A PROPOFOL SENSOR

Abstract

An enzymatic electrochemical sensor for the detection of blood propofol is provided.

Claims

1. A blood propofol concentration measurement sensor, wherein the sensor is enzymatic, and wherein the sensor is based on one or more members of the cytochrome P450 group of enzymes.

2. The blood propofol concentration measurement sensor of claim 1, providing discrete measurement of propofol.

3. The blood propofol concentration measurement sensor of claim 1, forming part of a blood-gas analyser.

4. The blood propofol concentration measurement sensor of claim 1, providing direct electrochemical measurement of propofol.

5. (canceled)

6. An enzyme-based electrochemical sensor for the detection of propofol, wherein the sensor is based on one or more members of the cytochrome P450 group of enzymes.

7. (canceled)

8. The enzyme-based electrochemical sensor of claim 6 in which the sensor is based on the enzyme cytochrome P450 2B6.

9. The enzyme-based electrochemical sensor of claim 6, in which enzymatic action converts propofol into a quinone/quinol redox pair.

10. A propofol detection system for point-of-care measurement of blood propofol concentration during general anaesthesia, comprising the enzyme-based electrochemical sensor of claim 6.

11. The propofol detection system of claim 10 comprising an analyte recovery system, the analyte recovery system allowing for continuous, real-time propofol monitoring without the need for drawing blood.

12. The propofol detection system of claim 11, in which the analyte recovery system comprises a molecular exchange means.

13. The propofol detection system of claim 11, in which the analyte recovery system comprises a microdialysis probe.

14. (canceled)

15. The enzyme-based electrochemical sensor of claim 6, comprising a working electrode, a counter electrode and a reference electrode.

16. The enzyme-based electrochemical sensor of claim 6, comprising an electrochemical reaction with a redox step, in which in a sequence of the electrochemical reaction, an oxidation occurs before the a reduction.

17. (canceled)

18. The enzyme-based electrochemical sensor of claim 6, in which the enzyme is expressed within deactivated yeast cells.

19. The enzyme-based electrochemical sensor of claim 18, in which the yeast cells are in turn immobilized within a chitosan film containing gold nanoparticles upon the surface of a screen-printed electrode.

20. The enzyme-based electrochemical sensor of claim 6, comprising an electrochemical reaction with a redox step in which in a sequence of the electrochemical reaction, a reduction occurs before an oxidation.

21. The enzyme-based electrochemical sensor of claim 6, comprising a single sensor measuring a reduction current of 2,6-diisopropylquinone.

22. The enzyme-based electrochemical sensor of claim 6, comprising two sensors in sequence with respect to a flow of a perfusate associated with the detection of propofol, the first measuring a reduction current of 2,6-diisopropylquinone and the second measuring an oxidation current of 2,6-diisopropylquinol.

23. The enzyme-based electrochemical sensor of claim 22, in which the two sensors in sequence are co-located on a single device or two separate devices in sequence.

24. The enzyme-based electrochemical sensor of claim 6 comprising a CYP enzyme, in which sensing occurs in the absence of direct electron transfer between an electrode and the CYP enzyme, using an indirect method via NADPH and electrochemically detecting the reaction products.

25. (canceled)

Description

MATERIALS AND METHODS

Materials

[0153] All materials were supplied by Sigma-Aldrich and used as supplied. β-Nicotinamide adenine dinucleotide phosphate sodium salt hydrate (NADP.sup.+) and D-glucose 6-phosphate dipotassium salt hydrate (G6P) were dissolved in 10 mM phosphate buffered saline (PBS), pH 7.4, to produce solutions in which the concentration of each was 50 .Math.g/ml. Unless stated otherwise, this is the testing solution for the electrochemical measurements described in section 2.4. 2,6-diisopropylphenol (97%) was diluted in dimethyl sulfoxide (99%) to create a 10 mM stock solution. This stock solution was further diluted with the NADP.sup.+/G6P solution to produce propofol solutions of varying concentrations as required for the tests described herein.

