WO2018220423A1 - Fouling-resistant pencil graphite electrode - Google Patents

Abstract

A Pencil Graphite Electrode (PGE) is herein disclosed. The electrode is characterized by a reduced content in graphite and an increased content in clay of the composite material composing it compared to the prior art PGEs. In particular, the PGE according to the invention is characterized by a clay/graphite ratio of at least 0.4. The PGE has a lower tendency to show a fouling effect when used in electrochemical analyses, particularly of phenolic compounds such as paracetamol and propofol present in a sample. Sensors comprising the PGE of the invention, as well as methods for using thereof, are also herein disclosed.

Description

Fouling-resistant Pencil Graphite electrode

Technical Field

[0001] The invention lies in the field of electrodes and electrochemical sensors. In particular, the invention lies in the field of graphite electrodes for electroanalytical analyses, preferably long-term electrochemical monitoring of chemical compounds.

Background Art

[0002] Anaesthetics are widely used compounds in surgical operations and in critical care settings. In order to obtain a suitable Depth Of Anaesthesia (DOA) in the patient, an effective and appropriate plasma concentration of anaesthetic agents are needed; typically, an anaesthetic is injected at a certain infusion rate depending on patient's physical characteristics (e.g. age, sex, weight, etc.) and on the type of surgical operation. Propofol (2,6-diisopropylphenol) is a very well- known intravenous anaesthetic agent. Its large popularity stems from its favourable properties: a short duration of action and a consequent rapid recovery, with the effective plasma concentration value ranging between 0.25 and 10 μg/L (1 - 60 μΜ).

[0003] Thanks to the progress in computing technology, anaesthetic compounds can be delivered to the blood through Target Controlled Infusion (TCI) pumps in order to maintain specific target concentrations (Guarracino, F. et al., Minerva anaestesiologica 2005, 71 , 335-337). The controlled drug infusion algorithms implemented on TCIs are based on Pharmaco-Kinetic (PK) models incorporated into an integrated computer that statistically predict the plasma drug concentration in patient's blood (Shafer, S. L, Gregg, K. M., Journal of Pharmacokinetics and Biopharmaceutics 1992, 20, 147-169). So far, several PK- based software tools simulating changes of propofol concentrations inside the blood have been developed.

[0004] Although these PK-models help predicting the target concentrations for an optimized infusion regimens, problems are still present as limitations associated with those models. The major difficulty in PK-model design for anaesthesia delivery relies on inter-patient variability in metabolism and drug tolerance (Anna, S.; Wen, P. Depth of anaesthesia control using internal model control techniques. Complex Medical Engineering (CME), 2010 IEEE/ICME International Conference on. 2010; pp 294-300). Additionally, most techniques are developed only for particular patient groups or anaesthetic techniques. All in all, this variability among patients and clinical conditions may affect the stability of the closed-loop control system due to uncertainty of their accuracy (Varvel, J. R. et al., Journal of pharmacokinetics and biopharmaceutics 1992, 20, 63-94).

[0005] To overcome these limitations and to avoid risk of over- or under-dosage, a continuous Therapeutic Drug Monitoring (TDM) (Balant-Gorgia, E. A.; Balant, L. P. CNS Drugs 1995, 4, 432-453) system should be integrated with the TCI pump to keep the concentration levels of drugs under control during anaesthesia and to identify patient specific injection/infusion dosage. In this way it would be possible to provide the anaesthesiologist with a closed-loop system where the infusion pumps are controlled by a PK algorithm corrected with the actual value of the drug measured into the patient's blood. At this point, the anaesthesiologist should only supervise the system to ensure a proper functioning.

[0006] Up to now, various technologies have been developed to determine the propofol concentration in human body fluids. Most of the studies are focused on chromatographic techniques based on High Performance Liquid Chromatography (HPLC) (Yarbrough, J. et al., Journal of chromatographic science 2012, 50, 162- 166) and Chromatography-tandem Mass Spectrometer (C-MS) (Vaiano, F. et al., Forensic science international 2015, 256, 1-6). Although their high sensitivity, this techniques are based on bulky instrumentation difficult to be integrated in portable systems. In addition to these techniques, detection of propofol by breath analysis has been proposed (Perl, T. et al., British journal of anaesthesia 2009, aep312; Akbar, M.; Agah, M., Journal of Microelectromechanical Systems 2013, 22, 443-451) but the correlation between blood and breath propofol concentrations is still not clear. For this reason, plasma samples are preferred as convenient media for propofol concentration monitoring.

[0007] To provide a long-term continuous monitoring sensors for propofol detection for clinical settings certain criteria should be met: (i) high specificity to propofol to be able to detect inside serum or blood with a very low LOD, (ii) amenability to miniaturization, (iii) independency from the temperature, and (iv) ability to ensure a reliable and accurate response over a long-term usage. With long-term monitoring, it is referred to the average duration of a common surgical operation, which corresponds to a few hours.

[0008] Electrochemical sensors are good candidates to fulfil these specifications and they have been already investigated (Kivlehan, F. et al., Analytical chemistry 2012, 84, 7670-7676; Langmaier, J. et al., Analytica chimica acta 201 1 , 704, 63- 67). From these studies it emerged that propofol, that is a phenolic compound, can be easily oxidized and sensed electrochemically, but free radicals produced by phenolic oxidation result in electro-polymerization. This reaction causes electrode fouling that decreases the sensor sensitivity upon subsequent measurements (Yang, X. et al., Electrochimica Acta 2013, 94, 259-268; Ferreira, M. et al., Electrochimica Acta 2006, 52, 434-442).

[0009] To address this undesirable phenomenon, in a recent study, a polymeric membrane-coated electrochemical sensor has been developed (Kivlehan, F. et al., Analyst 2015, 140, 98-106). This proved capable of detecting propofol inside serum-like media with a detection limit of 0.012 ± 0.004 μΜ. The results are promising, but the deposition of the membrane on the electrode surface is a critical step to obtain a proper thickness and uniformity, and, moreover, the major problem of PVC membrane sensors is their low physical and mechanical resistance for long-term usage. The difficulties in the fabrication process makes the reproducibility of the electrodes difficult and in a practical application this could lead to some problems, like over- or under-estimation of the propofol concentration in patient's blood.

