WO2025257531A1

WO2025257531A1 - Sensors

Info

Publication number: WO2025257531A1
Application number: PCT/GB2025/051257
Authority: WO
Inventors: Sara GHOREISHIZADEH
Original assignee: UCL Business Ltd
Current assignee: UCL Business Ltd
Priority date: 2024-06-12
Filing date: 2025-06-09
Publication date: 2025-12-18
Anticipated expiration: 2026-12-12
Also published as: GB202408388D0

Abstract

The present invention provides a sensor for use in detection of biomolecules, the sensor comprising an electrode and a film, wherein the electrode comprises a sensing surface having one or more biorecognition element, the film comprises one or more polyelectrolyte that dissolves in an aqueous solution upon application of an electrical stimulus, and the film is positioned above the sensing surface to form a barrier. The invention further provides sensing devices comprising said sensors, a method of forming said sensors and a method of detecting a biomolecule using said sensors.

Description

Sensors

Field of the Invention

The present invention relates to sensors for use in the detection of biomolecules, sensing devices comprising said sensors, a method of forming said sensors and a method of detecting a biomolecule using said sensors.

Background of the Invention

Real time and continuous sensing of biomolecules in biological fluids is a useful technique in the identification and profiling of various physiological processes, or responses of the body to external and internal stimuli.

For example, cortisol, known as the “stress hormone” play a vital role in the body’s stress response. Sustained abnormal levels of cortisol have detrimental effects on various physiological processes. It is well known that substantially high or low cortisol levels throughout the day are associated with Cushing’s syndrome and Addison’s disease, respectively. Studies suggest that not only genes and environmental factors (e.g., smoking and exercise) affect cortisol levels in individuals throughout the day, but also daily social and emotional experiences can lead to systematic day-to-day changes in cortisol levels in the body. It is therefore important to acquire longitudinal data across different time points to better understand the causal relationship between experience, cortisol and well-being.

Current cortisol measurement techniques include high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunosorbent assay (CLIA) and surface plasmon resonance (SPR). These techniques require bulky laboratory equipment, dedicated space, and expertise.

Electrochemical sensing is an alternative technique for cortisol measurement. This technique is low-cost and rapid. Cortisol sensors are typically fabricated by immobilizing a biological receptor molecule on the surface of a suitable transducer that converts the interaction between the receptor and target analyte into a quantifiable electronic signal. Such sensors are often single use or of very limited life span. Regeneration of such electrochemical sensors requires physical washing of the sensor which is difficult to perform economically or in situ. Most such sensors are therefore limited to a one-time measurement due to the challenge of regenerating the sensors in situ.

Furthermore, most sensors tend to degrade if left in situ for an extended period of time before use. This prevents on demand measuring, as sensors must be used shortly after they are put into position.

There is therefore a need for a sensor that does not degrade in situ and that can be integrated into a device for acquiring longitudinal data across different time points.

Summary of the Invention

The present inventors have identified that films comprising one or more polyelectrolyte can be used to protect sensing surfaces and selectively dissolved in situ to expose the sensing surface at the moment of measurement.

In a first aspect of the present invention, there is provided a sensor for use in detection of biomolecules. The sensor comprises an electrode and a film. The electrode comprises a sensing surface having one or more biorecognition element. The film comprises one or more polyelectrolyte that dissolves in an aqueous solution upon application of an electrical stimulus, and the film is positioned above the sensing surface so as to form a barrier.

The purpose of a biorecognition element is to provide analyte specificity for a biosensor. The biorecognition element provides strong and selective affinity for the target bioanalyte. In some embodiments the one or more biorecognition element is an antibody, antigen, molecularly imprinted polymer, enzyme, RNA, DNA or aptamer biorecognition element, or a combination thereof.

The film of the sensor protects the sensing surface from degradation by external factors before it is time to take a measurement with the electrode. When it is time to take a measurement, an electrical stimulus can be provided to the film via the sensing surface, causing a breakdown in the structure of the film such that it dissolves in an aqueous medium, such as the medium to be tested. The sensing surface of the electrode is then exposed to the aqueous medium and a measurement of the medium can be taken.

In some embodiments, the one or more polyelectrolytes may be selected from poly cations, poly anions, poly ampholytes or combinations thereof. Polyelectrolytes form films when exposed to a potential difference. When constructing the sensor this potential difference is between the electrode of the sensor and a counter electrode. The film of the present invention breaks down in response to an electrical stimulus and dissolves in an aqueous medium.

In some embodiments the film is a polyelectrolyte multilayered film comprising alternating layers of one or more poly cation or polyampholyte, and one or more poly anion or polyampholyte. For example, the film may comprise alternating layers of polycation and polyanion.

In some embodiments, the poly anion is selected from the group consisting of alginic acid or a salt thereof, hyaluronic acid or a salt thereof, carboxymethyl curdlan or a salt thereof, K-carrageenan, X-carrageenan, r-carrageenan, carboxymethyl cellulose or a salt thereof, pectic acid, poly(sodium phosphate), dextran, dextran sulfate, poly(styrene sulfonate), sulfonated poly(ether ether ketone), sulfonated polyether sulfone, sulfonated polyphenyl sulfone, sulfonated poly(2,6-dimethyl-l,4-phenylene oxide), sulfonated poly(4-phenoxybenzoyl-l,4-phenylene), sulfonated polyphenylene oxide, sulfonated poly(phenylene), sulfonated poly(phthalazinone), sulfonated poly(vinyl toluene), poly(acrylic acid) or a salt thereof, poly (malic acid) or a salt thereof, poly(vinylsulfonic acid sodium), poly(acetic acid) or a salt thereof, poly(lactic acid) or a salt thereof, poly(aspartic acid) or a salt thereof, heparin or a salt thereof, and combinations thereof.

