus for determining the concentration of an anion selected from a halide ion and a pseudohalide ion in a sample of interest, the apparatus comprising a working electrode having a substance in a first oxidation state on a surface thereof, the apparatus adapted to carry out a method involving:

contacting the working electrode with the sample of interest;

carrying out a voltammetry or chronoamperometry experiment that involves increasing the potential applied to the working electrode to a first potential to oxidise the substance to a second oxidation state, wherein, at the first potential, the electrochemical response of the substance is different when the anion is present in the sample compared to when the anion is absent in the sample, and obtaining electrochemical information during the voltammetry or chronoamperometry experiment; using a determined relationship between the electrochemical information and the concentration of the anion in a reference sample to calculate the concentration of the anion in the sample of interest.

The apparatus may be adapted to carry out at least part of the method as described herein in an automated way. In an embodiment, the apparatus may be adapted to carrying out the voltammetry or chronoamperometry experiment and using the determined relationship to the calculate the concentration of the anion in the sample of interest in an automated way; optionally the device may be adapted to contact the working electrode with the sample of interest in an automated way. All features as described above in relation to the method are equally applicable to the device. The apparatus may be programmed to carry out at least part of the method, e.g. carrying out the voltammetry or chronoamperometry experiment and using the determined relationship to the calculate the concentration of the anion in the sample of interest; and optionally contact the working electrode with the sample of interest. The apparatus may be a portable apparatus. In a third aspect, the present invention provides a use of an electrode having silver on a surface thereof in an electrochemical test method to determine the concentration of chloride ions in a sweat sample, wherein the electrochemical test method involves oxidation of the silver from Ag (0) to Ag(l) on the electrode in the sweat sample.

The features of the method described above may be applied equally to the use of the third or fourth aspects; in the use, the electrode may be the working electrode described in the method; and the silver in the use is the substance in the first oxidation state, which in the use is 0. The electrochemical test method may be the voltammetry or chronoamperometry experiment described herein. The sweat sample in the use of the third or fourth aspect may be the sample of interest in the method. Determining the concentration of chloride ions in a sweat sample may be as described for the method using the determined relationship described herein. Determining the concentration of cyanide ions in a sample may be as described for the method using the determined relationship described herein, and in an analogous manner to that exemplified for chloride ions. In the fourth aspect, the sample may comprise a liquid, e.g. water, The sample in the fourth aspect may, for example, be selected from sea water, river water, drinking water, waste water and effluent. The sample in the third and fourth aspects may be a biological sample, which may be selected from a sweat sample, a blood sample, saliva and a urine sample.

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

In the Examples below, the present inventors use silver nanoparticle modified screen printed electrodes to detect chloride levels in sweat, artificial sweat and in other liquids demonstrating the possibility of using the method for a cystic fibrosis screening system. The basis of such a procedure is depicted in Figure 1 ; layers of silver nanoparticles are deposited onto the working electrode of a screen printed electrode or glassy carbon electrode and anodically stripped off to generate silver cations. In the absence of chloride ions, the anodic stripping voltammetry of silver nanoparticles yields a single silver oxidation peak. Conversely, in the presence of chloride ions, the favourable conditions of formation of silver chloride causes two stripping peaks to be voltammetrically recorded, one for formation of silver chloride and another for the oxidation of silver to silver ions. Herein, it is demonstrated that the silver chloride peak may be used for the quantitative determination of chloride concentration in the target sample.¹- Silver nanoparticles are chosen for the experiments as they provide a facile method for producing a finite surface coverage on carbon electrodes which is needed to give the peaked responses. The usage of silver nanoparticles further ensures that the signal for the formation of silver chloride from silver is not dominated by the signal for silver oxidation to silver cations.^™' ²² In the following sections, chloride ion quantification is performed using a silver nanoparticle modified screen printed electrode.