[0010] Pencil Graphite Electrodes (PGE) are an attractive solution for developing electrochemical sensors, since the main material composing them, graphite, is cheap, readily available, possesses ease to make renewable surface, and is relatively stable. In principle, PGEs are suitable for sensing organic compounds in general (including phenolic compounds such as propofol), metabolites or macromolecules, and some attempts have been done in the past in this sense. For instance, Vishnu et al. (Anal. Methods, 2015, 7, 1943) reported the production of an ultra-low cost 6B grade pencil graphite, pre-anodized at 2 V vs. Ag/AgCI, as a novel electrochemical sensor for surface fouling-free and efficient Differential Pulse Voltammetry (DPV) detection of phenols (meta-cresol and phenol) in pH 7 Phosphate Buffer Solution (PBS) for diabetes application. This electro-analytical approach was validated by testing total phenolic contents in three different insulin formulations, but its behaviour was never tested for long- term monitoring (e.g. from 0.5 to several consecutive hours) of phenolic compounds.

[0011] Kariuki et al. (Sensors 2015, 15, 18887-18900) described a pencil electrode derived from a regular HB #2 pencil. PGEs were used by Square-Wave Voltammetry (SWV) in several biological applications including gallic acid determination, determination of total antioxidant capacity in samples of fruits, vegetables, tea, and coffee, determination of uric acid and determination of dopamine. However, this kind of electrodes has to be treated with a hydrophobic coating and polished before each use, which is not compatible for a long-term analysis.

[0012] US 2015/0090601 describes a cathodized gold nanoparticle graphite pencil electrode (AuNP-GPE) providing an enzymeless electrochemical glucose sensor that is based on it. Again, such PGEs require various treatments for their manufacturing and before their use.

[0013] It is therefore highly desired to develop new, alternative tools for electrochemically sensing chemical compounds, such as phenols, which are ready-to-use, fouling resistant and sensitive enough to allow precise and possibly long-term concentration monitoring either in vivo, ex vivo or in vitro. Moreover, cheapness and disposability of the electrochemical sensing element would be further desired assets, particularly for repetitive analyses setups.

Summary of invention

[0014] The present invention aims at solving the drawbacks of the prior art concerning electrochemical electrodes and sensors as reported in the previous section. In particular, one aim of the invention was to provide an electrode adapted to be used in a long-term (for instance, between 10 minutes and several hours, such as 4 hours) monitoring of a chemical compound, such as in the case of the monitoring of e.g. propofol during a surgical operation.

[0015] A further aim of the present invention was to develop an electrode for electroanalytical analyses and electrochemical sensing having a reduced tendency to fouling, especially when used to analyse phenolic compounds.

[0016] Still a further aim of the invention was to develop an electrode for electroanalytical analyses and electrochemical sensing adapted to be used with biological fluids such as serum or blood, either in vitro, in vivo or ex vivo.

[0017] A further aim of the invention was to develop an electrode for electroanalytical analyses and electrochemical sensing that was cheap enough to be disposable (for instance, for in vivo purposes) without sacrificing the performances.

[0018] An additional aim of the invention was to provide an electrode for electroanalytical analyses and electrochemical sensing through a non-expensive, simple and repeatable manufacturing process. [0019] All these aims have been accomplished by the present invention, as described hereinafter and in the appended claims.

[0020] In a non-limiting exemplary application, the direct electrochemical detection of propofol, used as a prototypical phenolic compound, has been possible thanks to an electrochemical sensor comprising a Pencil Graphite Electrode. As briefly described previously, the electrochemical oxidation of this intravenous anaesthetic produces free radicals that result in electrode fouling by an electro- polymerization process. This effect decreases the sensor sensitivity in long-term monitoring. Therefore, the effect of propofol oxidation at the interface of PGE has been studied, together with the problem of electrodes passivation. Thanks to the developed sensor and methods for using thereof, the passivating issues have been overcome, and ready-to-use, low-cost and robust point-of-care sensors including such PGEs have thus been validated for long-term anaesthesia practices. Indeed, the sensors were tested by performing 25 Cyclic Voltammetry (CV) measurements, corresponding to more than 4 hours of monitoring, so having demonstrated the possibility for continuous monitoring.

[0021] The direct detection of propofol in full human serum has been achieved by adopting PGE with Differential Pulse Voltammetry (DPV) obtaining a Limit Of Detection (LOD) of 0.82 ± 0.08 μΜ. Interestingly, the obtained LOD was lower than the physiological usually used range of propofol ([1 - 60] μΜ).

[0022] Most importantly, what has been extensively sought by the present inventors, in order to reduce the costs and facilitate the manufacturing of electrochemical sensors, was a simple solution to avoid any pre-treatment of PGEs such as doping with metal nanoparticles, membrane coating or pre- anodization/cathionization, while having a fouling-resistant, sensitive sensor adapted for electrochemical analyses, and particularly to long-term monitoring of phenolic compounds. In this context, they came up with the intuition of using PGEs having a reduced graphite content compared to those used in the prior art approaches. One consideration on which the invention is based is that pencils are made up of a homogeneous clay-graphite composite, and is expected that the graphitic part is a key for the electrochemical activity. Without being bound to this theory, the rationale behind the inventive concept of the invention relies on the idea that the clay contained in PGEs could act as a "protective shield" against fouling, thus avoiding the need of treating in any way the electrodes, and therefore rendering them particularly suitable for phenolic compounds' electrochemical sensing. [0023] Accordingly, one object of the present invention is to provide a Pencil Graphite

Electrode (PGE) for use in electroanalytical analyses, characterised in that it has a clay to graphite ratio of at least 0.4.

[0024] In preferred embodiments, the Pencil Graphite Electrode is characterized in that it is a doping-free and coating-free electrode.

[0025] Another object of the present invention relates to the use of the Pencil Graphite

Electrode as previously described for the manufacturing of an electrochemical sensor.

[0026] Another object of the present invention relates to an electrochemical sensor comprising the Pencil Graphite Electrode as previously described.

[0027] In one embodiment, the Pencil Graphite Electrode in the electrochemical sensor is the working electrode.

[0028] Still another object of the present invention relates to the use of an electrochemical sensor comprising the Pencil Graphite Electrode as previously described for electroanalytical analyses.

[0029] In one embodiment, the electroanalytical analysis is a potentiometric or voltammetric measurement.

[0030] In one embodiment, the electroanalytical analysis is a voltammetric measurement of the concentration of phenolic compounds such as propofol or paracetamol.

[0031] In one embodiment, the voltammetric measurement of the concentration of phenolic compounds is performed in vitro, in vivo or ex vivo.

[0032] In one embodiment, the voltammetric measurement of the concentration of phenolic compounds is performed for at least 10 minutes up to 4 hours.

[0033] Still another object of the present invention relates to a method of sensing, measuring or monitoring phenolic compounds, comprising the steps of:

[0034] a) contacting an aqueous sample containing phenolic compounds with a electrochemical sensor comprising the Pencil Graphite Electrode as previously described; and

[0035] b) obtaining a voltammetric response of the phenolic compounds.