In some embodiments the poly anion is selected from the group consisting of K- carrageenan, X-carrageenan, r-carrageenan, carboxymethyl cellulose or a salt thereof, dextran, dextran sulfate, heparin or a salt thereof, and combinations thereof.

In some embodiments, the one or more poly cation is selected from the group consisting of protonated chitosan, polybiguanide, quaternary ammonium polyethylenimine, quaternary ammonium polypropylenimine, quaternary ammonium poly amidoamine (PAMAM), poly(vinylamine hydrochloride) (PVH), poly(allylamine hydrochloride) (PAH), poly(amidoamine hydrochloride)), poly(N-isopropylallylamine hydrochloride), poly(N-tert-butylallylamine hydrochloride), poly (N-l,2-dimethylpropylallylamine hydrochloride), poly(N-methylallylamine hydrochloride), poly(methyl allylamine hydrochloride), poly(2-vinylpiperidine hydrochloride), poly(4-vinylpiperidine hydrochloride), poly(diallyldimethylammonium chloride), poly(methacrylate), poly(acrylamide-co-diallyldimethylammonium chloride), poly (diallyl methylaminehydrochloride), copolymer of 2-propen-l-amine-hydrochloride with N-2- propenyl-2-propen-l-aminehydrochloride, poly(N -alkyl-4-vinylpyridinium) salt, polyvinylpyrollidone, poly(lysine), poly ornithine, polyarginine, poly(ethylene oxide)- block-poly(vinyl benzyl trimethylammonium chloride), poly(ethylene oxide)-block- poly(l-lysine), poly(2-methacryloyloxy ethyl phosphoiylcholine methacrylate)-block- poly (vinyl benzyl trimethylammonium chloride), poly[2-(dimethylamino)-ethyl methacrylate, poly [3-(dimethylamino)-propylmethacrylate], poly [2-(dimethylamino)- ethyl methacrylamide], poly [3-(dimethyl amino) propyl methacrylamide], poly[2- (trimethylamino) ethyl methacrylate chloride], poly[2-(diethylamino)ethy 1 methacrylate], poly [2-(dimethylamino)ethyl acrylate], or combinations thereof.

In some embodiments the polyanion is selected from the group consisting of poly(lysine), poly(ornithine), poly(arginine), or combinations thereof.

In some embodiments the film is a polyelectrolyte multilayered film, comprising alternating layers of poly(lysine) and heparin.

In some embodiments, the film has a thickness of at least 1 nm.

In some embodiments the electrode is a gold-plated electrode.

In a second aspect of the present invention, there is provided a sensing device for the detection of a biomolecule, the device comprising one or more sensor of the first aspect.

The sensing device may be a semiconductor chip, such as a complementary metal oxide semiconductor (CMOS) chip, a silicone-germanium (SiGe) chip, a gallium-nitride (GaN) chip, or a gallium arsenide (GaAs) chip. The sensing device may be a CMOS chip. Semi-conductor chips have low static power consumption, making them ideal for on body, wearable or battery powered systems.

The sensing device may comprise a counter electrode. To dissolve the film of the sensor, an electrical stimulus may be applied, such as a current or potential difference applied between the electrode and a counter electrode. By integrating the counter electrode into the sensing device fewer auxiliary components are required to activate the device, and the device is therefore easier to incorporate into on-body or wearable technology. However, the counter electrode may be provided separately, for example the counter electrode may be provided on a separate substrate.

In some embodiments the device further comprises integrated instrumentation electronics component having signal amplification and signal processing capabilities. This integrated instrumentation electronics component may be an electrical readout circuit. In preferred embodiments the sensing device is a semiconductor chip further comprising integrated instrumentation electronics component having signal amplification and signal processing capabilities, such as an electrical readout circuit. Traditional sensing technology requires interconnection between the sensor chip and the instrumentation electronic chip. By integrating these systems into the same device, more electrodes of the first aspect can be incorporated into the sensing device. The scalability of the device is therefore improved as the number of sensors (i.e., electrodes) is not limited by the number of interconnects. Furthermore, a system wherein the device further comprises an integrated instrumentation electronics component having signal amplification and signal processing capabilities has a higher signal to noise ration due to in-pixel amplification.

In some embodiments the device comprises a plurality of sensors of the first aspect. The film of each sensor can be individually controlled. By providing a plurality of sensors, the sensing device is able to provide data sets showing the concentration of a biomolecule in a sample over time by uncovering each electrode in turn to expose the sensing surface and take a measurement. The device can therefore comprise any number of sensors required to provide data of the required resolution and timescale required for the application. For example, the device may comprise three or more sensors, five or more sensors, 10 or more sensors, 100 or more sensors or 1000 or more sensors. In some embodiments, the device comprises a plurality of sensors and integrated instrumentation electronics component having signal amplification and signal processing capabilities for each sensor.