Apparatus

[0154] The screen-printed electrodes used in these experiments were purchased from BVT Technologies. They constitute a three-electrode cell consisting of a 1 mm diameter graphite working electrode, a graphite counter electrode and a silver/silver chloride (Ag/AgCl) pseudo-reference electrode. All measurements were performed using a PalmSens EmStat.sup.3 potentiostat.

Electrode Preparation

[0155] The electrodes were pre-treated by immersing them in a 10 mM K.sub.3[Fe(CN).sub.6], 1 M KNO.sub.3 solution and performing cyclic voltammetry between -0.6 and +0.8 V at a scan rate of 100 mV/s for a total of 10 cycles.

[0156] Gold nanoparticles were produced using a standard sodium citrate reduction technique. Briefly, 10 mg of gold chloride hydrate (HAuCl.sub.4) (99.995%) were dissolved in 20 ml of deionised water and brought to boiling point under magnetic stirring. Trisodium citrate dihydrate (99%) was dissolved in deionised water to produce a 2.5% (w/v) solution and 1 ml of this solution was added to the HAuCl.sub.4 solution and the mixture kept at boiling point for 5-10 minutes until it had undergone a colour change to deep red, before being allowed to cool to room temperature.

[0157] CypExpress 2B6 (a deactivated, permeabilised yeast, expressing cytochrome P450 2B6) was suspended in phosphate buffer (pH 7) at a concentration of 25 mg/ml. This suspension was mixed with the gold nanoparticle solution and a 1 % (w/v) chitosan solution (1% acetic acid) in a ratio of 1:1:2 by volume. 1 .Math.l of this mixture was drop-cast onto the working electrode of the screen-printed electrodes and left to dry at 4° C. Once dry, the electrodes were immersed in 10 mM PBS for 30 minutes at room temperature, before being re-dried and stored at 4° C. until use.

Electrochemical Measurement

[0158] Cyclic voltammetry measurements were performed by depositing 50 .Math.l of propofol solutions of varying concentration upon the functionalised electrodes and cycling the potential between -0.8 and + 1.0 V at a rate of 100 mV/s.

[0159] Chronoamperometry measurements were performed by immersing the functionalised electrodes in 20 ml of 50 .Math.g/ml NADP.sup.+ and G6P solution (10 mM PBS) and measuring the current at +0.5 V. The solution was stirred with a magnetic stirrer and 60 .Math.l of 1 mM propofol solution were injected into the solution at regular intervals.

[0160] Control measurements were carried out by performing chronoamperometry as above in PBS solutions without NADP.sup.+ or G6P, and injecting 20 .Math.l of 1 mM propofol solution at regular intervals. These experiments were carried out three times using the same electrode. Between each run the electrode was rinsed with 10 mM PBS, dried and stored at 4° C. overnight.

RESULTS AND DISCUSSION

[0161] Cyclic voltammetry with varying propofol concentration (FIG. 4 - cyclic voltammograms of solutions of propofol concentrations: a) 0.18, b) 0.89, c) 1.78, d) 2.67, e) 3.57 and f) 4.47 .Math.g/ml, all solutions contain 50 .Math.g/ml NADP.sup.+ and G6P, 10 mM PBS. Scan rate is 100 mV/s. Potentials are vs. Ag/AgCl.) shows concentration dependant peaks at approximately + 0.6 V and -0.25 V. This behaviour is as would be expected for a quinone/quinol redox pair (Quan et al., 2007), with the peaks corresponding to the reduction of 2,6-diisopropylquinone and the oxidation of 2,6-diisopropylquinol respectively. This tells us that the enzyme within the yeast cells is converting propofol to its products as expected.

[0162] It has been found that oxidation of the quinol only occurs after reduction of the quinone. Monitoring at -0.2 V alone would provide no signal.