Experimental (i) Chemicals

Sodium nitrate (>99.5%, NaN0₃) was obtained from Fisons Scientific Equipment, Loughborough, UK. Trisodium citrate (>99%, Na₃C₆H₅0₇) was supplied from BDH Laboratory Supplies, Poole, UK. Potassium chloride (>99.5%, KCI), silver nitrate (>99%, AgN0₃), urea (>99%, CH₄N₂0), lactic acid (solution in water, >85%, C₃H₆0₃), nitric acid (70%, HN0₃), hexaamineruthenium(lll) chloride (98%, Ru(NH₃)₆CI₃) and sodium chloride (>99%, NaCI) was obtained from Sigma-Aldrich. Concentrated sulphuric acid (>95%, H₂S0₄) and hydrogen peroxide (>30%w/v, H₂0₂) were supplied from Fisher Scientific, Loughborough, UK. Synthetic sweat sample 1 was obtained from Synthetic Urine e.k., Eberdingen, Germany (DIN 53160-2). The solid given was dissolved in water. The solution was tuned to the required pH of 6.5 ± 0.1 with the given ammonia hydroxide solution and 0.1 M nitric acid. The final volume was ensured to be 100 ml_. Synthetic sweat sample 1 had a sodium chloride concentration of 5 g/L, which equates to 85.5 mM of chloride ions in the sample. Synthetic sweat sample 2 was prepared following British Standard (BS EN 1811-1999). However, a reduced sodium chloride concentration (10% of original value) was used.²¹ It was then tuned to pH 6.5 with the same ammonia hydroxide solution and 0.1 M nitric acid used for synthetic sweat sample 1. It contained a sodium chloride concentration of 1.08 g/L, which equates to 18.5 mM of chloride ions present in the sample. The sweat samples were then diluted by a factor of four with sodium nitrate solution (0.133 M). The diluted samples contained a quarter of the original chloride concentration and 0.1 M of sodium nitrate. All solutions were made with ultrapure water from Millipore with resistivity not less than 18.2 MQ.cm at 298K.

(ii) Silver Nanoparticle Synthesis and Characterisation

Silver nanoparticles capped with citrate were synthesised based on a method devised by Lee and Miesel.²² In summary, 25 mL of silver nitrate (0.1 M) solution was added to a flask containing 25 mL of ultrapure water. The solution mixture was heated in an 110°C oil bath with violent magnetic stirring. 1 g of trisodium citrate was dissolved in 100 mL ultrapure water. 5 mL of the trisodium citrate solution was added into the boiling silver nitrate solution. The solution was left to boil for an hour before it was cooled in a cold water bath. The resulting solution of silver nanoparticle obtained was washed with ultrapure water twice and the supernatant was removed each time. The solution was centrifuged at 4200 rpm for 27 minutes for each step. The pellet which remained after the second wash from 13.5 mL of unwashed silver nanoparticle suspension was made into a 3 mL washed silver nanoparticle suspension. All glassware was clean thoroughly with "piranha" solution (hydrogen peroxide and concentrated sulphuric acid) prior to the synthesis of silver nanoparticles. Figure 2. Nanoparticle illustrated tracking analysis for silver nanoparticles. Mean diameter = 35 nm.

Figure 3 illustrates scanning electron microscopy image for silver nanoparticles. The washed silver nanoparticle suspension was characterised by UV-vis spectroscopy (U-2001 , Hitachi, Mannheim, Germany). For the analysis, the washed nanoparticle suspension was diluted by a factor of 30 with ultrapure water. Both tungsten iodide and deuterium light source were used to run a wavelength scan from 600 nm to 250 nm with a scan rate of 400 nm min^"1. A broad surface plasmon peak at 419 nm was observed and indicated the presence of silver nanoparticles. Nanoparticle tracking analysis (LM 10, NanoSight Ltd, Wiltshire, UK) were performed to size the silver nanoparticles in solution-phase. Figure 2 shows the nanoparticle tracking analysis for the sample. The mean diameter of the nanoparticles was 35 nm. Scanning electron microscopy (LEO Gemini 1530, Zeiss, Oberkochen, Germany) was used to confirm that the synthesized nanoparticles were about 40 nm in diameter, see Figure 3.

