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WO2019186128A1 - Biocapteur - Google Patents

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Publication number
WO2019186128A1
WO2019186128A1 PCT/GB2019/050845 GB2019050845W WO2019186128A1 WO 2019186128 A1 WO2019186128 A1 WO 2019186128A1 GB 2019050845 W GB2019050845 W GB 2019050845W WO 2019186128 A1 WO2019186128 A1 WO 2019186128A1
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Prior art keywords
sensor
process according
solution
ttf
linker
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Inventor
Kar Seng TENG
Siamak SAMAVAT
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Swansea University
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Swansea University
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Priority to GB2015112.2A priority Critical patent/GB2586543A/en
Publication of WO2019186128A1 publication Critical patent/WO2019186128A1/fr
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/004—Enzyme electrodes mediator-assisted
    • C—CHEMISTRY; METALLURGY
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C12N11/087—Acrylic polymers
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • C12N11/089—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/18—Multi-enzyme systems
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
    • C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12N9/0048—Uricase (1.7.3.3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements

Definitions

  • 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.
  • 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 and biotechnological applications. 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 are well known and suitable devices for the diagnosis of analytes, especially in complicated and complex samples including blood, urine and serum. 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. 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.
  • amperometric sensors are the most commercially used devices.
  • the use of enzymes predominates, owing to their specific binding capabilities and biocatalytic activity.
  • the reaction under investigation generates a measurable amperometric current when a constant electrical potential is applied between the working and counter electrodes.
  • Amperometric devices continuously measure the current produced from a series of redox reactions between electroactive species.
  • the first generation of amperometric glucose sensors are heavily dependent on oxygen.
  • the glucose oxidase enzyme (GOx) which naturally exists in its oxidised form reduces after reacting with glucose and oxidises again in the presence of oxygen to produce hydrogen peroxide.
  • the hydrogen peroxide produced will then break down to oxygen and water donating electrons at the positively charged working electrode causing an increase in the amperometric signal.
  • the resulting current flow is then proportional to the analyte concentration.
  • the redox mediators provide three main advantages such as enhancing the selectivity of the sensors, independence of oxygen and less production of hydrogen peroxide which can damage the enzyme.
  • the redox mediated biosensors also show a better sensitivity than the non- mediated ones due to enhanced electron transfer throughout the sensing layer.
  • Electrochemical growth, chemical binding and physical adsorption are three common methods used for the immobilisation of the sensing material onto the working electrode. Amongst the three methods, chemical binding and physical absorption are less complex and more cost effective for mass production. Moreover, in the case of microneedle sensor arrays the covalent binding of the sensing material within the sensing layer is particularly important where a robust sensing layer is required to stand physical stress due to skin insertion. In terms of sensor fabrication, drop casting is the most commonly used method for the deposition of the sensing compounds in the sensing layer.
  • this method may lead to a ‘coffee ring’ effect and non-uniform/non- homogeneous distribution of the drop cast compounds on the surface especially when complex 3D structures are being coated.
  • the sensor compounds used to form the sensing layers are not suitable for routine manufacture due to their poor solubility in aqueous and organic solvents. Consequently, a uniform/homogeneous mixture of such compounds is not achievable via conventional drop casting or even a spray layering methodology (see figure 1 ).
  • our spray coating can provide good sensor reproducibility and control over the sensing layer thickness by changing the volume of the sprayed components: e.g. GOx and TTF, and the density of the sprayed layer by varying the concentration of the material used.
  • volume of the sprayed components e.g. GOx and TTF
  • density of the sprayed layer by varying the concentration of the material used.
  • such parameters can be used to tune the linear range of the sensors.
  • a process for the manufacture of a biosensor comprising: a) suspending at least one first sensing component and/or a linker for immobilising said or a component(s) onto the surface of a sensor substrate in a first solution;
  • said components form part of a biological reaction or a biochemical pathway.
  • said components comprises all but at least one component of the biological reaction or the biochemical pathway with the remaining component(s), when present in a sample, participating in the said reaction or pathway, when said sensor is in contact with said sample, to complete the reaction or pathway and so yield a measurable parameter.
  • This parameter is typically, but not exclusively, detected as electrical current flow.
  • said biosensor is an amperometric biosensor.
  • said sensor substrate of part c) comprises an electrically conducting part or layer onto which said solutions are coated.
