Field of the invention

The present invention relates to SARS-CoV-2 detection devices. More particularly, the invention concerns a sensor device for in-vitro detection SARS-CoV-2, and an electrode device for a sensor device.

Background of the invention

SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, is the strain of coronavirus that causes coronavirus disease 2019 (COVID-19), the respiratory illness responsible for the COVID-19 outbreak that was declared a pandemic by The World Health Organization on 11 March 2020. A strain of severe acute respiratory syndrome- related coronavirus (SARSr-CoV), SARS-CoV-2 has a simple structure and composition. It is a protein-coated single-stranded positive-stranded RNA virus, which particles are round or elliptical, often pleomorphic, with a diameter of about 60-140nm. The virus is believed to have zoonotic origins, possibly having emerged from a bat SARS-like coronavirus.

SARS-CoV-2 infects higher animals including humans and is highly infectious and harmful, leading to an increasing number of patients with severe symptoms and deaths. The virus primarily enters human cells by binding to the receptor angiotensin converting enzyme 2 (ACE2). Epidemiological studies estimate that when no members of the community are immune and no preventive measures taken, each infection results in 5.7 new infections. The virus primarily spreads between people through close contact and via respiratory droplets produced from coughs or sneezes, but studies suggest that aerosols and indirect contact via contaminated surfaces also are causes of infection. For most respiratory viruses, patients are typically most contagious when they are most symptomatic. For SARS-CoV-2, on the other hand, there are indications that subclinical infections are a source of many infections.

Hence, early detection and diagnosis followed by isolation and treatment are highly important in order to control the disease, prevent the spread of the virus and ultimately put an end to the COVID-19 pandemic. The infection should be detected as early and as fast as possible with a sensitive, reliable test, ideally not only available in the clinic, but also at the point of concern or care. Currently used methods for the detection of the SARS-CoV-2 virus generally rely on the polymerase chain reaction (PCR), i.e. nucleic acid testing, which includes the two steps of nucleic acid extraction and detection. Such methods are often cumbersome, requiring trained personnel, and the detection times are long. Other methods rely on ELISA, which is associated with limited sensitivity and is difficult to perform at the point of care.

Thus, there is a clear need for a point of care SARS-CoV-2 screening device that is reliable, selective and sensitive while allowing rapid diagnosis and use by nontechnicians. As the virus may undergo mutations, the device should be flexible enough to be usable for such mutations. Such a device will highly useful in managing the COVID-19 pandemic.

Brief summary of the invention

In one aspect, the present invention according to claim 1 concerns an electrode device for use as a part of a sensor device, wherein the electrode device comprises an electrode, a first layer arranged on a surface of the electrode, the first layer comprising one or more proteins selected from the group consisting of Protein A/G/L, Protein A/G, Protein A, and Protein G, and a second layer arranged on the first layer, the second layer comprising the rabbit SARS-CoV-2 Spike glycoprotein polyclonal antibody and recombinant fc-tagged ACE2 protein.

In another aspect, the present invention according to claim 7 concerns a sensor device for in-vitro detection SARS-CoV-2, wherein the sensor device comprises a substrate; an electrode device as defined above arranged on the substrate, an auxiliary electrode arranged on the substrate; and a redox indicator arranged on the electrode device or on the substrate.

In yet another aspect, the present invention according to claim 12 concerns a method for in-vitro detection of SARS-CoV-2, the method comprising the steps of providing a sensor device as defined above, setting the sensor device in electrical contact with an analog front-end, and contacting the sensor device with a sample.

Further advantageous features of the present invention are defined in the dependent claims. Figures

Figure 1 is a schematic representation of an electrode device according to the invention, comprising a first and a second layer.

Figure 2 is a schematic representation of an electrode device according to the invention further comprising a third layer arranged on and covering the first and second layers. Figures 3a and 3b each shows a schematic representation of an embodiment of a sensor device according to the invention, wherein the sensor device comprises two electrodes. Figure 4 shows a schematic representation of an embodiment of the invention wherein the sensor device comprises three electrodes.

Figure 5 shows a schematic representation of a sensor device according to the invention further comprising a flow channel.

Figure 6 shows a schematic representation of a sensor device according to the invention further comprising a microfluidic and/or capillary chamber.

Figure 7 shows the signals from an experiment wherein a sensor device according to the invention was used to detect SARS CoV-2.

Definitions

Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

As used herein, the term “layer” refers to a material, consisting of one or more components, disposed on at least a portion of one or more underlying surfaces in a continuous or discontinuous manner. Further, the term “layer” does not necessarily mean a uniform thickness of the disposed material, and the disposed material may have a uniform or a variable thickness.

The term "analyte" used herein shall mean viruses and their extracts, prions and their extracts, eucaryotic cells, fragments and extracts, prokaryotic cells (such as bacteria), fragments and extracts, prokaryotic spores, fragments and extracts, protein and peptide markers (biomarkers, especially biomarkers on cell surfaces), serum proteins, isolated proteins, synthetic chemicals, viral membranes, eukaryotic membranes, eukaryotic cell parts, including mitochondria and other organelles, prokaryotic membranes, DNA viruses (single and double stranded DNA viruses), RNA viruses (single and double stranded RNA viruses), point mutations (any organism), single nucleotide polymorphism (any organism), mRNA's, rTNAs, micro RNAs (from any organism).

