DESCRIPTION

Field of the Invention The present invention concerns the field of the electrochemical sensors, in particular for the determination of environmental pollutants. It describes a new molecularly-imprinted sensor for polyfluorinated compounds obtained by polymerization of ortho-phenylenediamine.

Prior Art The increasing presence of toxic or potentially toxic organic compounds in a variety of industrial products and products intended for consumption entails a serious risk for the environment and for human health. In the last forty years the perfluoroalkylated compounds (PFAS) have been produced for a wide range of applications. In particular, the perfluorooctansulfonate (PFOS) and perfluorooctanoic acid (PFOA) are synthetic surfactants. The C- F bond is one of the strongest, very stable to physical, chemical and biological degradation. The environmental risk for the health due to PFOS and PFOA is significant and gave rise to precautionary actions. In 2009 PFOS and similar compounds were inserted in the list of the persistent organic pollutants discussed in the Stockholm Convention (Miralles et at, Environmental International, 77 (2015) 148- 159). In particular, PFOS and derivatives are largely utilized as additives in inks, varnishes, waxes, fire- fighting and flame-retardant foams, cleaning products or products for metal plating, coating formulations (for walls, furniture, carpets, food), lubricants, repellents for leather, paper and tissues. Recently, as a consequence of analyses conducted on a large area of the region Veneto polluted by PFAS, the regional government has issued laws on the matter, the most recent thereof being the regional decree number 5 of July 22, 2016. Moreover, based on document n. 1584, January 16, 2014 of the "Istituto Superiore di Sanita", and the decree law n. 172 of October 13, 2015 "actualization of the directive 2013/ 39/ EU", the suggested maximum admissible limits of concentration are currently: PFOS < 30 ng/L (0.06 nM); PFOA < 500 ng/L; other PFAS < 500 mg/L. The very low concentration at which PFOS are present in the environment represents a challenge for their analytical quantification. The current technique for the determination of PFOS is the high-performance liquid chromatography (HPLC) coupled to mass spectrometry. Although a good sensitivity has been reached, this protocol requires complex and expensive instrumentation, as well as a complicated pre-treatment of the sample. The electrochemical analysis, thanks to the ease in measuring and to the large availability of instrumentation, to the excellent limits of detection at low cost with the possibility of easy miniaturization and automation, is a widely applied method for the determination of environmental pollutants. The development of innovative materials for electrodes is a key component for the future electroanalysis.

Molecularly-imprinted sensors (MIPs) are promising materials which are currently extensively studied as recognition elements or modification agents for sensors (Tran et at, Sensors and Actuators B 190 (2014) 745-751 ; Karimian et at, Biosensors & Bioelectronics 55 (2013) 492-498). Molecular imprinting is a process by which specific functional monomers and crosslinking agents are polymerized in the presence of a templating molecule to form a cast-like shell. Initially, the monomers form a complex with the templating agent through covalent or non-covalent interactions. The templating agent can be removed after polymerization. So the MIP contains binding sites which are complementary to the target molecule with respect to size, shape, position of functional groups, and which are kept in place by the crosslinked polymer matrix. Substantially, a molecular memory, able to selectively bind targets, is imprinted in the polymer (K.Haupt, Nature Materials, 9 (2010) 612-614). The above-mentioned monomers are polymerized on an electrically conductive surface (e. g. on a gold surface) thus obtaining a MIP coated electrode (here referred to as MIP electrode). During the analysis, the presence of the target analyte in the solution is detected through a variation of electrical signal (voltammetric, amperometric or coulometric measurement) through the MIP electrode. The electrical signal (redox peak) is generated by a substance which can undergo redox reaction (redox probe) present in solution which, upon applying a suitable potential, reaches the MIP electrode producing an electric current: the presence of analyte bound in the imprinted cavities of the MIP electrode opposes to the current flow through the electrode: therefore a decrease of the redox peak of the MIP electrode equilibrated with the sample, in comparison with the same MIP in a blank containing the same redox probe, but not the analyte, indicates the presence of the analyte itself, bound to MIP. The performance of MIP sensors is strictly dependent on the capability of interaction between the imprintable polymer and the specific used templating agent (analyte): such interaction varies in unpredictable manner depending on the chemical and physical characteristics of the respective molecules. The efficacy of such interaction determines the quality of the obtained mold, the ease in loading and unloading the analyte and, accordingly, the sensitivity, stability and reproducibility over time of the method of analysis. For example, an imprecise imprinting entails a reduced capability of binding to the target analyte and / or an increased interference caused by the binding of MIP with molecules different from the target. Therefore, the MIP sensors are always created "ad hoc", i. e. for the detection of a small and chemical- sterically homogeneous group of analytes and they require their own conditions of production process and measurement parameters, not a priori predictable or deducible from MIP sensors created for different analytes or based on different imprinted polymers. Often critical parameters are, for example, the thickness of the produced polymer matrix, the conditions for removal of the templating agent after polymerization, the amount of imprinted templating agent, in particular the amount/ accessibility of the binding sites obtained in the polymer matrix, the usage modes of the probe during measurement, the conditions of incubation with the target analyte (time, pH, etc.). A problem observed for many MIP sensors is the sensitivity decrease after a prolonged use: the cause could derive from the fact that the repeated loading and unloading of the analyte and/ or the working conditions to which the sensor is subjected, and/ or the simple exposition to environmental factors reduce the binding affinity between the analyte and the imprinted polymer. A further problem lies in that the polymer imprinting technique does not necessarily result in a product which is sensitive enough to detect low concentrations of the analyte: this problem is particularly felt for those analytes which are present in traces in the environment and nevertheless need precise detection. Among the different processes for MIP preparation, the most frequently used is the bulk polymerization. This technique creates monolithic structures, which are subsequently crushed and sieved in order to obtain the final product. These steps are time consuming and entail a low templating agent/MIP binding kinetics. To overcome these drawbacks, electropolymerization has proved to be an effective way to prepare MIPs directly on the surface of a transducer by simply controlling the thickness of the MIP layer through the amount of flown charge. This approach is particularly attractive to produce small devices for clinical diagnostics, environmental control and various determinations in the pharmaceutical industry. Ortho-phenylenediamine (o-PD) is easily electropolymerizable on various substrate material and forms film with a good chemical and mechanical stability (Malintesta et al, Analytical Chemistry 71 (1999) 1366- 1370). The presence of neutral or protonated -N¾ groups can be responsible for interactions with oligodeoxyribonucleotides, enzymes or molecules, when used as molecular templates (Losito et al, Analytical Chemistry, 75 (2003) 4988-4995). Thus, the o-PD is particularly suitable for molecular imprinting, providing hydrophilic, hydrophobic and basic recognition sites through electrostatic interactions (Malintesta et al, Analytical Chemistry, 62 (1990) 2735-2740). MIP sensors based on o-PD as imprinted polymer have been created for detecting various analytes, for example: kojic acid (Yang Wang et al, Talanta, 85(5), 201 1 , 2522-2527), folic acid (Karimian et al, Electrochemistry Communications, 36(2013), 92-95), ethylparaben (CN104458856), troponin T (Karimian et al, Biosensors and Bioelectronics, 50 (2013) 492-498), triclosan (Li et al, Microchemical Journal, 91 (2009) 222-226), theophylline (Kan et al Microchimica Acta, 171 , (2010) 423-429), sorbitol (Feng et al, Biosensors and Bioelectronics, 19 (2004) 1513- 1519), oxytetracycline (Li et al, Analytical Chemistry, 82 (2010) 6074-6078), glucose (Cheng et al, Biosensors and Bioelectronics, 16 (2001) 179- 185), dopamine (Song et al, Journal of Solid State Electrochemistry, 14 (2010) 1909- 1914, and CN-A- 102507697), 2,4-dichlorophenoxyacetic acid (CN-A- 104062331), thrombin (CN-A- 105259227), gibberellin A3 (CN-A- 103149268), streptomycin (CN-A- 103243367), kaempferol (CN-A- 104458857). For the detection of perfluorooctanoic acid, MIP sensors have occasionally been described in CN-A- 104327271 and CN-A- 104880495: these sensors are based on complex composite imprintable materials which are expensive (CdTa/CdS coated with TEOS, or Agl-BiOINF) and on different methodologies of analyte detection (fluorescence or photoelectric measurements). Shubo Deng et al. (Fronteers of Environmental Science & Engineering in China, 3(2), 2009, 171- 177) describes molecularly imprinted absorbents for removing millimolar concentrations of perfluorooctane sulfonates from water; after the PFOS removal step, the residual PFOS in water is analysed and quantified by traditional HPLC.