[0036] In preferred embodiments, said step of obtaining a voltammetric response further comprises the step of applying an electrical potential across the Pencil Graphite

Electrode to produce the voltammetric response.

[0037] In one embodiment, the method is performed in vitro, in vivo or ex vivo.

[0038] In one embodiment, the method is performed for at least 10 minutes up to 4 hours.

[0039] In one embodiment, the aqueous sample comprises blood or serum. [0040] In one embodiment, the phenolic compounds are propofol and/or paracetamol.

Brief description of drawings

[0041] In the Figures:

[0042] Figure 1 shows two embodiments of a Pencil Graphite Electrode according to the present invention. In the shown embodiments, a distal, working portion is operably connected with a connector such as a metallic wire (on the left), and the proximal portion includes means for electrically connecting the entire electrode to external devices such as a potentiostat;

[0043] Figure 2 schematically shows a typical three-electrode configuration for a voltammetric sensor according to the invention, as well as adapters for electrically connecting the electrodes to external devices;

[0044] Figure 3 shows a test of various cleaning parameters to identify the best procedure for PGE: a.) anodic CV scan without (full line) and with (dotted line) propofol in solution; b.) NaOH cleaning procedure by increasing the number of CV scans; c.) PBS cleaning by increasing the applied voltage and keeping constant the CA time;

[0045] Figure 4 shows a graph of sensitivity values for HB PGE electrodes evaluated for five calibrations, performed one after the other without intermediate cleanings. The errors bars are evaluated as one standard deviation obtained from the linear regression analysis for S evaluation (n=5, points in each calibration);

[0046] Figure 5 shows a scanning electron microscope (SEM) photography a HB PGE working portion in four conditions, a) bare electrode after 5 cycles of CV in only PBS (background electrolyte), b) after one propofol calibration, c) after NaOH cleaning and d) after PBS cleaning;

[0047] Figure 6 shows a Ferro-Ferrycianide Cyclic Voltammetry (CV) study of cleaning procedures on HB PGEs: a) PBS cleaning, b) NaOH cleaning;

[0048] Figure 7 shows a graph of Sensitivity (S) values for each of the five calibrations at PGE electrodes by implementing NaOH and PBS intermediary cleanings. Error bares are evaluated as one standard deviation from the linear regression analysis for S evaluation (n=5, points each calibration);

[0049] Figure 8 shows a continuous monitoring in time of a HB PGE with PBS cleaning;

[0050] Figure 9 shows a graph for propofol calibrations adopting Differential Pulse Voltammetry (DPV) in different background electrolytes: a) PBS and b) full serum solution; [0051] Figure 10 shows a diagram of the inter-electrode sensitivity for all PGE compositions. After 6B the softness of the lead brings to an immediate fouling effect of the electrode surface after first propofol measurement;

[0052] Figure 11 shows the comparison of the ratio Standard Deviation (StDev)/Sensitivity for all analyzed PGE performed in order to identify the best lead composition in terms of high sensitivity and small inter-electrode variation;

[0053] Figure 12 shows the sensitivity trend among different PGE lead compositions.

Five subsequent propofol calibrations are performed on each PGE;

[0054] Figure 13 shows the variability in sensitivity for subsequent propofol calibrations on same electrode respect to the average sensitivity of the electrode itself (StDev/Sensitivity);

[0055] Figure 14 shows the results of a continuous monitoring experiment by using four different kind of PGEs, 6H, 3H, 2B and 4B. Five calibrations (with five rising propofol concentrations each) were carried out resulting in 25 subsequent measurements, for a total of 4 hours, without any intermediate cleaning;

[0056] Figure 15 shows a SEM photography of 3H PGE lead composition. There is no evidence of fouling between before and after 5-points calibration measurement;

[0057] Figure 16 shows a SEM photography of a 8H PGE a) before and b) after its use, showing no signs of fouling, contrary to a 5B PGE in which clear fouling areas are present after its use (d) compared to before (c);

[0058] Figure 17 shows a SEM photography of a HB PGE a) before and b) after its use, showing an evident fouling layer on its surface after its use, contrary to a F PGE (before and after use, c and d), a H PGE (before and after use, e and f) and a 2H PGE (before and after use, g and h) in which no fouling evidences can be retrieved;

[0059] Figure 18 shows a graph of the peak area of DPV measurements for 9.9 μΜ

Propofol + 300 μΜ APAP (solid line), only 9.9 μΜ Propofol (dashed line) and only

300 μΜ APAP (dot line);

[0060] Figure 19 shows the Gaussian decomposition of peak current area isolated from

APAP DPV measurement;

[0061] Figure 20 shows the Gaussian decomposition for current peak area isolated from propofol DPV measurement;

[0062] Figure 21 shows the DPV peak area for 9.9 μΜ Propofol and 300 μΜ APAP solution. Description of embodiments

[0063] The present disclosure may be more readily understood by reference to the following detailed description presented in connection with the accompanying drawing figures, which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed disclosure.

[0064] As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise", "comprises", "comprising", "include", "includes" and "including" are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term "comprising", those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of" or "consisting of."

[0065] The invention provides for a Pencil Graphite Electrode (PGE) for use in electroanalytical analyses.

[0066] Electroanalytical analysis methods are a class of techniques in analytical chemistry which study an analyte by measuring the potential (volts) and/or current (amperes) in an electrochemical cell containing an analyte. These methods can be broken down into several categories depending on which aspects of the cell are controlled and which are measured. The three main categories are potentiometry (the difference in electrode potentials is measured), coulometry (the cell's current is measured over time), and voltammetry (the cell's current is measured while actively altering the cell's potential).

[0067] Potentiometry passively measures the potential of a solution between two electrodes, affecting the solution very little in the process. One electrode is called the reference electrode and has a constant potential, while the other one is an indicator electrode whose potential changes with the composition of the sample. Therefore, the difference of potential between the two electrodes gives an assessment of the composition of the sample. The most common potentiometric electrode is the glass-membrane electrode used in a pH meter. [0068] Coulometry uses applied current or potential to completely convert an analyte from one oxidation state to another. In these experiments, the total current passed is measured directly or indirectly to determine the number of electrons passed. Knowing the number of electrons passed can indicate the concentration of the analyte or, when the concentration is known, the number of electrons transferred in the redox reaction.

[0069] Amperometry is the term indicating the whole of electrochemical techniques in which a current is measured as a function of an independent variable that is, typically, time or electrode potential. Chronoamperometry is the technique in which the current is measured, at a fixed potential, at different times since the start of polarisation. Voltammetry is a subclass of amperometry, in which the current is measured by varying the potential applied to the electrode. Voltammetry applies a constant and/or varying potential at an electrode's surface and measures the resulting current with a three electrode system. This method can reveal the reduction potential of an analyte and its electrochemical reactivity. According to the waveform that describes the way how the potential is varied as a function of time, the different voltammetric techniques are defined.