  • said sensor also includes a counter electrode and, ideally a reference electrode. We prefer to use an Ag/AgCI reference electrode and a gold wire counter electrode.
  • part e) involves drying the sensor in a vaporised linker such as GA vapour to further enhance the crosslinking of the sensing layer.
  • said first solution is an inorganic solution, preferably water
  • said second solution is an organic solution, preferably an alcohol such as ethanol, or vice versa.
  • the inorganic solution is a polar solution and/or the organic solution is a non-polar solution.
  • Said linker is provided in at least one of said first and second solutions and is ideally provided in said first or second solution, although it can be provided in both.
  • said linker is a conventional linker and so one that is known to link any one or more of said components with said substrate or each other with a view to immobilising them on the substrate.
  • linkers suitable for working the invention 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 (disuccin
  • said sensor substrate is selected from the group comprising: silicon, solid polymers such as polyimide, SU-8 type material, 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.
  • solid polymers such as polyimide, SU-8 type material
  • injection mouldable polymers such as polyether ether ketone (PEEK) and conductive acrylonitrile butadiene styrene (ABS)
  • PEEK polyether ether ketone
  • ABS conductive acrylonitrile butadiene styrene
  • flexible material such as rubber silicon-based gels or other gel type materials.
  • said first and second solutions are sprayed onto said surface at the same rate, alternatively, said solutions are sprayed at different rates and in either case the relative amounts of said components is selectively controlled.
  • the concentration of each component in either said first or second or further solutions is selected so as to provide an amount of said component that is representative of its concentration in the natural biological reaction or the biochemical pathway.
  • the kinetics of the biological reaction or the biochemical pathway are to be manipulated, to generate an improved measurable parameter, at least one, and possible more than one or all of the components, are provided in said solutions in an amount that is selected so as to maximise the generation of the measurable parameter.
  • the relative proportions of the said first and the said further sensing components are adjusted in each of said solutions so that the amount of each component deposited on the sensor substrate is controlled to maximise sensor functionality.
  • the amount of mediator to enzyme may be increased to enhance the function of said enzyme; and/or the amount of enzyme to substrate may be increased or vice versa; and/or the amount of linker to mediator or enzyme may be increased.
  • Further step d) may be always part of the method and the solution of step d) may always contain linker as we have discovered that a final coat of linker immobilises the components on the substrate and so improves function.
  • each solution is sprayed using a different spray gun, alternatively a single gun with multiple nozzles may be used.
  • said method further includes insulating at least a part of the coated sensor with an electrically insulating material, ideally, by the application of an insulating material to said substrate.
  • This application of an insulating material serves to electrically isolate the backplane of the sensor.
  • said insulating material is an organic material that is solid at room temperature, such as a wax, ideally paraffin wax.
  • the method 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.
  • said wax is applied to a rear part of said sensor i.e. the part not participating in the biological reaction or the biochemical pathway.
  • paraffin wax was applied to electrically isolate the backplane of the sensor.
  • 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.
  • paraffin wax in its solid state paraffin wax is a great electrical isolator, water resistant and non-hazardous when in contact with skin. These characteristics make wax an ideal candidate as a backplane isolator for the mass production of sensors.
  • said sensor comprises a plurality of microneedles which are coated with said components. More preferably said microneedles comprise an electrically conducting part or layer onto which said solutions are coated. Preferably, an electrically insulating material is applied to the coated sensor at the base of said plurality of microneedles to electrically isolate the backplane of the sensor.
  • said at least one first sensing component comprises at least one reversible oxidising reagent also known as a mediator.
  • said at least one first sensing component comprises at least one enzyme.
  • said at least one further sensing component comprises at least one reversible oxidising reagent also known as a mediator.
  • said at least one further sensing component comprises at least one enzyme.
  • said first solution comprises said mediator and said second solution comprises said enzyme.
  • the enzyme component of the first and/or second solution is selected depending on the intended use of the biosensor end product.
  • GOx is selected as the enzyme for a glucose biosensor.
  • alternative enzymes suitable for working the invention include, but are not limited to, lactate oxidase (for the detection of lactate), lactate dehydrogenase (for the detection of lactate), uricase (for the detection of uric acid), glutathione oxidase (for the detection of glutathione), glutamate oxidase (for the detection of glutamate) and glutaminase (for the detection of glutamine), or the like.
