Copper Oxide Chitosan Nanocomposite - MDPI

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Sep 24, 2017 - chitosan yields a clear and stable dispersion of Copper Oxide .... Figure 4a,b shows respectively the current–voltage plot of a couple of cells developed as sketched ..... liquid/zinc oxide nanoparticles/chitosan/gold electrode.
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Copper Oxide Chitosan Nanocomposite: Characterization and Application in Non-Enzymatic Hydrogen Peroxide Sensing Antonella Arena *, Graziella Scandurra and Carmine Ciofi Department of Engineering, Messina University, Messina 98166, Italy; [email protected] (G.S.); [email protected] (C.C.) * Correspondence: [email protected]; Tel.: +39-90-397-7383 Received: 8 August 2017; Accepted: 22 September 2017; Published: 24 September 2017

Abstract: Electrochemical dissolution of metallic copper into slightly acidic aqueous solutions of chitosan yields a clear and stable dispersion of Copper Oxide nanoparticles into the organic polymer host. The electrochemically synthesized chitosan:CuOx nanocomposite is characterized by means of spectrophotometry, frequency domain electrical measurements and morphological analysis. Solid state electrochemical cells having pure chitosan as the electrolyte and using chitosan:CuOx as the electrode, are developed and characterized by means of electrical measurements performed in the ±1 V voltage window. The current-voltage loops of the cells, measured in deionized water, are found to reversibly change in response to hydrogen peroxide added to the water in 0.2 µM subsequent steps. Such changes, clearly distinguishable from changes recorded in response to other analytes, can be exploited in order to develop a hydrogen peroxide sensor able to work without the need for any supporting electrolyte. Keywords: electrochemical sensor; chitosan; modified electrodes

1. Introduction Hybrid materials, consisting of nanosized inorganic compounds dispersed into organic polymer hosts, have been the subject of renewed interest over the last few decades, owing to their facile preparation and to their potentiality in a variety of fields, going from fuel cells and supercapacitors [1,2], to photovoltaic energy generation [3,4], sensing [5,6], and catalysis [7,8]. Whenever the hybrid material has to be used in applications that involve interaction with living organisms, as is the case in the field of tissue engineering, in drugs delivery, and in food industry, the polymer host matrix is required to be biocompatible and to show no toxicity and allergenicity. All such requirements are fulfilled by chitosan [9–11]: an abundantly available biodegradable polymer, obtainable by partial deacetylation of chitin, a naturally occurring polymer found in crustacean shells, in fungal micelia and in other materials of biological origin [12]. Chitosan is a polysaccharide characterized by the presence of hydroxyl and amino functional groups in its chains. From the chemical point of view, it has the ability to interact with metal ions, organic halogen substances, and biological molecules, through a variety of mechanisms including chelation, electrostatic attraction, and ion exchange. For these reasons, chitosan has been successfully used in environmental applications, such as the removal of contaminants from wastewater [13]. In addition, chitosan is a substrate commonly used for enzymes immobilization [14]. Due to these properties, and to its ability to form stable films, insoluble in water, with good adhesion and high mechanical strength, chitosan is also an ideal candidate in sensing and biosensing applications. There are, in fact, several examples of gas [15–18] and humidity [19] sensors based on thin films of chitosan, and a wide variety of enzymatic [20–22] and enzyme-free [23–25] electrochemical sensors based on chitosan-transition metal complexes, and on chitosan dispersions of Sensors 2017, 17, 2198; doi:10.3390/s17102198