[0070] As used herein, a "Pencil Graphite Electrode" or "PGE" is an electrode comprising, as the electrically working portion (i.e. the portion directly in contact with an analyte), a pencil graphite lead. The pencil graphite leads are made up of a composite material containing graphite, clay and a binder (typically wax, resins or high polymer) in various percentages. According to the European Letter Scale, graphite pencils are marked with letters H (hardness) and B (blackness), as well as numbers indicating the degree of hardness or blackness, thus creating a scale spanning from 9H (the hardest) to 8B (the softest). B type leads contain more graphite and are softer, and the harder H type leads have more clay. This latter can have an influence on the chemical (e.g., ion exchange) and structural properties (e.g., degree of disorder and surface morphology) of the pencil graphite leads.

[0071] As used herein, the term "electrode" refers to an element used to connect an electric circuit with a sample comprising an analyte. An electrode is generally a conductive element that conducts an electric current toward or away from an electric circuit, said circuit being composed in its simplest embodiment of two electrodes and a material as a dielectric, an electrolyte, or a semiconductor placed in between. The term "electrically working portion" refers herein to the portion of the electrode which is directly in contact with a sample or an analyte, and responsible for the delivery of an electrical current from or to a conductor (for instance, a metallic wire assuring an electrical contact) operatively connected to said electrically working portion and connectable to an external device such as a power supply. Two embodiments of a Pencil Graphite Electrode according to the present invention are shown in Figure 1.

[0072] Besides the fact that they are cheap, PGEs are also easy to be used. These electrodes have proven to provide good sensitivity and reproducibility, being a viable, renewable, and economical tool, particularly when used in combination with voltammetric techniques. Despite these advantages, in certain instances there is a need for some time-consuming electrode surface cleaning/polishing step.

[0073] This is related to the fact that during the electrooxidation of phenolic compounds, a noticeable decrease in the current is observed owing to the formation of a polymeric film on the electrode surface, a phenomenon known as fouling. Electrode fouling in an amperometric chemical sensor leads to the decay of current during repetitive scans, continuous flow, or injections of samples, and is often caused by the formation of a passivating polymeric film on the electrode surface due to the electropolymenzation of phenolic compounds. This film may promote electrode passivation mainly by interfering in the supply of fresh reactants from bulk solution, removing products from the reaction zone or active sites, or decreasing the available overpotential to drive the reaction. The fouling is often a problem in electrochemical measurements and delays important electrode processes.

[0074] Pencil Graphite Electrodes are not immune to this phenomenon, and some previously outlined attempts have been done in the past to overcome this issue, by e.g. treating PGEs in various ways such as by anodization, doping with metal nanoparticles or preferably by polymer coating. In order to avoid these additional steps, the main inventive concept behind the present invention relates to the use of bare, untreated (e.g. doping-free and coating-free, including passivation) PGEs having a reduced content in graphite and an increased content in clay compared to the prior art PGEs for electrochemical analyses. In particular, the PGEs of the present invention are characterized by the fact of having a clay to graphite ratio of at least 0.4, wherein said ratio is calculated on a weight/weight basis. Without being necessarily bound to this theory, it is deemed that an increased clay content in a PGE leads to a reduced tendency to fouling, and therefore passivation of the electrode, thanks to some sort of shielding effect around the graphite particles present in the pencil lead.

[0075] What has been surprisingly assessed by the inventors is that, when reducing the graphite content and concomitantly increasing the clay content into the composite PGE material, there is a "switch point" in clay to graphite ratio for which the fouling effect cannot be revealed after electrochemical analyses. This switch point is set for a value of 0.4 in clay to graphite ratio, which in a real life PGE corresponds to the "F" pencil graphite lead having about 66% w/w in graphite content and about 28% w/w in clay content (assuming a fixed amount of a suitable binder such as wax at around 5% w/w, which is the industrial standard for e.g. pencil leads; see Table 1).

[0076] In preferred embodiments, the clay to graphite ratio is set on values above 0.4, such as 0.425, 0.492, 0.566, 0.62, 0.71 , 0.8, 0.9, 1 , 1.136, 1.29 and above, including intermediate values. A suitable range of the clay to graphite ratio for a reliable PGE could span from 0.4 to 1.5, such as from 0.425 to 1.29, corresponding to the range going from an "F" pencil graphite lead to a "9H" pencil graphite lead.

[0077] For the sake of clarity, the correspondent values in % w/w will be provided for the above-listed clay to graphite ratio values, and possible suitable ranges thereof.

For instance, the PGEs of the present invention could have a fixed binder (e.g., wax) content of 5% w/w, and a graphite content up to 66% w/w, while the clay content could go down to 28% w/w. Small variations in the binder content are acceptable, as long as the clay to graphite ratio is kept of at least 0.4 in a weight/weight comparison.

[0078] In some embodiments, the PGEs of the present invention are characterized by the fact of having a graphite content between 41 % and 66% w/w and a clay content between 28% and 53% w/w.

[0079] In some embodiments, the PGEs of the present invention are characterized by the fact of having a graphite content between 44% and 66% w/w and a clay content between 28% and 50% w/w.

[0080] In some embodiments, the PGEs of the present invention are characterized by the fact of having a graphite content between 47% and 66% w/w and a clay content between 28% and 47% w/w.

[0081] In some embodiments, the PGEs of the present invention are characterized by the fact of having a graphite content between 50% and 66% w/w and a clay content between 28% and 45% w/w. [0082] In some embodiments, the PGEs of the present invention are characterized by the fact of having a graphite content between 52% and 66% w/w and a clay content between 28% and 42% w/w.

[0083] In some embodiments, the PGEs of the present invention are characterized by the fact of having a graphite content between 52% and 60% w/w and a clay content between 34% and 42% w/w.

[0084] In some embodiments, the PGEs of the present invention are characterized by the fact of having a graphite content between 58% and 63% w/w and a clay content between 31 % and 36% w/w.

[0085] In one embodiment, the PGEs of the present invention are characterized by the fact of having a graphite content between 44% and 47% w/w and a clay content between 47% and 50% w/w.

[0086] In a preferred embodiment, the PGEs of the present invention are characterized by the fact of having a graphite content of 58% w/w and a clay content of 36% w/w.

