Electrochemical sensor

Abstract

An electrochemical sensor 20 comprises: a graphene-based working electrode 24 having a functionalised surface, wherein the functionalised surface is functionalised with organic molecules, such as phen

Description

This invention relates to an electrochemical sensor, an analytical apparatus and a method of manufacturing an electrochemical sensor. It is known to use electrochemical sensors as biosensors for detecting pathogens. According to a first aspect of the invention, there is provided an electrochemical sensor comprising: a graphene-based working electrode having a functionalised surface, wherein the functionalised surface is functionalised with organic molecules; and a receptor for binding one or more target analytes, wherein the receptor is combined with the functionalised surface. In embodiments of the invention, the organic molecules may be, but are not limited to, phenolic molecules. More preferably the phenolic molecules are, but are not limited to, aminophenol molecules. The organic molecules may be covalently or non-covalently bonded to the functionalised surface. In a preferred embodiment of the invention, the receptor may be configured to wet the functionalised surface. In other embodiments of the invention, the receptor may be combined with the functionalised surface in other ways, e.g. the receptor may form a discrete layer on top of the functionalised surface. The discrete layer may be in the form of a solid layer. In further embodiments of the invention, the receptor may be a bioreceptor. In still further embodiments of the invention, the receptor may include or may be made of free carboxyls, antibodies, enzymes, aptamers, polymers, peptides, ligands, proteins and / or small molecules. The use of other non-biological and biological materials in or as the receptor are envisaged. The electrochemical sensor may further include a reference electrode for providing a reference potential against which a potential of the working electrode is measured, and / or may further include a counter electrode for providing a path for the flow of electrical current. In embodiments of the invention, the electrochemical sensor may include a lipid layer that is formed on the functionalised surface. Preferably the lipid layer is a phospholipid layer. The lipid layer may function as an intermediary between the functionalised graphene and the receptor, so as to create a favourable environment for the receptor to interact with the or each target analyte and thereby enhance the sensitivity and specificity of the detection process. In a preferred embodiment of the invention, the functionalised surface is functionalised with aminophenol molecules, and the receptor is a bioreceptor that is configured to wet the functionalised surface, wherein the receptor includes or is made of free carboxyls, antibodies, enzymes, aptamers, polymers, peptides, ligands, proteins and / or small molecules, wherein the electrochemical sensor includes: a reference electrode for providing a reference potential against which a potential of the working electrode is measured; and a counter electrode for providing a path for the flow of electrical current. According to a second aspect of the invention, there is provided a breathalyser comprising an electrochemical sensor and at least one fluidic channel, wherein the electrochemical sensor is in accordance with any one of the first aspect of the invention and its embodiments, wherein the or each fluidic channel is configured to, in use, guide a breath sample to the electrochemical sensor. According to a third aspect of the invention, there is provided an analytical apparatus comprising an electrochemical sensor and a signal processing circuit, wherein the electrochemical sensor is in accordance with any one of the first aspect of the invention and its embodiments, wherein the signal processing circuit is configured to receive and process an electrical signal from or associated with the working electrode of the electrochemical sensor. The signal processing circuit may be configured as an amperometric signal processing circuit and / or may be configured as a voltammetric signal processing circuit. Other signal processing functions of the signal processing circuit are envisaged. The signal processing circuit may be or may form part of a computing device. The computing device may be, may include or may form part of one or more of an electronic device, a portable electronic device, a portable telecommunications device, a mobile phone, a personal digital assistant, a tablet, a phablet, a laptop computer, a server, a cloud computing network, a smartphone, a smartwatch, smart eyewear, and a module for one or more of the same. The computing device may include a processor and memory including computer program code. The memory and computer program code may be configured to, with the processor, enable the computing device to carry out one or more processing functions. It will be appreciated that references to a memory or a processor may encompass a plurality of memories or processors. According to a fourth aspect of the invention, there is provided a method of manufacturing the electrochemical sensor according to any one of the first aspect of the invention and its embodiments, the method comprising the steps of: providing a graphene-based working electrode; functionalising a surface of the working electrode with organic molecules; and combining a receptor with the functionalised surface, wherein the receptor is for binding one or more target analytes. The features and advantages of the preceding aspects of the invention and their embodiments apply mutatis mutandis to the features and advantages of the fourth aspect of the invention and its embodiments. It will be appreciated that the use of the terms "first" and "second", and the like, in this patent specification is merely intended to help distinguish between similar features, and is not intended to indicate the relative importance of one feature over another feature, unless otherwise specified. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, and the claims and / or the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and all features of any embodiment can be combined in any way and / or combination, unless such features are incompatible. The applicant reserves the right to change any originally filed claim or file any new claim accordingly, including the right to amend any originally filed claim to depend from and / or incorporate any feature of any other claim although not originally claimed in that manner. Preferred embodiments of the invention will now be described, by way of non-limiting examples, with reference to the accompanying drawings in which: Figure 1 shows an electrochemical sensor according to an embodiment of the invention; and Figures 2 to 5 show individual components of the electrochemical sensor. The figures are not necessarily to scale, and certain features and certain views of the figures may be shown exaggerated in scale or in schematic form in the interests of clarity and conciseness. It will be appreciated that the following specific embodiments of the invention apply mutatis mutandis to other embodiments of the invention within the scope of the claims. The electrochemical sensor of the invention is designed to detect a range of pathogens, fungi, bacteria, spike proteins and biomarkers relating to, but not limited to, respiratory diseases, and enzyme / protein detection. It is envisaged that the invention can be employed in various fields such as healthcare (including clinical settings, veterinary medicine, rapid screening, diagnostics and point-of-care testing), food safety, agriculture, environmental monitoring, and biodefense. The electrochemical sensor comprises a working electrode, a reference electrode, a counter electrode, a receptor and a signal processing circuit. The working electrode is graphene-based. In this embodiment, the working electrode is formed of pristine graphene having a functionalised surface that is functionalised with aminophenol molecules, particularly 4-aminophenol molecules. The aminophenol molecules are covalently bonded to the functionalised surface. Other type of organic molecules, such as phenolic molecules, may replace the aminophenol molecules in other embodiments. It is envisaged that, in other embodiments of the invention, the working electrode may be formed of a graphene derivative. 