Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template

Definitions

• This invention relates to a sensor and in particular to a sensor for the detection of biologically important species.
• PoC point-of-care
• biomolecules Due to their biological derivation, these biomolecules typically suffer from a number of limitations when used in sensing applications, for example, poor reproducibility, instability during manufacture, sensitivity to environmental factors, such as pH, ionic strength, temperature etc., and problems associated with the sterilisation process.
• MIPs molecular Iy imprinted polymers
• Synthetic receptors avoid many of the disadvantages associated with biological receptors.
• Molecular imprinting for example, is a generic and cost-effective technique for preparing synthetic receptors, which combine high affinity and high specificity with robustness and low manufacturing costs.
• MIP receptor materials have already been demonstrated for a wide range of clinically relevant compounds and diagnostic markers.
• synthetic receptors, and particularly MIPs typically are stable at low and high pH, pressure and temperature, are inexpensive and easy to prepare, tolerate organic solvents, may be prepared for practically any analyte, and are compatible with micromachining and microfabrication technology.
• Molecular imprinting may be defined as the process of template-induced formation of specific recognition sites (binding or catalytic) in a material, where the template directs the positioning and orientation of the material's structural components by a self- assembling mechanism.
• the material itself could be oligomeric, polymeric (for example, organic MIPs and inorganic imprinted silica gels) or two-dimensional surface assemblies (grafted monolayers).
• non-covalent MIPs are generally preferred, in particular in sensing applications.
• the template/analyte is only weakly bound by non-covalent interactions to these receptor materials, it can be relatively easily removed from the synthetic receptor and the sensor regenerated for a new measurement.
• non-covalent imprinting is easier to achieve and applicable to a wider spectrum of templates.
• Fig. 1 shows a schematic representation of the self-assembly of a MIP from monomeric starting materials to form a polymer having binding sites with specificity for the template and the subsequent elution or extraction of the template.
• MIPs for a range of chemical compounds, ranging from small molecules (up to 1200 Da), such as small organic molecules (e.g. glucose) and drugs, to large proteins and cells.
• small molecules up to 1200 Da
• small organic molecules e.g. glucose
• drugs drugs
• the resulting polymers are robust, inexpensive and, in many cases, possess affinity and specificity that is suitable for diagnostic applications.
• the high specificity and stability of MIPs render them promising alternatives to enzymes, antibodies, and natural receptors for use in sensor technology. See WO 2005/075995 for further details regarding MIPs and other synthetic polymers.
• the present invention provides a sensor for detecting an analyte comprising a substrate, a transducer disposed on the substrate, a receptor layer capable of binding to the analyte, and an intermediate layer between the transducer and the receptor layer wherein the intermediate layer comprises a first molecule bound both to the receptor layer and the transducer and which anchors the receptor layer to the transducer, and a second molecule bound both to the receptor layer and the transducer and which is conductive.
• the senor includes an intermediate layer between the receptor and the transducer which is composed of two chemically distinct materials, the first binding the receptor and transducer together and the second facilitating communication between the receptor and the transducer.
• This allows the use of simple, commercially available materials for the receptor layer thereby reducing manufacturing costs, whilst also achieving a beneficial balance of robustness and functionality.
• Fig. 1 shows a schematic representation of the self-assembly of a MIP
• Fig. 2 shows a representation of a sensor of the present invention
• Fig. 3 shows an example of an electrochemical transducer having three electrodes, namely a platinum working electrode (left), a platinum counter electrode (middle) and a AgCl reference electrode (right);
• Fig. 4 shows an example of an intermediate layer of the present invention between a transducer and a molecularly imprinted polymer receptor
• Fig. 5 shows an example of an electrochemical transducer of the type shown in Fig. 3 functionalised with a molecularly imprinted polymer
• Fig. 6 shows an example of a multi-parameter chip incorporating the sensor of the present invention
• Fig. 7 shows a sensor in accordance with the present invention incorporated into an intravascular monitoring system
• Fig. 8 shows a sensor response to consecutive injections of 30, 40 and 60 ⁇ M propofol samples, day 1 ;
• Fig. 9 shows a sensor response to consecutive injections of 12.5, 25, 50 and 100 ⁇ M propofol samples, day 2;
• Fig. 10 shows a repetitive injection of 25 and 50 ⁇ M propofol, day 3.
• the present invention relates to a novel arrangement for a chemical sensor.