[0163] The performance of the sensor was assessed by immersing it in a 10 mM PBS solution containing 50 .Math.g/ml of NADP.sup.+ and glucose-6-phosphate and performing amperometry at +0.6 V. The solution was spiked with a 1 mM propofol solution (10 mM PBS, 10% dimethyl sulfoxide) at regular intervals.

[0164] FIG. 5 - A: Chronoamperometric response of functionalised electrode with successive injections of propofol solution. Potential is +0.5 V vs. Ag/AgCl. Solution is 10 mM PBS containing 50 .Math.g/ml NADP.sup.+ and 50 .Math.g/ml G6P. B: Mean of the plateau current against propofol concentration. The error bars represent three standard deviations. Baseline correction has been applied.

[0165] Chronoamperometry measurements show a clear increase in current with each addition of propofol solution (FIG. 5A). The responses are fast, reaching a plateau in approximately 60 seconds, and stable throughout the experiment. Plotting the average plateau current against the propofol concentration (FIG. 5B) shows that the sensor produces a clear response to varying propofol concentration across the therapeutic range of 0.25 - 4 .Math.g/ml (Langmaier et al., 2011). The response is linear -between 0 and 1.4 .Math.g/ml, the sensitivity is 4.2 nA/.Math.g/ml/mm.sup.2 and the limit of detection is 49 ng/ml, well below the lower end of the therapeutic range.

[0166] The limit of detection (LoD) was calculated using the equation: LoD = 3.3 × σ.sub.low/gradient, where σ.sub.low is the standard deviation at a low propofol concentration.

[0167] FIG. 6 - A: Chronoamperometric response of electrode with successive injections of propofol solution in the absence of NADP.sup.+ and G6P for the first, second and third repetitions of the same electrode on successive days. Potential is +0.5 V vs. Ag/AgCl, solution is 10 mM PBS. B: Mean of the plateau current against the propofol concentration. The error bars represent three standard deviations.

[0168] FIG. 6A shows the result of the chronoamperometry measurements in the absence of NADP.sup.+ and G6P. The increases in current after the addition of propofol are the result of the direct electrochemical oxidation of the propofol, which in the absence of NADP.sup.+ and G6P will not have been converted by the enzyme. In the first run it can be seen that after the fourth injection of propofol (corresponding to a concentration of approximately 0.7 .Math.g/ml) there is a clear trend of decreasing current with time, suggestive of electrode fouling.

[0169] This is even more apparent in FIG. 6B in which it can be seen that the current response plateaus at approximately 1 .Math.g/ml of propofol, a value that is comfortably within the linear range of the sensor as discussed above. Repeated measurements, performed using the same electrode on successive days, show a similar response but at significantly reduced sensitivities. The third run with the electrode has a sensitivity that is 22% that of the first run. These results are clear evidence of fouling of the electrode by propofol oxidation, fouling that does not occur in the presence of NADP.sup.+ and G6P, where the propofol is converted by the enzyme. Similar experiments performed with the addition of NADP.sup.+ and G6P result in the sensor showing a sensitivity on the third run that is approximately 95% of that on the first run (not shown).

[0170] To improve the performance of the sensor, the electrode surface may be functionalised with nanocomposites. Various materials have been investigated, but the most promising are nanocomposites combining carbon nanotubes (CNT) and graphene oxide (GO) decorated with metal oxide nanoparticles, specifically copper oxide nanoparticles (CuONP) and iron oxide nanoparticles (FeONP).

[0171] FIG. 7A shows a sensor formed in accordance with the present invention and including a working electrode, a counter electrode and a reference electrode.

[0172] Reference and/or counter electrodes may be arranged aligned to the direction of flow, as illustrated in FIG. 7B.

[0173] FIGS. 8 to 17 show further embodiments of the present invention.

[0174] FIG. 8 is an illustration of functionalisation of an electrode surface with nanocomposite.

[0175] Planar carbon electrode with a film containing gold nanoparticles and a single type of yeast cells expressing a single CYP enzyme. Three electrode cell with a macroscale working electrode.