As used herein, the term "detect" includes identifying or determining the presence or absence of one or more analytes in a sample. It may also include quantifying the amount and/or concentration of one or more analytes in the sample.

As used herein, the term "immobilisation" refers to the attachment or entrapment, either chemically or otherwise, of a compound to one or more surfaces. A compound can be immobilised to a surface by any suitable method, including, but not limited to, absorption, adsorption, or covalent binding to the surface, or by attaching to another substance or particle that is immobilised to the surface.

As used herein, the term "adsorption" refers to a process that occurs when a liquid solute accumulates on the surface of a solid (adsorbent), forming a molecular or atomic film or layer (the adsorbate).

The term “redox indicator” as used herein refers to any compound or composition that may undergo any significant measurable change upon being oxidised and/or reduced.

As used herein, “subject” means any human or non-human animal from which a biological sample may be taken, and encompasses “patient”. None of the terms should be construed as requiring the supervision (constant or otherwise) of a medical professional (e.g., physician, nurse, nurse practitioner, physician's assistant, orderly, clinical research associate, etc.) or a scientific researcher.

A biological sample, also referred to herein merely as a “sample”, may be obtained from a subject. Biological samples from a subject include, but are not limited to, bodily fluids. As used herein the term “bodily fluid” refers to any fluid found in the body of which a sample can be taken for analysis. Non-limiting examples of bodily fluids include blood, plasma, serum, lymph, sudor, saliva, tears, sperm, vaginal fluid, faeces, urine or cerebrospinal fluid. Biological samples also encompass gaseous samples, such as volatiles of an organism. Techniques for obtaining different types of biological samples are known in the art. Detailed description of the invention

In the following, general embodiments as well as particular exemplary embodiments of the invention will be described. References will be made to the accompanying drawings. It shall be noted, however, that the drawings are exemplary embodiments only, and that other features and embodiments may well be within the scope of the invention as claimed.

In one aspect, the present invention relates to an electrode device for a sensor device. The sensor device may be an electrochemical biosensor.

Figure 1 illustrates an electrode device (100) according to the invention. The electrode device according to the invention comprises an electrode (E). The electrode may be made from various materials, such as gold, platinum, palladium, and/or a carbon material. In preferred embodiments, the electrode comprises a carbon material. Such carbon material may be of various types or allotropes, such as selected from the group comprising oxidized graphene, reduced graphene, graphite, multi-walled nanotubes, single walled carbon nanotubes, pyrolyzed carbon, etc. Preferably, the electrode is made from graphite. The electrode may be a bulk, thin-film, or thick-film electrode.

The electrode device according to the invention further comprises a first layer (10) arranged on the surface of the electrode (E). This first layer comprises one or more types of proteins (11) selected from the group consisting of Protein A/G/L, Protein A/G, Protein A, and Protein G. The first layer may be arranged on the whole surface of the electrode, such as fully covering the surface of the electrode. The first layer may be bound to the surface of the electrode through hydrophobic interactions of the protein with the surface by the use of an electrode material that enables such hydrophobic interactions, such as a carbon material. The first layer allows e.g. for the immobilisation of subsequently applied recognition molecules. In some embodiments, the first layer comprises Protein A/G/L. In preferred embodiments, the first layer consists of Protein A/G/L.

Protein A and Protein G are immunoglobulin binding proteins derived from Staphylococcus and Streptococcus species, respectively. Protein A has five binding sites and Protein G has three binding sites; both proteins target the Fc region of immunoglobulin regions. Protein L has five binding domains for the kappa light chain regions of immunoglobulins and is derived from the Peptostreptococcus species. Protein A, Protein G, and Protein L have varying affinity to immunoglobulins of different animal species, and have some regions that contribute to non-specific binding of albumin and Fab domains of IgGs. To overcome the non-specific binding, these proteins have been recombinantly engineered to expand their Ig binding profiles across species and eliminate regions of non-specific binding.

The electrode device according to the invention further comprises a second layer (20) arranged on the first layer (10), on the surface of the first layer that is opposite to the electrode (E). The second layer comprises a rabbit SARS-CoV-2 Spike glycoprotein polyclonal antibody (21) and a recombinant fc-tagged ACE2 protein (22). The rabbit SARS-CoV-2 Spike glycoprotein polyclonal antibody and the recombinant fc-tagged ACE2 protein are both recognition molecules for the SARS-CoV-2 virus. The second layer may be arranged on substantially all of the first layer. The second layer may fully cover the first layer. The second layer may be arranged on a part of the first layer. In some embodiments, the first layer fully covers the surface of the electrode, and the second layer fully covers the first layer.

ACE2 is an abbreviation for Angiotensin I Converting Enzyme 2, which is an membrane metalloproteinase. ACE2 is involved in the regulation of the renin-angiotensin system, and is ubiquitously expressed on the surface of multiple tissues such as the lungs, bronchi, nasopharynx, trachea, gut, and kidneys. The ACE2 receptor is the primary entry point for SARS viruses. Though both SARS-CoV-1 and SARS-CoV-2 interact with the ACE2 receptor via the viral Spike glycoprotein present on surface of these organisms, the binding affinity of the SARS-CoV-2 virus is at least 15-fold stronger than that of SARS- CoV-1 . This high binding affinity is exploited in using a recombinant version of Fc-tagged ACE2 receptor protein as one of the recognition molecules on the electrode device.