• the sensor has a substrate having a transducer disposed thereon.
• the sensor further comprises a receptor layer capable of binding an analyte when the sensor is exposed to a sample containing the analyte.
• the receptor layer is positioned proximal to the transducer so that it is in communication with the transducer. The nature of the communication will depend on the transduction principle being employed, but preferably the receptor layer is in electrical communication with the transducer.
• the analyte then selectively binds to the receptor and the binding event is detected by the transducer.
• the sensor has an intermediate layer between the transducer and the receptor layer which fulfils a dual function.
• the intermediate layer contains two different types of molecules, a first molecule which anchors' the receptor layer to the transducer and a second molecule which is conductive.
• the intermediate layer binds the receptor layer to the transducer to prevent physical separation of these components which would otherwise lead to communication between the components being reduced or lost.
• the first molecule need not be conductive. This provides a straight-forward means for detecting the presence of one or more analytes of interest in the sample being investigated and/or for measuring their concentration(s).
• One particular application area for this sensor relates to the detection and measurement of medical drugs, markers or medically relevant substances which are indicative of the health, status or treatment of a patient.
• the sensor can, for example, be used to analyse the current state of health of the patient (human or animal) and/or direct treatment of a medical condition suffered by the patient.
• Fig. 2 shows a typical sensor 1 of the type used in the present invention for detecting the presence of an analyte in a sample.
• the sensor 1 comprises a confinement structure
• the confinement structure 2 is disposed on the substrate 4.
• the confinement structure 2 comprises a first limiting structure defining a first interior space.
• the transducer 5 and the receptor layer 3 are disposed in the first interior space.
• the receptor layer 3 is in communication with the transducer.
• the first limiting structure is a continuous structure, i.e. the walls are continuous and fully surround/enclose the first interior space and most preferably is annular, i.e. a "well".
• a second limiting structure defining a second interior space which encloses the first limiting structure may also be provided as described in WO 2005/075995.
• the first and second limiting structures are preferably composed of polyimide.
• the sensor 1 may further comprise a channel to contain the sample and to define a flow path to direct the sample to the receptor layer 3.
• the intermediate layer is disposed within the first limiting structure and the receptor layer is disposed within the second limiting structure.
• the senor 1 is presented with the sample.
• the sample is typically a fluid sample, preferably a liquid and most preferably a bodily fluid, such as blood, urine, interstitial fluids or cerebro-spinal fluids.
• the sample is usually a "complex sample" in that it comprises the analyte being detected as well as one or more interferents which can interfere with the specific detection of the analyte.
• Any material having a high binding affinity and selectivity for the analyte and which may be immobilised on a microsensor chip may be used as the receptor material in the receptor layer 3.
• the receptor layer 3 comprises a synthetic polymer, an imprinted gel, or a biomolecule or a combination thereof.
• the biomolecule may be an enzyme or an antigen.
• the imprinted gel is typically an imprinted silica gel.
• the synthetic polymer may be any synthetic polymer provided the polymer is capable of selectively binding an analyte (i.e. it functions as a receptor). The selective binding may be a result of functional groups on the polymer which interact with a specific analyte.
• the synthetic polymer preferably comprises one or more functionalised monomers and one or more cross-linkers.
• a preferred polymer is a molecularly imprinted polymer.
• MIPs Molecularly imprinted polymers
• MIPs are essentially artificial macromolecular receptors prepared by molecular imprinting of synthetic polymers.
• MIPs are prepared by polymerising functional monomers or copolymerising functional and cross-linking monomers in the presence of an imprint molecule which acts as a molecular template.
• the functional monomers initially form a complex with the imprint molecule and, following polymerisation, their functional groups are held in position by the highly cross-linked polymeric structure. In this way, a molecular memory is introduced into the polymer which is then capable of binding the imprint molecule.
• the imprinting of small organic molecules is now well established in the art.
• the functional monomer should be capable of binding to the imprint molecule, via functional groups on the functional monomer. Binding may be via a covalent bond or by intramolecular forces, such as a hydrogen bond or van der Waals forces.
• a suitable functional group may be, for example, a carboxylic acid in a (meth)acrylic acid ester, although the nature of the functional group will depend on the nature of the imprint molecule.
• the monomer must, of course, be polymerisable and able to react with a cross-linker when present.