Considering the above-mentioned prior art, there is a need for new MIP sensors for PFOS which have a low production burden/ costs and optimal characteristics for the detection of such analytes. There is further still a need for identifying one or more parameters of MIP production and/ or analysis conditions useful to enhance the sensitivity/ selectivity of PFOS detection. It still remains the need for new MIP sensors, in particular for the PFOS detection, which provide a signal which can be reproduced over time also after a prolonged use. It still remains the need for sensors for PFOS that have optimal response characteristics within the typical PFOS concentration interval present in the environmental biological samples polluted by PFOS, so as to provide a tool of high practical value for safeguarding public health in respect of this specific group of pollutants. Summary

This study describes the manufacture and characterization of a new sensor for the analytic detection of PFOS based on a molecularly imprinted polymer preferably obtained by electrosyn thesis. PFOS is used as templating molecule for the electrochemical polymerization of the functional monomer o-PD. The analytic performance of the sensor is assessed by voltammetric, amperometric and coulometric measurements. Specifically, object of the present invention is a new molecularly-imprinted-polymer electrochemical sensor (herein referred to as "MIP sensor") comprising an electrically conductive surface covered with ortho-phenylenediamine (o-PD) polymerized in the presence of PFOS; after polymerization said PFOS is substantially removed. The so created sensor shows a high sensitivity for the detection of PFOS (from 0.1 nM to 1.5 μΜ, with a limit of detection of 0.038 nM), ad an excellent durability, highlighted by the constant current response even after prolonged use. Description of the drawings

Figure 1 : Cyclic voltammogram for the electropolymerization of o-PD on a gold electrode. (A) Preparation of the MIP electrode (10 mM o-PD, 1 mM PFOS in acetate buffer (pH 5.8), 33.3% methanol, 50 mV/s, 25 cycles). (B) Preparation of NIP electrode (same conditions as in (A) but without PFOS as templating agent).

Figure 2A: Cyclic voltammogram obtained with MIP-modified electrodes after removal of the templating agent with methanol/ water (1 : 1 v/v) mixture for 10+ 10 min (with moderate agitation) and then 5 min with pure ethanol. Solid line: in 0.5 mM FcCOOH, ammonia buffer solution (pH 8.46); Dashed line: in 0.5 mM K ₃ Fe(CN)6, 0.1 M KC1.

Figure 2B: Cyclic voltammogram obtained with MIP-modified electrodes after re-washing with methanol/ water (1 : 1 v/v) mixture for 10+ 10 min (with moderate agitation) and then 5 min with pure ethanol, after 7 PFOS additions for an analysis cycle through standard additions. Solid line: in 0.5 mM FcCOOH, ammonia buffer solution (pH 8.46); Dashed line: in 0.5 mM K ₃ Fe(CN)6, 0.1 M KC1.

