CCL17 Electrochemical biosensor Field of the disclosure The present disclosure relates to methods for the electrochemical detection of CCL17 and an electrochemical biosensor, as well as kits for detection of CCL17. Background to the disclosure The chemokine, CCL17 (also known as thymus and activation regulated chemokine (TARC)), has recently emerged as a highly promising candidate blood biomarker for classic Hodgkin lymphoma (cHL). It is an excellent marker of disease activity that could be utilised for early diagnosis, monitoring treatment response, and early detection of relapse. Presently, no specific point-of-care blood tests for cHL are available to primary and secondary care clinicians. cHL is a B-cell-derived malignancy characterised by the presence of Hodgkin and Reed- Sternberg cells, the tumour cells, in a background of inflammatory cells. The interaction between the Hodgkin and Reed-Sternberg cells and the inflammatory cells is crucial to the pathogenesis of cHL. CCL17 is highly expressed by Hodgkin and Reed-Sternberg cells and most likely plays a role in attracting T helper 2 (Th2) cells into the tumour microenvironment ¹ . CCL17 is elevated in blood samples from ≥90% of untreated cHL patients ² , and levels correlate with tumour burden ² , and Ann Arbor stage ³ . Importantly, in the present context, CCL17 levels fall rapidly in response to successful treatment ² . cHL has a good prognosis and with recent advances in patient management, cure rates of over 90% in patients with limited-stage disease and ~70% in patients with advanced-stage disease can be achieved ⁴ . The aim of treatment is to cure the disease whilst minimising late side-effects, which cause significant morbidity and mortality in cHL since most patients are young at the time of diagnosis and treatment ⁴ . Initial treatment decisions are based on the stage of the disease, and the frailty of the patient ⁴ . Many treatment protocols now incorporate response-adapted therapy where interim Fluorodeoxyglucose (18F) positron emission tomography, coupled with Computerised Tomography, scanning (PET-CT), is used to assess response to treatment after two or three cycles of chemotherapy and guide further treatment decisions. Although this approach has led to better outcomes, it is not perfect ⁵ . False positive PET-CT results, with subsequent escalation of treatment, can lead to potentially avoidable, delayed toxicity ⁶ , and false-negative results lead to under-treatment and disappointing outcomes ⁶ . This emphasises the need for additional, more sensitive markers to evaluate therapeutic response ⁵ . Pre-treatment CCL17 levels fall rapidly after initiation of successful treatment, and some studies have shown that interim CCL17 levels may provide a more accurate assessment of treatment response than interim PET/CT imaging ⁷ . Furthermore, CCL17 has shown potential to inform treatment response after one cycle of chemotherapy ⁷ , potentially allowing earlier identification of refractory patients compared to current interim PET- CT imaging strategies. CCL17 testing may, therefore, be useful as an adjunct or alternative to interim PET-CT to improve risk-adapted therapy. Electrochemical immunosensors permit characterisation and translation of antibody-antigen affinity interactions into quantifiable electrical signals ⁸ , and offer the possibility of a miniaturised, scalable and economical technology for point-of-care diagnostics with small blood sample volumes. Thus far, diverse approaches have been reported for electrochemical detection of clinical biomarkers, and feature a wide range of electrode substrates, detection strategies and functionalisation methods ⁸ . Previously, we demonstrated the clinical potential of an “unlabelled” electrochemical immunosensor for TREM-1, MMP-9, HSL and IL-6 biomarkers ⁹ . Recently, electrochemical immunosensors have increasingly utilised micro- and nano- technology for development of complex electrode formats and sophisticated surface chemistries to improve analytical performance for clinical testing. The Estrela group reported an electrochemical ELISA biosensor that utilised nanometre carboxylic functionalised popypyrrole films prepared via cyclic voltammetry on micro-array electrodes for blood serum testing ¹⁰ . Likewise, the Ingber group developed a three-dimensional porous antifouling assay supported by a network of nanomaterials for interleukin 6 detection in blood plasma ¹¹ . The Morgan group have also demonstrated an electrochemical ELISA biosensor based on a fully- integrated, bespoke microfluidic electrode system for cytokine detection in blood serum ¹² . Critically, however, clinical acceptance and widespread adoption of electrochemical immunosensors for clinical testing remains elusive, particularly for cancer diagnostic applications. Principally, there is an urgent need to translate proof-of-principle studies on spiked samples ¹³ , into clinical feasibility studies with cancer patient samples to validate clinical test performance. Additionally, proposed electrochemical biosensor strategies must be conducive to mass production and commercialisation to initiate widespread adoption and minimise test costs for healthcare providers. In one teaching, the present disclosure seeks to obviate one or more of the aforementioned disadvantages. Summary of the disclosure The present invention is based in part on the development of an electrochemical biosensor for use in detecting CCL17 in a sample obtained from a subject. In a first aspect there is provided an electrochemical biosensor system comprising: at least one electrode formed on a first substrate; and a CCL17 capture layer formed on the electrode or a further substrate, the CCL17 capture layer comprising a ligand molecule capable of specifically binding CCL17. In one embodiment, the CCL17 capture layer is formed on the electrode itself. Alternatively, other substrate, such as a membrane, or a micro particle or nano particle surface may be coated with the CCL17 capture layer. In this manner, any CCL17 binding to the electrode or further substrate may be detected by a change in electrical signal occurring due to capture of any CCL17 present in a sample. The ligand may, for example, comprise a receptor, such as CCR4, or a binding fragment thereof, which is