[0087] Within the frame of the present disclosure, a relative proportion in % w/w of graphite, clay and wax binder, and the correspondent European Letter Scale marking, is provided in Table 1 , together the correspondent clay to graphite ratio. However, since the content of the elements composing a pencil lead with relation to its hardness could vary from a manufacturer to another, in the present disclosure only the proportion in % w/w will be considered -when needed- for the sake of clarity, and when referring to hardness/blackness, it is understood that Table 1 shall be taken as a reference.

[0088] Table 1 - Graphite, clay and wax amount in the used PGEs (w/w)

[0089] As it will be evident for a person skilled in the art, the present invention also relates to the use of the Pencil Graphite Electrode as previously described for the manufacturing of an electrochemical sensor, as well as the electrochemical sensor obtainable therefrom. In particular, the electrochemical sensors of the invention comprise a Pencil Graphite Electrode of having a clay to graphite ratio of at least 0.4

[0090] According to a preferred embodiment of the invention, the electrochemical sensors are amperometric, particularly voltammetric, or potentiometric sensors for use in several kind of analyses. Therefore, still another object of the invention relates to the use of the PGE-based electrochemical sensors herein described for electroanalytical analyses, and preferably the use of the PGE-based electrochemical sensors of the invention for amperometric, particularly voltammetric, or potentiometric measurements. Voltammetric measurements can be performed as polarography, linear sweep voltammetry, differential staircase voltammetry, normal pulse voltammetry, reverse pulse voltammetry, differential pulse voltammetry or cyclic voltammetry.

[0091] As briefly described before, voltammetric sensors are a class of electrochemical sensors that determine the concentration of chemical and biochemical species by measuring the voltage-current relationship of an electrochemical cell. In cyclic sweep mode, the voltage is scanned linearly up and down repeatedly, then the height of the peak current quantifies the concentration of the species. In amperometric mode a fixed voltage is applied, and current is proportional to the redox reaction rate which depends on the concentration of the species.

[0092] Conventional electrochemical cells consist of three electrodes: a working electrode (WE), which possesses the interface to the sample of interest to be studied, a counter electrode (CE) that serves to collect the current to complete the electrochemical circuit, and a reference electrode (RE) that serves to establish a stable and well-known potential without passing current.

[0093] The voltage-current relationship of the electrochemical cell is measured with a potentiostat. This electronic circuit maintains the potential of the working electrode with respect to the reference electrode by adjusting the current through the counter electrode. In one embodiment, the voltammetric sensor can be miniaturized to fit at the tip of a catheter, syringes, stents or other implantable or semi-implantable devices. This is particularly useful and desired for in vivo measurements of biochemical species. For example, a miniaturized three- electrodes (WE, CE, RE) voltammetric sensor concept on the tip of a micro- catheter is well known for lactate analysis, used for early diagnosis of bacterial meningitis. In the preferred embodiment of the invention, the Pencil Graphite Electrode is used as the working electrode within an electrochemical sensor of the invention such as a voltammetric sensor. The size of the working electrodes of the present invention's sensor is such that the surface area in contact with a sample comprising an analyte is preferably of at least 10 mm².

[0094] Figure 2 schematically shows a typical three electrode configuration for a voltammetric sensor according to the invention. The sensor 1 comprises a PGE working electrode 10, a counter electrode 20 and a reference electrode 30. The working electrode 10 applies the desired potential in a controlled way and enables transfer of charges to and from a target analyte comprised in a sample 100, preferably an aqueous sample. The counter electrode 20 functions as the other half of a cell, and the voltammetry analysis essentially involves determining the half-cell reactivity of the analyte. The counter electrode 20 needs to have a known potential with which to gauge the potential of the working electrode 10 and to balance the charge added or removed by the working electrode 10.

[0095] The sensor of the invention can be applied to all known uses of voltammetric sensing, and can therefore be for example and without limitations a pH sensor, a nitric oxide sensor, a glucose sensor, a uric acid sensor, a lactic acid sensor or a creatinine sensor. In one embodiment, the electrochemical sensor of the invention can be a phenol sensor, i.e. it is adapted and suitable for voltammetric measurements of the presence or the concentration of phenolic compounds in a sample. Accordingly, one aspect of the invention relates to the use of the electrochemical sensor of the invention in voltammetric measurements of the presence or the concentration of phenolic compounds in a sample.

[0096] Phenolic compounds, such as phenol and cresol, have great importance in industry and are chemical pollutants widely present in the atmosphere, water systems, and many food products. Electrochemical sensing provides a valid technique for analysis since most phenolic compounds can be easily oxidized in an electrochemical cell. Advantageously, the electrochemical sensor of the invention is much less prone to electrode fouling, and therefore to degradation of sensor's signal, due to electro-polymerization of phenols on electrode's surface. The working life of the sensors is therefore lengthened before the electrode needs to be replaced or polished.

[0097] In one embodiment, the electrochemical sensor of the invention is used in voltammetric measurements of the presence or the concentration of propofol and/or paracetamol, and in some embodiments the electrochemical sensor is used for in vitro, in vivo or ex vivo measurements of the presence or the concentration of propofol and/or paracetamol.

[0098] In some embodiments, the voltammetric measurement of the concentration of phenolic compounds, such as propofol and/or paracetamol, either in vitro, in vivo or ex vivo, is performed for a lapse of time of at least 10 minutes up to 4 hours, such as for instance 30 minutes, one hour, two hours and so forth. This is particularly advantageous for propofol and/or paracetamol in a surgical operation scenario, in order to monitor the concentration of the said compounds over time without encountering, or at least reducing, fouling issues that would reduce the electrochemical signal. In this sense, the electrochemical sensor of the invention is much more reliable and suitable for a long term monitoring of propofol and/or paracetamol concentration. ι9] As will be evident, a further object of the present invention relates to a method of sensing, measuring or monitoring phenolic compounds, comprising the steps of:

00] a) contacting an aqueous sample containing phenolic compounds with a electrochemical sensor comprising the Pencil Graphite Electrode as previously described; and

01] b) obtaining a voltammetric response of the phenolic compounds.

02] As used herein, the word "sensing" relates to the ability of the sensor of the invention to detect the presence of a phenolic compound present in a sample through electrochemical analysis. The word "measuring" relates to the ability of the sensor of the invention to estimate one or more of the properties of a phenolic compound present in a sample, such as its concentration or its nature, through electrochemical analysis. Furthermore, in the frame of the present disclosure, the word "monitoring" relates to the ability of the sensor of the invention to control and/or record the trend of one or more variable properties of a phenolic compound present in a sample, such as its concentration, over time, through electrochemical analysis.