SARS-CoV-2 Spike glycoprotein has an important role in interacting with target receptors. The Spike glycoprotein consists of two subunits, S1 and S2, which form stable homotrimers containing a complex pattern of N-linked glycosylation critical to ACE2 interactions. Due to its important role in transmissibility, the spike glycoprotein is often used as an immunogen to generate antibodies to be used in vaccine development, diagnostic assays, and as research tools. The electrode device employs a rabbit polyclonal anti-spike glycoprotein antibody as a recognition molecule to which the SARS- CoV-2 organism binds.

The combination of the recombinant ACE2 protein and the anti-Spike antibody in the second layer of the electrode device according to the invention has several advantages. One advantage is that the combination of two recognition molecules results in a dual indicator/”bi-indicator” that may allow interaction of the SARS-CoV-2 virus with the electrode device with a higher affinity compared to having just a single recognition molecule. Hence, their presence may advantageously result in an electrode device having a high sensitivity and selectivity towards the SARS-CoV-2 virus compared to other devices for detecting the SARS-CoV-2 virus.

Further, a mutation in the SARS-CoV-2 virus that disrupts binding to one of the recognition molecules may not disrupt binding to the other, so that the mutated virus may still be able to bind to the electrode device. This may give the possibility of being able to detect future strains and mutations of SARS-CoV-2, including the case where future strains and mutations have an increase in infectivity and/or lethality.

The use of the ACE2 protein, the receptor that is the primary entry point for the SARS- CoV-2 into the human cells, as one of the two recognition molecules, inherently makes the electrode device particularly versatile. Due to the use of ACE2, all mutations that result in a virus that is able to enter human cells, can be expected to still be detectable by the electrode device invention. Further, any improvement of the virus with regards to its binding to ACE2 for entry into human cells will likely result in even more efficient detection. Thus, it may even be the case that if the virus becomes more infectious, it will become more easily detectable, due to improved binding to the ACE2 receptor.

Binding of a SARS-CoV-2 virus to the electrode device can have at least four components. A first component may be the binding of the virus to the ACE2 receptor of the electrode device, a second component may the binding of the virus to the antibody of the electrode device, a third component may be the binding of the virus to the antibody and the ACE2 receptor of the electrode device, and a fourth component may be virus attachment to two or more ACE2 and/or antibodies of the electrode device. The infectivity and lethality of SARS-CoV-2 virus and other SARS viruses start with the binding of the virus to the ACE2 receptor of the epithelial cells within the lung lining. As a component of the signal in the invention is the binding of virus to the ACE2 receptor of the electrode device, the level of binding to the electrode device - and also the signal strength if the electrode is used in a sensor device - can be proportional to the concentration of the virus and its binding constant. Any strain or mutant of SAR-CoV-2 which retains its binding to ACE2, and therefore infectivity and lethality via ACE2, may thus still be detectable using the electrode device according to the invention. Further, if strains and mutations develop that affect binding to ACE-2, which would likely confound virus assays based on RT-PCR, the anti-Spike antibody will still detect these strains and mutations.

The recognition molecules of the second layer may be bound to the first layer in an oriented manner. Oriented assembly may be achieved using an active or a passive method. In some embodiments, oriented assembly is achieved through the use of the Protein A/G/L which may bind the Fc region of the antibody, leaving the recognition regions exposed distally from the electrode surface. Oriented assembly though the use of Protein A/G/L is a passive method. In other embodiments, oriented assembly is achieved using an active method such as chemical crosslinking of the glycoproteins that primarily occurs at the Fc region of antibodies. Oriented assembly may advantageously increase the sensitivity of the electrode device compared to randomly oriented assembly, since oriented assembly means more recognition molecules are facing correct direction for binding sites to be made available to the virus.

As illustrated in Figure 2, an electrode device according to the invention (101) may further comprise a third layer (30) arranged on the surface of the second layer opposite the first layer and fully covering the second (20) layer and optionally the first layer (10). If the second layer does not cover the first layer, the third layer may cover the second layer and any part of the first layer that is not covered by the second layer. If the second layer covers the first layer, the third layer may cover the second layer. In some embodiments, the first layer fully covers the surface of the electrode, the second layer fully covers the first layer, and the third layer fully covers the second layer. This third layer comprises one or more types of blocking agent (31). The blocking agent may be present on the second layer and on the electrode device in areas between the ACE2 proteins and the anti-Spike antibodies, taking up space left unbound by these. A blocking agent is an agent, e.g. a molecule, that can inhibit, block, prevent or reduce interaction with the electrode by compounds which can be comprised in a sample to be tested. Non-limiting examples of blocking agents comprise one or more agents selected from the list comprising casein, bovine serum albumin, human serum albumin, nonfat dried milk, gelatin, polyethylene glycol, and detergents such as Tween-20, Tween-80, Triton X-100, and sodium dodecyl sulfate. The blocking agent of such third layer may thus reduce non-specific binding by molecules such as generic proteins which can be comprised in a sample and that may interfere with electrochemical detection. Hence, the sensitivity of the electrode may be improved, such as by reducing background interference and improving the signal-to-noise ratio. The presence of such third layer may be particularly useful for electrode devices suitable for use with complex samples for which the skilled person would expect a high level of non-specific binding, such as saliva samples.