capable of biding CCL17. Alternatively, the ligand may comprise an aptamer capable of specifically binding CCL17. Aptamers and methods of generating them, are well known in the art and are generally oligonucleotide or peptide molecules which specifically bind to a target molecule, in this case CCL17. However, conveniently, the ligand is an antibody or antibody fragment, which is capable of specifically binding to CCL17. The term “antibody” refers to an immunoglobulin molecule that recognizes and specifically binds to a target, in this case CCL17, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments), single-domain antibodies, camelid-derived heavy chain polypeptides (VHH) and fragments thereof (e.g., truncated VHH), single chain Fv (scFv) and mutants thereof, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determinating portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the ability to specifically bind CCL17. In one embodiment, the antibody is a monoclonal antibody, or a fragment derived therefrom. A monoclonal antibody refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to “polyclonal antibodies” that typically include different antibodies directed against different antigenic determinants. Furthermore, “monoclonal antibody” refers to such antibodies made by any number of ways including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals. The electrochemical biosensor may be used directly or indirectly. For direct use, the electrochemical biosensor may label free and is capable of detecting any physical or chemical change arising directly from complex formation between CCL17 and the ligand, without the use of a label. Alternatively, for indirect use, an electrochemical biosensor further employs a label or signal generating moiety, allowing highly sensitive and versatile detection. In indirect measurements enzymes, e.g., horseradish peroxidase, catalase and glucose oxidase, alkaline phosphatase, or electroactive compounds such as ferrocene, as a mediator, or Prussian blue may be used in the electrochemical bioosensor. In one embodiment, the electrochemical biosensor is employed in an enzyme linked immunosorbent assay (ELISA). Various forms of ELISA are known to the skilled addressee, including direct and indirect and competitive and non-competitive. Any of the known forms of ELISA may be used in accordance with the present teaching. In one embodiment, the ELISA is an electrochemical sandwich ELISA. An Electrochemical sandwich ELISA is a branch of electrochemical immunoassays where the recognition of a desired target is done using a traditional sandwich assay and detection is achieved using an electrochemical method. In accordance with the present disclosure, such an immunoassay involves generation of a sandwich complex between the electrochemical biosensor as described herein; CCL17; and further binding to CCL17 by a secondary recognition molecule, with an electrochemically active signal tag. For signal measuring, the electrochemical signal tag either provides the signal directly or a reaction, such as a Redox enzyme reaction, with a substrate is induced afterwards. Another suitable ELISA for use in accordance with the present invention, is a competitive ELISA. In a competitive ELISA, a known amount of labeled CCL17 would be included in the assay and the labeled CCL17 would compete with any CCL17 present in a sample, for binding to the CCL17 capture layer. If no CCL17 is present in the sample, or as would be observed in a control sample, known to be CCL17 free, a maximum value of labeled CCL17 would be detected. Any reduction from the maximum value is due to any CCL17, which is present in the sample, competing with the labeled CCL17 for binding to the CCL17 capture layer. Such a reduction from the maximum value can be equated with an amount of CCL17 being present in the sample. This is readily determined by the skilled addressee using know techniques. Signal detection from any of the electrochemical biosensors, as described herein, may employ electrochemical amperometric and/or voltammetric techniques including differential pulse voltammetry (DPV), linear sweep voltammetry (LSV), stripping voltammetry and square-wave voltammetry (SWV). The generated signal is directly proportional to the CCL17 (or when employed, labeled CCL17) concentration. For example, this type of sensing involving sandwiching of CCL17 between two highly specific capturing and recognition molecules, provides a high level of sensitivity. Moreover, several nanomaterials such as gold nanoparticles (Au NPs) or other metal nanoparticles, such as silver or copper, or different quantum dots (QDs) can be used to amplify the signal. In a labeled electrochemical biosensor, the label , as discussed above, may be attached to a competing CCL17. Alternatively, the label may be attached to an antibody, which is designed to bind CCL17 without disrupting the binding of CCL17 to the ligand formed on the surface of the electrode. In one preferred teaching of this disclosure, the electrochemical biosensor is used in conjunction with a label. The use of a label when the sample is a complex sample, such as a blood, serum or plasma sample, may be preferred as other proteins present in the sample may non-specifically bind to the electrode and/or further surface and impact any electrochemical measurement. Thus, the use of a label may serve to circumvent and/or improve sensitivity. In certain embodiments, at least one electrode of the electrochemical sensor is a working electrode. The electrochemical sensor may further comprise a counter electrode and a reference electrode. The working and counter electrode may be formed of either gold, platinum or copper material. The working electrode may also be formed of carbon material, or gold, platinum or copper, for example. The ligand for capturing any CCL17 present in a sample may, in one embodiment, be functionalised on to the carbon electrode, or further surface with application of a suitable cross-linker molecule, such as Sulfo-LC-SPDP. The reference electrode may be formed from, for example, carbon, gold, platinum, copper, silver/silver chloride and saturated calomel material. The working, counter and reference electrodes are formed on the first substrate, which may for example be any suitable