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nanoparticles. As far as electrochemical sensing behavior is concerned, chitosan has been applied in the been applied in the detection hydrogen peroxide[26,27] both in the [26,27] and in the nondetection of hydrogen peroxideof both in the enzymatic and inenzymatic the non-enzymatic versions [28–30]. enzymatic versions [28–30]. In this paper, we demonstrate that the presence of hydrogen peroxide in In this paper, we demonstrate that the presence of hydrogen peroxide in water, can be successfully water, canatbe successfullylevel detected at a micromolar levelcell byin means a solid state cellboth in which detected a micromolar by means of a solid state whichofchitosan is used as the chitosan is used both as thethe polyelectrolyte that provides the polyelectrolyte that provides electrical connection betweenthe the electrical electrodes,connection and as the between host polymer electrodes, and as the host polymer of the electrochemically active nanocomposite used as the of the electrochemically active nanocomposite used as the electrode. Compared to other hydrogen electrode. Compared to on other hydrogen peroxide sensors chitosan, cell described here peroxide sensors based chitosan, the cell described herebased takes on advantage ofthe chitosan being used as takes advantage of chitosan being used as the ion conducting bridge between the electrodes, and the ion conducting bridge between the electrodes, and therefore it is able to work without the need for therefore able to work without [31,32]. the need for any kind of supporting electrolyte [31,32]. any kind it ofissupporting electrolyte 2. Materials Materials and and Methods Methods 2. 2.1. Preparation Preparation of of the the Chitosan:CuOx Chitosan:CuOx Sensor Sensor 2.1. Chitosan(medium (mediummolecular molecularweight, weight,purchased purchasedfrom fromAldrich, Aldrich, Louis, MO, US), is soluble Chitosan St.St. Louis, MO, US), is soluble in in acidic aqueous environment, where, lower than6.5, 6.5,the theamino aminogroups groupsofofthe the polymer polymer are are acidic aqueous environment, where, at at pHpH lower than protonated to to NH NH33++, ,and andthe thepolymer polymerbehaves behavesas asaacationic cationicpolyelectrolyte. polyelectrolyte.Electrochemical Electrochemicalsynthesis synthesis protonated was performed performed by by placing placing two two copper copper wire wire electrodes electrodes (Aldrich) (Aldrich) inside inside aa beaker beaker containing containing 100 100 mg mg was chitosan solubilized in 150 mL water and acetic acid (pH ranging from 5 to 6), and by applying chitosan solubilized in 150 mL water and acetic acid (pH ranging from 5 to 6), and by applying aa constant electric electric field field between between the the two two electrodes electrodes while while the the solution solution was was agitated agitated through through aa magnetic magnetic constant stirrer, in in order order to to prevent prevent the the chitosan chitosan molecules molecules from from sticking stickingon onthe thesurface surfaceof ofthe thecopper coppercathode. cathode. stirrer, Depending on the acetic acid concentration, two different kinds of electrochemical processes, involving Depending on the acetic acid concentration, two different kinds of electrochemical processes, the releasethe of release copper of from the electrodes, were observed. At pH lower than 5.5, athan pale5.5, blue hydrogel involving copper from the electrodes, were observed. At pH lower a pale blue formed, similar to similar that obtained Geng et al. and by them as a copper/chitosan hydrogel formed, to thatbyobtained by [33], Geng et identified al. [33], and identified by them as a complex. As the complex. electrochemical proceeded, the pale blue solution darkened, copper/chitosan As theprocess electrochemical process proceeded, the slowly pale blue solutionreaching slowly the green after a fewthe hours (Figure pH higher than an applied electric field about darkened, reaching green after a1b). fewAthours (Figure 1b).5.5, Atunder pH higher than 5.5, under an of applied 5 V/cm, the copper anode started to quite rapidly consume, while the initially uncolored chitosan electric field of about 5 V/cm, the copper anode started to quite rapidly consume, while the initially solution began to darken and began becametounclear, assuming a darkfinally brown assuming color, afteraa dark few minutes uncolored chitosan solution darkenfinally and became unclear, brown (Figure 1c).a few minutes (Figure 1c). color, after Both the the electrochemically electrochemically obtained obtained materials materialswere were used used to to develop develop electrochemical electrochemical cells cells to to be be Both used to detect the presence of hydrogen peroxide in water. For each material, the cells were obtained used to detect the presence of hydrogen peroxide in water. For each material, the cells were obtained as follows. follows. We We started started from from parallel parallelrectangular rectangularshaped shapedgold goldelectrodes, electrodes,spaced spacedby byhalf halfaamillimeter, millimeter, as previously evaporated in vacuum onto copier grade transparency sheets (Tartan); a drop of the previously evaporated in vacuum onto copier grade transparency sheets (Tartan); a dropsolution of the (either the “green” the “brown” was deposited the top of onethe of the electrodes; after solution (either theor“green” or theone) “brown” one) wasonto deposited onto topgold of one of the gold all the residual solvent evaporated, chitosan was deposited in drops in such a way as to form an ion electrodes; after all the residual solvent evaporated, chitosan was deposited in drops in such a way conducting bridge between the exposed gold electrode and the one covered with the chitosan based as to form an ion conducting bridge between the exposed gold electrode and the one covered with material. A schematic view of cell is shown Figure 1d. the chitosan based material. A the schematic view ofinthe cell is shown in Figure 1d.

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(a) Figure 1. Cont.

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Figure 1. Absorption spectrum of the chitosan aqueous solution (a); photo and absorption spectrum Figure 1. Absorption spectrum of the chitosan aqueous solution (a); photo and absorption spectrum of of the green (b), and of the brown (c) materials electrochemically derived from copper wire electrodes the green (b), and of the brown (c) materials electrochemically derived from copper wire electrodes immersed in chitosan solutions; (d) schematic view of the solid state cells based on chitosan. immersed in chitosan solutions; (d) schematic view of the solid state cells based on chitosan.