Figure 3: Electrochemical characterization: (A): cyclic voltammograms registered with naked gold electrode (solid line a), MIP electrode (dashed line b) and NIP electrode (dashed line c). (B): differential pulse voltammograms registered with naked gold electrode (solid line a), MIP-modified (dashed line b) and NIP-modified (dashed line c) electrode. (C): cyclic voltammograms registered with MIP (solid line) and NIP (dashed line) electrode after removal of the templating agent. (D): differential pulse voltammograms registered with MIP electrode in 0.5 mM FcCOOH (pH 8.4), before (solid line) and after (dashed line) incubation in 5 nM PFOS.

Figure 4: Images of a golden slide subjected to 25 o-PD electropolymerization cycles: (A) macrophotograph (scale in mm); (B) microphotograph (25 x). In both images (A) and (B) the left area shows the naked electrode, the right area shows the coated electrode.

Figure 5: Profilometric plot on golden slide subjected to 25 o-PD electropolymerization cycles: The region at the left of the step refers to the naked electrode, the region at the right of the step refers to the coated electrode.

Figure 6: Dependence of the ratio between peak current and peak width at half height on: (A) increase E, (B) amplitude, (C) pulse width and (D) sampling width with a naked gold electrode in 0.5 mM FcCOOH (pH 8.4). Figure 7: Effect of different parameters on the response of the MIP electrode, expressed in terms of (i° -i), where i° and i are the current in the absence and in the presence of PFOS, respectively. (Al) and (A2): Effect of the rebinding time of PFOS on the response of the MIP (Al) and NIP (A2) electrode in a 0.5 mM FcCOOH solution containing 50.0 nM PFOS for various incubation times, (B) pH effect on the binding process of the templating agent, (C) effect of the number of cycles in the electropolymerization process, (D) effect of the molar ratio between o-PD and PFOS on the response of the MIP electrode in the electropolymerization process. Figure 8: Binding of PFOS with modified electrode.

(A) Differential pulse voltammogram of MIP-modified electrode after 15 minutes incubation at different PFOS concentration between 0.1 nM and 1.5μΜ containing 0.5 mM FcCOOH in an ammonia buffer solution (pH 8.4);

(B) current response of FcCOOH on the MIP electrode as a function of PFOS concentration and in the concentration ranges between (B) 0.1 and 4.9 nM and (C) 9.5xl0 ³ and about 1.5 μΜ; (D) relationship between PFOS concentration and current response of FcCOOH on the MIP electrode; (E) relationship between PFOS concentration and current response of FcCOOH on the NIP electrode. Figure 9: SEM-EDX spectrum for MIP film: (A) before removal of the templating agent, (B) after removal of the templating agent.

Figure 10: Variation of the differential pulse current peak of the 0.5 mM FcCOOH probe upon standard additions of PFOS in a sample of mineral water supplemented with 2 nM PFOS (error bars = ± standard deviation, n =3).

Detailed Description of the Invention

In the present invention, the term "molecularly-imprinted electrochemical sensor" or "MIP sensor" or "MIP electrode" identifies any electrically conductive material partially or totally covered by a polymer matrix obtained by polymerization of an appropriate monomer in the presence of an appropriate templating agent. The monomer used in the present invention is ortho-phenylenediamine (o- PD). In the operating conditions adopted herein, the latter forms a poly-o- phenylenediamine polymer matrix (film) (Ogura et ah, Electrochimica Acta, 40 (1995) 2707-2714; Dai et at, Journal of Electroanalytical Chemistry, 456 (1998) 47-59), which is electrically insulating (Losito et at, Journal of Materials Chemistry, 1 1 (2001) 1812- 1817).

The term "templating agent" refers to the molecule used, in the o-PD polymerization step, to obtain the appropriate imprinted cavities (herein also defined as molds or binding sites) which, after removal of the templating agent, in the step of analytical determination, are able to house the analyte.

The templating agent which can be used according to the present invention is perfluorooctansulfonate (PFOS). In the present invention, PFOS represents both the templating agent (in the step of sensor production) and the analyte to be determined (in the step of using the sensor). The term "PFOS" includes herein the perfluorooctanesulfonic acid and any salt thereof, preferably alkali or alkaline-earth metal salts. In particular sodium or potassium salts.

As analyte, PFOS can be searched, according to the invention, in any liquid environmental sample, preferably aqueous, which is desired to be assessed; environmental samples which were originally solid can also be analyzed, after their dispersion or extraction in a liquid medium, preferably aqueous.