In preferred embodiments, the electrode device comprises an electrode (E) made from a carbon material, a first layer (10) comprising protein A/G/L (11), a second layer (20) arranged on the first layer and comprising a rabbit SARS-CoV-2 Spike glycoprotein polyclonal antibody (20) and a recombinant fc-tagged ACE2 protein (21), and a third layer (30) arranged on the electrode (E) and all layers thereon (10, 20) as described above, the third layer comprising a blocking agent (31) selected from the list consisting of casein, bovine serum albumin, human serum albumin, nonfat dried milk, gelatin, polyethylene glycol, Tween-20, Tween-80, Triton X-100, sodium dodecyl sulfate, and any combinations thereof.

The concentrations of the components on the electrode device (protein, ACE2, anti-Spike antibody, blocking agent) are optimised to balance surface coverage (which enhances the blocking effect) with exposing sufficient electrode surface for detection of redox indicator. The first layer may comprise 250-1500 ng protein, such as 400-1200 ng protein, such as 500-1000 ng protein. The second layer may comprise 100-1000 ng Anti-Spike antibody, such as 100-400 ng Anti-Spike antibody. The second layer may comprise 100-1000 ng ACE2, such as 100-400 ng ACE2 antibody. The ratio between antibody and ACE2 may vary from 3:1 to 1 :3, such as from 2:1 to 1 :2, and is preferably 1 :1 . The skilled person will recognise that the bulk of the protein may determine the amount of recognition molecules that can be applied to the electrode device. The skilled person will further recognise that the amount of blocking agent is selected as known in the art.

The electrode device is suitable for electrical contact, such as for connection with an electrochemical point of care device.

In another aspect, the invention provides a sensor device (200) for in-vitro detection of SARS-CoV-2, wherein the sensor device comprises an electrode device (100, 101) as disclosed above. The sensor device may be an electrochemical biosensor.

Biosensors are analytical devices that couple biological recognition elements such as enzymes, antibodies, or nucleic acids with a transducer that can detect the interaction of an analyte. When the transducer is an electrode where electrochemical signal is measured, the biosensor is called a sensor device. Electrochemical biosensors detect viruses and/or bacteria based on the binding of pathogens to the electrode, which has been functionalised to be specific to the pathogen of interest. The binding of these pathogens to the electrode causes a modification of the electrode’s surface which can be detected through capacitance and/or resistance of the electrode, using various electrochemical techniques such as voltammetry, amperometry potentiometry, impedance, and field-effect, and is output as a measurable signal proportional to the presence of the target analyte in the sample. The modification of the surface can be detected either directly or indirectly by the inclusion of reporting molecules which interact with the analyte of interest. The electrode can be manufactured from a range of different materials including gold, carbon and platinum. It can be constructed as a either 1 D microelectrode, a 2D planar electrode, or a 3D featured electrodes, said features including nano-featured electrodes and porous electrodes.

Electrochemical biosensors are typically associated with a number of advantageous properties: Typically practical and economical in use, they are able to combine low response-times with high levels of sensitivity and selectivity. However, electrochemical biosensors for the detection of SARS-CoV-2 have not been available.

A person skilled in the art will appreciate that the sensor device according to the invention may further comprise a substrate (40) on which the electrode device may be configured for use as a working electrode, an auxiliary electrode (50) arranged on the substrate; and a redox indicator (60).

The use of the electrode device according to the invention in a sensor device may advantageously provide the effects discussed above for the electrode device. Hence the sensor device may be highly specific and sensitive towards SARS-CoV-2.

When the sensor device of the invention is an electrochemical biosensor, it may advantageously detect SARS-CoV-2 without the need for cell lysis or amplification of genetic material. Though biosensing based on molecular biological techniques such as RT-PCR/PCR are lauded as having high levels of specificity and also may be quite sensitive, the complicated workflow of sample isolation, purification and amplification mean that assay times can be long and the cost per test is high. The equipment is also frequently bulky and high cost. With the sensor device according to the invention, SARS- CoV-2 can be detected in its native state and within the original sample matrix. The sensor device according to the invention may detect SARS-CoV-2 by an electrochemical method. Hence, the sensor device can function in samples that have not otherwise undergone sample preparation such as dilution, purification, filtration, precipitation, chromatography, etc. The sensor device may thus function in samples such as blood, interstitial fluid, breath condensate, sputum, and saliva, contrasting methods such as fluorescence and absorption spectroscopy which may require a sample to be transparent and clear from particles.

The sensor device according to the invention comprises a substrate (40). The substrate may be selected from a suitable non-reactive, non-conductive material, such as quartz, sodalime glass, pyrex.

On the substrate there is arranged an electrode device as disclosed above (100, 101), such as configured for use as a working electrode, and an auxiliary electrode (50).