• Suitable monomers include, but are not limited to, acrylic monomers, such as (meth)acrylic acid, (meth)acrylic acid esters, (meth)acrylamide, (meth)acrylonitrile, 2-hydroxyethylmethacrylate (HEMA), N 5 N 5 N- triethylaminoethyl(meth)acrylate, trifluoromethyl acrylic acid, acrylamide, N 5 N'- methylenebisacrylamide, acrylonitrile, 2-acrylamido-2-methyl-l-propanesulfonic acid acrolein, ethylene glycol dimethacrylate, imidazole-4-acrylic acid ethyl ester, imidazole-4-acrylic acid, 2 -(diethyl amino)ethyl methacrylate; vinyl and allyl monomers, such as 2- and 4-vinylpyridine, m-and p-divinylbenzene, styrene, aminostyrene 1-vinylimidazo
• the cross-linker may be included to fix the template-binding sites firmly in the desired structure as well as to influence the porosity of the MIP.
• the cross-linker must be capable of reacting with the functional monomers to cross link the polymer chains and the cross-linker should preferably be of similar reactivity to the monomer.
• Suitable cross-linkers include, but are not limited to, ethylene glycol dimethacrylate (EDMA), glycerol dimethacrylate (GDMA), trimethylacrylate (TRIM), divinylbenzene (DVB) (which is particularly suitable for cross linking acrylate- and vinyl-containing functional monomers), methylenebisacrylamide and piperazinebisacrylamide (which are particularly suitable for cross linking acylamides), phenylene diamine (which is particularly suitable for cross linking amines such as aniline and aminophenyl boronate), dibromobutane, epichlorohydrine, trimethylolpropane trimethacrylate and N 5 N ' -methylenebisacrylamide .
• EDMA ethylene glycol dimethacrylate
• GDMA glycerol dimethacrylate
• TAM trimethylacrylate
• DVB divinylbenzene
• methylenebisacrylamide and piperazinebisacrylamide which are particularly suitable for cross linking acylamides
• the mole ratio of functional monomer to cross-linker is preferably from 1 :1 to 1:15. Mixtures of monomers and cross-linkers may also be used.
• the functional monomer and/or the cross-linker may act as a solvent for the polymerisation reaction or an additional solvent may be added. Suitable solvents are known in the art and include DMSO (dimethyl sulfoxide), formic acid, acetic acid, DMF (dimethylformamide), methanol, acetonitrile, dichloromethane, chloroform, THF (tetrahydrofuran), toluene and cyclohexane. Mixtures of these solvents may also be used to obtain the desired solvation and porogenic properties.
• the polymer preferably has a molecular weight from 1 to 100,000 kDa, more preferably 10 to 10,000 kDa and most preferably 10 to 5,000 kDa.
• MIP molecular imprinted polymers
• MAA methacrylic acid
• EDMA ethylenedimethylacrylic acid
• the receptor layer itself is also conductive.
• the receptor layer comprises a receptor material and a dispersed conductive material, such as a conductive powder.
• the dispersed conductive material is preferably an electrically conductive material.
• the conductive material may be selected from conductive carbon black, a metallic power (e.g. gold, silver, copper, platinum etc), metallic nanoparticles (e.g. gold, silver, platinum), carbon-based nanoparticles, such as fullerenes or carbon nanotubes, carbon powder and/or conductive organic molecules.
• Dispersion may be achieved by dispersing the conductive material; for example in the case of a synthetic polymer, the conductive material may be dispersed in a pre-polymer solution prior to polymerisation. In this manner the conductive material becomes integrated into the polymer matrix.
• the receptor layer obtainable by dispersing the conductive material in the pre-polymer prior to polymerisation is particularly preferred.
• the receptor layer obtainable in this way particularly preferably incorporates a synthetic polymer or a MIP as the receptor material.
• the pre-polymerisation mixture is treated with ultra-sound to aid dispersion.
• the receptor material may be provided as a powder and the conductive material is dispersed through the powder.
• the transducer is a well known component of the sensor of the present invention.
• the transducer of the present invention is preferably an electrochemical transducer although a large number of transduction principles can be used to realise the invention. These include potentiometric (in particular, ion selective field effect transistors, ISFETs, and chemically selective field effect transistor, CHEMFETs), conductimetric, optical, gravimetric, surface-acoustic waves, resonant, capacitive or thermal principles.
• the transducer is an amperometric transducer, a potentiometric transducer or a conductimetric transducer.