The electrically conductive surface of the sensor is any metal material (or at least metal on the surface or part thereof), said metal being preferably a noble metal (i. e. gold or platinum) or a metal resistant to an oxidation potential equal to about 1 V vs Ag/AgCl, or mixtures of said metals; the metal mixtures can be homogeneous (e. g. alloys) or derived from the assembly of structures or layers of different metals, on nanometric scale too (e.g. nanoparticles). The electrically conductive surface can be composed also by carbon in one of its conductive forms such as, for example, graphite, pastes or graphite-based inks, carbon fibers or nanotubes, graphene and reduced graphene oxide, carbon black, glassy carbon, doped diamond e.g. Boron-Doped Diamond (BDD), products pyrolized by polymers, including photoresist. An important characteristic of the present MIP sensors is the substantial absence of templating agent (PFOS) achieved thanks to the high capability of removal of the templating agent acquired with the production process of the invention described below. The substantial absence of templating agent corresponds to the maximal availability of the binding sites obtained by polymer imprinting. The substantial absence of templating agent is easily determinable by elementary analysis of the polymer matrix obtained: in particular, in a SEM-EDX spectrum, the substantial absence of the peaks corresponding to the F and/ or S atoms indicates successful removal of PFOS. The term "substantial absence" of PFOS or the term "substantially removed" takes into account that, in the experimental practice, an absolute removal of all the molecules of the templating agent from the MIP is never possible; therefore, the term "substantial absence of templating agent" or "substantially removed" used in the present text admit the presence of residual templating agent in traces; the presence of such traces is non influential from the point of view of the operation of the sensor; it is instead useful as a "marker" of a sensor created according to the present invention. The sensor is further functionally characterizable on the basis that (differently from a polymeric sensor non-imprinted or imprinted with templates different from PFOS), it has a significantly higher capability of binding to said PFOS. Advantageously, the sensors of the invention have shown a remarkably high sensitivity, with a PFOS limit of detection below 0.05 nM/liter: this allows an easy determination of PFOS at concentrations in the nanomolar range, i.e. at those levels in which PFOS may be present in the environmental water, thus allowing a precise quantification of such pollutants: lacking this fine sensitivity, they would be of no practical use for quantifying this analyte. Therefore the present sensors can be further characterized by having a PFOS limit of detection below 0.05 nM, typically between 0.01 and 0.05 nM/L; this allows an easy determination of PFOS concentrations in the nanomolar range. The high removability of the templating agent represents a further advantage not only in the step of MIP sensor production, but also during the reference service life thereof, allowing, in the case of multiuse sensors, a substantially complete unloading of the bound analyte after each performed analysis, thus eliminating a significant interference with the successive analysis. A preferred aspect of the present sensors is the degree of thickness of the polymerized o-PD on the electrically conductive layer. Such thickness is, as known in the art, easily controllable through the number of electropolymerization cycles applied. In the case of the present sensors it was experimentally verified that a number of cycles higher than 28 produced a quite thick matrix, full of binding cavities; however, the deepest part of them proved to be difficult to access for the analyte, so that a further increase in matrix thickness did not correspond to an increase in sensor efficiency; on the other hand, a decrease in the number of cycles under 22 caused an excessive reduction of the imprintable material and therefore a progressive reduction of sensor efficiency. Thus, for the purposes of the invention, the number of polymerization cycles is preferably from 22 to 28, more preferably from 24 to 26, ideally equal to 25: this allows to obtain a film with thickness generally from 50 to 200 nm (Cheng et at, Biosensors and Bioelectronics, 16 (2001) 179- 185); such thickness is non-limiting; for the purposes of the invention, thicknesses of the film in the range of about 10-500 nm, e.g. 30-400 nm or 40-300 nm or 60- 140 nm can be generally used. The MIP sensor of the invention is conveniently provided in form of an electrode (MIP electrode), in embodiments already widely known: to this end, it is connected (or connectable) to an electrically conductive wire, e.g. a copper wire, in turn connected (or connectable) to a suitable current detector composed for example by a potentiostat integrated with a function generator and suitable systems of sampling of the intensity of current/ electric charge and of data acquisition/ processing. To such end, any potentiostatic system of prototype or commercial type, suitable for the control of three electrode electrochemical cells, can be employed. Such electrodes include, without limitation: a working electrode (MIP); a counter electrode (generally made of Pt, C or another inert conductor); a reference electrode (e.g. Ag/AgCl/ KC1) or a pseudo reference electrode (ad es. Ag/AgO); thereto a further MIP electrode usable for comparison in a blank lacking the analyte can be assembled.

The MIP electrode of the invention can be provided together with the above- mentioned electrodes, as it is common knowledge for the arrangement of a potentiostatic detection system; such detection system, for the analysis of PFOS, is further object of the invention.

The electrochemical cell can be: a) of the classical type with the different electrodes separated and singularly insertable / replaceable in the cell which contains the electrolytic solution; b) in integrated and miniaturized form on chip on which the electrodes are imprinted by serigraphic processes (screen-printed electrodes), and/ or by deposition/ sputtering/ etching of conductors and insulators, and/ or by photolitography followed by pyrolysis (pyrolyzed photoresist carbon electrodes) and the like.

A further object of the invention is a system for the detection of PFOS comprising a MIP electrode as defined above and the further electrodes as defined above, conveniently connected to a current detector; such electrodes preferably comprise a MIP electrode as previously described, a counter electrode, a reference electrode and a possible further MIP electrode usable for comparison.

A further object of the invention is a process for the manufacture of the MIP sensors above described. Such process, in its general meaning, comprises the following steps: a) providing an electrically conductive surface; b) providing a solution comprising the monomer o-PD and the templating agent (PFOS); c) subjecting to polymerization, on said electrically conductive surface, the o-PD solution obtained in step b; d) remove said templating agent from the product of step c).

Further object of the invention is the process that creates the intermediate resulting from step c), i.e. the result of steps a), b) c), before the removal of the templating agent.

As previously indicated, the electrically conductive surface provided in step a) can be any metal material, or at least metal on its surface or part thereof), said metal being preferably a noble metal, such as e.g. gold or platinum, or carbon based conductive materials (graphite, glassy carbon, carbon black, nanotubes, graphene and derivatives, BDD etc.) pure or in mixtures; said mixtures can be homogeneous (e. g. alloys) or derived from the assembly of structures or layers of different metals. As known in the art, before polymerization, the electrically conductive surface is conveniently cleaned, e.g. with solvents and/or alumina suspensions or other abrasives and/or methods based on ultrasound cleaning and / or electrochemical cleaning as well as plasma, laser and similar techniques.

The solution prepared in step b) is preferably an hydroalcoholic solution, buffered at a suitable pH, preferably between 5 and 7. The o-PD concentration in the solution is between 6 and 14 mM, preferably 10 mM; the concentration of the templating agent, i.e. PFOS, in the solution is preferably between 0.1 and 1.4 mM.

An important aspect of the present process is the weight ratio between the used amounts of o-PD and PFOS. In the case of the present sensors it was in fact experimentally verified that a ratio higher than 20 produced a quite thick matrix, which had an insufficient number of binding sites for surface unit and/ or the binding sites were in too deep regions of the polymer matrix, which were difficult to access for the analyte. On the other hand, a ratio lower than 5 entailed an excessive reduction of the imprintable material and therefore an excessive reduction of sensor efficiency. Thus, the weight ratio between o-PD and PFOS present in the solution in step b) is preferably between 5 and 20, more preferably between 7 and 15, ideally equal to 10.