4-Aminophenol falls under the category of organic compounds. It is an aromatic amine, which is a type of organic compound that contains an amine group (-NH2) attached to an aromatic ring. The aromatic ring in 4-Aminophenol is a benzene ring (CeHe), and the amino group is attached to one of the carbon atoms of the benzene ring. The reference electrode is configured to provide a reference potential against which a potential of the working electrode is measured. The counter electrode is configured to provide a path for the flow of electrical current. In this way, the electrochemical sensor is configured as a three-electrode electrochemical sensor. The working, reference and counter electrodes are formed on a solid substrate. The working electrode may be made of graphene ink through screen printing on a substrate but may be made of other graphene-based materials. The functionalised surface of the working electrode is wetted by the receptor at the point of manufacture before, optionally, being covered by a protective laminate. The receptor is a receptor that is configured to wet the functionalised surface. The receptor functions as a highly loaded buffer material that is capable of binding target analytes such as MRNA. The interaction between the receptor and the target analytes may differ depending on the type of receptor and target analyte. The binding between the receptor and the target analytes may be biological or non-biological. The receptor may be a bioreceptor (also known as a biological recognition element or a biorecognition element). The binding of the target analyte to the receptor is a complex and specific molecular recognition event. It involves a careful interplay of various non-covalent forces and can result in conformational changes that facilitate a stable binding interaction. This binding is driven by the complementary shapes, sizes, and chemical properties of the receptor and the target analyte's molecules. The subsequent binding-induced changes can then lead to the generation of an electrical signal through transduction processes involving the functionalised graphene surface. Various non-covalent binding forces come into play during the binding event. These forces include: • Hydrogen bonding: Hydrogen bonds form between hydrogen donor and acceptor groups on the receptor and target analyte, respectively. These bonds can provide specificity and stability to the binding. • Van der Waals interactions: Weak attractive forces between electron clouds of adjacent molecules play a role in bringing the receptor and target analyte close together. • Electrostatic interactions: Charges on the receptor and target analyte can attract or repel each other, contributing to binding. For example, polar groups might interact through electrostatic forces. • Hydrophobic interactions: Nonpolar regions of the receptor and target analyte can cluster together to minimise exposure to water, driving binding in hydrophobic environments. When the target analytes are bound by the receptor, a change in the physicochemical properties of the local environment is triggered (such as an induced change in pH). The configuration of the electrodes enables a signal transduction of the change in physicochemical properties into a measurable electrical response which can be measured using electrical measurement techniques. In the embodiment shown, the receptor may include or may be made of free carboxyls, antibodies, enzymes, aptamers, polymers, peptides, proteins and / or small molecules. Preferably the receptor material may be protein-based. The receptor material can be tuned to be selective towards a specific analyte. In particular, the receptor material may include one or more components, each of which is designed to target a respective specific analyte. Such components include, but are not limited to, ligands and antibodies. Optionally the electrochemical sensor may include a phospholipid layer that is formed on the functionalised surface. The phospholipid layer enhances the sensing capabilities of the electrochemical sensor by creating a suitable environment for the receptor to interact with the target analytes. The electrochemical sensor forms part of an analytical apparatus, which further comprises a signal processing circuit. The signal processing circuit is configured to receive and process an electrical signal from or associated with the working, reference and counter electrodes of the electrochemical sensor. The signal processing circuit may be built into the same analytical device as the electrochemical sensor. Alternatively the signal processing circuit may be remote from the electrochemical sensor, whereby a signal transmission device is used to transmit the electrical signal from or associated with the working, reference and counter electrodes to the signal processing circuit. The signal transmission may be carried out through wired or wireless transmission. The combination of the functionalised graphene and the wetting receptor to form the electrochemical sensor not only enables high detection sensitivity and specificity of the electrochemical sensor but also enables tuning of the receptor for selectivity towards certain target analytes. Non-limiting further details of the electrochemical sensor are provided below. Background and significance of pathogen detection Pathogen detection plays a crucial role in various fields such as healthcare, food safety, environmental monitoring, and biodefense. Rapid and accurate identification of pathogens is essential for timely diagnosis, effective treatment, and containment of infectious diseases. Traditional methods for pathogen detection often involve timeconsuming culture-based techniques or complex laboratory procedures, which can delay diagnosis and treatment initiation. In recent years, there has been a growing demand for faster and more sensitive pathogen detection methods. The emergence of new infectious diseases, the spread of antimicrobial resistance, and the potential for bioterrorism have highlighted the need for advanced technologies that can rapidly identify and characterize pathogens. This has led to the development of various biosensing approaches for pathogen detection. A functionalised graphene electrode biosensor offers several advantages over standard testing methods for pathogen detection. Graphene, a two-dimensional carbon nanomaterial, possesses exceptional electrical, mechanical, and chemical properties, making it an ideal platform for biosensing applications. Here are some reasons why a functionalised graphene electrode biosensor is superior: • Sensitivity: Graphene-based biosensors have a high surface-to-volume ratio, allowing efficient biomolecule immobilisation and enhanced interaction with target pathogens. This increased sensitivity enables the detection of low pathogen concentrations that may be missed by conventional methods, leading to earlier and more accurate diagnosis. • Speed: Graphene biosensors can provide rapid results due to their ability to facilitate direct electrical detection of pathogens. The interaction between the target