polymer, silicon or glass material. The present inventors have observed that electrode surface roughness was found to have an impact on measurement reproducibility. Thus, is may be preferred in some embodiments to employ polycrystalline metal, such as gold "macro" electrodes, instead of screen-printed electrodes, for example. Although the use of a cross-linker molecule is described above, the ligand may be immobilized on the surface of the working electrode using a number of techniques, including physical adsorption, trapping, covalent bonding or affinity immobilization known in the art. Physical adsorption may be through electrostatic interactions, hydrogen bonding, van der Waals, and/or hydrophobic interaction. Trapping, for example, may be by encapsulation of the ligand in a polymer matrix such as a molecularly imprinted polymer (MIP) or a sol-gel known in the art. Covalent attachment of the ligand to the surface of the electrode may be via amine, carboxyl, carbohydrate, and/or thiol, for example. Generally, an electrode surface may first be coated with a thin film or self-assembled monolayer (SAM) providing functional groups in order to be able to covalently bond with amino groups of the ligand/antibody for example. For this purpose, reagents such as glutaraldehyde, carbodiimide succinimide ester, N- hydroxysuccinimide, periodate, or maleinimide may be used. Thus, in some embodiments, the working electrode is chemically functionalized prior to capture of the ligand molecule. In a preferred embodiment, the chemical functionalization comprises at least one SAM. In this manner, semi-covalent binding is formed between a sulfide group (disulfides, sulfides and thiols) and a metal electrode surface. One such suitable method employs a heterobifunctional agent, such as Sulfosuccinimidyl 6- (3'- [2-pyridyldithio]- propionamido)hexanoate (Sulfo-LC-SPDP). Sulfo-LC-SPDP is a heterobifunctional, thiol- cleavable and membrane impermeable crosslinker. It contains an amine-reactive N- hydroxysuccinimide (NHS) ester which is capable of reacting with lysine residues to form a stable amide bond. Further reaction with a reducing agent, such as dithiothreitol (DTT) reduces disulfide bonds within the cross-linker molecule to generate sulfhydryl bonds, which are capable of chemisorption with a metal electrode surface. The inventors observed that another molecule, thiolated protein G did not provide as reproducible results as sulfo-LC- SPDP, when used in a sandwich assay, as the protein G was observed to capture the secondary antibody and therefore provide poor discrimination between the control and positive CCL17 samples. Additionally, the inventors have observed that any unreacted DTT and/or non-reduced sulfo molecules may interfere with the assay. Thus, in one embodiment, in order to improve reproducibility, a cleaning step, such as a centrifugal filtration step, is carried out in order to remove any unreacted DTT or non-reduced sulfo molecules, prior to depositing on the surface of the electrode or further surface. This ensures that any excess DTT and/or non- reduced Sulfo molecules, do not interfere with immobilisation of the ligand on the electrode or further surface In some embodiments, the working electrode is cleaned prior to any surface modification. Conveniently, the cleaning comprises an electrochemical, chemical and/or mechanical cleaning process. In one embodiment, the working electrode is cleaned using a chemical, mechanical and electrochemical process. In a further aspect, there is provided a method of making an electrochemical sensor according to the first aspect, the method comprising cleaning the surface of the working electrode chemically, mechanically and electrochemically; and thereafter forming on the surface of the cleaned electrode a capture layer comprising a SAM, to which the ligand is covalently bonded. In one embodiment the SAM is formed using Sulfo-LC-SPDP. Sulfo-LC-SPDP may be further reduced by a reducing agent, such as DTT. Any unreacted reducing agent and/or non-reduced Sulfo-LC-SPDP, may be removed, such as by centrifugal filter, prior to depositing on the surface of the cleaned electrode. Following immobilization of the ligand to the surface of the electrode, by the above method, or any other of the methods described herein, any uncoated surface of the electrode may be blocked in order to prevent or minimize any non-specific binding of any CCL17 in a sample to the electrode. In a further aspect there is provided a kit for use in conducting an electrochemical bioassay, the kit comprising: an electrochemical biosensor according to the first aspect, or embodiments thereof; and optionally a receptacle for receiving a sample to be assayed and/or means, such as a finger stick device or swab, for obtaining a sample from a subject. The kit may separately further comprise an amount of labelled CCL17, or anti-CCL17 antibody and optionally reagents for reacting with the label in order to generate an electrochemically detectable signal. In another aspect, there is provided a method for detecting a CCL17 in a sample, comprising: a) providing an electrochemical biosensor according to the first aspect, or embodiments thereof; b) contacting the sensor with a sample; c) applying a voltage to the biosensor; and d) monitoring for any change in response of the sensor as a consequence of any CCL17 in the sample binding to the sensor. Step b) may further comprise adding a labelled anti-CCL17 antibody or labelled CCL17 and optionally (if the label is not directly electrochemically detectable) after a period of time providing one or more reagents which react with the label in order to generate an electrochemically detectable signal. The sample may be any suitable sample obtained from a subject. The sample may be a sample of a body fluid of an individual and may, for example, be whole blood or derived products, e.g., isolated mononuclear cells, plasma or serum, especially serum. For the purposes of the above aspects and embodiments, the subject may be a human or any other animal. In particular embodiments the subject is selected from the group consisting of human, non-human primate, equine, bovine, ovine, caprine, leporine, avian, feline or canine. The monitoring for any change in response may be carried out by any