The polymerization achieved in step c) is preferably performed with the electropolymerization technique. The parameters for the o-PD electropolymerization are known in the art and can be possibly modified if convenient. In a non-limiting way, it is possible to perform polymerization cycles by applying a suitable number of scans (preferably between 22 and 28, more preferably between 24 and 26, ideally 25) of cyclic voltammetry between 0 and 1.5 V (vs. Ag/AgCl) at a scan speed of 50 mV/sec, or the like, typically between 20 and 500 mV/sec, but not limited to these values. Alternatively, an electropolymerization at fixed and controlled potential (potentiostatic) or at controlled current intensity (galvanostatic) can be performed. The intermediate product of step c), i.e. the sensor as previously described, before the removal of the templating agent, is per se a further object of the present invention.

In step d) the removal of the templating agent is generally obtained by using an hydroalcoholic solution or another suitable solvent medium. To such end, the sensor obtained in step c) is immersed in the solution in agitation conditions for a suitable time, e.g. between 10 and 30 minutes. Preferably, the solution is a water: methanol solution in proportion between 1.5: 1 and 1 : 1.5, ideally 1 : 1. The above-mentioned solutions proved particularly effective in removing the templating agent, showing at the same time an optimal capability of: penetrating in the polymer matrix, dissolving and washing the PFOS incorporated therein, and being easily separable from the polymer matrix by evaporation. The solution has also proved to be inert with respect to the polymer matrix, avoiding to alter the cavities imprinted by the templating agent.

At the end of step d) the MIP sensor obtained in this way can be conveniently dried with standard techniques, e.g. under air or nitrogen flow, and/or vacuum exposition, and/ or heating.

A further object of the invention is a method of detection of PFOS characterized by the use of a MIP sensor as described above. As it is typical for this kind of sensors, the detection is mostly qualitative, so as to allow a rapid screening on an even large number of samples; nevertheless, the current sensors show a response which is proportional within specific analyte concentration ranges, such as to allow also a quantitative determination; they further display a high response sensitivity within a PFOS concentration range from 0.1 nM to 1.5 μΜ, said range including the concentrations at which these analytes are generally found in polluted environmental samples, with a very low limit of detection, equal to 0.038 nM. Advantageously, using the current MIP sensors it is possible to perform finer quantitative analyses (which are generally expensive and time consuming) only to the samples positive to the present analysis. The present MIP sensors can be used for the determination of PFOS present in any liquid sample, preferably aqueous, or in a solid sample conveniently dispersed in a liquid medium, preferably aqueous. In a preferred embodiment, the present MIP sensors are employed for the detection of PFOS in liquid media comprising food or biological samples, e.g. blood; said liquid media can be environmental water (e.g. lake water, river water, etc.) contaminated with the above-mentioned food or biological samples (in turn containing PFOS); or said liquid media can consist in the food or biological sample itself, if liquid, such as e.g. blood, milk, beverages, etc. In particular, the very high sensitivity shown by the present sensors, quite unusual for MIP sensors, makes them especially useful in the present methods, given the very low concentrations at which PFOS are present in the environment. They represent therefore an ideal tool for detecting PFOS concentrations which exceed the admissible limits established by current regulatory guidelines.

The use of the present MIP sensors occurs by generally known procedures. The MIP electrode is immersed in said liquid sample in which a suitable redox probe has been previously been solubilized; by means of the MIP electrode and the other two electrodes contained in the measurement cell (i.e. the reference electrode and the counter electrode) cyclic voltammograms or DPV (Differential Pulse Voltammetry) or SWV (Square Wave Voltammetry) are then registered; by way of a non-limiting example, for a typical DPV measurement the following measurement parameters can be used: potential range from 0 to 0.5 V (vs. saturated Ag/AgCl/KCl); pulse width equal to 25 ms; pulse amplitude equal to 25 mV; potential increase equal to 4 mV; scan speed equal to 20 mV/s, sampling amplitude equal to 0.0167 mV. The presence of the analyte in solution is indicated by a reduction of the redox peak generated by the probe and registered by the MIP electrode in the sample, with respect to the analogous signal previously registered with the same MIP in a reference blank, containing the same concentration of electrolyte, including the redox probe, but in absence of the analyte (PFOS).

Moreover, the Applicant has experimentally identified the best incubation conditions for the present sensors, i.e. the duration of the measurement and the pH of the solution to be tested, in order to further increase the efficiency (sensitivity) of the measurement. Useful values to this end were a duration between 5 and 25 minutes, preferably between 10 and 20 minutes, ideally 15 minutes, and a pH between 6.5 and 10.5, preferably between 7.5 and 9.5, ideally 8.4. The usable redox probe can be chosen among the ones currently available, e.g. ferrocyanide based compounds. However, in a particularly interesting development of the invention, it has been found that the current sensors do not show a sensitivity decay over time after repeated measurements (as it typically occurs for this kind of sensors) if, during such measurements, a ferrocenecarboxylic acid (FcCOOH) is used as a redox probe. Preferably, such probe is used within a concentration range from 0.1 and 1 mM, more preferably from 0.25 and 0.75 mM, ideally 0.5 mM, at a pH between 6.5 and 10.5, preferably between 7.5 and 9.5, ideally 8.4. This advantage, together with the fact that the analyte can be completely unloaded after each analysis using the previously described hydroalcoholic solutions, allows, in the case of multi-use applications, in each new analysis, even after multiple analyses performed over a long period of time, to have a sensor for PFOS essentially "as good as new" from the point of view of the sensitivity and reproducibility of the performance. The invention is described below by way of the following non-limiting examples of embodiments.

