WO2021001231A1

WO2021001231A1 - Graphene biosensor and process of manufacture

Info

Publication number: WO2021001231A1
Application number: PCT/EP2020/067636
Authority: WO
Inventors: Kar Seng TENG; Davide DEGANELLO; Siamak SAMAVAT; Wei Zhang
Original assignee: Swansea University
Current assignee: Swansea University
Priority date: 2019-07-02
Filing date: 2020-06-24
Publication date: 2021-01-07
Anticipated expiration: 2022-01-02
Also published as: GB201909502D0; GB202118127D0; GB2600030A

Abstract

The invention concerns a process for the manufacture of a biosensor; a biosensor manufactured according to said process; and method for taking a biological measurement comprising the use of said biosensor.

Description

Graphene Biosensor and Process of Manufacture

Field of the Invention

The invention concerns a process for the manufacture of a biosensor; a biosensor manufactured according to said process; a biosensor and method for taking a biological measurement comprising the use of said biosensor.

Background of the Invention

Sensors for detecting biological molecules, termed biosensors, are widely used. A large variety of biosensors have been developed for sensing or detecting biological molecules with increasing resolution and specificity. A biosensor is generally defined as an analytical device which converts a biological or biochemical response into a quantifiable and processable signal. Quantification of biological or biochemical parameters is commonplace in medical, biological, environmental, and biotechnological applications.

Besides optical and other approaches, many biosensors rely on the general principle of generating an electrical signal if the presence or absence of a biological molecule is detected. Also called affinity biosensors, they function on the principle whereby an immobilized receptor binds a target biomolecule, producing a measurable chemical change at a localized surface. This binding event is then transduced to a quantifiable output. For electrochemical biosensors, the measured signal is transduced into either a current or voltage output, which is then amplified and processed using peripheral electronics. Electrochemical biosensors provide an attractive way to analyse the content of a biological sample due to the direct conversion of a biological event into an electronic signal.

Electrochemical biosensors can broadly be classified into two groups: faradaic and non-faradaic. In faradaic biosensors, reduction and/or oxidation reactions among electroactive species functionalised to a sensor substrate take place at an electrode leading to the generation of an electrical current. Thus, the faradaic sensors require the presence of redox probes and the application of direct current (DC) conditions to promote the development of the electrochemical reactions. In contrast, non-faradaic sensors do not require the use of redox couples and, consequently, no reference electrode is necessary because no DC potential is required, as the impedance of a non-faradaic sensor arises from the double layer capacitance at the surface of the sensing layer. Binding between the receptor and target biomolecules will result in capacitance or phase change at the sensor. Therefore, the non-faradaic sensor is a promising candidate for real-time applications because it does not require the presence of redox couples making this sensor type more amenable to miniaturization and offering ease of use for direct detection of the target biomolecules.

Non-faradaic electrochemical biosensors are well known. However, the signal transduction and the general performance of electrochemical sensors are often determined by the surface architectures that connect the sensing element to the biological sample at the nanometre scale. For example, in the case of sensors that use antibodies as capture agents for the target analyte, a maximum retention of native target binding efficacy is desired as they are integrated into a surface architecture at a density that is also optimized for capture efficiency. Moreover, converting the biological information into an easily processed electronic signal is challenging due to the complexity of connecting an electronic device directly to a biological environment.

Graphene is an allotrope of carbon in the form of a two dimensional hexagonal crystal lattice with notable electronic properties and, as such, has gained increasing interests for use in biosensor technology; large surface-to-volume ratio, unique optical properties, excellent electrical conductivity, high carrier mobility and density, high thermal conductivity and many other attributes can be greatly beneficial for sensor function. Thus the structuring of graphene has advanced and the chemical modification of graphene has been investigated. To deal with biocompatibility issues or for applications in sensors, surfaces of material such as graphene are functionalised with biological molecules. However, graphene is today not a preferred choice for biological applications because of its low affinity for biological molecules. Further, owing to its surface architecture, typically its use has been limited to detect biological materials known to be present in larger concentrations owing to the fact that the effective surface area achievable for functionalisation with biological molecules (as receptors) is low.

Therefore a means to increase the effective surface area of graphene sheets, which are deposited on a substrate, is required for increased functionalisation with biological molecules, thereby improving sensor sensitivity and dynamic range.

We therefore herein disclose a process for development of non-faradaic electrochemical impedimetric sensor comprising vertically aligned graphene sheets, and a sensor comprising same. Notably, owing to the manufacture and sensor arrangement, these graphene sheets are very stable and provides extremely large specific surface area for biosensing applications thus providing an effective platform for use in bio-sensing applications

Statements of Invention

According to a first aspect of the invention there is provided a process for the manufacture of a graphene sensor for detecting a target molecule in a sample, the process comprising:

a) depositing at least one graphene ink onto the surface of a substrate to provide at least one sensing layer;

b) processing the at least one sensing layer by photonic curing to generate a graphene electrode comprising vertically aligned graphene sheets; c) functionalising at least a part of the surface of the sensing layer(s) by suspending at least one linker in a solution and spray coating same onto the surface of the sensing layer(s);

d) optionally coating at least a part of the sensing layer(s) with at least one anchor substance having a binding affinity for a capture reagent; and e) coating the sensing layer(s) of parts c) or d) with at least one capture reagent that bind said linker and/or said anchor substance to provide a sensor with a sensing layer comprising immobilized capture reagent on the surface.

Reference herein to vertically aligned graphene sheets refers to deposited graphene nanoplatelets (GNPs) that are provided in an oblique or perpendicular arrangement with respect to the plane of the substrate surface. Preferably, said GNPs are arranged perpendicular, or substantially perpendicular (i.e. ± 10°), to the surface of the substrate. Such non-parallel arrangements dramatically increase the surface area of the sensing layer(s) available for biosensing (in comparison to a conventional arrangement in which deposited GNPs would lay flat, i.e. parallel to the surface of the underlying substrate), thus improving sensor sensitivity and dynamic range. As will be readily apparent to the skilled person, such an oblique or perpendicular arrangement of the GNPs results in a consistent increase in surface roughness across the surface of the sensing layer(s). In contrast, and whilst a conventional (i.e. parallelly arranged) layer that includes one or more surface defect (e.g. a crease or wrinkle) may also be shown to have a high overall surface roughness (compared with a similar, parallelly arranged, pristine layer), this increase in surface roughness would be highly inconsistent across the entire surface of the layer.

Accordingly, the presence of vertically aligned graphene sheets can be verified by quantification of the surface roughness of the sensing layer(s). As is known to those skilled in the art, surface roughness is quantified by the deviations in the direction of the normal vector of a real surface from its ideal form. If these deviations are large, the surface is rough; if they are small, the surface is smooth. Surface roughness can be measured by various means including, but not limited to, a measurement of the surface profile made with a profilometer such as white light interferometer. In this manner, we have observed that a conventionally applied / parallelly arranged GNP surface layer has a surface roughness of no more than 2.5 ± 0.5 pm. In contrast, we have observed that analogous, vertically aligned, graphene sheets have a surface roughness of at least 5 pm, more preferably at least 10 pm, and more preferably still at least 15 pm. Further, the surface roughness was shown to be consistent across the surface of the sensing layer in that a variation of no more than ± 25%, and preferably no more than ± 15%, was observed across the surface of the sensing layer(s). Therefore, in preferred embodiments, the vertically aligned graphene sheets have a surface roughness of from 5-30 pm, more preferably 10-25 pm, and most preferably 15-20 pm variation of no more than ± 25%, and preferably no more than ± 15%.

Preferably, said sample is a biological sample. Reference herein to a biological sample refers to any sample isolated from the body of a subject including, but not limited to, a cell, a population of cells, a biopsy, a tissue, an organ, blood, plasma, serum, sputum, peritoneal fluid, CSF, synovial fluid, sperm, breast milk, bronchial lavage fluid, amniotic fluid, malignant ascites, pleural fluid, seminal fluid, tears, urine, faeces and saliva. In this preferred embodiment, as will be appreciated, the target analyte is a biological molecule which within the meaning of the present disclosure is an organic molecule produced by or occurring in living organisms. The term biological molecules includes, but is not limited to polymeric molecules occurring in nature and their analogues, such as proteins, polysaccharides, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products and may be at least one of a biomarker, a receptor molecule, an amino acid, an enzyme, an aptamer or an antibody or fragment thereof for the at least one biological molecule.