The sensors may be all configured to detect the same biomolecule, such as cortisol. Alternatively, the plurality of sensors is configured to detect a plurality of biomolecules, so that the concentrations of several different biomolecules may be measured over time. For example, the plurality of microelectrodes may comprise a first group sensors configured to detect a first biomolecule and a second group of sensors configured to detect a second biomolecule. In some embodiments from 10% to 50% of the sensors of the device may be configured to detect a first biomolecule and from 90% to 50% of the sensors may be configured to detect a second biomolecule.

In a third aspect there is provided a method of detecting the concentration of a biomolecule, the method comprising: i) providing a biofluid; ii) providing a sensor of the first aspect or a sensing device of the second aspect; iii) exposing the sensor to the biofluid iv) providing an electrical stimulus to the film of the sensor, causing it to dissolve in the biofluid; and v) detecting the concentration of the biomolecule using a voltametric or amperometric technique.

In some embodiments, the method is a time-resolved method. In such embodiments the method comprises providing a plurality of sensors according to the first aspect, or a sensing device of the second aspect having a plurality of sensors according to the first aspect. The time-resolved method further comprises the step of vi) wherein steps iv) and v) are repeated with a second sensor.

In some embodiments step vi) is performed after a set time-period relevant to the biofluid being measured or in response to an external instruction.

In some embodiments the concentration of the analyte is detected using a voltametric technique and the voltametric technique is cyclic voltammetry. Detailed Description of the Invention

The present invention provides a sensor for use in detection of biomolecules. The sensor comprises an electrode having a sensing surface and a film. The sensing surface comprises one or more biorecognition element. The film comprises one or more polyelectrolyte that dissolves in an aqueous solution upon application of an electrical stimulus, and the film is positioned above the sensing surface so as to form a barrier.

The Electrode

The electrode may be any suitable electrode. The use of electrodes in biomolecule detection is known.

The electrode comprises a sensing surface having a biorecognition element. The sensing surface is configured to detect an analyte. The sensing surface of the electrode may comprise a base layer. The base layer of the sensing surface may have other structures mounted upon it, such as nano structures or biorecognition elements. In some embodiments the sensing surface consists of a base layer and a biorecognition element mounted thereon. In some embodiments the base layer is a metal base layer, such as a layer formed from gold, silver, platinum, copper, or combinations thereof. In some embodiments the base layer is a metal oxide base layer, such as an indium tin oxide (ITO) layer. In some embodiments the base layer is a carbon layer, such as a base layer formed from graphene and derivatives of graphene including graphene oxide and reduce graphene oxide, derivatives of carbon including glassy carbon, carbon nanotubes and carbon yarns, or combinations thereof. In a preferred embodiment the base layer is formed from gold, silver or platinum, and most preferably the base layer is made from gold. The base layer may be an electroplated layer, preferably an electroplated gold layer.

The base may have a thickness of 2.5 micrometre or less. The base layer may have a surface roughness of about 600 nm or less.

In some embodiments the electrode is a microelectrode or a nanoelectrode. In some embodiment the sensing surface may have a surface area of less than 1 mm². For example, the sensing zone may have a surface area 900 pm² or less, 800 pm² or less, 700 pm² or less, 600 pm² or less, 500 pm² or less.

In some embodiments, the electrode may be formed on a semiconductor chip, such as a complementary metal oxide semiconductor (CMOS) chip, a silicone-germanium (SiGe) chip, a gallium -nitride (GaN) chip, or a gallium arsenide (GaAs) chip. In preferred embodiments the electrode may be formed on a complementary metal oxide semiconductor (CMOS) chip. CMOS chips are well known in microelectronics field. CMOS is a silicon technology for making integrated circuits. CMOS ensures very high accuracy of processing identical transistors (up to billions), high volume manufacturing, very low cost and high reproducibility at different levels (wafer level, wafer to wafer, and lot to lot). CMOS comes with high standards in quality and reliability.

In some embodiments the electrode is formed according to a method of preparing a CMOS chip having one or more electrodes. The method comprises the steps of providing a complementary metal oxide semiconductor having one or more sensor region. Each sensor region comprises a sensing zone and a passivation zone. The passivation zone encircles and directly abuts the sensing zone. The sensing zone comprises an active layer, an optional adhesive layer and a conductive substrate layer. The method further comprises the step of replacing at least part of the active layer with a metal sub-layer, which may be a gold sub-layer, by electroless deposition. The method then comprises the step of pulse current electroplating a base layer, which may form part of the sensing surface, onto the sub-layer. The base layer may be a platinum, silver or gold layer. In some embodiments, the pulse current electroplating is performed at a current density of from 1.5 to 5 mA/cm², with a deposition time of 1200s or less and a pulse frequency of greater than 10 Hz. This method provides sensing surfaces having base layers of low surface roughness. The method also provides control of the thickness of the base layer. Other methods of forming electrodes are known in the art.

The active layer is one that can be displaced by a metal sub-layer during the electroless deposition of gold. In some embodiments, the active layer is selected from a layer of copper, nickel phosphorous, nickel-boron, nickel, cobalt or iron. The skilled person will be aware of other suitable active layers. The conductive substrate layer is formed from the conductive substrate of the CMOS and is exposed when the CMOS is etched. Commonly, the conductive base layer is aluminium. However, the skilled person will be aware of CMOS with alternative conductive substrates, such as copper, which would be equally applicable.