03] In preferred embodiments, said step of obtaining a voltammetric response further comprises the step of applying an electrical potential across the Pencil Graphite Electrode to produce the voltammetric response. Voltammetric measurements can be performed as polarography, linear sweep voltammetry, differential staircase voltammetry, normal pulse voltammetry, reverse pulse voltammetry, differential pulse voltammetry or cyclic voltammetry.

04] In one embodiment, the method is performed in vitro, in vivo or ex vivo. In one embodiment, the method is performed for at least 10 minutes up to 4 hours, such as for instance 30 minutes, one hour, two hours and so forth. In one embodiment, the method is performed without any polishing step of the Pencil Graphite Electrode present in the sensor. In one embodiment, the aqueous sample comprises blood or serum. In one embodiment, the phenolic compounds are propofol and/or paracetamol.

05] In a prototypical setting according to some of the above-mentioned embodiments, an aqueous sample comprising phenolic compounds, preferably propofol and/or paracetamol, is monitored in a continuous fashion during a lapse of time of one to several (e.g., four) hours. As used herein, "continuous monitoring" refers to the practice of providing one measurement each 5 minutes (12 measurements for one hour), one measurement each 10 minutes (6 measurements for one hour) or one measurement each 15 minutes (4 measurements for one hour), preferably without any polishing step of the used electrodes. The sample can comprise a bodily fluid such as blood or serum, which can be taken periodically from a patient or can contact the PGE-based sensor of the invention via a blood vessel/sensor shunt or connection. A number of voltammetric measurements, such as 25 subsequent measurements (one measurement every 10 minutes) are carried out, resulting in up to four hours of total time monitoring. The applied electrical potential is comprised between +0.2 V and +2 V, with a scan rate of at least 0.1 V/s.

[00106] One of the technical advantages of the sensor of the invention, in the frame of a continuous monitoring of an analyte such as phenols, and particularly propofol and/or paracetamol, relies in its stability and minimally fluctuating characteristics, offering reproducible data and enabling reliable measurement. In this sense, the PGE-based sensor of the invention allows a sensitivity at least in the order of 10 ⁷ Α/μΜ, which can vary, e.g. diminishing, over time of no more than about 30%, preferably no more than about 25%, even more preferably of no more than 20% such as between 5% and 20%, particularly thanks to its fouling-resistant behaviour. Preferably, the PGE-based sensor of the invention allows to have a constant or even augmenting sensitivity over time, such as during a long-term monitoring electrochemical analysis.

Examples

[00107] The inventors validated a ready-to-use, low-cost and robust point-of-care sensor for long-term monitoring of propofol to be adopted during anaesthesia practices. PGEs were tested in terms of change in their sensitivity and surface characteristics upon long-term monitoring. Moreover, the effect on propofol electro-oxidation and fouling was widely analysed to fully understand the chemical reaction mechanism.

[00108] Additionally, two surface regeneration methods have been explored: regeneration in Phosphate Buffer Saline (PBS) and in sodium hydroxide (NaOH) have been developed. The sensitivity and reproducibility were monitored through 4 hours of data acquisition by measuring 1 sample each 10 minutes.

[00109] Chemicals

[00110] 2,6-Diisopropylphenol (propofol) was purchased from TCI chemical and and dissolved in 0.1 M NaOH to prepare the stock solution of 5.4 mM. Subsequent dilutions of propofol stock solution were done inside PBS (10 mM, pH:7.4) or serum to obtain concentrations of: [9.9 - 19.6 - 38.5 - 56.6 - 80.5] μΜ. The compounds KH2PO4, K2HPO4, NaOH, KNO3 and heat inactivated human male serum were purchased from Sigma Aldrich.

[00111] Electrochemical Analysis

[00112] Pencil mines with diameter of 0.5 mm were used as PGE Working Electrode (WE) in a voltammetric sensor and a commercial mechanical pencil was adopted as electrode's holder to establish electrical contacts with a power supply. A good electrical contact was obtained by soldering the metallic parts of pencil with a copper wire. The PGE used for this study comprised an HB mine, with a graphite content of 68% w/w and a clay content of 26% w/w, clay to graphite ratio: 0.38. The PGE was dipped for 8 mm into the solution in order to have an electro-active area of 12.6 mm². The Ag|AgCI|3.0 M KCI Reference Electrode (RE) was provided by Metrohm and a Pt wire was used as Counter Electrode (CE). All Cyclic Voltammetries (CVs) and Differential Pulse Voltammetries (DPVs) were performed with a Metrohm Autolab system (PGSTAT 302N) in conjunction with Nova 1.11 software.

[00113] Data analysis and plotting was done in Matlab R2013a. CV measurements were conducted in the range of [+0.2 - +1.4] V at a scan-rate of 0.1 V/s. DPV measurements were performed with PGE in the potential range of [0.0 - +1.1] V with interval time of 0.2 s and a scan-rate of 0.025 V/s. Electrochemical characterization of PGE electrodes was done via CV technique for the evaluation of sensitivity and LOD values over five subsequent set of 5-points-calibration experiments. Sensitivity (S) was considered as the slope of the calibration curve (concentration versus peak current) while LOD was calculated as LOD = 3 * ^(sD)Mank^ _w^_ere (SD) ian_k is the standard deviation at blank measurements (n = 3).

[00114] Cleaning Procedures

[00115] Two cleaning procedures were tested for reversing the effects of fouling caused by propofol oxidation:

[00116] · NaOH cleaning was done inside 10 ml of 0.1 M NaOH solution by carrying out ten cycles of CVs in the same potential range where the drug was sensed ([+0.2 -

+ 1.4] V for the tested PGEs);

[00117] · PBS cleaning consisted of a ChronoAmperometry (CA) performed by applying

+ 1.4 V for 30 s without stirring the solution. 10 ml of 10 mM PBS (pH:7.4) were used. [00118] The parameters of these two procedures has been chosen after having tested different combination of them. For NaOH cleaning an increasing numbers of CV scans (2, 5 and 10) has been tested while for PBS procedure different fixed potential (1.0, 1.2 and 1.4 V) has been applied for 30 s to the electrode. Results obtained from the different tested parameters are summarized in Figure 3.

[00119] Surface Analysis

[00120] The surface characterization of the electrodes was performed by using a XLF30- FEG Scanning Electrode Microscopy (SEM) instrumentation in the Interdisciplinary Center for Electron Microscopy (CIME) of EPFL. It has an 1 - 30 kV Schottky field emission gun and a Secondary Electrons (SE) imaging resolution of 2 nm at 30 kV; 8 nm at 1 kV. The samples have been inserted into the SEM vacuum chamber in dry conditions. The SE images were taken by applying an accelerating voltage of 20 kV and a resolution of 200 μιτι.