(iii) Voltammetry

All electrochemical experiments were performed using a three electrode system in a Faraday cage with a μΑυίο^ II from Metrohm-Autolab BV, Utrecht, The Netherlands. The working electrode was a 3.0 mm diameter glassy carbon electrode from CH instruments, Austin, TX, USA. The electrode was polished to a mirror finish on diamond sprays in the sequence of size 3.0 μηι, 1.0 μηι and 0.1 μηι from Kemet, Kent, UK. A homemade reference electrode of Ag/AgN0₃ (10 mM) & NaN0₃ (90 mM) was used. It was calibrated against a hexammineruthenium(lll) chloride solution (1 mM hexaamineruthenium(lll) chloride and 0.1 M sodium nitrate). The redox peaks were found to differ by +0.45 V compared to an experiment with a standard calomel electrode (SCE, +0.24 V vs NHE). Thus, the Ag/Ag(10 mM) & NaN0₃ (90 mM) reference had a potential of +0.69 V vs NHE. The reference electrode consists of a silver wire submerged in a solution of 10 mM silver nitrate and 90 mM sodium nitrate. The silver nitrate/sodium nitrate solution was changed regularly to maintain electrode stability. A platinum mesh functioned as the counter electrode. All experiments were generally performed under a nitrogen atmosphere and all solutions were degassed thoroughly with nitrogen. Screen printed electrodes were manufactured by Dropsens, S. L., Llanera, Spain and obtained from Metrohm Autolab, Runcorn, Cheshire, UK. They consist of a 4.0 mm diameter carbon working electrode, a silver pseudo reference electrode and a carbon counter electrode. 50 μΙ_ of solution was used to fully cover the electrode. Nanoparticle modification of the glassy carbon electrode involved drop casting of 3 μΙ_ of washed silver nanoparticle sample onto the electrode surface and drying the electrode in an oven at 50°C for 10 minutes. Five coatings of silver nanoparticles were applied to the glassy carbon electrode. Screen printed electrodes were modified in a similar manner with 5.3 μΙ_ of washed silver nanoparticle sample to compensate for the difference in working electrode area. Each coat was dried for 12 minutes in a 50°C oven. Three repeats were done for each experiment performed.

Results and Discussion

This part is separated into three different subsections. For section (i), the experiments performed on glassy carbon electrodes are summarised. Silver nanoparticles were coated onto the glassy carbon electrode and anodic stripping voltammetry were undertaken in sodium nitrate solutions spiked with different concentration of potassium chloride. A linear correlation of the voltammetric peak height and chloride ion concentration is found. In section (ii), experiments similar to the first section were performed on screen printed electrodes and similar correlation between peak height and chloride ion concentration was observed. In section (iii), repeat measurements and calculations of the sweat chloride levels for two synthetic sweat samples using the calibration plot obtained from the linear correlation of peak height and chloride ion concentration obtained from section (ii).

(i) Glassy Carbon electrode experiments

Figure 4 illustrates the oxidative stripping of silver nanoparticles from a glassy carbon electrode in 0.1 M sodium nitrate spiked with different amount of potassium chloride (KCI) at a scan rate of 0.05 V s^' Grey dashed line: No KCI; Black solid line: 2mM KCI; Black dashed Line: 8mM KCI; Black dotted line: 16mM KCI; Grey solid line: 32mM KCI. Reference electrode: Ag/AgN0₃ (10 mM) & NaN0₃ (90 mM) Counter electrode: Platinum mesh; Inlay: Calibration plot of peak height of peak A against the concentration of chloride ions. Each data point consists of a minimum of 3 repeats.