Experimental section

1. Materials and equipment o-phenylenediamine (o-PD,≥ 98%, Sigma Aldrich), ferrocenecarboxylic acid (FcCOOH≥ 97%, Aldrich), potassium salt of the perfluorooctanesulfonic acid (PFOS,≥ 98%, Sigma Aldrich), perfluorooctanoic acid (PFOA,≥ 96%, Sigma Aldrich), perfluorohexan sulfonate (PFHxS,≥ 98%, Sigma Aldrich), perfluorohexanoic acid (PFHxA, ≥ 97%, Sigma Aldrich), were used as received. FcCOOH (0.5 mM) was prepared at pH 8.4 in an ammonia buffer and ethanol. All the other reagents were of analytical grade and the solutions were prepared using double-distilled deionized water. All the electrochemical measurements were performed at room temperature with a potentiostat CH660B controlled by a software. A three electrodes standard configuration was used. A gold disk (2.0 mm diameter), a platinum wire and an Ag/AgCl/KCl electrode (3M) were respectively used as working electrodes, counter electrode and reference electrode. The scanning electron microscopy (SEM) was performed with an Hitachi TM3000 equipped with an analysis system EDX SwiftED-3000.

1.1 Electropolymerization In order to prepare a MIP electrode, the surface of a gold electrode was cleaned with a damp suspension of allumina of progressively finer (1.0, 0.3 and 0.05 μιη) granulometry and it was subsequently washed alternately in an ultrasound cleaner with double-distilled water for two minutes. Successively, the electrode was subjected to cyclic potential variations between 0.2 and 1.5 V with respect to Ag/AgCl in 0,5 M H2SO4 until obtaining a stable cyclic voltammogram. The electrosynthesis of the o-PD film was performed through cyclic voltammetry (25 scans) between 0 and 1.0 V with respect to Ag/AgCl at a scan speed equal to 50 mV/s in an acetate buffer solution (0. 1 M, pH 5.8) and methanol (33.3% v/v) containing 10.0 mM o-PD. PFOS was added in solution as templating molecule before polymerization at a concentration of 1.0 mM. A control electrode modified with a non-imprinted polymer (NIP) was obtained in the same way, without adding PFOS as templating agent. The modified electrodes were dried under air flow and stored at room temperature.

1.2 Removal of the templating agent

After polymerization, the modified electrode was washed with water and then with solution of water and methanol (1 : 1 , v/v) for 20 minutes under moderate agitation, followed by washing with methanol to remove the templating molecule trapped in the film.

1.3 Electrochemical measurements

The interaction between PFOS and the MIP electrode was assessed incubating the electrode in a solution containing appropriate concentrations of PFOS for 15 minutes under agitation. The electrochemical measurements to characterize the MIP electrode were performed in the presence of 0.5 mM solution of FcCOOH (pH 8.4). The cyclic voltammograms (CVs) of the imprinted membranes were registered in the potential range of 0.0 -0.5V with respect to Ag/AgCl with pulse duration equal to 25.0 ms, pulse amplitude equal to 25.0 mV, potential increase equal to 4.0 mV and scan speed equal to 20 mV/s.

2. Results and discussion

2.1 Evaluation of the electrodeposition cycles A typical cyclic voltammogram registered during the o-PD electro-polymerization in the presence of PFOS on gold electrode is shown in Figure 1A. A significant reduction of the anodic peak corresponded to the irreversible oxidation of the monomer to form an insulating thin film on the surface of the electrode during the continuous cyclization. Figure IB shows the voltammograms registered during o-PD electrodeposition in the absence of the templating agent. For the electropolymerization of the monomer alone (NIP) the same main oxidation peak was observed at about 0.4 V, followed by a second peak at 0.72 V, as typically observed for the polymerization of pure o-PD. The absence of any additional peak in the MIP voltammogram indicates that the templating agent PFOS is electrochemically inert in the potential range scanned. Both CV patterns of the Figures 1A and IB are characterized by a progressive current decrease progressing from the 1 st to the 25th cycle; this is in accordance with the deposition of the non conductor (insulating) polymer film. This fact drove us to prefer growing an ultrathin polymer film instead of a thick film; a thin polymer layer allows to improve the sensitivity of the device without too much increasing the ohmic resistance. It is to be noted that the thickness of the polymer can be easily modified by controlling the scan speed and the number of cycles during electropolymerization.

2.2 Evaluation of the redox probe

In previous studies on MIP sensors, the ferrocyanide have been used as redox probe suitable to monitor analyte capture/ release in/ from the imprinted cavities. When the cavities are bound to the analyte, the probe cannot penetrate to reach the underlying gold surface of the electrode; viceversa when the cavities are free (absence of the analyte) the probe generates its typical voltammetric signal.

Preliminary experiments indicated that ferrocyanide is not the best probe for repeatedly monitoring the capture/ release of PFOS by MIP of imprinted o-PD. As shown by the voltammograms (dashed lines) of Figures 2A and 2B, while the signal of ferrocyanide is well solved for a freshly prepared MIP (just after removal of the templating agent PFOS), after repeated loading/ unloading of the analyte the voltammetric signal of ferrocyanide becomes increasingly less distinguishable from the background current. This is not the case for the probe FcCOOH (see the solid lines in Figure 2), where the oxidation/ reduction signal of the redox probe remained practically remained unchanged after repeated loading/ unloading of the analyte PFOS on MIP. For this reason, in the following experiments we used FcCOOH as effective redox probe, suitable to monitor the process of loading of the analyte and to determine PFOS concentration through competition between FcCOOH and PFOS for MIP cavities.

2.3 Electrochemical characteristics

In order to characterize the prepared sensors, cyclic and differential pulse voltammograms were registered in the presence of FcCOOH. The formation of a non-conductive film on the electrode surface is confirmed by the disappearance of the redox peaks of FcCOOH (Figures 3A and 3B). This indicates that the o-PD film formed is very compact and there are no channels which allow the probe to come near to the electrode surface.