pathogen and the functionalised graphene surface leads to changes in electrical properties, such as conductivity or impedance, enabling real-time monitoring and fast detection. • Selectivity: Functionalising the graphene electrode with specific biomolecules, such as antibodies, aptamers, or peptides, enhances the biosensor's selectivity towards the target pathogen. The functionalisation enables the recognition and binding of the pathogen, resulting in a highly specific detection method that minimises false positives. • Miniaturisation and Portability: Graphene biosensors can be easily miniaturised and integrated into portable devices, enabling on-site and point-of-care testing. This portability is especially valuable in resource-limited settings or during outbreaks where immediate and decentralised pathogen detection is critical for effective containment and control measures. • Label-Free Detection: Unlike many traditional methods that require labeling or amplification steps, graphene biosensors can perform label-free detection. This eliminates the need for complex sample preparation and reduces the overall assay time, making the detection process more streamlined and cost-effective. • Multiplexing Capability: Functionalised graphene biosensors can be designed to detect multiple pathogens simultaneously, enabling multiplexed analysis in a single assay. This capability is advantageous for identifying co-infections or monitoring the presence of multiple pathogens in complex samples, saving time and resources. Overall, the use of a functionalised graphene electrode biosensor for pathogen detection offers significant advantages in terms of sensitivity, speed, selectivity, portability, label-free detection, and multiplexing capability. These attributes make it a promising technology for improving pathogen surveillance, early disease diagnosis, and effective control strategies. Importance of breath samples for non-invasive testing Non-invasiveness: Breath samples provide a non-invasive and painless method of sample collection. Unlike blood or tissue samples, which require invasive procedures, breath samples can be easily collected by individuals themselves or by healthcare professionals without causing discomfort. Convenience: Collecting breath samples is relatively simple and can be done quickly without the need for specialised medical personnel or equipment. This makes it a convenient option for large-scale testing, especially in settings such as airports, workplaces, or public spaces. Rapid results: Breathalyser devices equipped with graphene electrode biosensors can provide rapid results, often within minutes. This allows for quick screening and detection of spike proteins, which is crucial for timely identification and management of infectious diseases or other health conditions. Sensitivity and accuracy: Graphene electrode biosensors functionalised with aminophenol and free carboxyl receptors offer high sensitivity and accuracy in detecting specific molecules or proteins, such as spike proteins. The unique properties of graphene, including its large surface area and excellent electrical conductivity, make it an ideal material for biosensors, enabling reliable detection of target substances in breath samples. Real-time monitoring: Breathalyser-based detection systems allow for real-time monitoring of spike protein levels. This can be particularly useful for tracking the progression of an infection, evaluating the effectiveness of treatments or vaccines, and making informed decisions regarding patient care. Public health applications: Non-invasive breath testing using graphene-based biosensors can have significant implications for public health. It can be utilised for screening individuals in high-risk settings, conducting large-scale surveillance, and implementing early detection strategies for contagious diseases. This approach may contribute to the prevention and control of outbreaks by enabling swift identification of infected individuals. Overall, the utilisation of breath samples and graphene electrode biosensors functionalised with aminophenol and free carboxyl receptors in a breathalyser holds promise for non-invasive and efficient testing of spike proteins. This approach can offer numerous advantages in terms of convenience, speed, accuracy, and broader applications in public health. potential or eiectrocnernicai oiosensors in patnogen detection The electrochemical sensor of the invention has potential applications in various areas related to pathogen detection, including viruses, bacteria, fungi, and foodborne pathogens. Here are some potential areas of use: • Clinical Diagnostics: The electrochemical sensor can be utilised in clinical settings for the rapid detection of viral, bacterial, and fungal pathogens. By detecting specific biomarkers or genetic material associated with these pathogens, the biosensors can help diagnose infectious diseases more quickly and accurately. This can lead to timely treatment and better management of outbreaks. • Environmental Monitoring: The electrochemical sensor can be deployed in environmental monitoring systems to detect and monitor pathogens in air, water, and soil. This is particularly important in areas where waterborne or airborne pathogens pose a risk to human health. The electrochemical sensor can provide real-time data on the presence and concentration of pathogens, enabling early warnings and effective response strategies. • Food Safety: The electrochemical sensor can be employed in the food industry to detect foodborne pathogens such as salmonella, e. coli, listeria, and Campylobacter. By integrating the electrochemical sensor into the food production and supply chain, potential contamination can be identified quickly, preventing the distribution of contaminated food and reducing the risk of foodborne illnesses. • Veterinary Medicine: The electrochemical sensor can be used in applications in veterinary medicine for the detection of pathogens in animals. They can aid in the early detection and monitoring of diseases, allowing for timely intervention and preventing the spread of infections among livestock or pets. • Agriculture and Plant Pathology: The electrochemical sensor can be utilised in agricultural settings to detect and monitor plant pathogens, including bacteria and fungi. By detecting the presence of pathogens in crops or soil, farmers can take preventive measures to mitigate the spread of diseases and protect their crops. • Point-of-Care Testing: The portable and user-friendly nature of the electrochemical sensor makes them suitable for point-of-care testing in remote or resource-limited settings. They can be used in clinics, community healthcare centers, or even at home to provide rapid and accurate results, enabling early detection and containment of infectious diseases. • Biodefense and Biosecurity: The electrochemical sensor can contribute to biodefense and biosecurity efforts by providing rapid and sensitive detection of potential biological threats. They can be deployed in airports, border crossings, or high-security areas to screen individuals or environmental samples for the presence of dangerous pathogens. The implementation and effectiveness of the electrochemical sensor of the invention may vary depending on factors such as sensor design, target specificity, sample preparation requirements, and regulatory considerations. Overview of functionalised graphene electrodes Functionalised graphene electrodes offer several benefits when graphene nanotubes are used and stacked in a specific manner for the development of digital graphene inks on electrodes. These electrodes provide cyclic voltammetry results that can be utilised for the detection of specific pathogens. Here is an overview of the benefits: • Enhanced Sensitivity: Functionalised graphene electrodes exhibit high sensitivity towards the target pathogens. The unique structure of graphene nanotubes allows for efficient charge transfer, enabling the detection of even trace amounts of pathogens with excellent sensitivity. • Selectivity: The functionalisation process enables the modification of graphene electrodes to selectively detect specific pathogens. By incorporating specific functional groups onto the graphene surface, the electrodes can be tailored to recognise and respond to particular biomarkers or molecular targets associated with the pathogens of interest. This selectivity minimises false-positive or falsenegative results. • Rapid Detection: The utilisation of digital graphene inks on electrodes enables quick and efficient detection of pathogens. The cyclic voltammetry technique employed with these electrodes allows for fast and reliable electrochemical measurements, providing rapid results for pathogen detection. • Low Detection Limit: Functionalised graphene electrodes offer an exceptionally low detection limit. The combination of the high surface area of graphene nanotubes, their excellent electrical conductivity, and the functionalisation process results in electrodes capable of detecting pathogens even at extremely low concentrations, making them highly suitable for early-stage pathogen detection. • Stability and Reusability: Graphene-based electrodes exhibit remarkable stability and durability, ensuring reliable performance over extended periods. Additionally, they can be easily regenerated and reused, reducing the overall cost and enhancing the practicality of pathogen detection systems. • Miniaturisation and Integration: Functionalised graphene electrodes can be integrated into miniaturised devices, enabling portable and point-of-care pathogen detection. The unique properties of graphene, such as its flexibility and compatibility with various substrates, allow for the development of compact and integrated sensing platforms, making them ideal for on-site or field applications. Overall, functionalised graphene electrodes, when utilising graphene nanotubes and digital graphene inks, provide numerous advantages for pathogen detection. These electrodes offer enhanced sensitivity, selectivity, rapid detection, low detection limits, stability, and the potential for miniaturisation and integration, making them highly promising for various biomedical and environmental applications. Electrochemical sensor The electrochemical sensor of the invention leverages the unique properties of graphene to offer enhanced sensitivity, selectivity, and speed for detecting and analysing biological molecules. Graphene, a two-dimensional carbon material, possesses exceptional electrical, mechanical, and chemical properties. Its high surface-to-volume ratio, large electron mobility, and excellent electrical conductivity make it an ideal platform for biosensing applications. In the electrochemical sensor of the invention, the surface of the graphene is functionalised with specific biomolecules or nanomaterials, such as antibodies, enzymes, or aptamers, that can selectively bind to target analytes. The electrochemical sensor operates based on the principle of electrochemical sensing. When a target analyte interacts with the functionalised graphene surface, it induces a change in the electrical properties of the graphene, which can be detected and quantified using various electrochemical techniques, such as amperometry, voltammetry, or impedance spectroscopy. The integration of graphene into the electrochemical sensor enhances the sensitivity and signal-to-noise ratio, allowing for the detection of even trace amounts of analytes with high accuracy and precision. One of the major advantages of this biosensor is its potential for real-time monitoring. The rapid response time of the graphene-based electrochemical sensor enables on-site detection and analysis, making them highly suitable for point-of-care diagnostics and mass testing scenarios. Moreover, the high stability and robustness of graphene ensure long-term performance without compromising sensitivity or selectivity. In terms of applications, the electrochemical sensor has potential in various areas of diagnostics and mass testing, such as: • Disease diagnostics: The high sensitivity and specificity of the electrochemical sensor make it a valuable tool for diagnosing various diseases, including infectious diseases, genetic disorders, and cancer. By detecting specific biomarkers in patient samples, these biosensors can aid in early disease detection and personalised treatment strategies. • Environmental monitoring: The electrochemical sensor can be tailored to detect environmental pollutants, toxins, and contaminants in water, air, and soil samples. Their rapid response and sensitivity make them indispensable for monitoring and ensuring the safety of the environment and public health. • Food safety: By detecting harmful pathogens, allergens, and chemical contaminants in food samples, the electrochemical sensor can contribute to ensuring the quality and safety of the food supply chain. Their portability and ease of use make them suitable for field testing and monitoring in food production facilities. • Bioprocess monitoring: The electrochemical sensor can be employed for realtime monitoring of bioprocesses, such as fermentation and bioreactor operations. By continuously measuring key parameters and analytes, these electrochemical sensor enables precise process control, optimisation, and quality assurance in biopharmaceutical and biotechnological industries. a. Electrochemical sensor design and components An electrochemical sensor according to an embodiment of the invention is shown in Figure 1 and is designated generally by the reference numeral 20. Figures 2 to 5 show individual components of the electrochemical sensor 20. Substrate: The electrochemical sensor 20 comprises a substrate 22, which is a solid material that provides a solid support for the sensor components. It can be made of a wide range of materials like glass, silicon, or plastic. The substrate may be flexible. Working Electrode: The working electrode 24 is the electrode where the electrochemical reaction of interest takes place. The working electrode 24 is typically made of a conductive material, such as a glassy carbon electrode or a gold electrode, onto which graphene or another graphene-based material is deposited. In the embodiment shown, the working electrode 24 is a circular electrode and is connected to a first output electrical terminal 26a, which in turn is connected to the signal processing circuit 28. Graphene: Graphene is a two-dimensional carbon material consisting of a single layer of carbon atoms arranged in a hexagonal lattice. It has excellent electrical conductivity and a large surface area, making it an ideal material for sensing applications. The graphene used in the electrochemical sensor 20 is functionalised with aminophenol molecules. Graphene can be synthesised through various methods, such as chemical vapor deposition (CVD), mechanical exfoliation, or chemical reduction of graphene oxide. CVD is commonly used for large-scale production. In this method, a carbon-containing precursor gas, such as methane, is introduced into a heated chamber where it decomposes and forms graphene on a surface. Functionalisation: Functionalisation of graphene involves modifying its surface by attaching specific molecules or nanoparticles. Common functionalisation methods include chemical modification, covalent binding, or non-covalent interactions. After synthesizing the graphene, its surface is functionalised with aminophenol molecules. The functionalisation can be achieved through various