[00121] The experiments performed to design an electrochemical biosensor for long-term monitoring of propofol in serum, could be sub-grouped into two: (i) characterization of various electrode materials in terms of electrochemical and surface characterization techniques to check if the LOD and sensitivity (S) are affected by the time course and detection media and (ii) electrochemical detection of propofol inside human serum.

[00122] Electrode Fouling Phenomenon

[00123] Electro-oxidation of propofol causes free radicals that electro-polymerizes on the electrode surface forming a passivating layer that prevents the long-term stability of the sensor. The sensitivity values for the used PGEs and over five calibration curves (five points each) are shown in Figure 4. It is evident that the PGE reaches sufficiently high sensitivity values but there is a sharp sensitivity decrease after the third calibration. This is attributed to the fouling phenomena on the electrode surface.

[00124] To avoid the formation of the passivating layer and to stabilize the sensitivity values in time two cleaning procedures has been designed and validated. Furthermore, the electrode fouling phenomenon was further investigated through SEM surface characterization and electrochemical studies by analysing scan-rate and pH dependencies. [00125] Surface characterization on the fouling layer

[00126] SEM analysis was done to observe the effects of fouling and cleaning on PGE electrodes. Figure 5 shows SEM images of bare PGE, after one calibration set of experiments (five concentration values), after NaOH cleaning and PBS cleaning. Figure 5a shows that bare PGE is characterized by graphite striae on the lateral surface and small pointed tips at the base after CVs in PBS electrolyte solution. On the contrary, after a propofol calibration, a deposit layer is clearly seen on both lateral and base surface of the electrode (Figure 5b). This passivating layer of propofol fouling covers the superficial structures of the electrode and causes the decrease of sensitivity as it is shown by electrochemical characterization results. After performing cleaning procedures (NaOH cleaning Figure 5c and PBS cleaning (Figure 5d) this layer is removed and the morphology of the PGE is recovered back to its original bare structure. To support and integrate SEM results also ferro/ferricyanide analysis has been performed. CVs in 5 mM K3[Fe(CN)6]/K4[Fe(CN)6]24 after each propofol calibration have been carried out. Results are shown in Figure 6. The electrochemically active surface areas of the electrode has been evaluated from Randles-Sevick equation as follow: A = ip=(286:6 n(3=2) D(1 =2) C (1 =2)), where ip is the anodic peak currents from CV data, D is the diffusion coefficient (cm2=s), C is the concentration of the electro-active species in the bulk solution (mol=cm3) and is the scan-rate (V/s). CVs in Fig. 5 shows that after propofol calibration (n=5) the ferro/ferricyanide peak currents decrease. After PBS and NaOH cleaning the peaks increase again. That means that propofol reaction is reducing the active area of the electrode and the cleaning procedures are effectively polishing the electrode surface. By evaluating the active area from the anodic peak, we obtained a recover of 99:6% and 71 :2% for PBS and NaOH cleaning respectively.

[00127] The cleaning of the fouling layer

[00128] In order to provide a long-term monitoring of propofol, electrochemical cleanings of PGEs were tested and validated. Figure 7 shows the results of sensitivity for PGEs over each calibration curves (up to five) with cleaning procedures; i.e. PBS or NaOH cleaning, applied in between two subsequent calibration curves. It is clear from Figure 7 that, both PBS and NaOH cleaning prevent the fouling phenomenon that affects PGE electrode after the third calibration without intermediate cleaning (shown in Figure 4). [00129] A high sensitivity is recommended especially for direct detection in human serum. Five calibrations (five concentrations each) were carried out resulting in 25 subsequent measurements adding up to four hours of total time. From these results, it can be concluded that continuous monitoring is enabled for more than four hours. Continuous monitoring requires one measurement each 10 minutes (6 measurements for one hour). The measurements in time for the best electrode- cleaning combinations are shown in Figure 8, i.e. PGE with PBS cleaning.

[00130] If a final application in anaesthesia delivery and long-term continuous monitoring is considered, it could be more convenient not to add an external chemical compound, then PBS cleaning is preferred. Adding NaOH as a cleaning solution would complicate the system design and require extra time whenever cleaning is needed. Indeed, considering the final application, it has also been tested the cleaning in PBS with still 80 μΜ propofol concentration inside and comparable results with only PBS solution are obtained (data not shown). Therefore, the cleaning is successful also in presence of the analyte itself. Finally, the LOD was evaluated for PGEs with PBS cleaning, resulting in a value of 3:14 ± 1 :13 μΜ.

[00131] Serum Experiments

After the optimization of the parameters related to cleaning and the pH and scan- rate studies, the detection of propofol inside human serum was achieved in order to provide a system able to be adopted in anesthesia delivery practices. A successful detection of propofol has been done with DPV technique by using PGEs. First, calibrations of propofol were carried out by DPV in PBS to identify the oxidation peak position and the best potential range (Figure 9a). Then, the same set of calibrations were carried out in full serum. Figure 9b shows a visible oxidation peak in the same potential range within PBS. The peak current values increases linearly with increasing propofol concentrations that has a linear calibration equation with an R2 of 0.99 (n = 3). From this calibration curve, the LOD was calculated to be 0.82 μΜ with 9.3 Relative Standard Deviation (RSD)(%) and this concentration is in the physiological range ([1 - 60] μΜ).

[00132] PGE analysis on different leads

[00133] The sensitivity variation in propofol detection has been evaluated by comparing the performances of 20 different PGEs. The PGE lead composition varies in % w/w of clay, wax and graphite changes as summarized in Table 1. Accordingly to the composition the hardness of the PGE changes, growing from 9B (the softest) to 9H (the hardest).

[00134] The inter-electrode variability among all PGE lead compositions was evaluated by performing 5-points calibrations (9.9 - 19.6 -38.5 -56.6 - 80.5 μΜ propofol) on 3 different PGEs with same hardness. The general trend in Figure 10 shows that the sensitivity in propofol detection increases with the decrease in lead hardness. On the other hand, with softer lead than 6B, it is impossible to get any propofol signal. Indeed, the lead is too soft and the fouling effect due to propofol oxidation affects enormously the measurements. Therefore, it was impossible to calibrate propofol using 7B, 8B and 9B electrodes.

[00135] Moreover, the handling of soft PGEs, like 5B and 6B, is very challenging due to their fragility when cut. It is very difficult to obtain same surface as the others. That is the reason of the higher standard deviation got with these lead compositions.

[00136] Figure 1 1 shows the comparison of the ratio Standard Deviation (StDev)/Sensitivity for all analyzed PGE performed in order to identify the best lead composition in terms of high sensitivity and small inter-electrode variation. It easily identifiable that 3H composition (clay to graphite ratio: 0.62) ensures small inter-electrode variations and high sensibility to propofol.