Sweat chloride levels are crucial to the diagnosis of cystic fibrosis. Hence, a set of experiments in an electrochemical cell with a silver nanoparticle modified glassy carbon electrode was performed to determine if chloride detection is possible in an electrochemical system. Thus, the anodic stripping of silver nanoparticles was performed in a 0.1 M sodium nitrate solution with varied chloride ions concentration to observe silver nanoparticles' behaviour in the presence and absence of chloride ions. Silver nanoparticles were drop cast onto a glassy carbon electrode through five coatings of 3.0 μΙ_ of washed silver nanoparticle aliquot. Each of the coatings was dried in an oven at 50°C for 10 minutes. The modified electrodes were used in cyclic voltammetric experiment, which started at -0.7 V, swept towards +0.4 V before returning back to -0.7 V in a cell filled with 0.1 M sodium nitrate and varying concentration of potassium chloride (2 mM - 40 rtiM). For every new scan, the electrodes were polished to a mirror finish before silver nanoparticles were drop cast on. Each experiment was repeated for three times. Sodium nitrate was chosen as the electrolye as the nitrate ion is a weakly co-ordinating anion. Hence, it does not affect the peak positions as it does not form a strong ionic pair or precipitate with the silver ions. The nitrate ion is also not expected to alter the diffusion coefficient of silver ion or the rate constant of silver oxidation.

To determine the behaviour of silver nanoparticles in the absence of chloride ions, a control experiment was performed. As seen in the grey dashed line of Figure 4, without the presence of potassium chloride, only a broad peak at around +0.2 V (peak B) was observed. In the presence of potassium chloride (all the lines in Figure 4 except the grey dashed line), two broad peaks at around -0.25 V and +0.2 V (peaks A and B) were observed. In the absence of chloride ions, only peak B was observed. Peak B corresponds to the one electron oxidation of metallic silver:^™-

Ag -> Ag⁺ + e^" Equation (1)

The formal potential of the redox pair of Ag/Ag⁺ (equation (1)) is +0.80 V vs NHE.^~ Given that the reference electrode had a potential of +0.69 V vs NHE, the oxidation of metallic silver is expected to occur at +0.1 1 V under the experiment condition. The slight apparent overpotential of +0.1 V observed in experiments can be accounted by the high surface coverage of the silver nanoparticles. At high surface coverages, linear diffusion of silver ions away from the whole geometric surface of electrode surface takes place. Under this diffusion regime, it has been previously shown that the stripping peak potential varies with the silver surface coverage.— Since the oxidation of silver to silver cation occurs at +0.11 V under experimental conditions, peak A, which occurs around -0.25 V in presence of potassium chloride, does not account for oxidation of silver to silver cations. It is possible to deduce that at peak A, the following reaction occurs: Ag + CI^" AgCI + e^" Equation (2) First, this conclusion is evidenced by the observations in Figure 4 inlay. The peak height of peak A correlates linearly with the concentration of chloride ions. Second, the formal potential of the redox couple of Ag/AgCI (equation (2)) corrected for the concentration of chloride ions at 20 mM is +0.32 V vs NHE. Given that formal potential of Ag/Ag⁺ redox couple (equation (1)) is +0.80 V vs NHE, peak A and B should have a theoretical potential difference of -0.48 V; which is close to the experimentally measured difference of 0.45 V.²-⁴ Third, when peak A increases in height, the height of peak B decreases. This indicates that the process at peak A has consumed some of the silver nanoparticles attached to the working electrode, causing less charge to be passed under peak B. Due to the finite surface coverage of silver nanoparticles on the glassy carbon electrode, at higher chloride concentrations, the peak height no longer correlates linearly with chloride ion concentrations. As observed from the inlay of Figure 4, the linear range of the glassy carbon electrode system is between 2 mM and 40 mM of potassium chloride in the system.

(ii) Screen printed electrode experiments

Figure 5 illustrates the oxidative stripping of silver nanoparticles from a screen printed electrode in 0.1 M sodium nitrate spiked with different amount of potassium chloride at a scan rate of 0.05 V s^' The peak height of peak A (see Figure 4) is plotted against concentration of chloride. Each data point consists of a minimum of 3 repeats.

Having developed an electrochemical system capable to determine chloride levels accurately below 40 mM, to achieve the objective of developing a point-of-care sensor, the concept was reinvestigated using screen printed electrodes. The disposable screen printed electrode has a 4.0 mm diameter carbon working electrode, a silver pseudo reference electrode and a carbon counter electrode. In order to achieve comparable surface coverages of silver nanoparticles, each coat was changed from 3.0 μΙ_ to 5.3 μΙ_ of washed silver nanoparticle suspension. The five coatings of silver nanoparticles were dried in a 50°C oven for 12 minutes each. For the voltammetric measurements, 50 μΙ_ of analyte solution was used to cover the entire screen printed electrode. The analyte consisted of 0.1 M sodium nitrate and varying concentrations of potassium chloride from 2 mM to 40 mM. Each experiment was repeated for three times. The same trend for peak A and peak B was observed for the measurements done on the screen printed electrodes. The voltammetric signal height of peak A forms a direct linear correlation with concentration of chloride for up to 40 mM and the calibration graph has been plotted in Figure 5.