approaches, including covalent or non-covalent methods. Covalent functionalisation involves the attachment of aminophenol molecules to the graphene surface via covalent bonds, while non-covalent methods rely on n-n stacking interactions or other weak interactions. The electrode configuration for the electrochemical sensor 20 comprises three electrodes: the working electrode 24, reference electrode 30, and counter electrode 32. These electrodes 24,30,32 may be used as part of cyclic voltammetry (CV) tests to measure the electrochemical behavior of the electrochemical sensor 20. Reference Electrode: The reference electrode 30 is used as a reference point for the electrochemical measurements. It maintains a constant potential against which the working electrode's potential is measured. Commonly used reference electrodes 30 include the Ag / AgCI electrode or the saturated calomel electrode (SCE). The reference electrode 30 should have a stable and well-defined potential to ensure accurate potential measurements during the cyclic voltammetry test. In the embodiment shown, the reference electrode 30 is connected to a second output electrical terminal 26b, which in turn is connected to the signal processing circuit 28. Counter Electrode: The counter electrode 32 completes the electrochemical circuit by providing a pathway for the flow of current during the measurement. It provides a pathway for the flow of electrons to balance the oxidation or reduction reactions occurring at the working electrode 24. The counter electrode 32 is typically made of an inert material, such as platinum or graphite, but may also be made of a graphenebased material, non-limiting examples of which are described throughout the specification. It does not participate in the electrochemical reactions but serves to maintain electrical neutrality within the cell. In the embodiment shown, the counter electrode 32 curves partly around the working electrode 24 and is connected to a third output electrical terminal 26c, which in turn is connected to the signal processing circuit 28. Cover: The electrochemical sensor 20 includes a cover 34 that is made of a highly resistive material. The cover 34 is placed over the substrate 22. The cover 34 has a window 36 that overlays the working electrode 24, reference electrode 30, and counter electrode 32. Electrolyte Solution: The electrochemical sensor 20 requires an electrolyte solution to facilitate the movement of ions between the working and counter electrodes 24,32. The choice of electrolyte depends on the specific electrochemical detection technique employed. The structure, materials and configuration of the electrodes 24,30,32 enables the electrochemical sensor 20 to employ a label-free sensing mechanism based on the changes in electrical properties when target biomolecules bind to the receptor. The captured analytes (e.g. pathogens, fungi, or bacteria) introduce local changes in the local dielectric environment, resulting in measurable variations in electrical conductivity or impedance. These changes are then translated into quantifiable signals, enabling accurate and real-time detection. At the atomic and molecular level, the working electrode 24 exhibits excellent sensitivity due to graphene's exceptional surface-to-volume ratio, which allows efficient capture and detection of low-concentration biomolecules. The functionalisation further enhances selectivity by specifically binding to the target analytes while minimising false positives from other substances. Receptor material: In the context of a pathogen receptor material, components of the receptor material are utilised to target specific analytes. Non-limiting examples of such components include: • Ligands: Ligands are molecules that specifically bind to a receptor or target molecule. They can be small molecules, peptides, or other biological molecules. Ligands interact with their target analytes through various molecular interactions, e.g. hydrogen bonding, electrostatic interactions or hydrophobic interactions. In the context of a pathogen receptor material, ligands can be designed to bind to specific molecules on the surface of the pathogen, such as proteins or carbohydrates. By incorporating ligands into the receptor material, the material becomes capable of selectively capturing and recognising the targeted pathogen. • Antibodies: Antibodies, also known as immunoglobulins, are proteins produced by the immune system in response to foreign substances, called antigens. Antibodies are highly specific and can recognise and bind to particular antigens with high affinity. They are composed of two heavy chains and two light chains that form a Y-shaped structure. The variable regions of antibodies, known as the antigen-binding sites, are responsible for binding to specific antigens. In the context of a pathogen receptor material, antibodies can be employed to target and capture pathogens or pathogen-specific molecules, such as viral proteins or bacterial surface antigens. Antibodies can be immobilised or incorporated into the receptor material, allowing for the specific recognition and capture of the desired pathogens. In addition to ligands and antibodies, there are other components that can form part of a pathogen receptor material. Some non-limiting examples include: • Aptamers: Aptamers are single-stranded DNA or RNA molecules that can bind to specific targets with high affinity and selectivity. Similar to antibodies, aptamers can be generated against a wide range of analytes, including pathogens. Aptamers are selected through a process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and can be incorporated into the receptor material to enable specific pathogen recognition. • Lectins: Lectins are proteins or glycoproteins that have the ability to bind to specific carbohydrate structures. They are commonly found in plants and animals and can recognise and interact with various sugars and glycan structures. Lectins can be used as components in pathogen receptor materials to target specific carbohydrate moieties present on the surface of pathogens, such as bacteria or viruses. • Molecularly imprinted polymers (MIPs): MIPs are synthetic polymers that are designed to exhibit molecular recognition properties. They are created by polymerising monomers in the presence of the target analyte or a structural analog. As a result, the polymer network retains specific cavities or binding sites that are complementary to the target molecule. MIPs can be tailored to recognise pathogens or pathogen-specific molecules by imprinting them during the polymerisation process. These imprinted polymers can then be used as receptor materials for selective pathogen capture. The choice of component(s) of the receptor material depends on the specific target analyte and the desired selectivity and sensitivity of the receptor material. By combining multiple components, it is possible to create receptor materials that offer enhanced specificity and efficiency in capturing and detecting pathogens. Phospholipid layer: Typically, the phospholipid layer and the receptor material are separate entities, but they work together to facilitate the detection of a virus. The phospholipid layer, which consists of phospholipid molecules, forms on the aminophenol-functionalised surface of the working electrode 24. Phospholipids are amphiphilic molecules, meaning they have both hydrophilic and hydrophobic regions. When the electrode is wetted, the hydrophilic heads of the phospholipids interact with the aqueous environment, while the hydrophobic tails face inward, shielding the graphene surface. The primary function of the phospholipid layer is to create a suitable environment for the receptor material to interact with the virus. The phospholipids act