[0176] FIG. 9 - Planar gold electrode, functionalised with a single layer of carbon nanotubes, with a film containing a single type of yeast cells expressing a single CYP enzyme. Three electrode cell with a working electrode consisting of a microarray.

[0177] FIG. 10 - Planar platinum electrode, functionalised with a composite layer of gold nanoparticle functionalised carbon nanotubes, with a film containing silver nanoparticles and two types of yeast cells each expressing a single CYP enzyme. Two electrode cell with a macroscale working electrode and a combined counter/reference electrode.

[0178] FIG. 11 - Planar carbon electrode, functionalised with two layers of different nanomaterials, with a film containing gold nanoparticles and a single type of yeast cells expressing multiple CYP enzymes. Three electrode cell with a macroscale working electrode.

[0179] FIG. 12 - Planar platinum electrode, functionalised with a single layer of carbon nanotubes, with a film containing copper oxide nanoparticles and a single type of yeast cells expressing a single CYP enzyme. Multi-electrode device with multiple microscale working electrodes, each with a yeast expressing a different CYP enzyme, each with an associated counter electrode and sharing a common reference electrode (three electrode cells).

[0180] FIG. 13 - Planar platinum electrode, functionalised with vertically aligned carbon nanotubes partially encapsulated within and epoxy so as to form a nanoelectrode array. Film containing gold nanoparticles and a single type of yeast cells expressing a single CYP enzyme. Three electrode cell with a working electrode consisting of a nanoelectrode array.

[0181] FIG. 14 - Nanostrip electrode, with a silicon nitride passivation layer, with a film containing platinum nanoparticles and a single type of yeast cells expressing a single CYP enzyme. Three electrode cell with a nanostrip working electrode.

[0182] FIG. 15 - Gold nanostrip electrode coated with a conductive network of carbon nanotubes and a silicon nitride passivation layer. Film containing gold nanoparticles and a single type of yeast cells expressing a single CYP enzyme. Three electrode cell with a nanostrip working electrode.

[0183] FIG. 16 - Planar carbon electrode with a film containing gold nanoparticles and a single type of yeast cells expressing a single CYP enzyme. The film is coated with a cellulose membrane. Three electrode cell with a macroscale working electrode.

[0184] FIG. 17 - Planar carbon electrode, functionalised with a single layer of carbon nanotubes, with a film containing gold nanoparticles and a single type of yeast cells expressing a single CYP enzyme. The film is coated with a bilayer membrane consisting of a layer of polythene and a layer of Nafion. Three electrode cell with a macroscale working electrode.

[0185] The performance of nanocomposite functionalised sensors was assessed using the same amperometric measurements described previously.

[0186] The sensitivities and limits of detection of each variant of nanocomposite are shown in Table 1.

[0187] Table 1 shows the average sensitivities and limits of detection (LoD) for multiple replicates of CNT/GO, CNT/GO/CuONP and CNT/GO/FeONP electrodes (one example of each of which are shown in Error! Reference source not found.). As already discussed, electrodes prepared using metal-oxide nanoparticle decorated graphene oxide show significant increased sensitivity compared to electrodes prepared using non-decorated graphene oxide, with FeO nanoparticles resulting in the greatest improvement. However, the LoD of CNT/GO/CuONP is higher than that of CNT/GO, a fact that can be attributed to increased noise. However, the LoD for CNT/GO/FeONP electrodes is 7.0 ± 1.2, which is approximately half that of the CNT/GO electrodes, a significant improvement. In a previous publication [REF] we showed that the LoD for a sensor consisting of the type of enzyme film described here on a bare carbon screen printed electrode was 49 ng/ml. Therefore, it can be seen that these carbon nanocomposite functionalised electrodes offer significant improvements in sensitivity for propofol detection, with composites of carbon nanotubes and iron-oxide nanoparticle decorated graphene oxide offering the greatest improvement.