In a preferred embodiment, depositing at least one graphene ink is achieved by printing onto the substrate. Printing is advantageous as it permits high volume production at relatively low-cost and possibility of obtaining multifunctional electronics over large areas. Further, through printing, one can carefully control the print repeat to deposit multiple sensing layers which allows for careful control of surface architecture. Numerous printing techniques are known in the art and can broadly be classified into contact and non-contact printing methods. In a contact printing process, the patterned structures with inked surfaces are brought in physical contact with the substrate. In a noncontact process, the solution is dispensed via openings of nozzles and structures are defined by moving the stage (substrate holder) or nozzles in a pre-programmed pattern. Contact based printing technologies include, but are not limited to, gravure printing, gravure-offset printing, flexographic printing and roll-to-roll (R2R) printing. The prominent non-contact printing techniques include, but are not limited to screen-printing, slot-die coating, aerosol printing and inkjet printing. In a preferred embodiment, depositing at least one graphene ink is achieved by contact printing, most preferably flexographic printing, which has been found to offer greatest control of surface architecture of the graphene surface layer(s) and most importantly surface roughness which improves effective surface area for subsequent capture reagent immobilisation.

In a preferred process of the invention, said substrate is selected from the group comprising: silicon, solid polymers such as polyimide, SU-8 type materials, organic substrates such as polyethylene terephthalate (PET) or polyvinylchloride (PVC), injection mouldable polymers such as polyether ether ketone (PEEK) and conductive acrylonitrile butadiene styrene (ABS), paper, and flexible material such as rubber silicon-based gels or other gel type materials.

SU-8 is a commonly used epoxy-based negative photoresist material which, as shown below, comprises a statistical average of 8 epoxy groups per moiety.

SU-8 structure Preferably, said sensing layer of deposited ink is provided as interdigitated electrode(s). Multiple sensing or working electrodes are possible for multiplexing detections.

Reference herein to a graphene ink refers to a solution-processed graphene suitable for uniform deposition onto the substrate comprising graphene and a suitable solvent. In a preferred method said graphene ink is provided as graphene powder, most preferably as graphene nanoplatelets (GNPs) dispersed in a solvent. GNPs are stacks of graphene sheets in a two dimensional nano-particulate form but it has been found that GNPs can be dispersed into solvents to form an ink more suitable for deposition, particularly when deposited by printing methods such as flexographic printing.

More preferably still, the GNPs are provided with a particle size of less than about 10 pm, and more preferably less than about 1 pm. This has been found to be an important parameter for the graphene ink, as particle sizes above this were found to be prone to agglomeration, which was particularly relevant in the context of printing wherein agglomeration can seriously affect the ink transfer to the printing plate.

In yet a further preferred embodiment, said solvent can be an organic solvent such as, but not limited to, N-methyl pyrrolidone (NMP) and dimethyl formamide (DMF), isopropyl alcohol (IPA), diacetone alcohol (DAA), di(propylene glycol) methyl ether (DPGME), terpineol, ethanol, and cyclohexanone, or any combination thereof. More preferably still, said solvent is selected from isopropyl alcohol (IPA) and/or methyl ether (DPGME). IPA is extremely easy to evaporate due to the high vapour pressure of 4.4 kPa at 20°C while DPGME has a very low vapour pressure of 0.037 kPa under the same temperature. Most preferably, said solvent is a mixture of IPA and DPGME in a ratio of 1 :1 . Different loading ratios between these two solvents were evaluated to produce a solvent mixture with proper volatility, including 90/10, 80/20, 70/30, 40/60, and 50/50. As shown in Table 1 , evaporating time was found to increase with the increased addition of DPGME, with 50/50 IPA/DPGME offering a low evaporating rate (26min35s). This is important when undertaking printing to ensure that the printing plate maintains complete wetness during the whole printing process.

In yet a further preferred embodiment still, said graphene ink comprises a stabiliser to enable stable, high-concentration inks of pristine graphene with tunable viscosity and solvent composition for scalable and efficient production of graphene. Examples of stabilisers include, but are not limited to, polymer based stabilisers such as Ethyl cellulose (EC), poly(3,4- ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyaniline, and polypyrrole. More preferably, said stabiliser is EC, owing to its nontoxicity and hydrophobicity, which has been found to offer a favourable ink or deposition with tunable ink characteristics such as loading, viscosity, surface tension, and evaporation kinetics to meet the specific requirements of deposition. Also, advantageously, the provision of a stabiliser was found to further reduce the incidence of agglomeration of the graphene.

Further, it was found that the quality of sensor closely correlates with the GNP to stabiliser weight ratio. As proof of principle, for the stabiliser EC, three ratios were attempted, specifically GNP:stabiliser ratios of 2: 1 , 1 : 1 and 1 :2. Of the ratios tested, the weight ratio of 1 :2 was found as the optimum since higher EC loading improved the sensor printability and also preserved the resultant vertical graphene structures without any significant effect on sensor conductivity. Therefore, more preferably, said graphene ink comprises GNP to stabiliser at a ratio of at least 1 : 1 , and more preferably still at least 1 : 1 .15. Similarly, said graphene ink preferably comprises GNP to stabiliser at ratio of no more than 1 :5, more preferably no more than 1 :4, and most preferably no more than 1 :3.

In yet a further preferred embodiment still, graphene ink is deposited onto the surface of the sensor as a series of sequential deposited, or printed, layers wherein subsequent deposition or printing steps are undertaken to increase layer thickness. Through investigation of effect of multiple graphene layers it was found that the number of graphene layers, and so graphene thickness, determined electrical conductivity of the sensing layer. However, with increased layer thickness less vertically aligned graphene sheets were formed, yet with too few layers the vertical sheets were more brittle in nature. Therefore, the printed layers of graphene closely effects layer conductivity, brittleness and formation. In a preferred embodiment, said graphene ink is deposited as a plurality of layers, more preferably about 6-8 layers and most ideally 7 layers.

In a preferred method of the invention, step a) comprises depositing a conducting ink to provide at least one intermediary conducting layer, ideally at least 3 intermediary conducting layers, and subsequently depositing onto the surface of said intermediary conducting layer(s) the graphene ink to provide at least one sensing layer, more preferably about 6-8 sensing layers and most ideally 7 sensing layers (i.e. at least one intermediary conducting layer and from 6-8 sensing layers are provided).

In a preferred embodiment the conducting ink refers to any electrically conducting ink such as, but not limited to those containing conductive polymers; carbon, graphene or graphite carbon black; organic/metallic compounds such as silver, copper, silver-coated copper, silver-coated aluminium, coated mica, glass spheres, or mixtures thereof; metal precursors, and metal nanoparticles, wherein said conducting ink has been chemically functionalised to modify its surface properties. In particular, the incorporation of chemical species improves the compatibility/binding of graphene ink of the intermediary conducting layer(s) with the substrate, and also provides increased dispersion and compatibility in a selection of solvents and polymers. More importantly, this intermediary conducting layer improves lateral conductivity of the sensor by providing a conductive base of the graphene sheets with the substrate and further has also been found to provide increased stability to the sensor, in particular the vertically aligned graphene sheets of sensing/surface layer, which is important for long term stability of the sensor and the ability to photonically cure the sensing layer(s).

Most preferably, said conducting ink is a graphene ink and accordingly step a) comprises depositing at least a first graphene ink to provide at least one intermediary conducting layer and subsequently depositing a second graphene ink to provide at least one sensing layer, wherein said first graphene ink is as defined herein and has been further chemically functionalised to modify the surface of its GNPs.