(iii) Synthetic Sweat Sample Analysis

Table 1

Table 1 gives a summary of the expected chloride concentration and the measured chloride concentration of various synthetic sweat samples.

To evaluate the feasibility for a practical CF borderline sweat test, two synthetic sweat samples fulfilling industrial standards were tested against the system. Synthetic sweat sample 1 was prepared in accordance to the German industrial standard (DIN-53160- 2). Synthetic sweat sample 2 was prepared from a recipe from the British Standard (BS EN 181 1-1999) but the amount of sodium chloride used was 10% of its original value. This is because the original chloride amount was beyond the 160 mM recommended by authorities and beyond the sensing range of 2 mM to 40 mM.—^■—^■— Each sweat sample was diluted with sodium nitrate solution by a factor of four with final concentration of chloride ion being a quarter of the original and concentration of sodium nitrate being 0.1 M. The dilution of sweat sample by a factor of four ensures that the sensing range of 2 mM to 40 mM of chloride ions complies with the recommended range of 10mM to 160 mM of chloride ions. Dilution with sodium nitrate solution also brings about advantages of lowering the amount of sweat sample required to 12.5 μΙ_ and providing the electrochemical system the essential supporting electrolyte. 50 μΙ_ of the diluted synthetic sweat sample is dropped onto the silver nanoparticles modified screen printed electrode. Cyclic voltammogram was recorded from -0.2 V to +0.8 V before sweeping back to -0.2 V. Each experiment was repeated for three times. Figure 6 shows the voltammograms recorded from the synthetic sweat samples. By measuring the peak height of peak A in Figure 6, the measured concentration of chloride ion in the diluted sweat samples was calculated. The experimentally measured value was 18.9 ± 1.3 mM and 3.9 ± 0.6 mM for diluted synthetic sweat samples 1 and 2 respectively. The expected chloride ion concentrations for diluted synthetic sweat samples 1 and 2 are 21.4 mM and 4.6 mM respectively. Table 1 summarises the values of the expected and measured chloride ion concentration. The inlay in Figure 6 also displayed the synthetic sweat samples at the expected chloride concentration. As seen in Figure 6 inlay and Table 1 , the synthetic sweat samples had a slightly lower measured concentration compared to the expected chloride ion concentration. Since the system targets to give patients a pre-screening test, the resolution will not affect the answers of 'positive',' borderline' or 'negative'. The system could be employed as a pre-screening test before hospital testing for the high sweat chloride ion level and two genetic mutations for confirmation of CF.^{§ Z} In conclusion, cystic fibrosis is an important and common disease which can be diagnosed with sweat chloride levels. The present inventors have shown the sensitivity of oxidised silver nanoparticles towards chloride ions in anodic stripping experiments, the chloride content can be qualified from the analysis of voltammetry peaks. The experiments involving synthetic sweat samples indicated that sweat chloride content determination is possible on a screen printed electrode. The system exemplified was found to allow reliable chloride ion quantification in a measurement range from 2 mM to 40 mM of chloride ions. Thus, the dilution of sweat sample by a factor of four allows the recommendation range of 10 mM to 160 mM to be sampled. These Examples illustrate that the method and apparatus disclosed herein can be used, inter alia, in a point-of- care cystic fibrosis screening system.

References mentioned herein or otherwise useful for background:

1. B. Kerem, J. Rommens, J. Buchanan, D. Markiewicz, T. Cox, A. Chakravarti, M. Buchwald and L. Tsui, Science, 1989, 245, 1073-1080.