One of the most important elements of the manufacture of an efficient MIP electrode is the removal of the templating agent. An excellent extraction solvent should strongly interact with the templating agent so as to cause its release without damaging the backbone of the polymer. Thus, to study the effects of various post-treatments on the backbone of the polymer and the successful removal of the templating agent, each of the following post- polymerization treatments was performed on the NIP electrode as control. After examination of various strategies and solvents, the maximal difference in voltammetric signal between imprinted and non-imprinted polymers was observed after treatment in methanol-water (1 : 1 , v/v) solution for 20 minutes under light agitation, followed by washing with methanol to remove the templating molecules trapped in the film (Figure 3). To this end, after removal of the templating agent and performing measurements of background response, the MIP-modified electrode was immersed in a solution containing 5.0 nM PFOS for 15 minutes under agitation.

2.4 Physical and chemical-physical characteristics Figure 4A shows the MIP film deposited on a gold flat electrode obtained by 25 polymerization cycles in the presence of PFOS, while Figure 4B shows the same film observed by optical stereomicroscope. The thickness of said film was measured by means of an ALPHA step profilometer and a typical profile is depicted in Figure 5. The mean thickness of three films independently prepared by applying 25 electro-polymerization cycles was assessed from the mean height variation on the Y axis (in Angstrom in the graphic) between the portion of naked gold (on the left) and the portion covered with MIP (on the right); such thickness is 170 ± 12 nm. Both optical and profilometrical observations indicate a complete and homogeneous covering of the electrode surface with MIP.

To prove the nature of imprinting process on the surface of the transducer, the structural/ physical characteristics of the o-PD films and their chemical characteristics, analyses of scanning electron microscopy (SEM) and energy-dispersive x-ray spectroscopy (EDS) were performed. The SEM images showed the successful deposition of the polymer film on the gold surface and further, they revealed a pronounced difference in roughness of the surfaces and in the morphology between the films of MIP and NIP. Moreover, it was noted that the membrane surface of the MIP film becomes smoother and more uniform after the process of removal of the templating agent.

Studying the X rays emitted by the SEM analysis, information was obtained about the elementary composition of the sample. Figure 9 shows the patterns of EDX analysis confirming the existence of the grafted polymer on the surface of the transducer. In Figure 9A, the signals of sulfur S and of the fluorine F are relative to the elementary composition of the templating agent PFOS which, together with the signal of carbon C, clearly indicate the modification of the surface. Moreover, the slightly higher carbon level present on the MIP membrane with respect to the NIP membrane can be explained by the fact that the templating agent PFOS validates the successful synthesis of polymer nanoparticles molecularly imprinted with PFOS. The following table shows the chemical composition estimated by elementary analysis of the polymer film.

Before removal After removal

Element of templating agent (%)

of templating a gent (%)

Atomic weight Atomic weight Atomic weight Atomic weight

Carbon 21.350 67.615 7.037 42.948

Fluorine 4.813 9.637 0.369 1.423

Sulfur 0.173 0.206 - Since PFOS contains S and F atoms, the absence of S and the significant reduction of F and C in the EDX spectrum (Figure 9B and table) confirm the substantial removal of PFOS from the polymer matrix after washing process or removal of the templating agent and this is in accordance with the investigation of the electrochemical measurements.

2.5 Voltammetric parameters

Initially, in order to obtain the best sensitivity for the voltammetric determination, the influence of the differential pulse voltammetric (DPV) parameters on the characteristics of the peaks registered with a naked gold electrode in 0.5 mM FcCOOH was studied (Figure 6). The optimal conditions, indicated by the maximum of the ratios ip/Wl / 2 (wherein Wl/2 is the amplitude of the peak at half height), were obtained at an increase E equal to 0.004 V, an amplitude equal to 0.025 V, a pulse duration equal to 0.025 s and a sampling time equal to 0.0167 s. In order to create an efficient MIP sensor, different possible influencing factor were studied, including the number of cycles for o-PD electropolymerization, the molar ratio of monomer to templating agent (o- PD to PFOS), the incubation time and the pH. The change of response current of the redox process of FcCOOH of the ferrocenecarboxylic derivative on each electrode was calculated by subtracting the current registered in the presence of PFOS solution (50.0 nM) from the current registered in the absence of PFOS.

2.6 Effect of incubation time and pH

The incubation step is usually a simple and efficient way to increase sensitivity of the imprinted sensor (Xie et at, Microchim. Acta, 169 (2010) 145- 152). The time of accumulation of PFOS in solution using MIP- and NIP-modified electrodes was optimized. After an elution step using an extraction solvent, the imprinted electrode was incubated in an agitated solution containing 50.0 nM PFOS for various incubation times. Signal variation is expressed in terms of (i° -i), where i° and i are the current in the absence and in the presence of PFOS, respectively. The relationship between the changes of response current and rebinding time was studied in the range between 5 and 20 minutes. As shown in Figure 7A1 , the increase of the incubation time causes a clear increase of the current response of the MIP. The influence of the incubation time for the non- imprinted NIP electrode was studied in the same conditions (Figure 7A2) indicating also in this case an increase of the current response with the incubation time, but with normalized current intensities which are always an order of magnitude lower than the one measured on MIP. Such phenomenon is attributable to the adsorption of the templating agent on the surface of the polymer; the optimal incubation time for the electrochemical determination of PFOS on MIP was identified to be 15 minutes since such value allows to obtain a high value of the ratio (signal on MIP)/(signal on NIP) in a moderate time of analysis. The electrostatic interactions also play an important role in the recognition of the imprinting molecule (Kan et at, Sensors and Actuators, B, 168 (2012) 395-401). Therefore, the effect of the pH on the rebinding solution was studied. To this end the rebinding of the MIP-modified electrode was studied in solution containing a constant PFOS concentration (50.0 nM) in ammonia buffer, with pH value varying between 4.8 and 10.1 containing 0.5 mM ferrocenecarboxylic acid (Figure 7B). The results showed that pH 8.4 is the most appropriate for the incubation process.