Examples of functionalisation of inks are known in the art, for example commercially available HDPIas® graphene ink (Haydale Ltd) and include, but are not limited to, the formation of carboxyl, carbonyl, hydroxyl, amine, amide, oxo, oxide or halogen chemical group; surface modification with silicon, sulphur, selenium, or other metal such as silver, gold, platinum, copper and iron; doping with a dopant species such as N, B, S, Se, and/or a halogen atom, preferably F. Such chemical functionalisation can be achieved by numerous means well known in the art including, but not limited to, plasma treatment with suitable precursor gases including oxygen, water, hydrogen peroxide, alcohol such as methanol, nitrogen, ammonia, organic amines, halogens such as fluorine and chlorine, and halogenated hydrocarbons. Most preferably, conducting ink is functionalised by formation of oxygen or nitrogen chemical groups.

In a preferred embodiment, the graphene ink and/or conducting ink is dried in between subsequent layer deposition to ensure the layers are dry thus avoiding layer smudging. Numerous techniques can be used for the purpose of drying such as, but not limited to, air drying, oven drying, ultra-violet or near infrared illumination or a combination thereof.

Reference herein to photonic curing refers to high-temperature thermal processing using pulsed light from a flashlamp. Advantageously, it has been found that through application of photonic curing of the sensing layer can generate vertically aligned graphene sheets by at least partially eliminating the polymeric stabilisers (e.g. EC, PEDOTPSS). As stated herein, this dramatically increases the surface area of graphene which is available for biosensing through its vertical alignment (as opposed to graphene sheets lying flat on the surface of substrate), and thus improving sensor sensitivity and dynamic range. Indeed, when considering functionalisation with a capture agent we found that only photonic cured surface exhibited biosensor detection. Moreover, photonic curing permits one to attain a significantly higher temperature than the substrate can ordinarily withstand allowing use of wide range of substrates, especially for organic substrates (e.g. PET), that cannot withstand high temperature thermal annealing (i.e. oven). Additionally, the higher temperature processing afforded by photonic curing reduces the processing time exponentially, often from minutes down to milliseconds, which increases throughput while maintaining a small machine footprint.

Further, it has been found that the parameters of photonic curing, in particular the degree of absorbed flash power at the surface influences the nature of the vertical graphene alignment. In a preferred embodiment, the absorbed flash power emitted from the flashlamp is from about 2-6 kW/cm², and more ideally

2.1 kW/cm², 2.2 kW/cm², 2.3 kW/cm², 2.4 kW/cm², 2.5 kW/cm², 2.6 kW/cm²,

2.7 kW/cm², 2.8 kW/cm², 2.9 kW/cm², 3.0 kW/cm², 3.1 kW/cm², 3.2 kW/cm²,

3.3 kW/cm², 3.4 kW/cm², 3.5 kW/cm², 3.6 kW/cm², 3.7 kW/cm², 3.8 kW/cm²,

3.9 kW/cm², 4.0 kW/cm², 4.1 kW/cm², 4.2 kW/cm², 4.3 kW/cm², 4.4 kW/cm²,

4.5 kW/cm², 4.6 kW/cm², 4.7 kW/cm², 4.8 kW/cm², 4.9 kW/cm², 5.0 kW/cm²,

5.1 kW/cm², 5.2 kW/cm², 5.3 kW/cm², 5.4 kW/cm², 5.5 kW/cm², 5.6 kW/cm²,

5.7 kW/cm², 5.8 kW/cm², 5.9 kW/cm², and 6.0 kW/cm² including every 0.01 kW/cm² therebetween. Alternatively, as will be known from those skilled in the art, absorbed flash power can be measured by flash exposure power at the sensor, such as by using an optical meter. In this embodiment, the flash exposure power at the stage is from about 2-6 J/cm², and more ideally is from about 2-6 J/cm², and more ideally 2.1 J/cm², 2.2 J/cm², 2.3 J/cm², 2.4 J/cm²,

2.5 J/cm², 2.6 J/cm², 2.7 J/cm², 2.8 J/cm², 2.9 J/cm², 3.0 J/cm², 3.1 J/cm², 3.2 J/cm², 3.3 J/cm², 3.4 J/cm², 3.5 J/cm², 3.6 J/cm², 3.7 J/cm², 3.8 J/cm², 3.9 J/cm², 4.0 J/cm², 4.1 J/cm², 4.2 J/cm², 4.3 J/cm², 4.4 J/cm², 4.5 J/cm², 4.6 J/cm², 4.7 J/cm², 4.8 J/cm², 4.9 J/cm², 5.0 J/cm², 5.1 J/cm², 5.2 J/cm², 5.3 J/cm², 5.4 J/cm², 5.5 J/cm², 5.6 J/cm², 5.7 J/cm², 5.8 J/cm², 5.9 J/cm², and 6.0 J/cm² including every 0.01 J/cm² therebetween. As will be appreciated by those skilled in the art, flash power or flash exposure power (and thus absorbed flash power) can be varied according to numerous parameters of photonic curing, such as pulse voltage, pulse duration, pulse cycle, and working distance. In a preferred embodiment, photonic curing is undertaken at a pulse voltage from about 200-450V, and every 1V integer therebetween. More preferably, said curing is undertaken at about 300-400V, including every 1 V integer therebetween, and most preferably 375V. In yet a further preferred embodiment, said photonic curing is undertaken for a pulse duration from about 1000-5000ps, and every 1 ps therebetween, and more preferably 2000- 4000, and every 1 ps therebetween, and most ideally about 3000ps. In yet a further preferred embodiment still, said photonic curing is undertaken for a single pulse cycle. Alternatively, multiple pulse cycles can be undertaken. Advantageously, with these parameters it has been found that the stabiliser is at least partially decomposed to produce vertically aligned graphene sheets yet retain a thin layer of stabiliser at the lower sensing layers to provide stability to the GNP vertical sheets, with the intermediary conductive layer remaining intact for electrical conductivity. More importantly, there is limited or no damage to the substrate while using the photonic curing technique.

In yet a further preferred embodiment of the invention said process further includes a further optional step after step c) and prior to step d), said step comprising insulating at least a part of sensing layer with an electrically insulating material, ideally, by the application of an insulating material to part of said graphene sheets. In this manner, one can produce a substantially insulated graphene electrode with a part exposed region thereof to provide a sensing window for subsequent functionalisation and/or immobilisation. Numerous materials are known in the art that may be used as suitable insulating materials, including those that are also used to form the sensor substrate, such as but not limited to silicon, solid polymers such as polyimide, SU-8 type materials, organic substrates such as polyethylene terephthalate (PET) or polyvinylchloride (PVC), and flexible material such as rubber silicon- based gels or other gel type materials. More preferably, said insulating material is an organic material that is solid at room temperature, such as a wax, ideally paraffin wax. Thus, the process further involves, heating a wax until it forms a liquid and then dipping a part of said sensor in said wax or applying said molten wax to a part of said sensor whereby at least a part of said sensor is provided with an insulating material.

Paraffin wax is low cost, and easy to process due to its relatively low viscosity in a liquid state. Also, the low melting point of wax is greatly advantageous compared to many other insulating agents that may require high curing temperatures for long periods which would denature biological enzymes, whereas wax melts and hardens at low temperatures in seconds. Further, other insulating agents normally possess high viscosity making them difficult to handle. Finally, in its solid state paraffin wax is a great electrical insulator, water resistant and non-hazardous when in contact with skin. These characteristics make wax an ideal candidate as an insulator to produce sensing window and is suitable for the mass production of sensors.

Reference herein to functionalising, when used in relation to the surface of the sensing layer(s), refers to modification of at least a part of the sensing layer such that the functionalized portion of the sensing layer has a binding affinity for at least one capture reagent. Numerous means of functionalisation can be used, such as but not limited to chemical and biological functionalisation. In all embodiments, functionalisation is achieved by spray coating, which has been found to be essential to preserve the vertically aligned graphene sheets of the sensing layer. There are other advantages of the technique, such as simple preparation and fast processing, more importantly it is ideal for functionalization of fragile surface.