2. M. R. Knowles and P. R. Durie, N. Engl. J. Med, 2002, 347, 439-442. 3. S. M. Rowe, S. Miller and E. J. Sorscher, N. Engl. J. Med., 2005, 352, 1992- 2001.

4. M. J. Goldman, G. M. Anderson, E. D. Stolzenberg, U. P. Kari, M. Zasloff and J. M. Wilson, Cell, 1997, 88, 553-560.

5. T. Z. Khan, J. S. Wagener, T. Bost, J. Martinez, F. J. Accurso and D. W. Riches, Am. J. Respir. Crit. Care. Med., 1995, 151 , 1075-1082.

6. B. P. O'Sullivan and S. D. Freedman, Lancet, 2009, 373, 1891-1904.

7. P. M. Farrell, B. J. Rosenstein, T. B. White, F. J. Accurso, C. Castellani, J.

Pediatr, 2008, 153, 54.

8. M. Seia, L. Costantino, V. Paracchini, L. Porcaro, P. Capasso, D. Coviello, C. Corbetta, E. Torresani, D. Magazzu, V. Consalvo, A. Monti, D. Costantini and C.

Colombo, Clin. Biochem., 2009, 42, 611-616.

9. A. Green and J. Kirk, Ann. Clin. Biochem., 2007, 44, 25-34.

10. J. Coakley, S. Scott and J. Doery, Clin. Biochem. Rev., 2006, 27, 1-7.

1 1. J. H. Baumer, Arch. Dis. Child, 2003, 88, 1 126-1 127.

12. V. A. LeGrys, J. R. Yankaskas, L. M. Quittell, B. C. Marshall, P. J. Mogayzel, Jr. and F. Cystic Fibrosis, J. Pediatr, 2007, 151 , 85-89.

13. I. Sermet-Gaudelus, A. Munck, M. Rota, M. Roussey, D. Feldmann and T.

Nguyen-Khoa, Arch. Pediatr, 2010, 17, 1349-1358.

14. J. R. Yankaskas, B. C. Marshall, B. Sufian, R. H. Simon and D. Rodman, Chest, 2004, 125, 1S-39S.

15. M. Wilschanski and P. R. Durie, Gut, 2007, 56, 1 153-1163.

16. A. Green, The Association for Clinical Biochemistry, Guidelines developed with ACB involvement, Guidelines for the Performance of the Sweat Test for the

Investigation of Cystic Fibrosis in the UK, Association for Clinical Biochemistry, United Kingdom, 2003.

17. I. J. Schulz, J. Clin. Invest, 1969, 48, 1470-1477.

18. Sherwood Scientific Ltd, Model 926 Chloride Analyzer, http://www.sherwood- scientific.com/chloride/chloridemeters.html, April 2013.

19. A. G. Brolo and S. D. Sharma, Electrochim. Acta., 2003, 48, 1375-1384.

20. M. Giovanni and M. Pumera, Electroanalysis, 2012, 24, 615-617.

21. K. Kulthong, S. Srisung, K. Boonpavanitchakul, W. Kangwansupamonkon and R. Maniratanachote, Part. Fibre. Toxicol., 2010, 7, 8.

22. P. C. Lee and D. Meisel, J. Phys. Chem-US., 1982, 86, 3391-3395.

23. S. E. Ward Jones, F. W. Campbell, R. Baron, L. Xiao and R. G. Compton, J. Phys. Chem. C, 2008, 112, 17820-17827. 24. W. M. Haynes, D. R. Lide, T. J. Bruno, CRC Handbook of Chemistry and Physics 2012-2013, Taylor & Francis Group, Florida, 93rd edn., 2012.

25. H. S. Toh, C. Batchelor-McAuley, K. Tschulik, M. Uhlemann, A. Crossley, R. G. Compton, Nanoscale, 2013, 5, 4884-4893

Each individual reference mentioned herein is incorporated herein by reference in its entirety.

Claims

1. A method of determining the concentration of an anion selected from a halide ion and a pseudohalide ion in a sample of interest, the method involving:

2. A method according to claim 1 , wherein, at the first potential, the substance in its second oxidation state forms a solid compound with the anion on the electrode or the substance in its second oxidation state forms a complex with the anion, and the complex may or may not be dissolved in the sample.