suitable means. Typically, any change in response may be detected by amperometric, potentiometric, impedimetric or conductometric means known in the art. In one embodiment the detection is an amperometric detection method. Detailed Description of the Disclosure The present disclosure will now be further described by way of example and with reference to the following figures, which show: Figure 1: Product concept for the proposed electrochemical sensor and overview of the developed sandwich immunoassay; Figure 2: (a) Nyquist plots for functionalised electrodes following specified heterobifunctional cross-linker assay depositions (n=3). (b) Charge transfer resistance, Rct, for electrodes were obtained through circuit fitting with Randles equivalent circuit and Levenberg-Marquardt model; Figure 3: (a) Characterisation of TMB at the working electrode when subjected to two one- electron transfer redox reactions with CV (n=3). (b) Amperometric current-time curves that show reduction of oxidised TMB products at Epc = -0.2 V for functionalised electrodes following incubation with different target antigen concentrations ranging from 0-50,000 pg/ml (n=3), (c) plot of current responses of electrodes at 120 s for different target antigen concentrations (n=3). Asterisk’s indicate significantly different responses from control measurements where P < 0.05 (d) Plot of calibration curve for current responses versus log CCL17/TARC concentration for statistically significant responses fitted with a four-parameter logistic regression model (n=3); Figure 4 (a) Amperometric current-time curves show reduction of oxidised TMB products at Epc = -0.2V for functionalised electrodes following incubation with clinical cHL patient serum samples (n=3), (b) Plot of current responses of electrodes at 120 s following incubation with clinical patient serum samples (n=3), (c) Plot of current responses of electrodes categorised into patient groups, based on known CCL17/TARC concentration; group A (1-5 ng/ml), group B (5-10 ng/ml), group C (10-50 ng/ml) and group D (>50 ng/ml). Asterisk’s indicate significantly different responses where P < 0.05, cHL denotes classic Hodgkin lymphoma; Figure 5: (a) Amperometric current-time curves show reduction of oxidised TMB products at Epc = -0.2V for functionalised electrodes following incubation with paired cHL patient serum samples obtained at pre-treatment and on-treatment clinical stages (n=3), (b) plot of current responses (mean ± SD) of electrodes at 120 s following incubation with clinical patient serum samples (n=3); and Figure 6: (a) Plot of biosensor performance compared to the ELISA test for estimation of CCL17/TARC concentration in pre-treatment patient serum samples, (b) Plot showing correlation of estimated CCL17/TARC concentration in pre-treatment cHL patient samples between the biosensor and ELISA tests, (c) Plot of biosensor performance compared to the ELISA test for estimation of CCL17/TARC concentration in paired pre-treatment (PT) and on- treatment (OT) patient serum samples, (d) Plot showing correlation of estimated CCL17/TARC concentration in paired pre-treatment (PT) and on-treatment (OT) cHL patient serum samples between the biosensor and ELISA tests. Materials & Methods Clinical Samples Samples from pre-treatment and on-treatment cHL patients were collected as part of the Investigation of The Cause of Hodgkin lymphoma (ITCH) and the Biomarkers And Classical Hodgkin lymphoma (BACH) studies. Samples were selected to represent the range of CCL17 values in serum samples from cHL patients. CCL17 levels, quantified using the Human CCL17 Quantikine ELISA Kit (R&D Systems, Inc. Minneapolis, USA), were available for all samples. Samples from healthy controls were collected as part of the SHARE study (Study of Healthy Adult Response to EBV). In total, 54 samples were subjected to electrochemical analysis including samples from five healthy volunteers, and 42 pre-treatment and seven on-treatment cHL patients. All studies were approved by Research Ethics Committees, and all participants gave written, informed consent. Materials & Reagents Concentrated sulphuric acid (98%), hydrogen peroxide (30%) in water and the CHI120 electrode polishing kit (CH Instruments, Inc. Austin, USA) were purchased for the electrochemical cleaning procedure. Sulfosuccinimidyl 6-(3'-(2-pyridyldithio) propionamido) hexanoate (Sulfo-LC-SPDP), DL-dithiothreitol (DTT), 6-mercapto-1- hexanol (MCH), tris(2-carboxyethyl)phosphine (TCEP), phosphate-buffered saline (PBS), deionised (DI) water (Millipore, Livingston, West Lothian), Human CCL17 DuoSet ELISA (R&D Systems, Inc. Minneapolis, USA) and Amicon Ultra-0.5 mL 3 KDa centrifugal filters were employed in the cross-linker sandwich assay. Lastly, potassium ferricyanide, potassium ferrocyanide and the DuoSet ELISA Ancillary Reagent kit 1 (R&D Systems) were purchased for appropriate testing of developed electrochemical immunosensors. Unless otherwise stated, reagents were purchased from Sigma-Aldrich (Sigma-Aldrich, Poole, Dorset). Electrochemical Instrumentation Electrochemical experiments were conducted on a PalmSens Emstat potentiostat (PalmSens, The Netherlands) that interfaced with one CH Instruments CHI101 gold 2.0 mm working, CHI111 silver silver/chloride reference and CHI115 platinum counter electrode arranged in a standard three-electrode configuration. Electrochemical Cleaning Procedure Working electrodes were chemically cleaned in hot piranha solution comprised of sulphuric acid and 30% hydrogen peroxide in water (3:1 ratio) for 10 minutes to remove organic contaminants. Electrodes were subsequently mechanically cleaned with 0.05 µm alumina MicroPolish powder on a Microcloth pad for 5 minutes and sonicated in DI water for 15 minutes to remove alumina particles. Thereafter, working electrodes were electrochemically cleaned in 0.5 M sulphuric acid in DI water by applying cyclic potential sweeps between vertices of - 0.2 V and +1.5 V with a scan rate of 100 mV/s for 20 consecutive scans. Heterobifunctional Cross-linker Sandwich Assay Working electrodes were functionalised with capture CCL17 antibodies through modification of a previously reported protocol ⁹ . Firstly, 40 mM Sulfo-LC-SPDP in DI water and 200 µg/ml primary CCL17 antibodies (2:1 ratio) in 1 × PBS were agitated