In a preferred embodiment, spray coating is achieved by any of numerous means as known to those skilled in the art, such as but not limited to aerosol and non-aerosol spraying. In a preferred embodiment, functionalisation is achieved by aerosol spray coating, which is undertaken at a compressed air pressure from about 5-50 psi, more preferably 15-25 psi, and at a distance between 5 and 50 cm, more preferably 20 and 25 cm, and every 1 psi integer and/or every 1 cm integer there between. These parameters can vary depending on the manufacturing setup. Generally, higher spray air pressures will require more material to be sprayed and at a larger distance to preserve the graphene structures. Preferably, the air pressure and spray nozzle distance are adjusted so that the air speed at the biosensor electrodes surface is less than 10.0 m/s, more preferably about 1 -7 m/s, more preferably still about 3-7 m/s, most ideally about 5.0 m/s to preserve the graphene structures. However, as will be appreciated, alternative arrangements are possible. Further, ideally, the spray coating is undertaken by rotating the spray nozzle and/or sample stage to improve uniformity. This aerosol spray coating process was found to achieve uniform coating without damaging the vertical graphene sheets.

In yet a further preferred embodiment still, said functionalisation comprises spray coating with a linker whose function is to act as thin insulating layer and cross-linking molecule. Preferably, said linker is selected from the group comprising: Aminopropyltriethoxysilane (APTES), polydopamine, polyethylene glycol (PEG) and cysteamine.

It has also been found that the thickness of linker as well as the extent of surface coverage can be controlled by its concentration. Indeed, in the case of APTES, concentrations of linker in deionised water solution composing of 0.5, 1 , 2.5, 5, 10 15, 20 and 30 % were tested. Electron transfer resistance (Ret) showed a sharp increase from 0.5% to 5% and begun to plateau between 5% and 10% Therefore, in a preferred embodiment, said linker is APTES and is spray coated at a concentration from 0.5-30%, and every 0.1 % therebetween, More preferably, 0.5-15% and yet more preferably 5-15% and every 0.1 % therebetween, and most preferably 15%. Reference herein to a capture reagent refers to any substance or reagent that can bind, reversibly or irreversibly, to a target analyte in said sample (by capturing it from the sample). In this manner, as will be appreciated by those skilled in the art, a sample having, or suspected of having, the target analyte is added by a user to the sensor wherein said analyte (if present) is specifically recognized and bound by the capture reagent, such that it can be used to identify and/or quantitate the analyte.

In a preferred embodiment, said capture reagent includes, but is not limited to, antibodies, aptamers and fragments thereof, oligonucleotides or other specific ligands or receptors comprising a specific binding partner for the target analyte in the sample. Multiple capture reagents may be used to allow multiplexing detections.

In a further preferred embodiment of the invention, said capture reagent is bound directly onto at least a part of the sensing layer by any means known to those skilled in the art, such as but not limited to, immobilisation by adsorption or by chemical coupling. For example, such chemical crosslinkers include Glutaraldehyde (GA), N-Hydroxysuccinimide (NHS) esters, imidoesters, DSG (disuccinimidyl glutarate), DFDNB (1 ,5-difluoro-2, 4-dinitrobenzene), BS3 (bis(sulfosuccinimidyl)suberate), TSAT (tris-(succinimidyl)aminotriacetate), BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate), BS(PEG)9 (PEGylated bis(sulfosuccinimidyl)suberate, DSP (dithiobis(succinimidyl propionate)), Lomant's Reagent, DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)), DST (disuccinimidyl tartrate), BSOCOES (bis(2- (succinimidooxycarbonyloxy)ethyl)sulfone), EGS (ethylene glycol bis(succinimidyl succinate)), DMA (dimethyl adipimidate), DMP (dimethyl pimelimidate), DMS (dimethyl suberimidate), DTBP (dimethyl 3,3'- dithiobispropionimidat; Wang and Richard's Reagent), and other known protein crosslinking reagents.

Alternatively, said capture reagent is bound indirectly onto at least a part of the sensing layer by the use of the anchor substance that binds the capture reagent. Therefore, in this embodiment at least a part of the sensing layer is coated with the anchor substance and said capture reagent comprises a binding partner that binds the anchor substance. Most ideally, said anchor substance is selected from the group comprising: avidins, oligo and polynucleotides, proteins or lectins which bind a binding partner on the capture reagent and therefore cause the capture reagent to bind to at least a part of a surface of the reaction chamber.

As is known to those skilled in the art, avidins are a member of a family of proteins, including avidin, streptavidin, and neutravidin, functionally defined by their ability to bind biotin with high affinity and specificity, which serves as their specific binding partner. The binding affinity of avidins to biotin, albeit noncovalent, is so high that it can be considered irreversible. Therefore, in this embodiment, said capture reagent is biotinylated. Many agents can be biotinylated typically by chemical or enzymatic means and are well known to those skilled in the art. Most ideally, said biotinylated capture reagent is a biotinylated antibody.

Lectins are naturally occurring carbohydrate specific binding proteins which selectively bind carbohydrate groups such as typical sugar moieties. Lectins perform recognition at the cellular and molecular level and play numerous roles in biological recognition phenomena involving cells, carbohydrates, and proteins. Therefore, in this embodiment, said lectins bind to carbohydrate groups of the capture reagent such as, but not limited to, those occurring on antibodies and fragments thereof.

Oligo and polynucleotides can bind to capture reagents through complementary nucleotide molecules and these complementary nucleotides can be used to modify capture reagents in order to elicit the requisite binding.

According to a further aspect of the invention there is provided a sensor manufactured according to the above process. According to yet a further aspect of the invention there is provided a sensor comprising a substrate, and at least one sensing layer wherein said sensing layer comprises vertically aligned graphene sheets immobilised with capture reagent.

Preferably, said at least one sensing layer is provided as at least one interdigitated electrode.

In a preferred sensor said sensor layer is manufactured from graphene provided as graphene nanoplatelets (GNPs) with a particle size of less than about 10 pm, and more preferably less than about 1 pm.

More preferably, said graphene comprises a stabiliser. Examples of stabilisers include, but are not limited to, polymer based stabiliser such as Ethyl cellulose (EC), poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyaniline, and polypyrrole. More preferably, said stabiliser is EC.

Further, it was found that the quality of sensor closely correlates with GNP to stabiliser weight ratio. As proof of principle, for the stabiliser EC, three ratios were attempted, specifically GNP:stabiliser ratios of 2: 1 , 1 : 1 and 1 :2. Of the ratios tested, the weight ratio of 1 :2 was found as the optimum since more EC loading improved the sensor printability and also preserved the resultant vertical graphene structures without any significant effect on sensor conductivity. Therefore, more preferably, said graphene comprises GNP to stabiliser at a ratio of at least 1 : 1 , and more preferably still at least 1 :1 .15. Similarly, said graphene comprises GNP to stabiliser at ratio of no more than 1 :5, more preferably no more than 1 :4, and most preferably no more than 1 :3. In yet a further preferred sensor still, the sensor comprises a plurality of sensing layers, preferably about 6-8 sensing layers and most ideally 7 sensing layers.

More preferably, said sensor comprises at least one intermediate conducting layer positioned between the substrate and the at least one sensing layer.

In a preferred embodiment the intermediary layer(s) is manufactured from a conducting ink. Reference to a conducting ink refers to any electrically conducting ink such as, but not limited to those containing conductive polymers; carbon, graphene or graphite carbon black; organic/metallic compounds such as silver, copper, silver-coated copper, silver-coated aluminium, coated mica, glass spheres, or mixtures thereof; metal precursors, and metal nanoparticles, wherein said conducting ink has been chemically functionalised to modify its surface properties. In particular, the incorporation of chemical species improves the compatibility/binding of graphene ink of the intermediary conducting layer(s) with the substrate, and also provides increased dispersion and compatibility in a selection of solvents and polymers. More importantly, this intermediary conducting layer(s) improves lateral conductivity of the sensor by providing a conductive base of the graphene sheets with the substrate and further has also been found to provide increased stability to the sensor, in particular the vertically aligned graphene sheets of sensing/surface layer, which is important for long term stability of the sensor and the ability to photonically cure the sensing layer(s).