  • the mediator component of the first and/or second solution can be any conventional reversible oxidising reagent that can react with the enzyme and act as an electron acceptor/donor.
  • the use of a combination of such conventional mediators is also envisaged.
  • mediator components that are suitable for working the invention include, but are not limited to, tetrathiafulvalene (TTF), ferrocene or derivatives thereof such as, for example, dimethylferrocene or ferrocenecarboxaldehyde; Tetracyanoquinodimethane (TCNQ); Conducting salts such as, for example, Tetrathiafulvalene-Tetracyanoquinodimethane (TTF-TCNQ), N- methylphenazinium-tetracyanoquinodimethane (NMP-TCNQ), and N- methylacridinium-tetracyanoquinodimethane (NMA-TCNQ); quinones; Ferrcyanide; and Ferrocyanide.
  • TTF tetrathiafulvalene
  • ferrocene or derivatives thereof such as, for example, dimethylferrocene or ferrocenecarboxaldehyde
  • TCNQ Tetracyanoquinodimethane
  • Said first and/or said second solution also comprise(s) a linking agent whereby said at least one first component and said at least one further components are immobilised onto said sensor substrate.
  • said first solution comprises mediator tetrathiafulvalene (TTF) and said second solution comprises glucose oxidase (GOx) enzyme.
  • said linker preferably comprises glutaraldehyde (GA).
  • said first solution comprises a mixture of said TTF and GA in an organic solution such as an alcohol e.g. ethanol
  • said second solution comprises GOx in an inorganic solution such as water.
  • said first solution comprises 300 to 600 pL TTF (40mM) in ethanol plus 1 .8 ml_ pl_ GA (5%) in ethanol to make a total of 2.1 to 2.4 ml_.
  • said second solution comprises 1200 mI_ GOx (10 mg/mL) in deionised (Dl) water.
  • said further sensing component comprises a linking agent and so is ideally 1 .2 ml_ GA (5%) in Dl water.
  • a sensor manufactured according to the above process comprise a sensing layer that comprises a homogeneous mixture of sensing components immobilized onto the surface of a sensor substrate.
  • a sensor comprising a homogenous mixture of sensing components immobilized onto the surface of a sensor substrate, manufactured according to the above process for use as a biosensor, for example, as a glucose sensor.
  • a microneedle array comprising a sensing layer that comprises a homogeneous mixture of sensing components immobilized onto the surface of said array, adapted to perform or function as a biosensor wherein said array has been exposed to the manufacturing process of the invention.
  • said sensor or said microneedle array has coated on at least a part thereof said components whereby said components are mixed there together homogeneously so as to maximise the measurement of said measurable parameter.
  • said components are mixed as herein described and so have the properties herein disclosed.
  • a method for taking a biological measurement comprising the use of said biosensor or said microneedle array.
  • any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.
  • Figure 1 Shows a schematic diagram of different sensor surface coating techniques: the drop cast in layers (DL), sprayed in layers (SL), and spray mixing (SM) of the sensing compounds;
  • FIG. 1 Shows scanning electron microscope (SEM) images (left) and energy-dispersive X-ray spectroscopy (EDX) maps of sulphur (right) showing the planar surface of the DL, SL and SM fabricated sensors;
  • SEM scanning electron microscope
  • EDX energy-dispersive X-ray spectroscopy
  • Figure 3 Shows the cross-sectional EDX analysis for a surface coated using a) SM and b) SL methods;
  • Figure 4 Shows the amperometric response to glucose using the a) DL b) SL and c) SM sensors and d) the average Lax and KM values for DL, SL, and SM sensors;
  • Figure 6 Shows a schematic diagram of the sprayed layers on a typical working electrode
  • Figure 7 Shows an image of a carbon sprayed electrode of a microneedle array (left) compared to a full working electrode with waxed backplane (right);
  • Figure 8 Shows the sensitivity of a glucose sensor manufactured in accordance with the invention when using different TTF volumes as well as an increased amount of GA as a measure of the sensor performance;
  • Figure 9 Shows the sensitivity of a glucose sensor manufactured in accordance with the invention when using different GOx volumes as well as an different amounts of GA as a measure of the sensor performance .
  • Figure 10 Shows the sensitivity of a glucose sensor manufactured in accordance with the invention when part d) is practised using two different GA volumes in the part d) spray.