as a biocompatible and stable interface between the receptor material and the graphenebased working electrode 24. They provide a hydrophilic surface that enables the receptor material, which is typically biomolecules such as antibodies or aptamers, to adhere and interact with viral particles that may be present in the sample. The receptor material, which can selectively bind to specific viral components, is responsible for the actual detection of the virus. It recognises and captures the target virus or its associated molecules, such as viral proteins or genetic material. By attaching to the receptor material, the virus becomes immobilised on the electrode's surface, allowing for further analysis or detection. In summary, the phospholipid layer serves as an intermediary between the aminophenol-functionalised working electrode 24 and the receptor material. It creates a favourable environment for the receptor material to interact with the virus, enhancing the sensitivity and specificity of the detection process. By facilitating the immobilisation of the virus, the phospholipid layer assists in concentrating and localizing the target, enabling subsequent detection methods to be employed effectively. Signal Amplification Systems: In order to enhance the sensitivity and detection limit of the electrochemical sensor, signal amplification systems may be used. These systems amplify the signal generated by the binding events between the target molecules and the working electrode 24, increasing the overall sensitivity of the detection. Various signal amplification strategies can be employed, such as enzyme-based amplification or nanoparticle-based amplification. For example, enzymes or nanoparticles can be functionalized with detection antibodies and introduced. These elements can catalyse chemical reactions or produce signal-generating molecules upon binding to the target molecules, resulting in an amplified signal that can be easily detected by the working electrode 24. Signal amplification systems play a crucial role in improving the detection sensitivity and ensuring that even trace amounts of pathogens or spike proteins can be accurately detected. pH sensing The pH sensing capabilities of such an electrode come into play once it comes into contact with a pathogen. The binding of a pathogen to the aminophenol-functionalised and receptor-wetted graphene-based working electrode 24 triggers a change in the local environment, including the pH. The specific binding event induces an electrochemical response that can be detected by measuring the changes in pH using various sensing techniques. The electrodes of the electrochemical sensor 20 are designed to exhibit changes in their electrochemical behaviour in response to pH changes. The functionalised graphene electrode acts as the working electrode 24 in an electrochemical cell setup. By applying a potential or current to the working electrode 24 and monitoring the resulting electrochemical response, the pH changes caused by the pathogen can be quantified. To enhance the sensitivity and accuracy of the pH sensing, signal amplification techniques can be employed. These techniques may include using redox mediators, signal amplification enzymes, or nanoparticles to amplify the electrochemical response. The amplified signal is then analysed using appropriate instrumentation and algorithms to quantify the presence and concentration of the pathogen. Overall, the graphene-based working electrode 24 functionalised with aminophenol molecules and wetted with the receptor material enables the detection of pathogens, spike proteins, and biomarkers. The pH sensing capabilities come into play when the functionalised working electrode 24 interacts with a pathogen, leading to changes in pH that can be measured and analysed to identify and quantify the presence of the target pathogen. This approach offers a potential platform for rapid and sensitive detection of respiratory diseases and other relevant biological markers. Signal transduction In the presence of the target pathogen, the receptor binds to the pathogen or its associated molecules. This binding event is specific to the target and leads to the formation of a receptor-pathogen complex. The aminophenol molecules serve as electroactive moieties that can undergo redox reactions. Thus, the aminophenol-functionalised graphene-based working electrode 24 acts as an electron transfer mediator. When the receptor-pathogen complex forms on the functionalised surface, it induces a change in the local electronic environment. This change facilitates the transfer of electrons between the receptor material and the aminophenol-functionalised graphene. The binding event between the receptor material and the pathogen causes a modulation in the electrical conductivity of the graphene layer. As a result, an electrical current can flow through the graphene material. The modulation of electrical conductivity can be measured by applying a voltage across the graphene layer and measuring the resulting current. This change in current is proportional to the concentration of the target pathogen or its associated molecules. The transduction effect refers to the conversion of the biological binding event into an electrical signal that can be quantitatively measured. By monitoring the changes in the electrical current, the presence and concentration of the pathogen can be detected and quantified. The greater the binding between the receptor material and the pathogen, the larger the change in the electrical current, allowing for sensitive detection of the pathogen. In conclusion the electrical current flows between the receptor material and the aminophenol-functionalised graphene by exploiting the binding-induced changes in the electrical conductivity of the graphene layer. This transduction effect enables the detection of the pathogen or its associated molecules in a sensitive and specific manner. Cyclic voltammetry During a cyclic voltammetry (CV) test, the electrochemical behavior of the electrochemical sensor is investigated by measuring the current response as a function of the applied potential. The CV test involves sweeping the potential of the working electrode 24 linearly between two defined values over a certain range and at a specific scan rate. The CV test proceeds as follows: • Initialisation: The electrochemical sensor 20 is assembled, and the three electrodes (working, reference, and counter) are connected to a potentiostat / ga Iva nostat instrument. • Potential Sweep: The potential of the working electrode 24 is swept linearly between two defined values, typically starting from a negative potential and increasing towards a positive potential. The scan rate determines the speed at which the potential is swept. • Current Measurement: As the potential is swept, the electrochemical reactions occur at the working electrode's surface. The resulting current flowing through the electrochemical sensor 20 is measured by the potentiostat / ga Iva nostat instrument. The measured current is directly proportional to the rate of the electrochemical reaction. • Data Collection: The measured current is recorded at different potentials throughout the potential range. Breathalyser The electrochemical sensor may be implemented as part of an analytical device in the form of a breathalyser for the detection of pathogens or spike proteins of viruses. Microfluidics is employed to handle and process a breath sample containing potential pathogens or spike proteins. One or more microfluidic channels are used to direct the flow of the breath sample to the electrochemical sensor, ensuring efficient interaction with the sensing element of the electrochemical sensors. The sample may undergo various treatments, such as filtration or