Ideally, the intermediary layer(s) is manufactured from a conducting ink wherein said conducting ink is a carbon, graphene or graphite carbon black ink.

Examples of functionalisation of inks are known in the art, for example commercially available HDPIas® graphene ink (Haydale Ltd) and include, but are not limited to, the formation of carboxyl, carbonyl, hydroxyl, amine, amide, oxo, oxide or halogen chemical group; surface modification with silicon, sulfur, selenium, or other metal such as silver, gold, platinum, copper and iron; doping with a dopant species such as N, B, S, Se, and/or a halogen atom, preferably F. Such chemical functionalisation can be achieved by numerous means well known in the art including, but not limited to, plasma treatment with suitable precursor gases including oxygen, water, hydrogen peroxide, alcohol such as methanol, nitrogen, ammonia, organic amines, halogens such as fluorine and chlorine, and halogenated hydrocarbons. Most preferably, conducting ink is functionalised by formation of oxygen or nitrogen chemical groups.

More preferably still, at least a part of the surface of the sensing layer comprises an insulating material. In this manner, one can produce a substantially insulated sensing layer with a part exposed region thereof to provide a sensing window for subsequent functionalisation and/or immobilisation. Numerous materials are known in the art that may be used as suitable insulating materials, including those that are also used to form the sensor substrate, such as but not limited to silicon, solid polymers such as polyimide, SU-8 type materials, organic substrates such as polyethylene terephthalate (PET) or polyvinylchloride (PVC), and flexible material such as rubber silicon-based gels or other gel type materials. More preferably, said insulating material is an organic material that is solid at room temperature, such as a wax, ideally paraffin wax.

In yet a further preferred sensor, said sensing layer is functionalised as defined herein. Most preferably, said sensing layer is functionalised with a linker whose function is to act as a thin insulating layer and cross-linking molecule. Preferably, said linker is selected from the group comprising: Aminopropyltriethoxysilane (APTES), polydopamine, polyethylene glycol (PEG) and cysteamine .

In yet a further preferred embodiment, said sensor comprises a capture reagent bound to the surface of the sensing layer, directly or indirectly. In a preferred embodiment, said capture reagent includes, but is not limited to, antibodies, aptamers and fragments thereof, oligonucleotides or other specific ligands or receptors comprising a specific binding partner for the target analyte in the sample.

In a further preferred embodiment of the invention, said capture reagent is bound directly onto at least a part of the sensing layer by any means known to those skilled in the art, such as but not limited to, immobilisation by adsorption or by chemical coupling. For example, such chemical linkers include Glutaraldehyde (GA), N-Hydroxysuccinimide (NHS) esters, imidoesters, DSG (disuccinimidyl glutarate), DFDNB (1 ,5-difluoro-2, 4-dinitrobenzene), BS3 (bis(sulfosuccinimidyl)suberate), TSAT (tris-(succinimidyl)aminotriacetate), BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate), BS(PEG)9 (PEGylated bis(sulfosuccinimidyl)suberate, DSP (dithiobis(succinimidyl propionate)), Lomant's Reagent, DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate)), DST (disuccinimidyl tartrate), BSOCOES (bis(2- (succinimidooxycarbonyloxy)ethyl)sulfone), EGS (ethylene glycol bis(succinimidyl succinate)), DMA (dimethyl adipimidate), DMP (dimethyl pimelimidate), DMS (dimethyl suberimidate), DTBP (dimethyl 3,3'- dithiobispropionimidat; Wang and Richard's Reagent), and other known protein crosslinking reagents.

Alternatively, said capture reagent is bound indirectly onto at least a part of the sensing layer by the use of the anchor substance that binds the capture reagent. Therefore, in this embodiment at least a part of the sensing layer is coated with the anchor substance and said capture reagent comprises a binding partner that binds the anchor substance. Most ideally, said anchor substance is selected from the group comprising: avidins, oligo and polynucleotides, proteins or lectins which bind a binding partner on the capture reagent and therefore cause the capture reagent to bind to at least a part of a surface of the reaction chamber. As is known to those skilled in the art, avidins are a member of a family of proteins, including avidin, streptavidin, and neutravidin, functionally defined by their ability to bind biotin with high affinity and specificity, which serves as their specific binding partner. The binding affinity of avidins to biotin, albeit noncovalent, is so high that it can be considered irreversible. Therefore, in this embodiment, said capture reagent is biotinylated. Many agents can be biotinylated typically by chemical or enzymatic means and are well known to those skilled in the art. Most ideally, said biotinylated capture reagent is a biotinylated antibody.

According to a yet further aspect of the invention there is provided a method for taking a biological measurement comprising the use of said biosensor.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

Throughout the description and claims of this specification, the words “comprise” and“contain” and variations of the words, for example“comprising” and“comprises”, mean“including but not limited to” and do not exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

The Invention will now be described by way of example only with reference to the Examples below and to the following Figures wherein:

Figure 1. (A) The size of graphene nanoplatelets (GNPs) after different ball milling time, (B) Scanning Electron Microscopy (SEM) image regarding to 24 hours ball milled GNPs, left is low magnification (*90) while right is high magnification (*5k), (C) Long-term stability of 24 hours ball milled GNPs. ;

Figure 2. (A) Schematic illustration of a flexographic printing arrangement for printing the electrode as defined according to the invention (B) Schematic cross-sectional illustration of different printed graphene layers using flexographic printing of the electrode as defined herein, (C) Top planar view of an interdigitated electrodes according to the invention; Figure 3. (A) SEM images of different printing recipes after 350V, 3000ps and single cycle of photonic curing: 1 and 2 are cross-sectional and tilted view on 8 GNP layer prints; 3 and 4 are cross-sectional and tilted view on 7 GNP layer prints; 5 and 6 are cross-sectional and tilted view on 6 GNP layer prints, (B) Bar chart on conductivity measurements for different printed GNP layers after various voltage applied involving: before, 200, 250, 300 and 350V coupled with 3000ps and single cycle photonic curing;

Figure 4. (A) Visible comparison of non-photonic cured and photonic cured area, (B) Tilted SEM image of non-photonic curing compared with photonic curing under conditions of 350V, 3000ps and single cycle, (C) SEM image of cross-sectional viewing on non-photonic curing, (D) SEM image of cross- sectional viewing on the surface photonic curing under conditions of 350V, 3000ps and single cycle, (E) insert SEM image of high magnification (X600) for GNP vertical sheets;

Figure 5. (A) Raman Spectroscopy after different plasma duration, (B) Id/lg ratio of different plasma duration, (C) Raman Spectroscopy after different water vapour incubation time, (D) Id/lg ratio of different water vapour incubation time, (E) Charge transfer resistance (Ret) after spray coated with different concentrations of APTES solution, (F) Electrochemical impedance under different concentrations of ATPES solution analysed through faradaic EIS by involving 100 pL of [Fe(CN)6]3-/4- as a redox probe, (G) Results on SEM coupled with EDX: 1 is top-down SEM image without APTES coating, 2 is corresponding EDX for SEM-1 , 3 is top-down SEM image with ATPES coating under conditions of 20 cm, 10 psi, 10% APTES, 4 is corresponding EDX for SEM-3, 5 is high magnification cross-sectional SEM image with ATPES coating under conditions of 20 cm, 10 psi, 10% APTES and 6 is corresponding EDX for SEM-5, insert image is chemical element intensity (silicon signal observed); Figure 6. Nonfaradaic biosensing results showing a change in phase for different concentrations of pp65 immediate early nuclear antigen of cytomegalovirus as exemplar capture reagents.

Figure 7. Schematic of a preferred sensor of the invention comprising substrate and deposited thereon a sensing layer comprising vertically aligned graphene sheets bound by capture agents in the form of antibodies. The provision of vertically aligned graphene sheets dramatically increases the effective surface area of the sensing layer. Further, positioned between the sensing layer and substrate is shown an intermediary conducting layer, manufactured from an electrically conducting functionalised ink, that improves lateral electrical conductivity but also improves binding of the sensing layer and the stability of the vertical graphene sheets.