TABLE-US-00001 Sensitivities and limits of detection for electrodes functionalised with various different nanomaterials Electrode functionalisation Sensitivity (nA/.Math.g/ml/mm.sup.2) LoD, pre- processing (ng/ml) LoD, post processing (ng/ml) CNT/GO 12.2 ± 0.5 18.9 ± 0.6 14.2 ± 0.5 CNT/GO/CuO NP 18.6 ± 3.8 48.6 ± 15.3 19.9 ± 8.1 CNT/GO/FeO NP 29.9 ± 6.4 17.7 ± 6.6 7.0 ± 1.2

TABLE-US-00002 Sensitivity and limit of detection for sensors functionalised with nanocomposites Electrode functionalisation Sensitivity (nA/.Math.g/ml/mm.sup.2) Limit of detection (ng/ml) No nanocomposite 4.1 ± 0.2 67 ± 7 CNT/GO 12.2 ± 0.5 14.2 ± 0.5 CNT/GO/CuONP 18.6 ± 3.8 19.9 ± 8.1 CNT/GO/FeONP 29.9 ± 6.4 7.0 ± 1.2

[0188] The nanocomposite functionalised electrodes present a significant improvement compared to the non-functionalised electrode, with the CNT/GO/FeONP composites offering the greatest improvement. This improvement in performance is a result of a combination of increased surface area, improved electron transfer and catalytic effects of the nanocomposite. A moving average filter (with a ten second bin) is applied to the current response as a means of noise reduction, providing additional improvement to the limit of detection.

[0189] FIG. 18: A) Amperometric response of sensor to successive injections of propofol solution for i) CNT/GO, ii) CNT/GO/CuONP, and iii) CNT/GO/FeONP functionalised sensors B) Mean of plateau current against resultant propofol concentration. Potential is +0.5 V vs. screen printed Ag/AgCl. Solution is 10 mM PBS containing 50 .Math.g/ml NADP.sup.+ and 50 .Math.g/ml G6P. Error bars represent three standard deviations. Baseline correction and moving average filter have been applied.

[0190] 18A shows the results of amperometry measurements for electrodes functionalised with CNT/GO (i), CNT/GO/CuONP (ii), and CNT/GO/FeONP (iii) for an increasing propofol concentration (propofol solution is injected every five minutes). The average plateau currents versus the resultant propofol concentration is shown in 18B. It can be seen that all three sensors produce clear increases in current with increasing propofol concentration. These responses are fast, occurring within one minute, and stable throughout the experiment. In all cases the current response is linear with respect to propofol concertation over the range investigated. [0191] CNT - Carbon nanotube [0192] CuONP - Copper oxide nanoparticle [0193] FeONP - Iron oxide nanoparticle [0194] GO - Graphene oxide

[0195] The sensitivity of the electrodes prepared using metal-oxide decorated graphene oxide appears much greater than that of the sensor prepared using non-decorated graphene oxide. As discussed previously, the cyclic voltammetry results do not suggest improvements in terms of surface area or electron transfer are achieved through the inclusion of the metal-oxide nanoparticles, suggesting that the improvements insensitivity are the result of catalytic properties of the metal-oxide nanoparticles.

[0196] It is clear that the nanocomposite functionalised electrodes present a significant improvement compared to the non-functionalised electrode, with the CNT/GO/FeONP composites offering the greatest improvement. This improvement in performance is a result of a combination of increased surface area, improved electron transfer and catalytic effects of the nanocomposite. A moving average filter (with a ten second bin) is applied to the current response as a means of noise reduction, providing additional improvement to the limit of detection.

[0197] FIG. 19: A) Amperometric response of CNT/GO/FeONP functionalised sensor to successive injections of propofol solution in serum-like solution (5 wt% BSA, 10 mM PBS), B) Mean of plateau current against resultant propofol concentration. Potential is +0.5 V vs. screen printed Ag/AgCl. Solution is 10 mM PBS containing 50 .Math.g/ml NADP.sup.+ and 50 .Math.g/ml G6P. Error bars represent three standard deviations. Baseline correction has been applied.