An electrode can be provided on the substrate using various techniques known to the person skilled in the art, such as screen printing, printed circuit board assembly, sputtering/vapour deposition, spray coating, dip coating, drop casting, electroplating, and/or electro-deposition. E.g. screen printing is known as a low-cost method for providing electrodes. Methods can be borrowed from classic glucose strip manufacturing, and techniques such as electrodeposition which are not commonly utilised outside of the lab can also be implemented.

Figures 3a and 3b illustrate two embodiments of the invention (200, 201) wherein the sensor device comprises two electrodes: an electrode device as disclosed above (100, 101) configured for use as a working electrode, and an auxiliary electrode (50). As used herein, the terms “auxiliary electrode” and “counter electrode” are used interchangeably to refer to an electrode that may be a part of the electrochemical cell of the sensor device of the invention and is distinct from the working electrode and from any “reference electrode”, which establishes the electrical potential against which other potentials may be measured.

Figure 4 illustrates an embodiment of the invention wherein the sensor device (203) comprises three electrodes: an electrode device as disclosed above (100, 101) configured for use as a working electrode, an auxiliary electrode (50), and a further electrode (70). The further electrode may be a non-specific electrode, such as a reference electrode, such as for measuring a background signal. The further electrode may be a further working electrode. The further working electrode may be functionalised for a specific pathogen, such as a bacteria, virus, protein, DNA, RNA, small molecule, ion, or enzyme, so that the sensor device may test, in parallel or sequentially, for one or more additional analytes in addition to SARS-CoV-2. The further working electrode may be adapted for quality assurance, such as for a known positive and/or negative control, such as for quality control to ensure the correct functioning of the system. The further electrode may a reference electrode so that the sensor device is a classic three electrode electrochemical biosensor with a working electrode, a reference electrode, and a counter electrode.

The sensor device of the invention may comprise four or more electrodes. Each working electrode may have its own dedicated auxiliary electrode, or two or more working electrodes may share a common auxiliary electrode.

Advantageously, if the electrode device of the invention used in the sensor device of the invention comprises a third layer (30) comprising a blocking agent (31), the auxiliary electrode and/or any further electrodes may also comprise a layer comprising a blocking agent.

The electrodes of the sensor device are suitable for electrical contact, such as for connection with an electrochemical point-of-care device for use as a diagnostic tool.

The principle of the sensor device according to the invention is based on the reduced electron transfer of the redox indicator (60) at the electrode surface measured by one or more electrochemical techniques. The redox indicator may be arranged on a surface of the substrate, such as in the vicinity of the electrode device (100, 101 ), adjacent to the electrode device (100, 101 ), near the electrode device (100, 101 ), as illustrated in Figure 3a, embodiment 200. The redox indicator may then be for dissolving into a sample when the sensor device is in use. The change in the surface of the electrode device through specific binding can then be measured by a change in the redox indicator molecules’ ability to exchange electrons with the electrode. Alternatively, the redox indicator may be arranged as a permanent layer on the electrode device according to the invention, as illustrated in Figure 3b, embodiment 201 . The binding of the SARS-CoV-2 virus to the recognition molecules of the sensor device then acts as a steric barrier preventing the redox indicator molecules from accessing the surface of the electrode. The reduced faradaic current is shown as a decrease in peak height as compared to the pre-sample curve. In some embodiments, the redox indicator is chosen from the group of ferricyanide, ferrocyanide, ferrocene, methylene blue, ruthenium hexamine, anthraquinone, Nile blue, thionine, viologen, and any combinations thereof. In preferred embodiments, the redox indicator is potassium hexacyanoferrate(ll) trihydrate (Fe(CN) ₆ ] ^{3 /4-} ).

When the sensor device is in use, a potential may be applied between the at least two electrodes using a potentiometer to control the voltage between the electrodes. The current that results as the voltage is changed may be measured by a potentiostat. The voltage range applied to a given sensor may depend on the redox indicator used, as known to the skilled person. As an example, for a hexacyanoferrate(ll) redox indicator a voltage range of 0V to 0.5V may advantageously be used. The output from the sensor device may be a voltage against current curve, which may be analysed using a calibration algorithm to determine the viral concentration. The results may be displayed, such as on a dedicated screen, such as with dedicated software, such as on an auxiliary smart phone with a dedicated app.

The electrochemical technique used to monitor the specific binding to the surface can be one of more techniques selected from the group of voltammetry, open-circuit potential (OCP), amperometry, field effect, and coulometry. In some embodiments, the electrochemical technique is voltammetry. Preferably the technique used is data rich, such as a technique selected from cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, staircase voltammetry, and/or other hyphenated voltammetric techniques, so that a multiple variable analysis can be used. Then data can be analysed, such as by tradition methods such as, current, peak position and peak height, and/or by more advanced Al machine learning.