Figure 8. White light interferometry on the printed graphene electrode before [A] and after photonic curing [B] The average surface roughness was 1 .61 ± 0.14 and 15.88 ± 2.12 pm before and after photonic curing respectively, which increased by almost an order of magnitude after the photonic curing.

Table 1. Evaporating time related to different loading ratio of IPA to DPGME.

MATERIALS AND METHODS

Manufacture of Graphene Nanoplatelets

Graphene Nanoplatelets (GNPs) are prone to agglomerate together, which can seriously affect the ink transfer from the anilox to the printing plate. To solve this problem, ball miller (FRITSCH, Germany) was used to improve the printability of ink and sensor conductivity. 2 g of GNP (Advanced Chemicals Supplier, USA) mixed with 40 ml of solvent, such as isopropyl alcohol (IPA), diacetone alcohol (DAA), di(propylene glycol) methyl ether (DPGME), was loaded into a 250 ml steel bowl with 400 g milling balls (diameter 5mm) followed by undergoing different milling time including 0, 4, 8, 12, 16, 20 and 24 hours. Ink samples were extracted every four hours during this process and subsequent size measurement was carried out using Zetasizer Nano ZS (Malvern Panalytical, UK).

Manufacture of Graphene Ink

To avoid agglomeration of graphene ink, water-insoluble polymeric stabiliser was added during the ink formation. As proof of concept, Ethyl cellulose (EC) was used in this experiment because of its nontoxicity, hydrophobicity and commercial availability. Other potential polymer stabilisers or binders can be used including poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), polyaniline, and polypyrrole. Mixed solvent for this experiment was made up of isopropyl alcohol (IPA) and Dipropylene glycol monomethyl ether (DPGME). IPA is extremely easy to evaporate due to the high vapor pressure of 4.4 kPa at 20°C while DPGME has a very low vapor pressure of 0.037 kPa under the same temperature. Different loading ratios between these two solvents were evaluated to produce a solvent mixture with proper volatility, including 90/10, 80/20, 70/30, 40/60, and 50/50. As shown in Table 1 , evaporating time was found to increase with the increased addition of DPGME. Consequently, 50/50 was selected as the best loading ratio of IPA to DPGME because of the relatively low evaporating rate (26min35s). This was to ensure that the printing plate maintained complete wetness during the whole printing process. The quality of sensor was closely correlated with GNP to EC weight ratio. Three ratios were attempted, specifically GNP:EC ratios of 2: 1 , 1 : 1 and 1 :2. The weight ratio of 1 :2 was found as the optimum since more EC loading would improve the sensor printability and also preserve the vertical structures without any significant effect on sensor conductivity.

Printing

Flexographic printing is a roll-to-roll (R2R) printing technique featuring simple operation, high volume low-cost production. Firstly, a printing plate with interdigitated patterns was attached to the rotating plate cylinder. Polyimide was used as the printing substrate, other substrates, such as polyethylene terephthalate (PET), polyvinyl chloride (PVC) and paper, can also be used as substrate. The substrates were sonicated in IPA for 10 min and followed by sonication in acetone for another 10 min to clean the substrates prior to printing. After the surface cleaning treatment, the substrates were blow-dried with compressed nitrogen. Flexographic printing was carried out using RK Flexiproof 100 printer (RK Print-Coat Instruments Ltd. Hertfordshire, UK). Flexible printing plate was taped on the flexo plate roller while polyimide substrate was fixed on the impression roller. Doctor blade engaged with the anilox roller produce a reservoir for the ink which was transferred to the printing plate using a 24 cm³/m² anilox, and simultaneously transferred to polyimide substrate from the printing plate. To control the amount of ink transferred, two working distances from anilox to printing plate and from printing plate to substrate were optimized as 260~280pm and 340~360pm respectively, at a printing speed of 20 to 30 m/m in.

To ensure good lateral conductivity, polyimide substrate was firstly printed using highly conductive ink, e.g. HDPIas® (Haydale) or other conductive inks, as the base of the electrode. In this case, HDPIas® graphene paste was diluted to form a suitable ink by adding 41 g of the paste in a 9 ml solvent composed of 50/50 v/v IPA/DPGME coupled with 2 hours ball milling.

Photonic Curing

Photonic curing (carried out using PulseForge 1300, NovaCentrix, Austin, Texas, USA) was used to produce vertically aligned graphene sheets by eliminating the polymeric stabilisers (e.g. EC, PEDOT:PSS) at the top printed layer, thereby increasing the surface area of graphene which is available for biosensing through its vertical alignment. Photonic curing is a high- temperature processing of a thin-film using pulsed light from a flash-lamp.

This novel technique is easy to use and more importantly, it is applicable to a wide range of substrates, especially for organic substrates (e.g. PET), that cannot withstand high temperature thermal annealing (i.e. oven). Parameters to be considered for photonic curing are (1 ) pulse voltage, (2) pulse duration, (3) pulse cycle and (4) working distance. Different pulse voltages (e.g. 200, 250, 300, 350, 400 and 450 V), pulse duration (1000 to 5000 ps) and numbers of pulse cycle (1 to 3), have were studied, respectively.

Surface Hydroxylation

Hydroxyl groups were first generated at the graphene surface through oxygen plasma (Henniker HPT-100, flowrate 20 s.c.c.m.) coupled with water vapor incubation carried out in a desiccator. Raman spectroscopy was applied to investigate the presence of hydroxyl groups under different plasma durations, such as 5, 10, 20, 30 and 60 min, followed by water vapour incubation for 30 min to 2 hours.

Spray Coating

Aminopropyltriethoxysilane (APTES) is a type of dielectric self-assembled monolayer (SAM), which has been considered as a cross-linking molecule in the application of electrochemical biosensing. Other SAM candidates include polydopamine, and cysteamine. Instead of dipping the vertically aligned graphene in APTES solution, spray coating technique was applied in this work to preserve the vertically aligned graphene sheets. There are other advantages of the technique, such as simple preparation and fast processing, more importantly it is ideal for functionalization of fragile surface. The sensor was secured on a working stage that constantly rotates during the whole spray coating process. APTES solution was introduced into the spray gun chamber of an IWATA Eclipse HP-CS Airbrush (IWATA Eclipse, Japan) and then promptly transformed into fine droplets (e.g. aerosol) due to the compressed air. Working distance (10 to 50 cm between the spray nozzle and the desired area of stage) and the compressed air pressure (psi) from 5 to 25 psi were investigated in order to optimize the spray coating technique.

The thickness of APTES as well as the extent of surface coverage can be controlled by its concentration, which is another essential parameter in this APTES spray coating. Different concentrations of ATPES in deionised water solution composing of 0.5, 1 , 2.5, 5, 10, 15, 20 and 30 % were studied, although higher concentrations can be utilised. Surface functionalisation

APTES-coated graphene sensor surface was annealed at 120 °C for 20 min under continuous N2 flow in order to densify the layer through promotion of condensation reactions between the side arms of the APTES molecules. Paraffin wax was then used to define and seal the sensing window (5 mm ^c 8 mm). The sensing window was spray coated with 125 pL of 2.5% glutaraldehyde in phosphate buffered saline (PBS) and incubated for 1 hour and then washed with PBS. This was followed by spray coating of 100 pl_ of 10 pg mL-1 anti-pp65 antibody (Virusys-Corporation, Maryland, United States) in deionised water and incubated for 1 hour. The sensing window was subsequently washed with deionised water. The sensing window was then spray coated with 100 mI_ of 50 mM ethanolamine (pH 1 1 .5) for 15 mins to deactivate the unreacted aldehyde binding sites of the glutaraldehyde and was washed as previous. The elevated pH of the ethanolamine solution also aids in removing unbound antibody from the surface. Finally, 100 mI_ of 5% bovine serum albumin (BSA) was incubated onto the sensing window for 30 mins and washed as previous. The BSA blocks sites where proteins could non- specifically absorb onto the sensor surface, improving the selectivity of the sensor.