[0198] In order to assess the performance of the sensor in conditions closer to physiological conditions, solutions were prepared containing 5 wt% of bovine serum albumin (BSA), 137 mM NaCl, 2.7 mM KCl and 10 mM phosphate buffer (pH 7.4), in addition to 50 .Math.g/ml NADP.sup.+ and glucose-6-phosphate. These solutions are considered “serum-like” as they have physiological salinity (Opoku-Okrah, 2015), pH (Brørs, 1985) and albumin concentration (Kim, 2020). In these solutions the sensor produces a linear current response across the therapeutic range of propofol (1-10 .Math.g/ml) (Rengenthal, 1999). The sensitivity is 3.1 ± 0.2 nA/.Math.g/ml/mm.sup.2 and the limit of detection is 143 ± 27 ng/ml (FIG. 20). The sensitivity is lower and the limit of detection is higher than that obtained in PBS, which is to be expected as the majority of the propofol will be bound to the albumin, as discussed previously, and therefore the sensor is only detecting the free-fraction. However, this limit of detection is still comfortably below the lower end of the therapeutic range.

[0199] FIG. 20: Current response against propofol concentration for CNT/GO/FeONP sensors tested 1, 2, 5 and seven days after fabrication.

[0200] The nanocomposite sensors have been shown to produce consistent results up to seven days of storage after fabrication (FIG. 20), with what little variation in sensitivity and limit of detection observed accountable for by inter-device variation. The sensors were stored at 4° C. between fabrication and testing. Longer term shelf-life tests (over periods of months) are currently being carried out.

[0201] In order to assess the specificity of the sensor towards potential interfering perioperative drugs, amperometry measurements were performed as previously, injecting various different drugs at regular intervals (FIG. 21). The sensor produces no discernible response to the common perioperative drugs: lidocaine (Altermatt, 2012), ibuprofen (Rainsford, 2009), morphine (Pinky, 2020), midazolam (Wong, 1991), cistricurium besilate (Guo, 2017) and fentanyl (Peng, 1999). Each drug is injected at the appropriate concentration to produce a final concentration that shares the same approximate proportionality to the final propofol concentration as their expected therapeutic ranges (Regenthal, 1999).

[0202] FIG. 22: Amperometric response of CNT/GO/FeO NP/enzyme functionalised electrode with successive injections of A: lidocaine solution (i-iii; 0.23, 0.47 and 0.70 .Math.g/ml respectively), ibuprofen solution (iv-vi; 0.21, 0.41 and 0.62 .Math.g/ml respectively), morphine solution (vii-ix; 0.0028, 0.0057 and 0.0085 .Math.g/ml respectively) and propofol solution (x-xii; 0.18, 0.35 and 0.53 .Math.g/ml respectively) B: Midazolam solution (i-iii; 0.0033, 0.0065 and 0.0097 .Math.g/ml respectively), cisatracurium besilate solution (iv-vi; 0.12, 0.25 and 0.37 .Math.g/ml respectively), fentanyl solution (vii-ix; 0.0018, 0.0035 and 0.0053 .Math.g/ml respectively) and propofol solution (x-xii; 0.18, 0.35 and 0.53 .Math.g/ml respectively). Potential is +0.5 V vs. screen printed Ag/AgCl. Solution is 10 mM PBS containing 50 .Math.g/ml NADP.sup.+ and 50 .Math.g/ml G6P.

[0203] One potential perioperative drug that does result in interference is the commonly used anti-inflammatory drug paracetamol (Colsoul, 2019), as a result of the similarities in the chemical structure between it and propofol. Various attempts to mitigate this potential source of interference were investigated including Nafion membranes and molecularly imprinted polymers. One relates to the fact that paracetamol can be oxidised at a lower potential than propofol or the products of the enzymatic reaction. This can be seen in FIG. 22 which shows amperometric measurements at +0.25 V for electrodes functionalised with CNT/GO/FeONP nanocomposite (but without enzyme film) with successive injections of both propofol and paracetamol solutions. It is clear that at this potential, the electrode produces a response for paracetamol but not for propofol, allowing the possibility of a second electrode at a lower potential selectively measuring paracetamol concentration, which can then be subtracted from the final signal. Dual-potential methodology may be used to account for potential paracetamol interference.