The interaction between the SARS-CoV-2 virus and the electrode though the specific binding surface may be interrogated at multiple voltages and so a current (I) as a function of voltage (V) spectrum can be constructed. In some embodiments, more than one voltage is used determine whether specific binding has taken place and the relative change in the peak compared to a standard curve is used to determine quantitatively the amount of pathogen or analyte. Hence the sensor device according to the invention may be used to quantify the amount of SARS-CoV-2, or to give a binary response as to whether SARS-CoV-2 is present in a sample or not. The sensor device and electrode device according to the invention may advantageously be dry formulations, i.e. they may dry after assembly and remain dry until the sensor contacts a sample. In some embodiments, the removal of liquids as part of manufacturing is performed at reduced pressures to assist with evaporation.

In some embodiments, the electrode device and/or the sensor device comprise a dissolvable coating, such as dextrose and/or sucrose, for protecting the electrode device and/or sensor device, such as during shipping and storage. The use of such coatings may extend the shelf life of the electrode device and/or the sensor device.

The sensor device according to the invention may be used in a method for in-vitro detection of SARS-CoV-2, the method comprising the steps of setting the sensor device in electrical contact with an analogue front-end, and contacting the sensor device with a sample. An analogue front-end (AFE or analogue front-end controller AFEC) is a set of analogue signal conditioning circuitry that uses sensitive analogue amplifiers and other circuits to provide a configurable and flexible electronics functional block needed to interface a variety of sensors to digital back-end, analog-to-digital converter or, in some cases, to a microcontroller.

An electrode device (100, 101) according to the invention may, as an example, have an area of 1 -6 mm ² , such as 1 -4 mm ² , such as a length of 4 mm and a width of 1 mm. A sensor device (200, 201 , 202, 203, 204) according to the invention may, as an example, have an approximate area of 150-200 mm ² , such as 170-190 mm ² , such as 180 mm ² . In one embodiment, a sensor device (202) as shown in Figure 4 has a length of 27 mm and a width of 7 mm, the electrode device (100, 101 ) has an area of 4 mm ² , the auxiliary electrode (50) has an area of 3 mm ² , and the further electrode (70) is a reference electrode with an area of 6 mm ² , the electrodes and the electrode device being spaced apart by a distance of 0.3 mm.

A sample to be analysed can be contacted with the sensor device in different manners, including but not limited to direct addition, e.g. by the dropping of a sample onto the sensor, or flowing the sample onto the sensor device, e.g. by using a flow channel (80) as illustrated in Figure 5. The presence of an active means for bringing the sample onto the sensor is not required; passive means such as capillary fill or gravity fill are viable options. In some embodiments, the sensor device according to the invention is used in conjunction with, or further comprises, a microfluidic channel or microfluidic capillary (80) for use as a flow channel to channel the sample onto the electrodes.

The volume of a sample to be analysed may be 4-50 pL, such as 10-30 pL, such as 4-20 pL, such as 4-10 pL. Hence, the sensor device according to the invention may allow a high ratio between the working electrode area and the sample volume. Consequently, the time taken for the virus to be transported to the surface is relatively fast when compared to microwell or batch type assays, allowing shorter detection times than for these assays. The assay time may be further enhanced by constraining the sample as a thin film on top of the electrode device. This thin film configuration of the sample on top of the working electrode reduces the time taken for the pathogen to be mass-transported though the sample and to be bound on the electrode.

Because of the high ratio between the working electrode area, i.e. the electrode device, and the sample volume, the sensor device according to the invention may be suitable for use in very small sample volumes, such as such as in sample volumes of 20 microliters or less, such as such as in sample volumes of 10 microliters or less, such as in sample volumes of 4 microliters or less.

As illustrated in Figure 6, the sensor device according to the invention may be used in conjunction with, or further comprise, a microfluidic and/or capillary chamber (90) that envelopes all electrodes and the electrode device. The chamber may be arranged to draw samples onto the sensor in a repeatable manner and/or to control the volume of sample on the sensor and/or to control the distribution of sample on the sensor device. A channel (80) may act as a chamber above the electrode(s) and the electrode device, or a channel may guide sample into a chamber (90). The size and/or material properties of a channel and/or chamber may be selected to aid self-filling of the chamber by capillary fill. The height of a chamber and/or a channel may be 1 mm or less and the width 1 mm or wider, and the length may be 5 mm or longer. The material may advantageously be hydrophilic. A hydrophilic material may aid self-filling of the sensor. The chamber may both promote the flow of a sample onto the electrodes and control the volume of the sample and the placement of the sample, such as above the electrode. In some embodiments, the microfluidic chamber can create a thin film of sample on the electrodes and electrode device so that the there is a low aspect ratio between the depth of the sample and the width/length of the sample. In some embodiments, the chamber comprises an air vent for allowing air to be vented from the chamber as it fills with sample. In some embodiments, a further material that can aid sample preparation and/or improve the subsequent electrochemical assay is arranged on the substrate to dissolve into the sample when the sensor device is used. Non-limiting examples of such materials are salts, buffers, electrolytes, further redox molecules, reporting molecules. In preferred embodiments, such materials are arranged in the proximity of the electrodes and/or within a microfluidic chamber. Materials that are not necessarily compatible with an electrode and/or with one another can be strategically arranged, separating materials that are mismatched, for example separating an oxidising material from carbon electrodes.

The ability of the sensor device of the invention to analyse non-processed sample, the high ratio of electrode area to sample volume, and, in some embodiments, also the thin film of sample created on the electrode, are factors that may contribute to low detection times compared to commonly used detection methods for SARS-CoV-2.