The optional adhesive layer, when present, improves adhesion between the conductive base layer and the active layer or gold layer, compared with disposing the active layer directly onto the conductive base layer. In some embodiments, the adhesive layer is zinc. The skilled person will be aware of other suitable adhesive layers. Where the adhesive layer is zinc, is may be disposed on the conductive base layer by the double zincation process.

Electrochemical sensors typically employ biorecognition elements (also known as biological receptors or probes) in their structure. The sensing surface comprises one or more biorecognition element. The sensing surface may comprise one or more biorecognition element selected from an antibody, an antigen, a molecularly imprinted polymer (MIP), an enzyme, an RNA, a DNA or an aptamer biorecognition element, or combinations thereof. The biorecognition element may be immobilised on the sensing surface of the electrode.

In some embodiments, the one or more biorecognition elements are disposed on a base layer of the sensing surface. In some embodiments, the biorecognition element is selected from an antibody, an antigen, a molecularly imprinted polymer (MIP), an enzyme, an RNA, a DNA or an aptamer biorecognition element, or combinations thereof and is immobilised on the gold, silver, platinum or copper base layer of the electrode, preferably a gold base layer.

The selective recognition sites in MIPs are achieved through the polymerization of monomers in the presence of a template molecule (target analyte). Once the template is removed, cavities of the same size bearing structural similarity to the template are left behind in the polymer matrix.

Aptamers are single-stranded nucleic acid sequences that can be made to have a selective affinity toward a certain biomarker. They are generated via a process called systematic evolution of ligands by exponential enrichment (SELEX). Antibody and antigen sensing rely on the formation of antigen-antibody complexes.

Electrochemical cortisol sensors typically employ antibodies, antigens, molecularly imprinted polymers (MIPs), aptamers or combinations thereof. Therefore, in some embodiments the biorecognition element is selected from antibodies, antigens, molecularly imprinted polymers (MIPs), aptamers or combinations thereof.

The sensing surface may further comprise nanostructures. Nanostructures may be mounted on the base layer of the sensing surface. In some embodiments, the nanostructures may be selected from the group consisting of ZnO, multiwalled carbon nanotubes, single walled carbon nanotubes, nanoparticles such as silicon nanoparticles, silver nanoparticles, gold nanoparticles, samarium molybdate nanoparticles or platinum nanoparticles, nanowires such as silicon nanowires, silver nanowires, gold nanowires, platinum nanowires, gold nanorods, graphene structures such as graphene quantum dots, or combinations thereof.

Therefore, in some embodiments the electrode comprises a sensing surface having a base layer, preferably a metal base layer and most preferably a gold base layer, upon which are disposed one or more biorecognition elements as described above. In alternative embodiments the electrode comprises a base layer, preferably a metal base layer and most preferably a gold base layer, upon which are disposed nanoparticles as described above with one or more biorecognition elements disposed on these nanoparticles.

The sensing surface has a surface area of less than 1 mm². For example, the sensing zone may have a surface area 900 pm² or less, 800 pm² or less, 700 pm² or less, 600 pm² or less, 500 pm² or less. In some embodiments, the sensing zone may have a surface area of from 1 pm² to 999 pm², such as from 10 pm² to 900 pm².

The Film

The sensor comprises a film. The film is positioned above the sensing surface so as to form a barrier. The barrier is between the sensing surface and the external environment, so as to prevent contact between the sensing surface and the medium to be analysed when the sensor is exposed to this medium.

In some embodiments, the film is coated onto the sensing surface. In some alternative embodiments there is a gap between the sensing surface and the film.

Biorecognition elements are typically highly sensitive and degrade when exposed to external factors. For example, the biorecognition element may immediately bind to the target analyte when exposed to a medium containing the analyte, making time resolved measurements impossible. In another example, biofouling may occur on the sensing surface leading to unreliable measurements.

Electrodes can also be sensitive to halide-mediated passivation. When exposed to a solution containing halide ions, the electrode surface is first etched by the halide ions with the formation of a soluble metal halide complex. Subsequently, a portion of the dissolved metal ions is redeposited onto the electrode surface, initiating a process which leads to the passivation of the electrode surface. This process is particularly relevant in gold electrodes.

The film comprises one or more polyelectrolyte that dissolves in an aqueous solution upon the application of an electrical stimulus.

The film of the sensor protects the sensing surface from degradation by external factors until a measurement at the sensing surface is needed, preventing degradation of the sensing surface through mechanisms such as biofouling or halide-mediated passivation. When a measurement is to be taken at the sensing surface, an electrical stimulus can be applied via the electrode of the sensor, causing the film to dissolve in the medium to be tested.

Polyelectrolytes have charged functional groups. In some embodiments, polyelectrolytes 5% or greater of the functional groups of the polyelectrolyte dissociate in solution, such as in an aqueous solution. For example, 5% or greater of the functional groups of the polyelectrolyte dissociate in solution, such as 7% or greater or 10% or greater. In some embodiments from 5% to 25% of the functional groups of the polyelectrolyte dissociate in solution, such as from 10% to 15%. The one or more polyelectrolytes may be selected from poly cations, poly anions, poly ampholytes or combinations thereof. Poly cations, also known as poly bases, have positive charges. Poly anions, also known as poly acids, have negative charges. Poly ampholytes have both positive and negative charges.

The film may be a polyelectrolyte multilayered film comprising alternating layers of one or more poly cation or polyampholyte, and one or more poly anion or polyampholyte. For example, the film may comprise alternative layers of polycation and poly anion.