[00137] The fouling effect due to propofol oxidation is a very well known problematic because it decreases the sensitivity of the electrode. Inventors have also evaluated the fouling for all the PGE compositions (except 7B, 8B and 9B that are already affected by fouling after one measurement due to their softness). Figure 12 reports all sensitivities obtained for 5 calibrations performed one after the other in time with same PGE hardness. In this way it is possible to evaluate the trend in time of the sensitivity for each composition. To better identify which electrode is less affected by the fouling effect, Figure 13 reports the variability in sensitivity for subsequent propofol calibration on same electrode respect to the average sensitivity of the electrode itself (StDev/Sensitivity). Also for this analysis, the 3H lead results to have high sensitivity for propofol detection with smaller variation of this sensitivity in time.

[00138] In an attempt to compare the sensivity of PGEs with different content of graphite and clay in a long-term analysis setting, the inventors have performed a continuous monitoring experiment by using four different kind of PGEs. Also in this case, five calibrations (with five rising propofol concentrations each) were carried out resulting in 25 subsequent measurements, adding up to four hours of total time; however, no intermediate cleaning was done in this setting, in order to 1) create a real-life scenario in which propofol content can be continuously monitored during surgical procedures without the need to clean up the electrodes, and 2) verify the stability of the electrodes and the resistance to the fouling effect. To this aim, a 6H pencil lead (50% in graphite and 45% in clay, w/w; ratio: 0.9), a 3H lead (58% in graphite and 36% in clay, w/w; ratio: 0.62), 2B lead (74% in graphite and 20% in clay, w/w; ratio: 0.27) and a 4B lead (79% in graphite and 15% in clay, w/w; ratio: 0.19) were used as working portions of a working PGE electrode (2 mm in diameter) included into a voltammetric sensor according to the invention. As shown in Figure 14, the 3H PGE is very sensible and stable over time, as demonstrable by the reproducibility of the obtained signals during the analyses. The 4B PGE reaches higher current values, but the fouling effect reduces the current peaks obtained over time, as for the 2B PGE. The 6H-based PGE reaches lower current values compared to the other PGEs used, but it shoes less fouling of the working electrode portions as shown by the reproducibility of the signals over time.

[00139] SEM analysis of PGE 3H lead composition shows no evidence of fouling between before and after 5-points calibration measurement (Figure 15). The same SEM analysis was also performed on other kind of PGE electrodes to highlight the influence of the clay and graphite content in the electrode on the fouling effect. As shown in Figure 16, an 8H PGE (44% in graphite and 50% in clay, w/w; ratio: 1.13) show no signs of fouling after its use, contrary to a 5B PGE (82% in graphite and 12% in clay, w/w; ratio: 0.14) in which clear fouling areas are present. Moreover, additional SEM analyses were put in place in order to define the switching point in clay to graphite ratio in a PGE between a fouling-prone behaviour toward a fouling-resistant one. As clearly highlighted in Figure 17, after its use a HB graphite lead shows clear signs of fouling on its surface, while F, H and 2H show none, defining the F graphite lead (66% w/w in graphite content and about 28% w/w in clay content, ratio: 0.425) as the sought switching point.

[00140] Propofol detection: APAP interference study at 3H PGE

[00141] To evaluate the selectivity of a 3H electrode, Propofol measurements have been performed also in presence of paracetamol (APAP), considered as main potential interfering compounds. In particular, Differential Pulse Voltammetry (DPV) with PGE 3H in PBS 10 mM pH 2.5 as background electrolyte has been used for studying the interference between APAP and Propofol oxidation processes.

[00142] As clearly visible from Figure 18, the obtained peak area in the DPV measurement obtained for 9.9 μΜ Propofol and 300 μΜ APAP, is the sum of Propofol and APAP contributions separately.

[00143] From the DPV graph, the current peak region was first isolated and then straightened and fitted by applying Gaussian decomposition. As first step, Gaussian decomposition was evaluated for the current peak area isolated from DPV graphs of only APAP and only propofol, as shown in Figures 19 and 20. In this way it is possible to identify the single contributions of each of the two compounds. Finally, by summing the Gaussian contributions from APAP and Propofol a complex peak is obtained. From this shape it is possible to evaluate concentration for both Propofol and APAP.

[00144] Since the two Gaussian components are well identifiable, it is possible to separate and classify the contributions of APAP and Propofol from the current peak (Figure 21). The same experiment was performed also at physiological (pH 7.4) with similar results (not shown).

Claims

Claims

1. A Pencil Graphite Electrode (PGE) for use in electroanalytical analyses, characterised in that it has a clay to graphite ratio of at least 0.4.

2. The Pencil Graphite Electrode of claim 1 , characterized in that it is a doping-free and coating-free bare electrode.

3. The use of the Pencil Graphite Electrode of claim 1 for the manufacturing of an electrochemical sensor.

4. An electrochemical sensor characterized in that it comprises the Pencil Graphite Electrode of claims 1 or 2.

5. The electrochemical sensor of claim 4, characterized in that the Pencil Graphite Electrode is the working electrode.

6. Use of the electrochemical sensor of claims 4 or 5 for electroanalytical analyses.

7. The use according to claim 6, wherein the electroanalytical analysis is a potentiometric or voltammetric measurement.

8. The use according to claim 7, wherein the electroanalytical analysis is a voltammetric measurement of the concentration of phenolic compounds such as propofol or paracetamol.

9. The use according to claim 8, wherein the voltammetric measurement of the concentration of phenolic compounds is performed in vitro, in vivo or ex vivo.

10. The use according to claims 8 or 9, wherein the voltammetric measurement of the concentration of phenolic compounds is performed for at least 10 minutes up to 4 hours.

11. A method of sensing, measuring or monitoring phenolic compounds, comprising the steps of:

a) contacting an aqueous sample containing phenolic compounds with a electrochemical sensor comprising the Pencil Graphite of claims 4 or 5; and b) obtaining a voltammetric response of the phenolic compounds.

12. The method of claim 11 , wherein said step of obtaining a voltammetric response further comprises the step of applying an electrical potential across the Pencil Graphite Electrode to produce the voltammetric response.

13. The method of claims 1 1 or 12, characterized in that it is performed in vitro, in vivo or ex vivo.

14. The method of claims 1 1 to 13, characterized in that it is performed for at least 10 minutes up to 4 hours.

15. The method of claims 1 1 to 14, characterized in that the aqueous sample comprises blood or serum.

16. The method of claims 11 to 15, characterized in that the phenolic compounds are propofol and/or paracetamol.