Sensor Measurements

For typical EIS measurements, AC signals of different frequencies are applied between the two electrodes while the voltage and current are monitored. This allows the frequency-dependent impedance to be measured. For non-faradaic measurements, the attachment of biomolecules to the surface would change the dielectric constant at the surface due to displacement of water and the addition of target biomolecules, hence changing the surface capacitance and resulting in phase change. To perform the EIS measurements, a potentiostat (Gamry Reference 600+) was used and 100 mI_ of PBS was added to the sensing window to acquire a baseline. Non-faradaic EIS measurements were performed over a frequency range of 50 mHz to 1 kHz with an applied AC potential of 10 mV. After acquiring baseline, sensor was incubated in 100 pl_ pp65 antigens in PBS for 20 min and then a non-faradaic EIS measurement was carried out. This procedure was repeated for different concentrations of pp65 antigen after the same incubation period.

RESULTS

Fabrication of graphene biosensor

Normalized results with respect to the size of GNPs under different durations of ball milling can be seen in Figure 1 A. The nominal peak location shifted to the left from 3129 nm (0 h) to 692 nm (24 h) indicating successful size reduction of GNPs. Long-term stability was also studied using 24 hours ball milled sample which can be seen in Figure 1 C. Results showed that nominal peak was not significantly shifted after one and two weeks storage, indicating an excellent stability. Furthermore, the morphology of GNPs after 24 hours ball milling was characterized by means of Hitachi S4800 Scanning Electron Microscopy (SEM) (Hitachi Ltd., Japan). SEM images of 24 hours ball milled sample can be found in Figure 1 B as the size of GNPs was reduced down to a few hundreds of nanometres. Therefore, 24 hours was chosen as the optimal duration of ball milling.

As proof of principle, sensor electrodes were printed by flexographic printing owing to its simple operation, high volume and low-cost production (figure 2A). Firstly, a printing plate with interdigitated patterns was attached to the rotating plate cylinder. To ensure good lateral conductivity, polyimide substrate was firstly printed with highly conductive chemically functionalised ink. As proof of principle FIDPIas® ink was used as a conductive base, followed by printing to produce a layer of GNPs (figure 2B). Multiple layers of GNP flexographic ink printing (i.e. 6, 7 and 8) as herein disclosed were then printed to provide a suitable electrode thickness. Corresponding SEM images and electrical resistance of photonic cured sensors are shown in Figure 3. 8 layers of GNP printing resulted in less formation of GNP vertical sheets while 6 layers GNP printing produced a very brittle vertical sheet structure. Flowever, 7 layers GNP printed sample produced the best conductivity. Thus, the optimized printing recipe consisted of a printed highly conductive graphene ink, e.g. HDPIas® graphene ink or other similar conductive ink, layer with subsequent 7 layers of GNP/EC ink at the speed of 30 m/min (see Figure 3A).

Increased surface area achieved by photonic curing

As stated herein, graphene electrodes are limited for biological applications owing to their reduced effective surface area for functionalisation when the graphene sheets are lying flat at the surface. To overcome this, photonic curing was employed to generate vertically aligned graphene sheets by eliminating the polymeric stabilisers (e.g. EC, PEDOT:PSS) at the top printed layer, thereby increasing the effective surface area of graphene which is available for biosensing through its vertical alignment (see Figure 4). This novel technique is easy to use and more importantly, it is applicable to a wide range of substrates, especially for organic substrates (e.g. PET), that cannot withstand high temperature thermal annealing (i.e. oven). Parameters to be considered for photonic curing are (1 ) pulse voltage, (2) pulse duration, (3) pulse cycle and (4) working distance. Different pulse voltages (e.g. 200, 250, 300, 350, 400 and 450 V), pulse duration (1000 to 5000 ps) and numbers of pulse cycle (1 to 3), have been studied, respectively. It was observed that the density of vertical graphene sheets was dominantly determined by the pulse voltage, and thus one cycle of 375V with duration of 3000 ps was chosen as the optimal setting for the photonic curing. EC stabiliser was partially decomposed during this high-temperature, short-pulse-interval process, leading to vertically aligned graphene sheets with a thin layer of EC at the bottom to hold the GNP, while the highly conductive FIDPIas® (or similar conductive ink) layers remain much intact. More importantly, there is limited damage to the organic substrate while using the photonic curing technique.

The surface roughness of the graphene layer was measured using white light interferometry, which provides a non-destructive characterisation on the surface topography. An average surface roughness before and after photonic curing was measured to be 1 .61 ± 0.14 pm and 15.88 ± 2.12 pm, respectively as shown in Figure 8. This represented almost an order of magnitude increase in the surface roughness after photonic curing caused by the formation of vertically aligned graphene sheets. Due to stabiliser coating around graphene, graphene sensor without photonic curing did not show any biosensing capability.

Functionalisation of graphene sensor surface by spray coating preserves the vertically aligned graphene sheets

Prior to APTES coating, hydroxyl groups were first generated at the graphene surface through oxygen plasma coupled with water vapor incubation carried out in a desiccator. The ratio of Id and l_g increased after 10 min of plasma surface treatment hence implying the introduction of functional groups on the activated surface and the ratio began to plateau beyond 10 min (Figure 5 A and B). Moreover, the duration of water vapour incubation was also optimized by analysing Id and l_g ratio after 20 min of oxygen plasma treatment with subsequent 0.5, 1 , 2 and 3 hours incubation in water vapour. The plotted graph indicates a saturation of hydroxyl groups on the graphene surface after an hour of incubation as shown in Figure 5 C and D.

Sensor surface was then functionalised with cross-linker by a spray coating technique applied in this work to preserve the vertically aligned graphene sheets. There are other advantages of the technique, such as simple preparation and fast processing, more importantly it is ideal for functionalization of fragile surface. Based on the SEM images coupled with energy-dispersive X-ray spectroscopy (EDX), 20 cm and 10 psi presented the best coverage of ATPES on the planar surface of the vertically aligned graphene as evidenced by the highest silicon signal either in top-down or cross-sectional SEM images (see Figure 5 G 3, 4, 5 and 6) as compared to the one without ATPES coating (see Figure 5 G 1 and 2). The thickness of APTES as well as the extent of surface coverage can be controlled by its concentration, which is another essential parameter in this APTES spray coating. Different concentrations of ATPES in deionised water solution composing of 0.5, 1 , 2.5, 5, 10 and 15% have been studied. Electron transfer resistance (Ret) showed a sharp increase from 0.5% to 5% and begun to plateau between 5% and 10% (see Figure 5 E and F). 15% APTES was picked to ensure comprehensive coverage at the surface.

Nonfaradaic electrochemical impedance spectroscopic measurements were performed on the graphene biosensor comprises of functionalised vertically aligned graphene sheets with antibodies that recognise pp65 immediate early nuclear antigen of cytomegalovirus as exemplar capture reagents. Figure 6 shows the change in phase at the biosensors in response to different concentrations of pp65-antigens. The plot shows a linear regression between the concentration of the target biomolecules and electrochemical impedance response. The excellent performance of the biosensor is due to the enhanced surface areas of the vertically aligned graphene sheets generated after photonic curing as both sides of the sheet are immobilised with the capture reagents (e.g. antibodies) as shown in Figure 7.

Summary

A novel production process, comprising photonic curing of printed graphene- based sensing layers, has been developed to prepare improved non-faradaic electrochemical impedimetric sensors. In particular, said sensors comprise vertically aligned graphene sheets, which greatly enhance the effective surface area of the sensing layer, enabling increased functionalisation with biological molecules, and thus improved sensor sensitivity and dynamic range.

Further, owing to the manufacture process, the sensor arrangement (in particular the vertically aligned graphene sheets) not only provide an increased effective surface area of the sensing layer but are also very stable. Accordingly, such an arrangement provides a highly effective platform for use in bio-sensing applications. Table 1 .