For example, the poly anion may be selected from the group consisting of K- carrageenan, X-carrageenan, r-carrageenan, carboxymethyl cellulose or a salt thereof, dextran, dextran sulfate, heparin or a salt thereof, and combinations thereof. In a preferred embodiment the poly anion is heparin or a salt thereof.

In some embodiments, the poly cation is selected from the group consisting of protonated chitosan, polybiguanide, quaternary ammonium polyethylenimine, quaternary ammonium polypropylenimine, quaternary ammonium poly amidoamine (PAMAM), poly(vinylamine hydrochloride) (PVH), poly(allylamine hydrochloride) (PAH), poly(amidoamine hydrochloride)), poly(N-isopropylallylamine hydrochloride), poly(N-tert-butylallylamine hydrochloride), poly (N- 1,2- dimethylpropylallylamine hydrochloride), poly(N-methylallylamine hydrochloride), poly(N-methyl allylamine hydrochloride), poly(2-vinylpiperidine hydrochloride), poly(4-vinylpiperidine hydrochloride), poly(diallyldimethylammonium chloride), poly(methacrylate), poly(acrylamide-co-diallyldimethylammonium chloride), poly (diallyl methylaminehydrochloride), copolymer of 2-propen-l-amine-hydrochloride with N-2- propenyl-2-propen-l-aminehydrochloride, poly(N-alkyl-4-vinylpyridinium) salt, polyvinylpyrollidone, poly(lysine), poly(ornithine), poly(arginine), poly(ethylene oxide)-block-poly(vinyl benzyl trimethylammonium chloride), poly(ethylene oxide)- block-poly(l-lysine), poly(2-methacryloyloxy ethyl phosphoiylcholine methacrylate)- block- poly(vinyl benzyl trimethylammonium chloride), poly[2-(dimethylamino)-ethyl methacrylate, poly[3-(dimethylamino)-propylmethacrylate], poly [2-(dimethylamino)- ethyl methacrylamide], poly[3“(dimethyl amino) propyl methacrylamide], poly[2- (trimethylamino) ethyl methacrylate chloride], poly[2-(diethylamino)ethy 1 methacrylate], poly[2-(dimethylamino)ethyl acrylate], and combinations thereof.

In some embodiments the poly cation may be a poly amino acid or salts thereof. For example, the poly cation may be selected from the group consisting of poly(lysine), poly(ornithine), poly(arginine), or combinations thereof. In a preferred embodiment the poly cation is poly(lysine) or a salt thereof.

In some embodiments the polyampholyte is formed from monomeric residues selected from (acrylamide), A-[3-(Dimethylamino)propyl] acrylamide (DMAPAA), 2- (Dimethylamino)ethyl methacrylate (DMAEM), 2-(Diethylamino)ethyl methacrylate (DEAEM), [2-(Methacryloyloxy)ethyl] trimethylammonium chloride (TM), 2- (Acryloyloxyethyl)trimethyl ammonium chloride, (TMA), [3- (Methacryloylamino)propyl] trimethylammonium chloride (MAPTAC), 2-Carboxyethyl acrylate (CAA), Methacrylic acid (MAA), Acrylic Acid (AA), Carboxylated poly-1- lysine (COOH-PLL), 3-Sulfopropyl methacrylate potassium salt (SA), 2-Sulfoethyl methacrylate (SE) or combinations thereof. In some embodiments the polyampholyte is selected from modified chitosan, wherein the chitosan has been modified to include charged functional groups. Examples of amphoteric modified chitosan include carboxymethyl chitosan.

In some embodiments, the film is a polyelectrolyte multilayered film, comprising alternating layers of poly(lysine) and heparin. In some embodiments, the film comprises alternating layers of the poly cation, consisting of poly(lysine), and the poly anion, consisting of heparin.

Polyelectrolyte multilayered films of the present invention are obtained by selfassembly of polyelectrolytes using a layer-by-layer method, which is by successive adsorption of the polyelectrolyte onto the sensing surface of the electrode. A first layer of polyelectrolyte is formed by adsorption of the polyelectrolyte onto the sensing surface having an opposing charge. Subsequent layers can be absorbed onto the first layer according to the charge of the sensing surface of the electrode resulting in a film having the structure (polycation/polyanion)_n or (polyanion/polycation)_n.

The polyelectrolyte multilayered films of the present inventions may be built up using electrochemical methods. The electrode is immersed in a first solution comprising one or more polyelectrolytes, wherein the polyelectrolytes have the same charge. A potential difference is then applied between the electrode and a counter electrode such that the electrode has the opposite charge of the one or more polyelectrolytes to induce the formation of a polyelectrolyte film. The electrode is then removed from the solution and washed. The electrode is then immersed in second solution comprising one or more polyelectrolytes, wherein the polyelectrolytes have the opposite charge those of the first solution. A potential difference is then applied between the electrode and a counter electrode such that the electrode has the opposite charge of the one or more polyelectrolytes of the second solution, thereby inducing the formation of a second polyelectrolyte layer. The electrode is then removed from the second solution and washed. This process is repeated until a polyelectrolyte multilayered film of appropriate thickness and complexity is formed.