• the transducer has two or more different regions, typically on the surface, i.e. a first surface region and a second surface region, the first and second surface regions having different conductivities and/or surface compositions to one another.
• the first region is conductive and the second region is insulating.
• the first surface region may be composed of silver, gold, platinum, carbon, stainless steel, preferably platinum, and the second surface region may be composed of silicon nitride.
• the surface may be modified in selected areas using standard photolithographic techniques.
• the present invention provides an intermediate layer between the receptor layer and the transducer. The intermediate layer firstly anchors the receptor layer to the transducer and secondly facilitates communication between the receptor layer and the transducer.
• the intermediate layer uses two different molecules to allow a simplified approach to achieving the dual properties of the intermediate layer, without having to rely upon more complex molecules having a dual functionality.
• the first molecule anchors the receptor layer to the transducer.
• the chemical nature of the first molecule will depend on the nature of the receptor and the transducer. However, it will need to form strong bonds between itself and both the receptor layer and transducer. Typically the bonds will have a bond enthalpy of 300 KJ/mol.
• the bond between the first molecule and the receptor layer will be a C-N or C-O bond, preferably a C-N bond, and between the first molecule and the transducer will be a Si-O bond.
• the first molecule does not need to be conductive, and in a preferred embodiment is not conductive. Conductive herein means conductive in the same manner as the transduction principles used.
• the second molecule is conductive (i.e. it facilitates communication between the receptor layer and the transducer). To achieve this function, the second molecule needs match the properties of the transduction principle. That is, the nature of the conductivity will be dependent on the transduction principle being used. For example, where the transducer is an electrochemical transducer relying on current as the measurement principle, e.g. an amperometric transducer, the second molecule will be electrically conductive. This is typically achieved by the molecule containing delocalised electrons, e.g. by containing C-C double bonds. Where optical means are used, the second molecule will be optically conductive, for example by matching refractive indices. Alternative, it may be based on changes in dielectric constant.
• the intermediate layer is a self-assembled monolayer (SAM).
• SAMs are known in the art and comprise a single layer of molecules on the surface of the transducer. SAMs may be prepared simply by adding a solution or suspension of the first and second molecules on to the surface of the transducer and washing off the excess. Forming a SAM is preferable to other techniques, such chemical vapour deposition or molecular beam epitaxy as it is considerably less complex, does not require such expensive equipment, and provides greater control over the thickness of the intermediate layer.
• the first molecule is bound both to the receptor layer and the first surface region of the transducer and a second molecule bound both to the receptor layer and the second surface region of the transducer.
• the first surface region is preferably insulating and the second surface region is preferably conductive, more preferably electrically insulating and electrically conductive, respectively.
• the first and second surface regions may have different surface compositions.
• a chip-based amperometric transducer is functionalised in accordance with the present invention.
• An example of this transducer is shown in Fig. 3.
• the transducer has three electrodes, namely a working electrode made from platinum, a counter electrode made from platinum and a
• AgCl reference electrode The chip surface in the immediate vicinity of the electrodes is covered in a silicon nitride coating. Due to this surface composition, aminopropyltriethoxysilane (APTES) and aminomercaptophenol (AMP) self-assembled monolayers are used to immobilised the polymerisation initiator for MIP photografting.
• APTES aminopropyltriethoxysilane
• AMP aminomercaptophenol
• APTES is firstly immobilised onto the silicon nitride (Si 3 N 4 ) component of the transducer, followed by the derivatisation of the transducer's platinum electrodes with AMP.
• Figs. 4 and 5 show the resulting sensor.
• the receptor layer is represented as a "MIP” and the platinum and silicon nitride regions of the transducer are shown accordingly.
• Fig. 5 is a photograph of the sensor.
• aminomercaptophenol is exemplified, other difunctional unsaturated compounds may be used, for example aromatic compounds based on pyrrole (PPY), aniline or thiophene.
• the first molecule preferably has a conjugated core, a first functional group capable of binding to the receptor layer and a second functional group capable of binding to the transducer (preferably the first surface region thereof).
• the first functional group is preferably an amino or hydroxyl group, preferably an amino group, and the second is preferably a thiol.
• the nature of the groups will depend on the nature of the receptor layer and the transducer.
• the second molecule preferably has a linker, a first functional group capable of binding to the receptor layer and a second functional group capable of binding to the transducer (preferably the second surface region thereof).