3. A method according to claim 1 or claim 2, wherein the electrochemical information is the height or area of a peak of current at the first potential, or peak potential.

4. A method according to any one of the preceding claims, wherein a first peak of current is observed at the first potential, and the method further involves raising the potential above the first potential, so that a second peak in current is observed at a second potential.

5. A method according to claim 4, wherein the difference between the first and second potentials at which there is a peak in current is used to identify the anion and/or used as the electrochemical information to calculate the concentration of the anion in the sample of interest.

6. A method according to claim 4, wherein a ratio of (i) the area under the peak of current at the first potential and (ii) the area under the peak of current at the second potential is used as the electrochemical information to calculate the concentration of the anion in the sample of interest.

7. A method according to any one of the preceding claims, wherein the substance is selected from silver, gold, platinum, palladium, iron, copper, zinc, mercury, tin, lead, bismuth, gallium, thallium and indium, and the first oxidation state is 0.

8. A method according to any one of the preceding claims, wherein the substance is selected from silver, lead, mercury, copper, and thallium, and the first oxidation state is 0.

9. A method according to any one of the preceding claims, wherein the substance is present in a finite amount.

10. A method according to any one of the preceding claims, wherein the substance is in a form selected from nanoparticles and a film.

1 1. A method according to any one of the preceding claims, wherein the anion is a halide selected from chloride, bromide and iodide.

12. A method according to any one of the preceding claims, wherein the anion is a pseudohalide selected from CN^", N₃ ^",NCO^", SCN^", NCS^", SeCN^", NCSe^", CS₂N₃ ^" and TeCN^".

13. A method according to any one of the preceding claims, wherein the substance is silver, the first oxidation state is 0 and the anion is chloride.

14. A method according to any one of the preceding claims, wherein the sample is a biological sample selected from a sweat sample, a blood sample, saliva and a urine sample.

15. A method according to any one of the preceding claims, wherein the working electrode forms part of a screen printed electrode having the working electrode, and one or both of a counter electrode and a reference electrode thereon.

16. A method according to any one of the preceding claims, wherein the substance is silver, the anion is chloride, the sample is a sweat sample from a human subject.

17. A method according to claim 16, wherein the method gives an indication of whether the concentration of the chloride in the sweat sample is above a threshold value, and, if above a threshold value, further tests are carried out to determine if the subject has cystic fibrosis.

18. A method according to claim 16, wherein the method gives an indication of whether the concentration of the chloride in the sweat sample is above or below a threshold value, and, if above or below this threshold value, this gives an indication of whether or not the subject is sufficiently hydrated.

19. An apparatus for determining the concentration of an anion selected from a halide ion and a pseudohalide ion in a sample of interest, the apparatus comprising a working electrode having a substance in a first oxidation state on a surface thereof, the apparatus adapted to carry out a method involving:

contacting the working electrode with the sample of interest;

20. The apparatus according to claim 19, wherein the substance is silver, the anion is chloride, the sample of interest is a sweat sample from a human subject, and apparatus is adapted to give an indication of whether the concentration of the chloride in the sweat sample is above or below a threshold value.

21. The apparatus according to claim 20, wherein, if the concentration is above a threshold value, the apparatus indicates that further tests should be carried out to determine if the subject has cystic fibrosis.

22. The apparatus according to claim 20, wherein, if the concentration is above a threshold value, the apparatus indicates whether or not the subject appears to be sufficiently hydrated.

23. Use of an electrode having silver on a surface thereof in an electrochemical test method to determine the concentration of chloride ions in a sweat sample, wherein the electrochemical test method involves oxidation of the silver from Ag (0) to Ag(l) on the electrode in the sweat sample.

24. Use of an electrode having silver on a surface thereof in an electrochemical test method to determine the concentration of cyanide ions in a sample, wherein the electrochemical test method involves oxidation of the silver from Ag (0) to Ag(l) on the electrode in the sample.

25. The use according to claim 24, wherein the sample is a sample is selected from sea water, river water, drinking water, waste water and effluent.