The film of the sensor protects the sensing surface from degradation by external factors. The durability and permeability required of the film will depend on the specific application, but in some embodiment the films of the present invention may have a thickness of at least 1 nm, such as at least 2 nm, at least 5 nm, at least 10 nm, at least 50 nm, at least 100 nm, at least 500 nm or at least 1 pm. The thicker the film, the more durable and less permeable the film will be. Films of the present invention may have a thickness of 1 mm or less, such as 500 pm, or 250 pm or less. For example, the film of the present invention may have a thickness of from 1 nm to 1 mm, such as from 10 nm to 250 pm. As the skilled person would understand, the thickness of the film can be controlled by the applied potential difference, the contact time or the concentration of the polyelectrolyte solutions used in the electrodeposition method.

Sensing Device

Also provided herein is a sensing device. The sensing device comprises one or more sensor as defined herein.

One benefit of the sensor is that the sensing surface remains isolated from the fluid or medium to be measured until the film is removed. This allows measurements to be taken at the desired time point rather than when the sensor is positioned in the fluid or medium. This further allows an array of sensors to be positioned in the fluid or medium and together provide time-resolved data sets.

Therefore, in some embodiments the sensing device comprises a plurality of sensors, such as five or more sensors, ten or more sensors, 50 or more sensors, 100 or more sensors or 1000 or more sensors.

In some embodiments, the sensors are developed on a chip that is fabricated in a standard semiconductor technology. For example, the sensors may be developed using CMOS, SiGe, GaN, or GaAs technology.

In some embodiments, the sensing device is a semiconductor chip, such as a complementary metal oxide semiconductor (CMOS) chip, a silicone-germanium (SiGe) chip, a gallium -nitride (GaN) chip, or a gallium arsenide (GaAs) chip. In some embodiments, the sensing device is a CMOS chip.

Dissolution of the film in situ requires the presence of a counter electrode in electrical communication with the sensing surface of the sensor via the film and medium to be analysed. In some embodiments the sensing device comprises a counter electrode.

The counter electrode may be an electrode such as a microelectrode or nanoelectrode. The counter electrode may be formed on the CMOS chip. The surface material of the counter electrode may be any suitable surface material. In preferred embodiments, the surface material of the counter electrode is platinum or gold. In some embodiments, the counter electrode is developed on a chip that is fabricated in a standard semiconductor technology. For example, the sensors may be developed using CMOS, SiGe, GaN, or GaAs technology. In some embodiments the counter electrode is developed using the same technology as the sensor or the electrode of the sensor described herein. In some embodiments, the counter electrode is formed on a semiconductor chip, such as a complementary metal oxide semiconductor (CMOS) chip, a silicone-germanium (SiGe) chip, a gallium -nitride (GaN) chip, or a gallium arsenide (GaAs) chip. In some embodiments the counter electrode is formed on the same type of semiconductor chip, or on the same semiconductor chip, as the sensor or electrode of the sensor of the present invention.

In some embodiments the counter electrode is provided separately. For example, the counter electrode may be provided on a separate substrate.

The sensing device may comprise an integrated instrumentation electronics component. The integrated instrumentation electronics component may have signal amplification capabilities, signal processing capabilities, or a combination thereof.

Traditional sensing technology requires interconnection between the sensor chip and the instrumentation electronic chip. By integrating these systems into the same device, the scalability of the device is improved as the number of sensors is not limited by the number of interconnects. Furthermore, a system wherein the device further comprises an integrated instrumentation electronics component having signal amplification and signal processing capabilities has a higher signal to noise ration due to in-pixel amplification.

Where a plurality of electrodes is present, the device may comprise a circuit configured to individually address the electrodes, such that the film can be dissolved from each individually. However, at least part of the circuit configured to individually address the electrode may be provided separately, for example provided on a separate substrate.

Also provided herein is a wearable device comprising one or more sensing device as described above. The wearable device may be an item of clothing, a watch, glasses, a bracelet, a personal monitor such as heart monitors used by cyclists, or any other item of jewellery or wearable personal item. Method of Detecting an Analyte

Also provided is a method of detecting the concentration of an analyte in a medium. The method comprises: i) providing a medium to be analysed; ii) providing a sensor or a sensing device comprising a sensor as described herein; iii) exposing the sensor to the medium to be tested; iv) providing an electrical stimulus to the film of the sensor, causing the film to dissolve in the medium; and v) using the sensor of step iv) to detect the concentration of the analyte using a voltametric or amperometric technique.

In some embodiments the medium is a biofluid, such as saliva, sweat, urine, blood, or any other biofluid.

In some embodiments, the analyte is a protein, an amino acid, a steroid, an eicosanoid, a gas or any other suitable bioanalyte. For example, the analyte may be cortisol, dopamine, testosterone, insulin, adrenalin, an oestrogen, or a progestogen. The sensing surface of the sensor is configured to detect the specific analyte to be detected using the method.

In some embodiments the concentration of the analyte is determined using a voltametric technique, which may be selected from cyclic voltammetry or Fast Scan Cyclic voltammetry. In some embodiments the concentration of the analyte is determined using an amperometric technique, which may be selected from chronoamperometry.