2.7 Effect of the cycles of o-PD polymerization It is known that the thickness of the imprinted membranes of o-PD can influence the amount of PFOS molecules trapped and this in turn influences the sensitivity of the sensor. Changing the number of electropolymerization cycles can be done in order to modify the thickness of the imprinted polymer membrane. Figure 7C shows the effect that the number of cycles has on the responses of the MIP. The volume of imprinted sites increases with increasing thickness of the imprinted membrane. However, if the imprinted membranes are too thick, the imprinted sites located in the middle are difficult to reach due to the high resistance to the mass transfer. Further, such sites can be physically isolated from the underlying metal surface, thus, in any case, they do not contribute to the signal generated by the redox process at the expense of the probe molecule. As a result, the thick imprinted membranes offer poor accessibility to the binding site and to the metal surface of the underlying electrode, de facto reducing the useful signal (Kong et at, Sensors and Actuators, B, 185 (2013) 424-431). Therefore, in this study we decided that 25 cycles was the most appropriate number to produce a membrane with optimal analytical characteristics. 2.8 Effect of the ratio between o-PD and PFOS

The amount and the quality of the recognition sites of the molecularly imprinted polymer is a direct function of the amount of templating agent (Nezhadali et al, Sensors and Actuators, B, 171 (2012) 1 125- 1 131). On the other hand, the monomer concentration in the polymerization process influences the thickness of the deposit and the amount of imprinted molecules in the polymer matrix, which in turn further influences the electrochemical behavior of the sensor (Kan et at, Sensors and Actuators, B, 168 (2012) 395-401). Thus, in order to determine the optimal concentration of templating agent and monomer, the effect of the ratio between o-PD and PFOS on the response of the MIP electrode was studied. To this end the MIP films were electropolymerized in solution at a constant PFOS concentration (1 mM) and varying o-PD concentrations in the range 0.5-20 mM. As shown in Figure 7D, after PFOS extraction, the response of FcCOOH to the modified electrode is increased with increasing ratio of o- PD/PFOS from 0.5: 1 to 10: 1 , which was possibly attributed to an unstable MIP film on the surface of the electrode since too little o-PD was used. However, when the ratio raised to 20, the current response decreased, since when the membranes of imprinted polymer are too thick due to the high monomer concentration, the molecules of the templating agent located in the central area of the polymer membrane can not be completely removed from the polymer matrix (Li et ah, Sensors and Actuators, B, 186(2013) 96- 102). Thus, the optimal ratio o-PD/PFOS is chosen at 10.

2.9 Analytical performance of the MIP electrode To study the practical feasibility of the MIP electrode for the determination of PFOS, linear range and limit of detection were obtained from calibration curves. In optimal conditions, differential pulse voltammetry was used to monitor the response of the probe ferrocene/ ferricinium, since influenced by the binding of PFOS on the MIP-modified electrode. After removing the templating agent and measuring the background response, the MIP- modified electrodes were immersed for 15 minutes under agitation in a solution containing different PFOS concentrations. Figure 8A shows the reduction of the redox peak currents of the couple ferrocene / ferricinium for increasing PFOS concentration of the binding solution. With increasing concentration, the number of imprinted sites which re-bound to PFOS increases. Thus, the reaction of the electrode at the couple ferrocene/ ferricinium was prevented by the binding of PFOS with MIP. The results showed that the reduction of the peak current due to FcCOOH was proportional to the logarithm of the concentration of PFOS in two ranges from 0.1 to 4.9 nM and from 9.5 x 10-3 to 1.5 μΜ, with a correlation coefficient equal to 0.993, as shown in Figures 8B and 8C. The Limit Of Detection (LOD) was assessed as the ratio between three times the standard deviation of the control signal divided by the sensitivity of the method (sensitivity = slope of the straight calibration curve); LOD was found to be equal to 0.038 nM. On the contrary, the reduction of the peak current of FcCOOH proved to be much more moderate and independent from the PFOS concentration when the NIP-modified electrode was used as working electrode (against the MIP electrode as shown in Figure 8D), due to the lack of imprinted cavities in the polymer film (Figure 8E). 2.10 Reproducibility of the preparation of the sensor

The reproducibility of manufacture of the imprinted electrodes was estimated by determining the PFOS level using three electrodes produced independently in identical experimental conditions. The relative standard deviation (RSD) was 7.7% at the PFOS concentration of 5.0 nM. 2.1 1 Sample analysis

To provide an evaluation of the accuracy of the analyses performed with the sensor, some samples from different waters were analyzed, supplemented with known amounts of PFOS, assessing then the recoveries calculated as percent difference between expected and measured value. Initially the samples were diluted with an ammonia buffer (pH 8.4) containing a redox probe, supplemented with a known PFOS concentration and analyzed quantifying the PFOS concentration in the sample supplemented (spiked) using the standard addition method. From the registered differential pulse voltammograms values (iO - i) were calculated, wherein iO is the peak differential current of the blank and i are the values of peak differential current measured after the standard additions of known PFOS concentrations (CaddedPFOS). The graphic (iO - i) vs. CaddedPFOS was drawn, wherein the signal of the spiked sample corresponds to (iO - i) for CaddedPFOS =0. As known from the standard addition method, the concentration measured in the sample corresponds to the intercept on the X axis, changed in sign, of the calibration curve. Figure 10 depicts by way of example the graphic obtained applying such methodology to a sample of mineral water supplemented with a spike of PFOS at a 2 nM concentration. The results on different water samples supplemented with different amounts of PFOS are summarized in Table 1.

Table 1

Measured value

Expected

Sample (nM) (mean ± range, Rescue (%) value (nM)

n = 3)

1.75 ± 0.20 2.00 87.5

Distilled water

4.70 ± 0.28 4.96 94.7

1.92 ± 0.25 2.00 96.0

Mineral water

4.84 ± 0.36 4.96 97.6

Tap water 1.71 ± 0.28 2.00 85.5