at room temperature for 1 hour to allow amide bond formation between N-hydroxysuccinimide (NHS) esters and lysine residues of the cross-linker and antibody respectively. Secondly, 150 mM DTT in 1 × PBS was introduced to the assay for 45 minutes to enable reduction of disulfide bonds within the cross- linker molecule to sulfhydryl groups for improved chemisorption to gold. Thirdly, Amicon Ultra- 0.5 mL 3 KDa centrifugal filters were used to remove unreacted Sulfo-LC-SPDP and DTT molecules prior to assay deposition since both feature free sulphur atoms that may inadvertently attach to electrode surfaces. Lastly, the assay filtrate was reconstituted in 1 × PBS and subsequently pipetted on to working electrode surfaces and incubated in a humidity chamber at 4 ^o C for ~15 hours to facilitate chemisorption between sulphur atoms of the cross- linker and gold electrode surfaces. Working electrodes were then thoroughly rinsed with 1 × PBS solution to remove excess antibody solution. Remaining bare gold electrode surfaces were blocked with 1 mM MCH in 1 × PBS and 5 mM TCEP for 30 minutes to prevent non-specific binding and subsequently rinsed in 1 × PBS to remove unbound MCH particles. Electrodes were then incubated with 25 µL of either target CCL17 antigen (24-50,000 pg/ml) in 1 × PBS or clinical cHL samples, diluted ten-fold in 1 × PBS, for 1 hour at room temperature. Electrodes were then thoroughly rinsed in 1 × PBS to remove unbound sample constituents prior to incubation with 25 uL of 200 ng/mL biotinylated CCL17 secondary antibody solution in 1 × PBS for 1 hour at room temperature. Electrodes were thoroughly rinsed with 1 × PBS and incubated out of direct sunlight for 20 minutes with 25 µL of ×40 diluted streptavidin-conjugated horseradish-peroxidase in 1 × PBS. Lastly, electrodes were thoroughly washed with 1 × PBS to remove unbound streptavidin- conjugated horseradish-peroxidase and individually placed in 500 µL substrate solution comprised of equal volumes of stabilised 3,3',5,5'-Tetramethylbenzidine (TMB) and stabilised hydrogen peroxide solution for 20 minutes. Amperometric measurements were subsequently obtained by subjecting working electrodes to a constant potential of -0.2 V for 120 s with 0.1s time intervals. Self-Assembled Monolayer Characterisation Working electrodes were subjected to electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measurements to ensure capture CCL17 antibodies and blocking agents were successfully immobilised prior to antigen exposure. Electrochemical measurements were conducted by immersing all three electrodes in a solution of 10 mM ferri/ferro potassium cyanide in 1 × PBS following thorough washing of respective assay depositions. EIS measurements were recorded with respect to the open circuit potential with an applied ac potential of 10 mV rms amplitude over a frequency range of 0.1-100,000 Hz. CV experiments were conducted between -0.4 V and 0.6 V with a step of 0.01 V at scan rates of 50 mV/s. All measurements were performed on three functionalised electrode surfaces. Reproducibility Study A reproducibility study was conducted on functionalised electrodes with EIS to evaluate inter- assay variability of SAM formation prior to antigen exposure. EIS was conducted with electrodes immersed in a solution of 10 mM ferri/ferro potassium cyanide in 1 × PBS following respective assay depositions. EIS measurements utilised previously specified parameters and considered five functionalised working electrodes per measurement day over a 7-day period. Scanning Electron & Atomic Force Microscopy Set-up Scanning electron microscopy (SEM) experiments were performed with a JEOL JSM-IT100 InTouchScope configured in high vacuum mode with an acceleration voltage of 10-20 kV. Images were acquired from three random sites on working electrodes at x1700-10,000 magnification at a working distance of 10-14 mm to provide accurate representations of substrates. Atomic force microscopy (AFM) experiments were conducted with the Asylum Research MFP-3D instrument with triangular AFM cantilevers configured in contact mode. Images were acquired from three random sites on working electrodes to provide accurate topographical information on substrates. Data Analysis & Statistics One-way ANOVA studies were conducted on EIS and CV measurements obtained during immobilisation experiments, with respect to percentage differences in charge transfer resistance, R _ct , and peak currents, I _pa and I _pc . Similarly, one-way ANOVA analyses were conducted on steady-state amperometric currents at 120 s for all concentrations evaluated during spiked CCL17/TARC studies. Additionally, four-parameter logistic regression analysis was performed to observe the relationship between CCL17 concentration and current for all concentrations statistically significant relative to control measurements where p-values < 0.05. Thereafter, one-way ANOVA studies were conducted on currents at 120 s intervals for clinical samples to determine whether differences between healthy volunteer and cHL patient samples were statistically significant. All one-way ANOVA studies utilised the Tukey post hoc test to confirm the origin of statistical significances between groups. All amperometric data consisted of three replicate and one repeat measurements. Amperometric and ELISA measurement data were compared for all patient samples. A comparative analysis was also performed for all cHL patient samples that had a predicted CCL17 concentration for both electrochemical and ELISA tests to establish the correlation between predicted CCL17 concentration using the different test platforms. Reproducibility data were subjected to one- way ANOVA analysis by comparing R _ct values associated with assay depositions over a 7- day period to establish variability in SAM formation. Post-hoc studies with Fisher least significant difference testing were performed on statistically significant results where p-values <0.05. All electrochemical results describe mean values ± standard deviation. Results & Discussion Electrochemical Characterisation of Functionalised Electrodes Working electrodes were subjected to electrochemical cleaning prior to assay immobilisation to improve chemisorption between thiol head groups of cross-linker molecules and gold atoms of