  • Table 1 Shows the performance of the sensors of the invention when compared to a number of TTF mediated enzymatic glucose sensors disclosed in the literature that were manufactured using the conventional drop cast method.
  • Aspergillus Niger GOx (270 U/mg) was purchased from BBI solutions Ltd, UK. TTF, 0.01 M sterile phosphate buffer solution (PBS used as the supporting electrolyte for the electrochemical studies), D glucose anhydrous, and extra pure deionised water were purchased from Fisher Scientific, UK. Glutaraldehyde (25%) and diacetone alcohol were purchased from Sigma Aldrich, UK. Graphene based carbon ink (HDPIas® IGSC02002) was purchased from Haydale, UK. Iwata Eclipse CS spray guns were purchased from Airbrushes UK. All the reagents were of analytical grade and used without further purification.
  • a stock solution of 40 mg/mL GOx was prepared in deionised (Dl) water by weighing GOx powder at room temperature. This was further diluted to 10mg/ml_ so that it could be sprayed without blockage at the nozzle.
  • Glutaraldehyde stock solutions of 5% were prepared in Dl water and ethanol. All of the above-mentioned solutions were stored at 4°C.
  • 40 mM TTF stock solutions were prepared in ethanol and stored at -20°C. Before use, the TTF solution was ultrasonicated for 10 min and vortexed re-dissolving the TTF crystals in the solution. 3M glucose stock solution was made in PBS and left for at least 24 hours at 4°C to allow mutarotation.
  • the glucose sensors were fabricated on flexible polyimide substrate by spray coating a conductive carbon layer and subsequently depositing the sensing compounds.
  • the conductive layer of the working electrodes 0.15 mm thick polyimide sheets were cut into 1 .4 x 0.7 cm rectangular shapes; 5 g of the carbon paste was mixed in 20 g of diacetone alcohol to achieve a 4: 1 solvent to carbon paste ratio and spray deposited to form the conductive layer for the working electrodes. After spray deposition, carbon electrodes were annealed on a hot plate at 250°C for 10 min. Resistance of the conductive layer was measured using a standard multimeter which was approximately 200 W across 1 .4 cm along the electrodes.
  • the conductive carbon electrodes were coated with TTF and GOx sensing compounds via three different fabrication methods ( Figure 1 ): drop casting TTF and GOx in subsequent layers (DL method); spraying TTF and GOx in subsequent layers (SL method); and spray mixing of the TTF and GOx compounds simultaneously (SM method).
  • DL method drop casting TTF and GOx in subsequent layers
  • SL method spraying TTF and GOx in subsequent layers
  • SM method spray mixing of the TTF and GOx compounds simultaneously
  • the last layer, involving GA was applied to ensure that the GOx was well cross-linked especially at the surface; consequently, this layer was not studied as a part of the sensing layer in the current study.
  • Distance between the spray outlet and the samples in all the three cases was 13.2 cm.
  • Spray rate for GOx and GA was found to be optimum around 1 pL/sec which was the fastest spray rate without forming any visible droplets (wetting) on the surface.
  • Spray rate of TTF/GA in ethanol was adjusted to allow simultaneous spray of both compounds over the same period of time.
  • a 3x3 mm window was created on the carbon electrodes using paraffin wax. Subsequently, 1 .13 pl_ TTF (4 mM) in ethanol, 1 .42 mI_ GOx (20mg/ml_) in Dl water, and 2.47 mI_ GA (5%) in Dl water were drop-cast one at a time onto the carbon window under intensive ventilation. These volumes were chosen to achieve equal quantities of GOx and TTF for all three types of sensors to allow direct comparison between them. Sensors were allowed to completely dry between each layer and were then left overnight at 4°C to maximise cross- linking of the GOx and GA. Before performing the amperometric measurements these sensors were rinsed thoroughly with Dl water to remove any loosely bound compounds and blow dried with nitrogen.
  • the carbon electrodes were spray coated with 1 .2 mL TTF (20mM) in ethanol followed by 450 pL GOx (13.33 mg/mL) in Dl water and finally 1 .05 pL GA in Dl water. Fabricated sensors were then left at 4°C overnight for GOx and GA to crosslink and for the sensing layer to dry. Sensors were then rinsed with Dl water and blow dried with nitrogen. A 3x3 mm window was then created using paraffin wax before sensor testing. SM Sensors
  • Fabrication of the SM sensors was carried out in two steps.