concentration, to isolate and enrich the target molecules of interest prior to introduction into the breathalyser. The microfluidics enables precise control of fluid movement, allowing for rapid and accurate analysis of the breath sample. The working electrode's functionalised surface is wetted with a receptor, e.g. in the form of specific ligands or antibodies, with affinity for the pathogen or spike protein. When the breath sample passes over the working electrode, any target molecules present in the sample will bind to the receptor, leading to a measurable change in electrical properties. The invention permits high detection sensitivity and selectivity, enabling the accurate detection of the pathogens or spike proteins in the breath sample. In summary, the breathalyser utilises microfluidics to handle and process the breath sample, and an aminophenol-functionalised and receptor-wetted graphene-based working electrode to selectively capture the target molecules of interest and provide a signal transduction effect. Signal amplification systems may be used to enhance the detection sensitivity. These supporting components work together to enable the accurate detection of pathogens or spike proteins of viruses in the breath sample. a. Breath sample collection and preparation Breath sample collection and preparation may be performed as follows: • Breath Sample Collection: The first step is to collect a breath sample from an individual using the breathalyser. The individual is asked to exhale into a designated volume of the breathalyser, allowing the breath sample to be captured. The individual may be asked to undertake the following steps. Pre-collection: The individual undergoing the breath sample collection should be instructed to follow certain guidelines to obtain a representative sample. These guidelines may include avoiding food, drink, smoking, or vigorous physical activity for a specific period before the test. Such instructions aim to minimise potential interference with the breath analysis. Deep Breath: The individual takes a deep breath, filling their lungs with air. Breath Holding: The individual holds their breath momentarily to allow for stabilisation of the breath composition. Exhalation: The individual exhales forcefully or steadily into the breathalyser. The device may have an attached mouthpiece into which the individual can exhale. Collection Time: The individual may be required to exhale into the device for a specific duration, typically a few seconds, to ensure an adequate sample volume for analysis. • Breath Sample Handling: The breath sample is temporarily and securely stored inside a collection chamber or cartridge of the breathalyser. The chamber or cartridge maintains the integrity of the breath sample until it is to be further processed. • Sample Transfer to Biosensor: The breath sample is then transferred, via one or more microfluidic channels, from the collection chamber or cartridge to the electrochemical sensors within the breathalyser. • Pathogen Detection: The breath sample comes into contact with the functionalised surface of the working electrode, which is coated with specific molecules or antibodies that can bind to the pathogens of interest. If any of the target pathogens are present in the breath sample, they will bind to the functionalised surface. • Electrochemical Analysis: Once the pathogens bind to the functionalised surface, an electrochemical analysis is performed. This analysis involves measuring the electrical properties or signals generated by the interaction between the captured pathogens and the electrochemical sensor. • Signal Processing and Interpretation: The electrical signals generated by the electrochemical sensor are processed and interpreted by the signal processing circuit. Software is used to analyse the signals and compare them to known patterns or signatures associated with specific pathogens. This step helps identify and quantify the presence or absence of pathogens in the breath sample. • Signal Amplification and Analysis: The electrical signal generated by the electrochemical sensor may be weak and thereby require amplification for accurate detection. Signal amplification techniques such as enzymatic reactions, redox cycling, or signal enhancement through additional electrodes may be employed. The amplified signal is then analysed using appropriate algorithms or analytical methods to quantify the presence and concentration of the target pathogen. • Diagnostic Output: Based on the analysis and interpretation of the signals, the breathalyser generates a diagnostic output. This output provides information about the presence or absence of specific pathogens in the breath sample, and the concentration level of the detected pathogens. The output can be displayed on a digital screen, transmitted to a connected device or saved to a data storage medium for further analysis and interpretation by healthcare professionals. Overall, the breath sample collection and preparation in combination with the breathalyser with the electrochemical sensor offers a non-invasive and rapid method for detecting pathogens in a breath sample. It will be appreciated that the breath sample can be collected independently of the breathalyser as follows. • Breath Sample Collection: The first step involves collecting the breath sample from the individual by using a specific collection device designed for breath analysis, such as a cartridge or collection tube. The individual is instructed to exhale into the collection device to capture the breath sample. • Sample Transport: Once the breath sample is collected, it is transported to the testing facility or the location where the breathalyser is situated. It's essential to maintain the integrity and stability of the sample during transportation to avoid any potential contamination or degradation. • Sample Introduction: At the testing facility, the breath sample is introduced into the breathalyser. The specific mechanism for sample introduction may vary depending on the device design. It could involve direct injection of the breath sample into the system or using a sample collection cartridge or module that interfaces with the breathalyser. • Pre-processing: Pre-processing steps may be carried out to prepare the breath sample for analysis. These steps can include: • Moisture Removal: Breath contains moisture, which can interfere with the sensing mechanism. Moisture removal techniques, such as condensation or desiccation, may be employed to eliminate excess water content from the breath sample. • Particle Filtration: Filtering the breath sample helps remove large particles or impurities that could affect the sensor's performance. This can be achieved by using filters or membranes with specific pore sizes to retain particles while allowing the passage of analytes. • Dilution or Concentration: Depending on the concentration of the target pathogen or analyte, the breath sample may need to be diluted or concentrated. This step ensures that the concentration of the analyte falls within the sensor's detection range, enabling accurate measurements. • Sample Conditioning: In some cases, additional conditioning steps may be required to enhance the performance of the electrochemical sensor. This can include adjusting pH levels, temperature, or applying specific chemicals or reagents to optimise the interaction between the target pathogen and the electrochemical sensor. The listing or discussion of an apparently prior-published document or apparently prior-published information in this specification should not necessarily be taken as an acknowledgement that the document or information is part of the state of the art or is common general knowledge. Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention.