electrodes. Electrodes were subsequently immersed in the redox couple, Fe(CN)6 ^-3/4 , and EIS and CV experiments were performed to determine electrode cleanliness. Electrodes measured small R _ct and large I _pa / I _pc values characteristic of Fe(CN)6 ^-3/4 species, which indicates that the redox couple can freely participate in electron transfer events, and implies electrodes are appropriately clean for immobilisation. Electrodes immobilised with CCL17 antibodies conjugated to Sulfo-LC-SPDP measured significant increases in Rct (p-value < 0.05) and decreases in I _pa (p-value < 0.05) and I _pc (p-value < 0.05), which suggests [Fe(CN)6]- ^3/4 ions have reduced ability to participate in electron transfer due to antibody SAM formation on electrodes. Electrodes functionalised with MCH further increased Rct (p-value < 0.05) and decreased I _pa (p-value = 0.208) and I _pc (p-value < 0.05), indicative of chemisorption between sulphur atoms of MCH and gold atoms of electrodes. Electrodes measured consecutive increases in ΔE during respective assay depositions, from ΔE = 0.110V to ΔE = 0.809V, indicative of slow electron transfer kinetics due to increased density of SAM and antibody films. Assay reproducibility was evaluated over a 7-day time period to determine whether SAM formation varied between electrodes and assay batches. Primary antibody immobilisation markedly increased R _ct on all days relative to R _ct for bare electrodes (p-value < 0.05). However, Rct varied between electrodes on a specific day, with an inter-assay co-efficient of variance (COV) of 21.7% over the 7-day period. Furthermore, Rct differed significantly between electrodes on different days for antibody depositions (p-value < 0.05), indicative of varied antibody immobilisation between assay batches. Backfilling increased R _ct of electrodes and represented varied thiol attachment on a specific day, reflected by an inter-assay COV of 20.0% over the 7-day period. Additionally, backfilled electrodes recorded significant variation in R _ct on different days over the 7-day period (p-value < 0.05), further suggestive of variability in MCH formation between assay batches. Thiol SAM formation on gold occurs in a two-step process, a high and slow growth phase, where the density and packing of molecules is influenced by several external and internal factors. The high growth phase is heavily influenced by surface cleanliness, concentration of adsorption molecules, immersion time, temperature and humidity ¹⁴ . Therefore, fluctuations in experimental conditions may likely explain R _ct variability between days. The slow growth phase partly determines SAM morphology and is influenced by hydrogen bonding and Van der Waals forces between alkane thiol chains ¹⁵ . Hence, the stability and density of SAM formation may be improved by selecting alkane thiols of increased chain length, and may be considered for future experiments. Nevertheless, current findings indicate successful assay immobilisation for detection of CCL17 antigen. Electrochemical Detection of TMB Oxidation States Electrochemically-cleaned electrodes were immersed in substrate solution consisting of hydrogen peroxide and TMB and subjected to CV to determine whether it was possible to electrochemically detect TMB products. In sandwich assays, horseradish peroxidase enzymatically catalyses reduction of hydrogen peroxide to water in the presence of the native TMB diamine that acts as a proton donor. Consequently, TMB is subjected to a one-electron transfer oxidation reaction, and the resultant TMB diimine is measured at λ = 650 nm with ELISA. The first oxidised TMB product can be detected electrochemically, where I _pa = 3.69 µA ± 0.21 at E _pa = 0.06 V, indicated in Figure 3a. The oxidised TMB product is typically subjected to a second oxidation reaction initiated by sulphuric acid, and the product is then measured at λ = 490 nm with ELISA. Similarly, the two-electron transfer product can be characterised electrochemically, where I _pa = 5.66 µA ± 0.14 at E _pa = 0.21 V. The oxidised product is proportional to target antigen concentration and measurable electrochemically by reducing oxidised species with an appropriate Epc. The oxidised products gain one electron during subsequent reduction reactions and were observed at Ipc = -1.79 µA ± 0.12 at Epc = 0.17 V and I _pc = -4.38 µA ± 0.09 at E _pc = 0.01 V. Hence, electrodes successfully identified redox reactions characteristic of electrochemical TMB detection ¹⁶ . A potential of Epc = -0.20 V was selected for amperometric experiments as the applied potential does not coincide with oxidation potentials, E _pa , of TMB. Amperometric curves showed reduction of oxidised TMB species at electrodes, where consumption of reactants with time produced a constant concentration gradient and mass- transfer limited currents, shown in Figure 3b. Immunosensors showed significant discrimination between control and sample mass-transfer limited currents at 120 s for spiked CCL17 concentrations ≥194 pg/ml (p-value < 0.05), indicated in Figure 3c. Signals were not linear over the specified concentration range, which is unsurprising given the wide range of tested concentrations. Therefore, electrode responses and CCL17 concentrations were plotted on logarithmic scales and fitted with a four-parameter logistic regression model. Mass- transfer limited currents at 120 s displayed a strong positive relationship with CCL17 concentration, R ² = 0.986, for samples with statistically significant differences from control measurements, shown in Figure 3d. Four-parameter logistic regression was deemed suitable for analysis, as the model has been employed in electrochemical immunoassays ¹⁷ , and better represents biological systems that display non-linear behaviour over wide concentration ranges. Inter-assay COV was calculated as 14.6% and 12.6% for all tested and statistically significant CCL17 concentrations, respectively. Lower and upper limits of quantification were calculated as 387 pg/ml and 50,000 pg/ml (based on COV ≤ 20%), respectively, with a limit of detection of 194 pg/ml. Comparatively, the biosensor demonstrates a superior dynamic range, with an upper limit of quantification of 50,000 pg/ml CCL17 compared to 2,000 pg/ml for the ELISA platform. The biosensor demonstrates