  • two solutions i.e. solutions A and B
  • Solution A consisted of 600 pL TTF (40mM) in ethanol, 450 pl_ GA (5%) in ethanol and 150 mI_ of ethanol to make a total of 1 .2 ml_.
  • Solution B consisted of 450 mI_ GOx (40 mg/mL) in Dl water.
  • the quantities of GOx, TTF, and GA used in SL and SM sensors were determined via UV Vis absorption and used to fabricate the DL sensors. Based on the UV results 9 pg TTF, 8 active units GOx and 2.47 pL GA (5%) were deposited within the active region of each sensor for the SM, SL, and DL sensors. An extra 450 pL GA (5%) was mixed with TTF to enhance the immobilisation of the GOx and TTF for the SM case.
  • paraffin wax is low-cost and easy to process due to its low viscosity (below 3 mpa.s at 75 °C) and relatively good wetting (contact) on the sensing materials.
  • FIG. 6 shows all the layers sprayed on a microneedle (complex 3D) working electrode.
  • Figure 7 a typical carbon sprayed electrode and a full working electrode with waxed backplane are compared together.
  • the uniformity of the TTF and GOx in the fabricated sensors was studied using SEM and EDX techniques. All studies were performed on the sensing layer deposited onto carbon coated silicon substrates, instead of polyimide substrate to avoid interference in the EDX carbon peak from the polyimide and to minimise charging effects. GOx and GA quantities for all samples were kept the same in the DL, SL and SM sensors.
  • the deposited TTF was increased by 10-fold to enhance the EDX signal from sulphur in TTF for both SM and SL samples to a detectable range in order to study the uniformity throughout the sensing layer. Also this allowed a much thicker TTF layer which could be observed more clearly in the SEM images.
  • the carbon ink was not deposited for the cross sectional studies to avoid interference in the EDX signal on the carbon especially in the SM case.
  • Amperometric measurements were performed using an Ivium CompactStat from Alvatek and an electrochemical cell consisting of a fabricated working electrode (area 0.09 cm 2 ), an Ag/AgCI reference electrode and a gold wire counter electrode at 0.3 V which is the established oxidation potential for TTF mediator [1,2] All three electrodes were immersed in 10mL of sterile PBS buffer. After applying the electrical potential, the cell was allowed to stabilise electrochemically until the change in the electrical current was smaller than 100 pico amps/sec. The amperometric measurements were performed whilst stirring at 350 rpm at which rate the noise associated with the magnetic stirrer was minimised to ⁇ 5 nA.
  • the following section investigates the uniformity of TTF throughout the sensing layer to support the postulations made in the section above.
  • the distribution of the sensing elements, i.e. TTF and GOx, at the surface of the electrodes (planar surface) and through the sensing layer (cross section) was investigated.
  • the presence of TTF in the sensing layer is indicated by observing the sulphur EDX peak.
  • the biological components, GOx and GA are indicated by the presence of carbon and oxygen.
  • TTF also contains carbon in its molecular structure; however, the EDX results on the SL sample in Figure 3 b shows negligible carbon peak in the pure TTF region on the right-hand side of the image. Also, there is a good correlation between the carbon and oxygen was observed in Figure 3 b which confirmed the carbon peak was dominated by the presence of GOx and GA rather than TTF.
  • the Si peak is due the underlying silicon substrate in all EDX results.
  • FIG. 2 shows the SEM images and EDX mapping analysis for sulphur (i.e. associated with TTF) at the planar surface in typical DL, SL and SM samples.
  • the EDX map for the DL samples exhibits much stronger Sulphur signal intensity near the edges of a waxed window which indicates the presence of a ‘coffee ring’ effect.
  • the formation of the TTF crystals, especially around the edges of a silicon substrate was observed by an optical microscope. Around the edges of the sensing window, TTF crystals are highlighted by a red circle in the EDX map in figure 2.
  • the cross-sectional EDX analysis allowed the study of the mixing of the biological component and the mediator throughout the sensing layer for the SM sample in comparison to the SL sample (figure 3a and 3b, respectively).
  • the DL sample in contrast led to formation of TTF large crystals and non- uniform distribution of TTF across the surface.
  • the EDX results for SM sample in figure 3 a show a uniform distribution of sulphur (representing TTF), carbon and oxygen (both representing the biological components) from top to bottom of the sensing layer (i.e. left to right of the image).