The method of the present invention may provide time-resolved data sets. To provide a time-resolved data set the method comprises providing a plurality of sensors or a sensing device comprising a plurality of sensors. The method then comprises the additional step (vi) of repeating steps (iv) and (v) with a second sensor following a delay. The delay may be of any period suitable for the investigation being conducted. In some embodiments, the delay is of 0.1 s or greater, 0.5 s or greater, 1 s or greater, 5 s or greater, 60 s or greater, 300s or greater, 600 s or greater, 900s or greater 1 hr or greater, 2 hrs or greater, 3 hrs or greater, 12, hrs or greater 24 hrs or greater, or 72 hrs or greater. In embodiments, the delay is of 5s or greater, preferably 300s or greater. For example, if cortisol levels in sweat are being measured in situ over the course of 24 hrs, the delay of step (vi) may be between 60 s and 600 s to provide a data set containing from 144 to 1440 data points over the 24 hr period.

In a preferred embodiment, measurements are taken once every 300s to 1800s.

In some embodiments, step iv) occurs following a set period. In other embodiments, step iv) occurs in response to external stimulus. For example, the user or investigator may instruct the sensor to take a measurement, causing step iv) to occur and allowing progression onto step v). In these embodiments the delay is determined by the user or investigator.

In some embodiments the electrical stimulus is the application of a potential difference across the film or the application of an electric current across the film.

The dissolution of the film may be brought about by the application of a potential difference across the film. The electrical stimulus of step iv) may therefore comprise applying a potential difference between the sensing surface of the sensor and a counter electrode. It will be appreciated that the potential difference can be varied to achieve the desired dissolution time. In some embodiments, this potential difference is 0.1 V or greater, such as 0.2 V or greater, 0.5 V or greater, 0.7 V or greater, 1 V or greater, 1.2 V or greater, 1.4 V or greater, 1.6 V or greater, or 1.8 V or greater. The sensing surface of the electrode may act as the anode or the cathode, most preferably the anode.

In some embodiments, the dissolution of the film may be brought about by the application of an electric current across the film. The electrical stimulus of step iv) may therefore comprise applying an electric current between the sensing surface of the sensor and a counter electrode. It will be appreciated that the electric current can be varied to achieve the desired dissolution time. In step iv), complete dissolution of the film is not necessary. The film may become nanoporous following the application of the electrical stimuli. The medium may pass through the nanopores to the sensing surface, allowing detection of the analyte to occur. In some embodiments however, complete dissolution of the film is achieved in step iv).

Description of the Drawings

Embodiments of the present invention will now be described by way of example with reference to the accompanying drawings, in which:

Figure 1 is a schematic view of a sensor having a biorecognition element as part of the sensing surface;

Figure 2 is a schematic view of a sensor having nanostructures as part of the sensing surface;

Figure 3 is a schematic view of a sensing device comprising a plurality of sensors, each sensor having a biorecognition element as part of the sensing surface;

Referring firstly to Figure 1 of the accompanying drawings, the figure shows a schematic view of a sensor 100 according to an embodiment of the present invention. The sensor is formed on a CMOS chip 101 and comprises a passivation layer 103. The sensing surface comprises a base layer 105 and a biorecognition element 107, represented by an antibody. It will be appreciated that any biorecognition element could be used. Underneath the surface layer the sensor comprises an active layer 109, an adhesive layer 111 and a conductive substrate layer 113. The sensor further comprises a film 115 forming a barrier between the external environment and the sensing surface.

Referring now to Figure 2 of the accompanying drawings, the figure shows a schematic view of a sensor 200 according to an embodiment of the invention. The sensor is formed on a CMOS chip 201 and comprises a passivation layer 203. The sensing surface comprises a base layer 205 and nanostructures bearing a biorecognition element 207. Underneath the surface layer the sensor comprises an active layer 209, an adhesive layer 211 and a conductive substrate layer 213. The sensor further comprises a film 215 forming a barrier between the external environment and the sensing surface. Referring now to Figure 3 of the accompanying drawings, the figure shows a schematic view of a sensing device 300 comprising a plurality of sensors 301, 303 and 305 formed on a CMOS Chip 307. Each sensor comprises a sensing surface having a base layer 315, 317 and 319 a biorecognition element 321, 323 and 325. Each sensor further comprises a film 327, 329 and 331 comprising at least one polyelectrolyte. Films 327 and 331 are intact and form a barrier between the external environment and the sensing surfaces of sensors 301 and 305. The dissolution of film 329 has been caused by an electrical stimulus exposing the sensing surface of sensor 303 to the external environment.

Claims

1 . A sensor for use in detection of biomolecules, the sensor comprising an electrode and a film, wherein the electrode comprises a sensing surface having one or more biorecognition element; and the film comprises one or more polyelectrolyte that dissolves in an aqueous solution upon application of an electrical stimulus, and the film is positioned above the sensing surface to form a barrier.

2. The sensor of claim 1, wherein the one or more biorecognition element is an antibody, antigen, molecularly imprinted polymer, enzyme, RNA, DNA or aptamer biorecognition element or a combination thereof.

3. The sensor of claim 1 or claim 2 wherein the one or more polyelectrolytes are selected from a poly cation, a poly anion, a poly ampholyte or combinations thereof.

4. The sensor of any preceding claim, wherein the film is a polyelectrolyte multilayered film, comprising alternating layers of one or more poly cation or polyampholyte, and one or more poly anion or polyampholyte.