• the first functional group is preferably an amino or hydroxyl group, preferably an amino group, and the second is preferably a silane or a thiol.
• the linker is preferably an alkylene, e.g. C 1-10 or C 1-6 alkylene. Again, the nature of the groups will depend on the nature of the receptor and the transducer.
• the present invention also provides a sensor for the detection of propofol functionalised with a molecularly imprinted polymer imprinted with propofol (i.e. synthesised in the presence of propofol) having the above-defined components.
• the propofol is detected by oxidising propofol on the working electrode of the amperometric sensor. This can, for example, be achieved by operating the sensor as an amperometric sensor and applying a voltage of 0.8 V or larger between the working electrode and the reference electrode. In this case a reference electrode external to the transducer may be used.
• An amperometric sensor may be of two electrode or three-electrode design.
• the electrodes may be made of a number of materials, including silver, gold, platinum, carbon, stainless steel. Other materials are known to those skilled in the art.
• the amperometric sensor may also be functionalised through the deposition of enzymes (for example, glucose oxidase, lactate oxidase, or other suitable chemicals), membranes (for example, comprising silicone, HEMA, PVC, Nafion, polyurethane) in order to detect preferentially the analyte of interest and remove the effects of interferents.
• enzymes for example, glucose oxidase, lactate oxidase, or other suitable chemicals
• membranes for example, comprising silicone, HEMA, PVC, Nafion, polyurethane
• the senor is a micromachined device.
• the transducers employed in the chemical sensor can be realised on the same die or different dies.
• One particular example of a silicon-based microsensor chip with multiple transducers is shown in Fig. 6. It includes a range of transducers based on potentiometric (in particular, ion selective field effect transistors, ISFETs, and chemically selective field effect transistor, CHEMFETs), amperometric and conductimetric principles.
• the adsorption or reaction of the analyte(s) of interest will release another agent into the sample, which is subsequently detected by a transducer which is capable of detecting the agent.
• a transducer which is capable of detecting the agent.
• the increase in the sensor signal associated with the release of the agent in the fluid is measured.
• sensors based on the invention can be applied to the following areas: measurement of anaesthetic agents, in particular those administered intravenously, such as propofol (propofol can be administered in a variety of forms, for example as an emulsion or in an aqueous forms); detection and measurement of antibiotic agents, e.g.
• the sensor of the present invention may be adapted to detect any of the above analytes. The adaptation is apparent from the breadth at which the signal is detected, e.g. the voltage range selected for a potentiometric transducer.
• the sensor of the present invention is a propofol sensor.
• the sensor of the present invention is typically incorporated into a sampling system and a signal processing unit. Accordingly, the present invention also provides a sampling apparatus comprising a housing coupled to a sampling port and incorporating the sensor as described herein and a signal processing unit in electronic communication with the sensor.
• a sampling apparatus comprising a housing coupled to a sampling port and incorporating the sensor as described herein and a signal processing unit in electronic communication with the sensor.
• An example of such a system is shown in Fig. 7.
• the system is equipped with a housing 7 incorporating the sensor 1 coupled to a sampling port 8 in an intravascular line 9 above the sensor 1.
• a sampling device 10 for example a syringe, is coupled to the sampling port 8.
• the user will withdraw blood flushing it across the sensor 1 in order to take a measurement. After the measurement is completed, the blood may be flushed back into the patient or it may be flushed to waste.
• the sensor can be incorporated into the intravascular-flushing line, for example, along with one or more other sensors
• the sensor 1 is connected to a local display and signal processing unit 11 which may be connected to a patient monitoring device 12.
• the sensor 1 is also connected to the housing 7 electronically using techniques known in the art.
• the senor may be employed in a range of other sensing systems, known to those skilled in the art.
• a sample may be taken from the patient and transported to and injected into an analyser, into which the sensor is integrated, for sample analysis.
• the senor of the present invention provides feedback for the treatment of the patient based on the results of the analysis made.
• This feedback may be provided either directly to the user or it may be part of a closed-loop control system including the device administering the treatment to the patient.
• an anaesthetic agent such as propofol
• the concentration of the anaesthetic agent in one or more bodily fluids or body compartments e.g. blood or blood plasma
• the subsequent delivery of the anaesthetic agent e.g. by controlling the rate of delivery to the patient via a syringe pump.
• the sensor may also be used with systems which monitor other parameters which characterise the health of a patient, monitor particular markers indicating disease states or direct the patient's treatment, e.g. blood gases, pH, temperature etc.