  • figure 3 b shows strong EDX signal for carbon and oxygen (associated with the GOx and GA) at the top of the sensing layer (i.e. left hand side of the image).
  • the EDX signal for carbon and oxygen dropped almost to zero while the signal for sulphur reached maximum.
  • the EDX signal for C 0, and S decreases and Si increases.
  • the uniform mixing of the TTF mediator and the biological components i.e. GOx and GA
  • the technique is relatively simple, low cost and scalable to allow market demands for such devices to be met.
  • FIG 4 illustrates the amperometric response of the DL, SL, and SM glucose sensors by means of the average Lax and KM parameters representing maximum amperometric current and the Michaelis-Menten constant respectively.
  • the DL sensors exhibited the smallest amperometric response to glucose compared to both the SL and SM sensors.
  • the weak performance of the DL sensors (especially the average Lax) shown in figure 4a would suggest the lack of redox reactions between the GOx and the TTF mediator given their quantities were the same as SL and SM sensors.
  • the SL sensors in figure 4b show a larger average Lax (6.5 times) and KM (250 times) than the DL sensors. This significant increase in the performance of the SL sensors (especially the average Lax) could indicate the enhanced redox reaction as a result of better proximity between the enzyme and the mediator due to the spray coating technique.
  • the SM sensors in figure 4c show a much larger average Lax (20 times) and KM (12 times) than the DL sensors.
  • the average Lax of the SM sensors was also about 3 times larger than the SL sensors. Furthermore, the SM sensors exhibited the smallest sensor to sensor variation as compared to the DL sensors which showed the largest variation for Lax and KM parameters. This is evident in the standard deviations of the mean Lax and KM values shown in figure 4d. The KM parameter is (21 times) larger for the SL sensors compared to the SM sensors (figure 4d).
  • the sensitivity of the SM glucose sensors were studied and compared to the SL and DL sensors.
  • the selectivity and stability of the SM sensors were also studied.
  • the DL sensors showed the least performance in terms of lma X as well as a very large sensor to sensor variation.
  • sensitivity of the SL sensors was very small compared to the SM sensors and the smallest r 2 value with the largest standard deviation was observed. From both studies we can confirm that the DL sensors are not suitable for biosensing applications mainly due to their inconsistency between the sensors and they will no longer be discussed in this study.
  • the sensitivity of the SM TTF mediated glucose sensors of the invention are better than some of the sensors developed by other groups (some of which involve the use of nanomaterials) as stated in Table 1 .
  • the r 2 value for the linear regression was larger than 0.9 for the SM and SL cases.
  • the SM sensors possess an average sensitivity of 38.7uA/mM/Cm 2 , which compares well with the previously reported sensors in the literature.
  • the KM value on the other hand is smallest for the SM sensors showing high affinity for the enzyme glucose reaction.
  • Sensing layer optimisation for SM sensors
  • V is volume or part expressed in spray volume
  • Figure 8 shows that an increase in sensitivity (slope of the curve at zero mM of glucose) was achieved as a result of increasing TTF deposition during the simultaneous spraying of a solution of GOx and a solution of TTF when 1V, 2V, and 3V TTF was deposited keeping the GOx and GA constant. Also it was observed that an increase in GA mixed with TTF increased the sensitivity for the 3Vol TTF case. Any more TTF would cause the sensing layer to become too fragile to withstand the skin insertion process.
  • Figure 9 shows the increase in sensitivity as a result of increasing GOx deposition during the simultaneous spraying of GOx and TTF where 0.5V, 1V, and 2V GOx & GA are mixed with 1 V of TTF.
  • the optimum spray parameters are 2V GOx sprayed simultaneously with a mixture of 3V TTF and 2V GA. Additionally, 2V GA is sprayed after the simultaneous spraying of components to ensure an excess of GA is present on the surface for covalent crosslinking of the GOx based sensing layer.
  • German, N., et al. The use of different glucose oxidases for the development of an amperometric reagentless glucose biosensor based on gold nanoparticles covered by polypyrrole. Electrochimica Acta, 2015. 169: p. 326.

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L'invention concerne un processus de fabrication d'un biocapteur; un biocapteur fabriqué selon ledit processus; et un procédé de prise d'une mesure biologique comprenant l'utilisation dudit biocapteur.
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