5. The sensor of claim 3 or claim 4, wherein the poly anion is selected from alginic acid or a salt thereof, hyaluronic acid or a salt thereof, carboxymethyl curdlan or a salt thereof, K-carrageenan, X-carrageenan, r-carrageenan, carboxymethyl cellulose or a salt thereof, pectic acid, poly(sodium phosphate), dextran, dextran sulfate, poly(styrene sulfonate), sulfonated poly(ether ether ketone), sulfonated polyether sulfone, sulfonated polyphenyl sulfone, sulfonated poly(2,6-dimethyl-l,4-phenylene oxide), sulfonated poly(4-phenoxybenzoyl-l,4-phenylene), sulfonated polyphenylene oxide, sulfonated poly(phenylene), sulfonated poly(phthalazinone), sulfonated poly(vinyl toluene), poly(acrylic acid) or a salt thereof, poly (malic acid) or a salt thereof, poly(vinylsulfonic acid sodium), poly(acetic acid) or a salt thereof, poly(lactic acid) or a salt thereof, poly(aspartic acid) or a salt thereof, heparin or a salt thereof, and combinations thereof.

6. The sensor of any one of claims 3-5, wherein the polyanion selected from the group consisting of K-carrageenan, X-carrageenan, r-carrageenan, carboxymethyl cellulose or a salt thereof, dextran, dextran sulfate, heparin or a salt thereof, and combinations thereof

7. The sensor of any one of claims 3-6, wherein the one or more poly cation is selected from comprises protonated chitosan, polybiguanide, quaternary ammonium polyethylenimine, quaternary ammonium polypropylenimine, quaternary ammonium poly amidoamine (PAMAM), poly(vinylamine hydrochloride) (PVH), poly(allylamine hydrochloride) (PAH), poly(amidoamine hydrochloride)), poly(N-isopropylallylamine hydrochloride), poly(N-tert-butylallylamine hydrochloride), poly (N-1,2- dimethylpropylallylamine hydrochloride), poly(N-methylallylamine hydrochloride), poly(N-methylallylamine hydrochloride), poly(2-vinylpiperidine hydrochloride), poly(4-vinylpiperidine hydrochloride), poly(diallyldimethylammonium chloride), poly(methacrylate), poly(acrylamide-co-diallyldimethylammonium chloride), poly (diallyl methylaminehydrochloride), copolymer of 2-propen-l-amine-hydrochloride with N-2-propenyl-2-propen-l-aminehydrochloride, poly(N -alkyl-4-vinylpyridinium) salt, polyvinylpyrollidone, poly(lysine), poly ornithine, polyarginine, poly(ethylene oxide)-block-poly(vinyl benzyl trimethylammonium chloride), poly(ethylene oxide)- block-poly(l-lysine), poly(2-methacryloyloxy ethyl phosphoiylcholine methacrylate)- block- poly (vinyl benzyl trimethylammonium chloride), poly[2-(dimethylamino)-ethyl methacrylate, poly [3-(dimethylamino)-propylmethacrylate], poly [2-(dimethylamino)- ethyl methacrylamide], poly [3 “(dimethyl amino) propyl methacrylamide], poly[2- (trimethylamino) ethyl methacrylate chloride], poly[2-(diethylamino)ethy 1 methacrylate], poly [2-(dimethylamino)ethyl acrylate], or combinations thereof.

8. The sensor of any one of claims 3 to 7, wherein the polycation is selected from the group consisting of poly(lysine), poly(ornithine), poly(arginine), or combinations thereof.

9. The sensor of any preceding claim, wherein the film is a polyelectrolyte multilayered film, comprising alternating layers of poly(lysine) and heparin.

10. The sensor of any preceding claim, wherein the film has a thickness of at least

11. The sensor of any preceding claim, wherein the sensing surface comprises a base layer of electroplated gold.

12. A sensing device for the detection of a biomolecule, the device comprising one or more sensor according to any one of claims 1 to 11.

13. The sensing device of claim 12 wherein the sensing device is a CMOS chip.

14. The sensing device of any one of claims 12 or 13, wherein the sensing device further comprises a counter electrode.

15. The sensing device of any one of claims 12 to 14, wherein the device further comprises an integrated instrumentation electronics component having signal amplification and signal processing capabilities.

16. The sensing device of any one of claims 12 to 15, wherein the device comprises a plurality of sensors as defined in any one of claims 1 to 11.

17. A wearable device comprising one or more sensing device of any one of claims 12 to 16.

18. A method of detecting the concentration of an analyte, the method comprising: i) providing a medium to be analysed; ii) providing a sensor according to any one of claims 1 to 11 or a sensing device according to any one of claims 12 to 16; iii) exposing the sensor to the medium to be analysed; iv) providing an electrical stimulus to the film of the sensor, causing the film to dissolve in the medium; and v) using the sensor of step iv) to detect the concentration of the analyte using a voltametric or amphoteric technique.

19. The method of claim 18, wherein the method is a time-resolved method and the method comprises providing a plurality of sensor according to any one of claims 1-11, or the sensing device of claim 16, wherein step iii) comprises exposing the plurality of sensors to the medium and steps iv) and v) are performed on a first sensor, the method further comprising: vi) providing an electrical stimulus to the film of a second sensor, causing the film to dissolve in the medium; and vii) using the second sensor to detect the concentration of the analyte using a voltametric or amphoteric technique.

20. The method of claim 19, wherein step vi) is delayed for a period following the completion of step v).

21. The method of any one of claims 18-21, wherein the concentration of the analyte is detected using a voltametric technique, and the voltametric technique is cyclic voltammetry.