Claims

1 . A process for the manufacture of a graphene sensor for detecting a target molecule in a sample, the process comprising:

b) processing the at least one sensing layer by photonic curing to generate a graphene electrode comprising vertically aligned graphene sheets;

c) functionalising at least a part of the sensing layer(s) by suspending at least one linker in a solution and spray coating same onto the sensing layer(s);

d) optionally coating at least a part of the sensing layer(s) with at least one anchor substance having a binding affinity for a capture reagent; and

e) coating the sensing layer(s) of parts c) or d) with at least one capture reagent that bind said linker and/or said anchor substance to provide a sensor with a sensing layer comprising immobilized capture reagent on the surface.

2. The process according to any preceding claim wherein depositing is achieved by printing onto the substrate.

3. The process according to any preceding claim wherein depositing is achieved by flexographic printing.

4. The process according to any preceding claim wherein said substrate is selected from the group comprising: silicon, solid polymers such as polyimide, SU-8 type materials, organic substrates such as polyethylene terephthalate (PET) or polyvinychloride (PVC), injection mouldable polymers such as polyether ether ketone (PEEK) and conductive acrylonitrile butadiene styrene (ABS), paper, and flexible material such as rubber silicon-based gels or other gel type materials.

5. The process according to any preceding claim wherein said sensing layer of deposited ink is provided as at least one interdigitated electrode(s).

6. The process according to any preceding claim wherein said graphene ink is provided as graphene nanoplatelets (GNPs) dispersed in a solvent.

7. The process according to claim 6 wherein the GNPs are provided with a particle size of less than about 10 pm.

8. The process according to any one of claims 6-7 wherein said solvent is selected from the group comprising: N-methyl pyrrolidone (NMP), dimethyl formamide (DMF), isopropyl alcohol (IPA), diacetone alcohol (DAA), di(propylene glycol) methyl ether (DPGME), terpineol, ethanol, and cyclohexanone, or any combination thereof.

9. The process according to claim 8 wherein said solvent is selected from isopropyl alcohol (IPA) and/or methyl ether (DPGME).

10. The process according to claim 9 wherein said solvent is a mixture of

IPA and DPGME in a ratio of 1 :1.

11. The process according to any preceding claim wherein said graphene ink comprises a stabiliser.

12. The process according to claim 11 wherein said stabiliser is selected from the group comprising: Ethyl cellulose (EC), poly(3,4- ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyaniline, and polypyrrole.

13. The process according to any one of claims 11 -12 wherein said graphene ink comprises GNP to stabiliser at a ratio from about 1 :1 to 1 :5.

14. The process according to any preceding claim wherein said graphene ink is deposited onto the surface of the sensor as a series of sequential deposited layers wherein subsequent deposition steps are undertaken.

15. The process according to any preceding claim wherein step a) comprises depositing a conducting ink to provide at least one intermediary conducting layer and subsequently depositing the graphene ink to provide at least one sensing layer.

16. The process according to claim 15 wherein about 6-8 sensing layers are deposited.

17. The process according to claim 16 wherein 7 sensing layers are deposited.

18. The process according to any one of claims 15-17 wherein conducting ink comprises an electrical conductive material selected from the group comprising: conductive polymers; carbon, graphene or graphite carbon black; organic/metallic compounds such as silver, copper, silver-coated copper, silver-coated aluminium, coated mica, glass spheres, or mixtures thereof; metal precursors; or metal nanoparticles, wherein said conducting ink has been chemically functionalised to modify its surface.

19. The process according to claim 18 wherein said conducting ink is chemically functionalised by one or more of the following: formation of carboxyl, carbonyl, hydroxyl, amine, amide, oxo, oxide or halogen chemical group; surface modification with silicon, sulphur, selenium, or other metal such as silver, gold, platinum, copper and iron; doping with a dopant species such as N, B, S, Se, and/or a halogen atom.

20. The process according to claim 19 wherein said conducting ink functionalisation is achieved by plasma treatment.

21 . The process according to any one of claims 1 -20 wherein the graphene ink and/or conducting ink is dried in between depositing a further layer.

22. The process according to any preceding claim wherein the photonic curing comprises an flash exposure power from about 2-6 J/cm².

23. The process according to any preceding claim comprising a further step after step c) and prior to step d), wherein said step comprises insulating at least a part of the sensing layer with an electrically insulating material.

24. The process according to claim 23 wherein said insulating material is paraffin wax.

25. The process according to any preceding claim where said linker is selected from the group comprising: Aminopropyltriethoxysilane (APTES), polydopamine, polyethylene glycol (PEG) and cysteamine.

26. The process according to claim 25 where said linker is APTES and is spray coated at a concentration from 0.5-30%.

27. The process according to any preceding claim where said capture reagent(s) is selected from the group comprising: antibodies, aptamers and fragments thereof, oligonucleotides or other specific ligands or receptors comprising a specific binding partner for the target analyte in the sample.

28. The process according to any preceding claim wherein the capture reagent is bound directly onto at least a part of the sensing layer(s) by use of a crosslinker selected from the group comprising: Glutaraldehyde (GA), N-Hydroxysuccinimide (NHS) esters, imidoesters, DSG

(disuccinimidyl glutarate), DFDNB (1 ,5-difluoro-2, 4-dinitrobenzene), BS3 (bis(sulfosuccinimidyl)suberate), TSAT (tris- (succinimidyl)aminotriacetate), BS(PEG)5 (PEGylated bis(sulfosuccinimidyl)suberate), BS(PEG)9 (PEGylated bis(sulfosuccinimidyl)suberate, DSP (dithiobis(succinimidyl propionate)), Lomant's Reagent, DTSSP (3,3'- dithiobis(sulfosuccinimidyl propionate)), DST (disuccinimidyl tartrate), BSOCOES (bis(2-(succinimidooxycarbonyloxy)ethyl)sulfone), EGS

(ethylene glycol bis(succinimidyl succinate)), DMA (dimethyl adipimidate), DMP (dimethyl pimelimidate), DMS (dimethyl suberimidate), and DTBP (dimethyl 3,3'-dithiobispropionimidat; Wang and Richard's Reagent).

29. A sensor manufactured by the process according to any preceding claim.

30. A sensor comprising a substrate, at least one sensing layer wherein said graphene layer comprises vertically aligned graphene sheets immobilised with capture reagent.

31. The sensor according to claim 30 wherein said at least one sensing layer is provided as at least one interdigitated electrode.

32. The sensor according to any one of claims 30-31 wherein sensing layer is manufactured from graphene provided as graphene nanoplatelets (GNPs) with a particle size of less than about 10 pm.

33. The sensor according to claim 32 wherein said graphene comprises a stabiliser.

34. The sensor according to claim 33 wherein said graphene comprises GNP to stabiliser at a ratio from about 1 : 1 to 1 :5.

35. The sensor according to any one of claims 30-34 wherein said sensor comprises about 6-8 sensing layers.

36. The sensor according to any one of claims 30-35 wherein said sensor comprises at least one intermediate conducting layer positioned between the substrate and the at least one sensing layer.

37. The sensor according to claim 36 wherein the at least one intermediary layer is manufactured from a conducting ink.

38. The sensor according to claim 37 wherein the conducting ink is comprises an electrical conductive material selected from the group comprising: conductive polymers; carbon, graphene or graphite carbon black; organic/metallic compounds such as silver, copper, silver-coated copper, silver-coated aluminium, coated mica, glass spheres, or mixtures thereof; metal precursors, or metal nanoparticle containing ink and further wherein said conducting ink has been chemically functionalised to modify its surface.

39. The sensor according to claim 38 wherein said conducting ink is chemically functionalised by one or more of the following: formation of carboxyl, carbonyl, hydroxyl, amine, amide, oxo, oxide or halogen chemical group; surface modification with silicon, sulphur, selenium, or other metal such as silver, gold, platinum, copper and iron; doping with a dopant species such as N, B, S, Se, and/or a halogen atom.

40. A method for taking a biological measurement comprising the use of the sensor according to any one of claims 29-39.