reduced test sensitivity at 194 pg/ml when compared to ELISA at 7 pg/ml. Nevertheless, test sensitivity is well below the proposed clinical cut-off for elevated CCL17 in cHL patients at 1000 pg/ml ¹⁸ , and therefore shows significant promise for clinical testing. Electrochemical immunosensors demonstrated the ability to discriminate between healthy volunteers and all cHL pre-treatment patient samples. Electrodes measured significant increases in current signals at 120 s for all 42 cHL patients relative to healthy volunteers (p- value < 0.05), depicted in Figures 4a & 4b, with an inter-assay COV of 19.5% across all patients. The increase in COV compared to concentration studies is likely attributable to the complexity of biologically rich serum derivatives. Nevertheless, the biosensor demonstrates the capacity to discriminate between healthy volunteers and cHL patients that have raised CCL17 levels, which reflects ~90% of all cHL patients ¹ . Electrode responses were characteristic of CCL17 antigen capture at functionalised surfaces and were significantly different between healthy volunteer and all cHL patient groups (p-value < 0.05), stratified according to known CCL17 concentration into 1-5 ng/ml, 5-10 ng/ml, 10-50 ng/ml and >50 ng/ml patient categories. Hence, the biosensor also shows the potential to quantify serum CCL17 in cHL patients, which is important clinically as CCL17 correlates with tumour burden ² . However, further testing may be conducted on cHL patients with known CCL17 levels around the proposed clinical cut-off, to confirm the test identifies all cHL patients with elevated CCL17. To the best of our knowledge, our findings demonstrate for the first time, sensitive electrochemical detection of serum CCL17 from pre-treatment cHL patients. Electrochemical detection of serum CCL17 may facilitate introduction of a rapid, economical point-of-care biosensor for triage of cHL patients and treatment response monitoring. Clinically, the test would provide a minimally invasive and highly accessible platform to promote triage of cHL patients and enable timely referral for secondary care diagnosis with lymph node biopsy. Practically, discriminatory signals can be generated in seconds to minutes, and require minimal user interpretation, allowing the possibility of a streamlined pathway for patients and clinicians. Presently, time-to-result from blood sample introduction is 2 hours and 42 minutes. A significant reduction in assay time is required to facilitate while-you-wait results, and will be a primary motivation for future studies. Economically, electrochemical biosensors offer a scalable technological platform well-suited to high-volume manufacture, minimising overall costs per test to health care providers. Biosensor reproducibility must be improved to enable clinical translation. It is recognised an inter-assay COV ^15% is acceptable for bioanalytical tests, although ^20% is deemed acceptable for measurements approaching the lower limit of quantification. Current test COV of 19.5% on patient samples is likely attributable to varied antibody immobilisation between electrodes, highlighted in assay reproducibility studies. Electrode topography was also evaluated prior to antibody immobilisation, to understand whether surface characteristics contribute to signal variability. SEM revealed reduced surface roughness with small scratches on electrodes likely attributable to repeated mechanical polishing procedures. AFM confirmed superficial electrode scratches, although substrates were relatively smooth with good surface homogeneity. Electrode measurements are re-assuring for functionalisation procedures as high surface roughness promotes poor thiol organisation and hinders SAM immobilisation ¹⁹ . Indeed, intra-assay COV for 23 patient samples was ≤15%, providing evidence that the current technology has potential for clinical testing. Testing was performed on cHL patient serum samples acquired pre- and on-treatment to determine biosensor utility for patient treatment response monitoring. Immunosensors measured significant decreases in current signals at 120 s for all paired cHL patient samples (p-value <0.05), indicated in Figures 5a and 5b. Therefore, the biosensor has potential for monitoring treatment response, provided CCL17 is clinically utilised in the future. Recently, decreases in CCL17 in cHL patients have been shown to inform treatment response after one cycle of chemotherapy ⁷ , which would facilitate earlier identification of refractory patients, and addresses issues regarding false positives/negatives associated with interim PET/CT imaging ⁶ . Likewise, CCL17 has clinical potential to identify cHL patients unresponsive to allogeneic stem cell transplants ²⁰ . Therefore, the biosensor represents a promising tool as an adjunct or alternative to interim PET-CT, with potential to support development of personalised treatment strategies. Optimisation of sample dilution should be considered to address reduced test performance for patients with very high CCL17 levels. Nevertheless, biosensor quantification of CCL17 in pre- treatment patient samples positively correlated (Pearson’s r = 0.910) with ELISA, depicted in Figure 6b. Biosensor results also under-estimated serum CCL17 for pre- and on-treatment cHL samples compared to the ELISA test, indicated in Figure 6c. However, measurements still positively correlated (Pearson’s r = 0.880) with ELISA results, shown in Figure 6d. The biosensor was unable to estimate serum CCL17 for three healthy volunteers and five cHL patients during chemotherapy, which were all quantified by ELISA. Signals for these patients were below the lower interpolation limit of the calibration curve (section 4.4), and therefore it was not possible to accurately quantify CCL17 concentrations. However, the concentration of serum CCL17 in these samples was in the low normal range, well below the proposed clinical cut-off for raised CCL17, and therefore would not affect the clinical usefulness of the assay. Conclusion The need for earlier diagnosis of cHL in the clinical pathway is well-recognised and forms part of a wider key healthcare strategy to detect 75% of all cancers at early disease stage in the UK by 2028. The developed biosensor for serum CCL17 detection has potential to