In many biosensing diagnostics it is common practice to have multiple processes, such as washing of the sensors, the addition of liquid samples, etc. For instance, the use of a traditional ELISA-type biosensor may require several steps, including adding the sample, washing the sensor to remove non-specific binding elements, and adding a second reporting antibody. Such multi-step processes are not required for use of the sensor device according to the invention; the only steps necessary are contacting the sample with the sensor device and the passive step of measurement.

An additional first, passive step that is independent of the user may be included, such as the dissolution of dry reagents into the sample, such as to remove a dissolvable coating used to protect the formulations during shipping and storage, or to dissolve a further material for aiding sample preparation and/or improving the subsequent electrochemical assay and that is arranged on the sensor device. The sensor device is thus easy to use, also by non-technicians.

In some embodiments, the dissolution of such a further material into the sample and/or the homogenisation of the sample can be measured by a passive technique, such as open circuit potential, using a reference electrode. As the sample homogenises, the potential differences at the working and reference electrodes can be measured in the absence of current. When a plateau is reached, indicating that the reference and working electrodes have similar potentials, this will then indicate homogeneity. The sample can be analysed for whether it heterogeneous or homogenous as a function of time, and the electrochemical assay can be started either accounting for the heterogeneity, or at a point where the open circuit potential is stable and homogeneity of the sample is reached.

Many biosensors are endpoint assays, where the sensor has reached a plateau in the signal and the final reading is taken. These endpoint assays can be time-consuming due to the time it takes to reach a stable signal. For the sensor device according to the invention, the assay can still be in a dynamic phase where the signal is changing with time, but the rate of change is still correlated to the analyte or pathogen of interest. This can be termed a kinetic assay. For the sensor device according to the invention both endpoint and kinetic methods work. Changes of the signal may be measured over time as a kinetic measurement. Alternatively, the assay can be analysed after waiting a fixed amount of time, i.e. an endpoint analysis. A kinetic assay is preferred for a low assay time, such as two minutes or less. The kinetic assay is enabled by the use of the SARS-CoV-2 Spike glycoprotein polyclonal antibody and recombinant fc-tagged ACE2 protein as recognition molecules. Without being bound to any mechanism of action, the inventors suggest that the specific binding constants of the SARS-CoV-2 virus to these specific recognition molecules lead to controlled kinetics that enable kinetic assays.

The sensor device according to the invention can be manufactured from a substrate that has been provided with a working electrode region and an auxiliary electrode using a method known to the skilled person, using the following method:

Firstly, an aqueous protein composition comprising Protein A/G/L, Protein A/G, Protein A, or Protein G, is prepared and applied to the working electrode region and bound to the working electrode through active or passive means, preferably passive, such as by adsorption, such as by hydrophobic interactions. In preferred embodiments, the working electrode is a carbon electrode, and the protein is passively adsorbed onto carbon working electrode via hydrophobic interactions. Active immobilisation may be performed by crosslinking the protein to the activated carbon surface using a chemical crosslinker. In some embodiments, the chemical crosslinker is selected from the group of carbodiimide linkers, dimethylpimelimidate crosslinkers, disuccinimidyl suberate crosslinkers. In some embodiments, the protein is bound to the working electrode by using a carbodiimide crosslinker.

The sensor device is then dried. As used herein, the term “drying” refers to partial or complete removal of any solvents. Non-limiting examples of useful drying conditions are 2-8°C for approximately 12 hours, 37 °C for 20-60 minutes, ambient temperature for 1 -4 hours.

Secondly, the working electrode region of the sensor device is coated with the recognition molecules; the rabbit SARS-CoV-2 Spike glycoprotein polyclonal antibody and recombinant fc-tagged ACE2 protein. The coating is achieved by preparing aqueous solutions of the SARS-CoV-2 Spike glycoprotein and the ACE2 protein and applying these to the working electrode region of the sensor device, either together or separately. The SARS-CoV-2 Spike glycoprotein antibody and the recombinant fc-tagged ACE2 bind to the protein coating on the sensor working electrode.

The sensor device is then dried. Non-limiting examples of useful drying conditions are 2- 8°C for approximately 12 hours, 37 °C for 20-60 minutes, ambient temperature for 1-4 hours.

Optionally, the working electrode may be coated with a blocking agent by using a blocking buffer. The term “blocking buffer” as used herein is a solution that comprises one or more blocking agents. The sensor is treated with a blocking buffer, optionally after a wash step, such as to remove excess unbound antibody and/or ACE2 protein, before it is dried, such as at 2-8 °C overnight, 37 °C for 20-60 minutes, ambient temperature for 1-4 hours. A range of different blocking agents, and thus blocking buffers, can be used. In some embodiments, the blocking agent comprises one or more agents selected from the list comprising casein, bovine serum albumin, human serum albumin, nonfat dried milk, dextrose, gelatin, polyethylene glycol, and detergents such as Tween-20, Tween-80, Triton X-100, and sodium dodecyl sulfate. In some embodiments, the blocking agent comprises a combination of bovine serum albumin, dextrose, and a detergent such as Tween-20.

The sensor is then rinsed and briefly left to dry.