[0204] FIG. 22: Current response for CNT/GO/FeONP functionalised electrodes (no enzyme film) for amperometric measurement at + 0.25 V with successive injections of propofol (black) and paracetamol (red) solutions.

Nanoparticle Synthesis

[0205] Metal oxide nanoparticles were synthesised using methods adapted from Jamzad et al.

[0206] Bay leaf extract was prepared by grinding 20 g of dried bay leaves to a powder using a mortar and pestle. The powdered bay leaves were then added to 200 ml of deionised water and stirred at 90° C. for 10 minutes. The resultant solution was filtered and then centrifuged to remove any remaining plant material. This bay leaf extract solution was stored at 4° C. until required, and used within four weeks.

[0207] For copper oxide and iron oxide decorated graphene oxide (GO), GO was added to 0.1 M metal salt solution (either FeCl.sub.3 or CuCl.sub.2) at a concentration of 1 mg/ml and sonicated for 30 minutes to disperse the GO. This dispersion was added to bay leaf extract solution at a ratio of 1:1 and left at room temperature overnight to allow metal oxide nanoparticles to form.

[0208] The metal oxide nanoparticle-decorated graphene oxide was then extracted from solution by centrifuging at 5000 rpm for 15 minutes and washed by re-suspending in de-ionised water and re-centrifuging three times. The metal oxide nanoparticle-decorated graphene oxide was then suspended in de-ionised water once again and added to MWCNTs and the mixture sonicated for 1 hour to produce a dispersion with 2 mg/ml MWCNT and 1 mg/ml GO. The dispersion is then diluted with de-ionised water to a concentration of 0.1 mg/ml MWCNT and 0.05 mg/ml GO.

Electrode Functionalisation

[0209] The MWCNT/GO/MONP dispersions described are sonicated for 1 hour to ensure maximal dispersion. 1 .Math.l of the dispersion is then drop-cast onto the working electrode of the SPEs and allowed to evaporate before a second 1 .Math.l is drop-cast and dried in the same manner. The electrodes are then rinsed with deionised water to remove any unbound nanomaterials.

[0210] Preparation of the enzyme film has been described previously. Briefly, CypExpress 2B6 is suspended in a phosphate buffer (pH7) at a concentration of 25 mg/ml and this suspension is mixed with a gold nanoparticle solution and a 1% chitsosan solution (1% acetic acid) in a ratio of 1:1:2 by volume. 1 .Math.l of this mixture is deposited on the WE of the SPEs and left to dry at 4° C. Once dry, the electrodes are immersed in 10 mM PBS for 30 minutes and then dried in air at room temperature. The functionalised electrodes are stored at 4° C. until use.

Results

[0211] FIG. 23 - Examples of A: Baseline correction, and B: smoothing by moving average filter. Inset - zoomed in section of between 63 and 64 minutes.

[0212] In order to counteract the effects of drift and noise that are common for sensors of this type, some simple signal processing was applied. Firstly, baseline correction was applied by performing a liner fit to the current response from the five minutes prior to the first injection of propofol solution and correcting all of the data so that it is measured relative to this baseline. An example of this is shown in 23A in which the raw data is depicted by the solid line and the calculated baseline is depicted by the dotted line. The sensor drift is evidenced by the slight downward trend of the baseline.

[0213] Secondly, smoothing was performed by applying a moving average filter to the current data with a bin size of ten seconds. The bin size was decided upon as being a reasonable compromise between degree of smoothing and induced time-lag. An example of this is shown in 23B in which the raw data (depicted by the black line) is plotted alongside smoothed data (depicted by the blue line). The reduction in the noise by the filter is clearly evident.

[0214] Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiments shown and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention.

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