• the present invention further provides a method of detecting an analyte in a sample comprising providing a sample potentially containing the analyte, contacting the sample with the sensor as described herein and obtaining a signal indicative of the concentration of analyte present in the sample.
• the sample is a fluid sample and more preferably a bodily fluid.
• a transducer having three electrodes, namely a working electrode made from platinum, a counter electrode made from platinum and a AgCl reference electrode (see Fig. 3).
• the chip surface in the immediate vicinity of the electrodes was covered in a silicon nitride coating.
• the transducer was initially immersed in an anhydrous solution of APTES (3% v/v in dry toluene) for 3 hours to functionalise the silicon nitride component. This reaction was carried out in the dark. After this time the transducer was washed in a stream of acetone to remove any excess of the silane and dried in a flow of nitrogen gas. In a second functionalisation step the transducer was then immersed in a solution of 50 mM AMP dry toluene for 15 hours. Again, this functionalisation was carried out in the dark. Following the 15 hour time period, the transducer was again washed in a stream of acetone to remove any excess of the thiols and dried in a flow of nitrogen gas.
• APTES 3% v/v in dry toluene
• the polymerisation initiator was then grafted on to the free amine groups of the immobilised molecules by exposing the chip to a solution of 58 mg of 4,4'-(azo-bis- cyanovaleric acid), 32 mg of carbon diamide and 32 mg of HBOT in 10 mL of dry DMF. This reaction was carried out in the dark and at room temperature. The reaction was allowed to continue for approximately 5 hours.
• the propofol MIP was then photochemical Iy grown on the derivatised transducer by immersing the transducer in a mixture of 200 mg of propofol, 840 mg of DEAEM monomer, 2.8 g of cross-linker ethylene glycol dimethacrylatedissolved in 3.68 g of dry dimethylformamide.
• the pre-polymerisataon mixture was further bubbled with nitrogen for 5 mins in order to remove any dissolved oxygen present in the mixture.
• the radical polymerisation is initiated upon UV irradiation of the photo initiator for 245 sec. In order to ensure MIP porosity, nitrogen was blown in the polymerisation chamber during UV curing step.
• the resulting MIP was then rinsed with methanol.
• the propofol sensor was conditioned by cyclic voltammetry between +1.5 and -1 V in a phosphate buffer of pH 3.
• Example 1 In order to assess the sensitivity of the propofol sensor, the sensor prepared in Example 1 was used to detect propofol in an acetonitrile: water mixture (30:70 v/v, pH 5).
• Figs. 7, 8 and 9 illustrate the detection of propofol using the present sensor over a testing period of 3 days.
• the sensor was exposed to samples of different propofol concentrations (day 1 : 30, 40 and 60 ⁇ M; day 2: 12.5, 25, 50 and 100 ⁇ M; and day 3: 25 and 50 ⁇ M).
• the sensor was able to detect propofol over this period of time and provided a linear response to the propofol concentration over the whole testing period.
• the measurement was proven to be repeatable with less than 9% variation between repetitive and consecutive injections of propofol (Fig. 9).
• This example relates to the preparation of a sensor containing a conductive receptor layer.
• a sensor was prepared by microfabricating a sensor chip and depositing a MIP on the transducer using the methodology discussed in WO 2005/075995 and WO 2006/120381. Specifically, 50 mg of propofol, 210 mg of DEAEM (monomer), 1.3 g of ethylene glycol dimethacrylate (cross linker), and 31 mg of 2,2-dimethoxy-2- phenylacetophenone (free-radical polymerisation photoinitiator) were dissolved in 1.55 g of dimethylformamide. The pre-polymerisation mixture was further bubbled with nitrogen for 5 mins in order to remove any dissolved oxygen present in the mixture.
• Vulcan XC72R conductive carbon black
• an ultrasound homogeniser in order to disperse the carbon particles.
• Approximately 40 nL of the pre-polymerisation mixture was then deposited onto a transducer comprising a platinum electrode and irradiated with UV radiation for 10 mins.
• the sensor was finally washed with 5 mL of 0.1 M HCl / 20% methanol, rinsed with water, and washed with 5 mL of 0.1 M NaOH / 20% methanol, rinsed with water, and finally blow dried in a stream of compressed air.
• the transducer is pre-treated with the first and second molecules of the intermediate layer as described hereinabove.

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