facilitate rapid triage of patients who have a differential diagnosis that includes cHL in primary care, and permit monitoring of chemotherapy response in secondary care. The electrochemical biosensor has demonstrated quantitative detection of CCL17 with high sensitivity, linearity and a large dynamic range (194-50,000 pg/ml). The biosensor demonstrated successful discrimination between serum samples of all tested cHL patients and healthy volunteers, which shows considerable promise for clinical translation of a point-of-care triage strategy. Additionally, the biosensor showed the ability to qualitatively measure decreases in serum CCL17 between all seven paired pre- and on-treatment cHL patient samples, which provides potential to measure treatment response during chemotherapy. Overall, our preliminary findings have demonstrated considerable potential for electrochemical detection of serum CCL17 in clinical samples, and represents an important step towards development of a rapid triage and treatment response test for cHL. References (1) Hnátková, M., Mociková, H., Trnený, M., Zivný, J. (2009) The biological environment of Hodgkin's lymphoma and the role of the chemokine CCL17/TARC. Prague Med Rep.2009;110(1):35-41. (2) Plattel, W. J., Alsada, Z. N. D., van Imhoff, G. W., Diepstra, A., van den Berg, A., & Visser, L. (2016). Biomarkers for evaluation of treatment response in classical Hodgkin lymphoma: comparison of sGalectin-1, sCD163 and sCD30 with TARC. British Journal of Haematology, 175(5), 868–875. doi:10.1111/bjh.14317 (3) Niens, M., Visser, L., Nolte, I. M., van der Steege, G., Diepstra, A., Cordano, P., … van den Berg, A. (2008). Serum chemokine levels in Hodgkin lymphoma patients: highly increased levels of CCL17 and CCL22. British Journal of Haematology, 140(5), 527–536. doi:10.1111/j.1365-2141.2007.06964.x (4) Connors, J. M., Cozen, W., Steidl, C., Carbone, A., Hoppe, R. T., Flechtner, H.-H., & Bartlett, N. L. (2020). Hodgkin lymphoma. Nature Reviews Disease Primers, 6(1).doi:10.1038/s41572-020-0189-6 (5) Hutchings M. (2019). PET-adapted treatment of Hodgkin lymphoma. Blood, 134(15), 1200–1201. https://doi.org/10.1182/blood.2019002420 (6) Sauer, M., Plütschow, A., Jachimowicz, R. D., Kleefisch, D., Reiners, K. S., Ponader, S., … von Strandmann, E. P. (2012). Baseline serum TARC levels predict therapy outcome in patients with Hodgkin lymphoma. American Journal of Hematology, 88(2), 113–115. doi:10.1002/ajh.23361 (7) Plattel, W. J., van den Berg, A., Visser, L., van der Graaf, A. M., Pruim, J., Vos, H., Hepkema, B., Diepstra, A., & van Imhoff, G. W. (2012). Plasma thymus and activation-regulated chemokine as an early response marker in classical Hodgkin's lymphoma. Haematologica, 97(3), 410–415. https://doi.org/10.3324/haematol.2011.053199 (8) Mollarasouli, Kurbanoglu, & Ozkan. (2019). The Role of Electrochemical Immunosensors in Clinical Analysis. Biosensors, 9(3), 86. doi:10.3390/bios9030086 (9) Ciani, I., Schulze, H., Corrigan, D. K., Henihan, G., Giraud, G., Terry, J. G., … Mount, A. R. (2012). Development of immunosensors for direct detection of three wound infection biomarkers at point of care using electrochemical impedance spectroscopy. Biosensors and Bioelectronics, 31(1), 413– 418. doi:10.1016/j.bios.2011.11.004 (10) Arya, S. K., Estrela, P. (2020) Electrochemical ELISA Protein Biosensing in Undiluted Serum Using a Polypyrrole-Based Platform. Sensors (Basel). 20(10):2857. doi: 10.3390/s20102857. PMID: 32443483; PMCID: PMC7287672. (11) Sabaté del Río, J., Henry, O.Y.F., Jolly, P. et al. (2019) An antifouling coating that enables affinity-based electrochemical biosensing in complex biological fluids. Nat. Nanotechnol.14, 1143–1149 https://doi.org/10.1038/s41565-019- 0566-z (12) Evans, D., Papadimitriou, K.I., Vasilakis, N., Pantelidis, P., Kelleher, P., Morgan, H., Prodromakis, T. A. (2018) Novel Microfluidic Point-of-Care Biosensor System on Printed Circuit Board for Cytokine Detection. Sensors. 18(11):4011. https://doi.org/10.3390/s18114011 (13) Marques, R. C. B., Viswanathan, S., Nouws, H. P. A., Delerue-Matos, C., & González-García, M. B. (2014). Electrochemical immunosensor for the analysis of the breast cancer biomarker HER2 ECD. Talanta, 129, 594– 599. doi:10.1016/j.talanta.2014.06.035 (14) Wang, Y., Solano Canchaya, J. G., Dong, W., Alcamí, M., Busnengo, H. F., & Martín, F. (2014). Chain-Length and Temperature Dependence of Self-Assembled Monolayers of Alkylthiolates on Au(111) and Ag(111) Surfaces. The Journal of Physical Chemistry A, 118(23), 4138–4146. doi:10.1021/jp412285v (15) Vericat, C., Vela, M. E., Benitez, G., Carro, P., & Salvarezza, R. C. (2010). Self- assembled monolayers of thiols and dithiols on gold: new challenges for a well- known system. Chemical Society Reviews, 39(5), 1805. doi:10.1039/b90730 (16) Lee, G.-Y., Park, J.-H., Chang, Y. W., Cho, S., Kang, M.-J., & Pyun, J.-C. (2018). Chronoamperometry-Based Redox Cycling for Application to Immunoassays. ACS Sensors, 3(1), 106–112. doi:10.1021/acssensors.7b00681 (17) Bettazzi, F., Romero Natale, A., Torres, E., & Palchetti, I. (2018). Glyphosate Determination by Coupling an Immuno-Magnetic Assay with Electrochemical Sensors. Sensors, 18(9), 2965. doi:10.3390/s18092965 (18) Plattel, W. J., Visser, L., Diepstra, A., Glaudemans, A., Nijland, M., van Meerten, T., Kluin-Nelemans, H. C., van Imhoff, G. W., & van den Berg, A. (2020). Interim thymus and activation regulated chemokine versus interim ¹⁸ F- fluorodeoxyglucose positron-emission tomography in classical Hodgkin lymphoma response evaluation. British journal of haematology, 190(1), 40–44. https://doi.org/10.1111/bjh.16514 (19) Butterworth, A., Blues, E., Williamson, P., Cardona, M., Gray, L., & Corrigan, D. K. (2019). SAM Composition and Electrode Roughness Affect Performance of a DNA Biosensor for Antibiotic Resistance. Biosensors, 9(1), 22. doi:10.3390/bios9010022 (20) Farina, L., Rezzonico, F., Spina, F., Dodero, A., Mazzocchi, A., Crippa, F., … Corradini, P. (2014). Serum Thymus and Activation-Regulated Chemokine Level Monitoring May Predict Disease Relapse Detected by PET Scan after Reduced- Intensity Allogeneic Stem Cell Transplantation in Patients with Hodgkin Lymphoma. Biology of Blood and Marrow Transplantation, 20(12), 1982– 1988. doi:10.1016/j.bbmt.2014.08.016