Claims

1. An electrochemical sensor comprising:a graphene-based working electrode having a functionalised surface, wherein the functionalised surface is functionalised with organic molecules; anda receptor for binding one or more target analytes, wherein the receptor is combined with the functionalised surface.

2. An electrochemical sensor according to Claim 1 wherein the organic molecules are phenolic molecules.

3. An electrochemical sensor according to Claim 2 wherein the phenolic molecules are aminophenol molecules.

4. An electrochemical sensor according to any one of the preceding claims wherein the organic molecules are covalently bonded to the functionalised surface.

5. An electrochemical sensor according to any one of Claims 1 to 3 wherein the organic molecules are non-covalently bonded to the functionalised surface.

6. An electrochemical sensor according to any one of the preceding claims wherein the receptor is configured to wet the functionalised surface.

7. An electrochemical sensor according to any one of the preceding claims wherein the receptor is a bioreceptor.

8. An electrochemical sensor according to any one of the preceding claims wherein the receptor includes or is made of free carboxyls.

9. An electrochemical sensor according to any one of the preceding claims wherein the receptor includes or is made of antibodies.

10. An electrochemical sensor according to any one of the preceding claims wherein the receptor includes or is made of enzymes.

11. An electrochemical sensor according to any one of the preceding claims wherein the receptor includes or is made of aptamers.

12. An electrochemical sensor according to any one of the preceding claims wherein the receptor includes or is made of polymers.

13. An electrochemical sensor according to any one of the preceding claims wherein the receptor includes or is made of peptides and / or proteins.

14. An electrochemical sensor according to any one of the preceding claims wherein the receptor includes or is made of ligands.

15. An electrochemical sensor according to any one of the preceding claims wherein the receptor includes or is made of small molecules.

16. An electrochemical sensor according to any one of the preceding claims further including a reference electrode for providing a reference potential against which a potential of the working electrode is measured.

17. An electrochemical sensor according to any one of the preceding claims further including a counter electrode for providing a path for the flow of electrical current.

18. An electrochemical sensor according to any one of the preceding claims including a lipid layer that is formed on the functionalised surface.

19. An electrochemical sensor according to Claim 18 wherein the lipid layer is a phospholipid layer.

20. An electrochemical sensor according to Claim 1 wherein the functionalised surface is functionalised with aminophenol molecules, and the receptor is a bioreceptor that is configured to wet the functionalised surface, wherein the receptor includes or is made of free carboxyls, antibodies, enzymes, aptamers, polymers, peptides, ligands, proteins and / or small molecules, wherein the electrochemical sensor further includes:a reference electrode for providing a reference potential against which a potential of the working electrode is measured; anda counter electrode for providing a path for the flow of electrical current.

21. An electrochemical sensor according to Claim 20 including a lipid layer that is formed on the functionalised surface.

22. A breathalyser comprising an electrochemical sensor and at least one fluidic channel, wherein the electrochemical sensor is in accordance with any one of the preceding claims, wherein the or each fluidic channel is configured to, in use, guide a breath sample to the electrochemical sensor.

23. An analytical apparatus comprising an electrochemical sensor and a signal processing circuit, wherein the electrochemical sensor is in accordance with any one of the preceding claims, wherein the signal processing circuit is configured to receive and process an electrical signal from or associated with the working electrode of the electrochemical sensor.

24. An analytical apparatus according to Claim 23 wherein the signal processing circuit is configured as an amperometric signal processing circuit.

25. An analytical apparatus according to Claim 23 or Claim 24 wherein the signal processing circuit is configured as a voltammetric signal processing circuit.

26. A method of manufacturing the electrochemical sensor according to any one of Claims 1 to 21, the method comprising the steps of:providing a graphene-based working electrode;functionalising a surface of the working electrode with organic molecules; and combining a receptor with the functionalised surface, wherein the receptor is for binding one or more target analytes.

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