Finally, the working electrode may be coated with the redox indicator, using a solution of said redox indicator. Various redox indicators known to the skilled person may be used. Non-limiting examples of redox indicators include ferricyanide, ferrocyanide, ferrocene, methylene blue, ruthenium hexamine, anthraquinone, Nile blue, thionine, viologen, phenanthraquinones, osmium complexes. In preferred embodiments, the redox indicator is potassium hexacyanoferrate(ll) trihydrate. After a brief period of drying, the sensor device is ready for use.

The same method, mutatis mutandis, may be used for an electrode device according to the invention.

Optionally, a further material for dissolving into the sample to aid sample preparation and/or improve the electrochemical assay when the sensor device is in use, may be applied on the substrate.

Optionally, a dissolvable coating, such as dextrose or sucrose, for protecting the sensor device during shipping and/or storage, may be applied to the sensor in a final step.

The invention shall not be limited to the shown embodiments and examples. While various embodiments of the present disclosure are described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications and changes to, and variations and substitutions of, the embodiments described herein will be apparent to those skilled in the art without departing from the disclosure. It is to be understood that various alternatives to the embodiments described herein can be employed in practicing the disclosure.

It is to be understood that every embodiment of the disclosure can optionally be combined with any one or more of the other embodiments described herein.

It is to be understood that each component, compound, or parameter disclosed herein is to be interpreted as being disclosed for use alone or in combination with one or more of each and every other component, compound, or parameter disclosed herein. It is further to be understood that each amount/value or range of amounts/values for each component, compound, or parameter disclosed herein is to be interpreted as also being disclosed in combination with each amount/value or range of amounts/values disclosed for any other component(s), compound(s), or parameter(s) disclosed herein, and that any combination of amounts/values or ranges of amounts/values for two or more component(s), compound(s), or parameter(s) disclosed herein are thus also disclosed in combination with each other for the purposes of this description. Any and all features described herein, and combinations of such features, are included within the scope of the present invention provided that the features are not mutually inconsistent. It is to be understood that each lower limit of each range disclosed herein is to be interpreted as disclosed in combination with each upper limit of each range disclosed herein for the same component, compound, or parameter. Thus, a disclosure of two ranges is to be interpreted as a disclosure of four ranges derived by combining each lower limit of each range with each upper limit of each range. A disclosure of three ranges is to be interpreted as a disclosure of nine ranges derived by combining each lower limit of each range with each upper limit of each range, etc. Furthermore, specific amounts/values of a component, compound, or parameter disclosed in the description or an example is to be interpreted as a disclosure of either a lower or an upper limit of a range and thus can be combined with any other lower or upper limit or a range or specific amount/value for the same component, compound, or parameter disclosed elsewhere in the application to form a range for that component, compound, or parameter.

Examples

Experiment 1 - Manufacture of sensor device

A three-electrode biosensor with carbon electrodes was manufactured according to the following method:

A Protein A/G/L formulation was prepared in sterile PBS solution. The protein A/G/L formulation was stored at -20°C, briefly thawed and then kept cool on ice. 1 pL of the formulation is applied onto the working electrode region of the sensor. The protein A/G/L was bound passively to the working electrode. The sensor was then dried overnight at 2- 8°C.

The sensor was subsequently coated with the recognition molecules. A solution of SARS- CoV-2 Spike glycoprotein antibody was prepared in PBS, and a solution of ACE2 was prepared in ultrapure sterilized water. 1 pL of each was applied to the working electrode of the sensor. The sensor was then dried overnight at 2-8 °C.

Next the sensor was rinsed and coated with blocking buffer 1% Bovine Serum Albumin 1% Dextrose 0.005% Tween-20 mixture prepared in sterile PBS; 30pL of the 1% BSA 1% Dextrose 0.005% Tween-20 was coated onto the sensor covering all three electrodes. The sensor was again dried overnight at 2-8°C.

Subsequently the sensor was rinsed with sterile PBS and briefly left to dry, before it was coated with the redox indicator: 1 pl of 200mM Potassium hexacyanoferrate(ll) trihydrate prepared in 0.1 M potassium chloride 10mM Phosphate buffer (pH7.4). The senor was then dried at room temperature for 30 minutes. Experiment 2 - challenging sensor device with various amounts of SARS-CoV-2 Sensor devices according to Experiment 1 was used to detect various amounts SARS CoV-2 virus, ranging from 6.7x10 ⁵ PFU/ml to 6.7 PFU/ml.

A SARS-CoV-2 stock at 6.7x10 ⁶ PFU/mL was serially diluted in sterile culture media to form concentrations ranging from 6.7x10 ⁵ PFU/ml to 6.7 PFU/ml. 30 pL of each viral concentration was applied to sensors in three replicates and incubated for 5 minutes. The sensor was then tested using a laboratory potentiostat to administer square wave voltammetry. The results were presented on the scientific software displaying the current v potential graph as shown in Figure 7.

The data in Figure 7 shows the raw signal when the sensors were used to detect SARS CoV-2. It is clear that the signal changes linearly with the concentration of virus, indicating that the sensor device according to the invention does indeed detect the virus, and that the response is dependent on the virus concentration in a sample.