2.2. Characterization of Chitosan:CuOx Sensor 2.2. Characterization of Chitosan:CuOx Sensor The chitosan based materials developed as described in the previous section, were characterized chitosan based materials developed as described inin the previous section, byThe means of spectrophotometric measurements performed the visible range by were usingcharacterized an HR4000 by microspectrophotometer means of spectrophotometric measurements performed in the visible range by HR4000 (Ocean Optics, Largo, FL, US). Atomic Force Microscopy using (AFM)an analysis, microspectrophotometer (Ocean Optics,FlexAFM Largo, FL, US). Atomic (AFM) analysis, carried out by means of a Nanosurf equipped with aForce C3000Microscopy controller (Nanosurf AG, carried out by means of a Nanosurf FlexAFM equipped with a C3000 controller (Nanosurf AG, Liestal, Liestal, Switzerland), was performed to investigate the morphology of the chitosan based films, Switzerland), was performed to onto investigate morphology of the chitosan based deposited deposited from the solutions silicon the substrates. The electrical properties of films, the developed from the solutions onto siliconby substrates. The electrical propertiesperformed of the developed materials were investigated using impedance measurements, in air bymaterials means of were an Agilent 4284A LCRimpedance meter (Agilent Technologies, Santa Clara, CA,means US), in theAgilent frequency range investigated by using measurements, performed in air by of an 4284A LCR between 20 Hz and 1 MHz, with 100 mVCA, amplitude. Thefrequency impedancerange measurements were meter (Agilent Technologies, Santaa Clara, US), in the between 20 Hzperformed and 1 MHz, onasamples by drop-depositing materials fromwere the liquid phase, on onto the insulating gapby with 100 mVobtained amplitude. The impedancethe measurements performed samples obtained between parallel electrodes spaced by a few hundred microns, thermally on copier drop-depositing thegold materials from the liquid phase, onto the insulating gap evaporated between parallel gold grade sheets. electrodes spaced by a few hundred microns, thermally evaporated on copier grade sheets. Chitosan:CuOx Sensing Tests 2.3.2.3. Chitosan:CuOx Sensing Tests A 2400 source meter(Keithley, (Keithley,Cleveland, Cleveland, OH, OH, US) US) was was used A 2400 source meter used to to measure measurethe thecurrent currentofofthe the chitosan based solid state cells, in response to zero average triangular voltage inputs. Measurements chitosan based solid state cells, in response to zero average triangular voltage inputs. Measurements were carried different voltagetime timerates, rates,over overthe the±±1 V voltage voltage windows. were were carried outout at at different voltage 1V windows.Sensing Sensingtests tests were performed in deionized water, into which hydrogen peroxide, was injected by using a micro syringe, performed in deionized water, into which hydrogen peroxide, was injected by using a micro syringe, increasing its concentration in steps of 0.2 × 10−6 M. Ammonia and acetic acid where used for testing increasing its concentration in steps of 0.2 × 10−6 M. Ammonia and acetic acid where used for testing the behavior of the sensor in the presence of interferent analytes. the behavior of the sensor in the presence of interferent analytes. 3. Results and Discussion 3. Results and Discussion The electrochemically derived materials based on chitosan and copper were developed starting The electrochemically derived materials based on chitosan and copper were developed starting from chitosan acidic aqueous solutions transparent in the visible spectral range, as it is shown in the from chitosan acidic aqueous solutions transparent in the visible spectral range, as it is shown in the spectrum of Figure 1a. Depending on the pH of the chitosan acidic aqueous solution in which the spectrum of Figuresynthesis 1a. Depending on the of the chitosan are acidic aqueous in which the electrochemical takes place, twopH kinds of products obtained: thesolution clear green solution electrochemical synthesis takes place, two kinds of products are obtained: the clear green solution and and the dark brown suspension shown in the photos of Figure 1b,c. The nature of the two theelectrochemically dark brown suspension in the photos of Figure 1b,c. Thebasis nature theability two electrochemically derivedshown products can be understood on the of of the of chitosan to derived products can be understood on the basis of the ability of chitosan to coordinate coordinate transition metal ions, forming metal complexes, on the condition that the pHtransition of the metal ions, in forming complexes, on the condition that the pH ofrequirements. the solution Itin has which the solution which metal the chemical reaction takes place meets certain been chemical reactionthat takes place from meetscopper certain requirements. has beensolutions, demonstrated that starting demonstrated starting salts dissolved in Itchitosan’s in slightly acidic from copper salts in chitosan’s solutions, in slightly acidic copper condition [34], the polymeric condition [34], dissolved the polymeric amine groups coordinate bivalent ions, forming metal complexes. the contrary, in basic condition, complexes do not form,On andthe copper hydroxides amine groups On coordinate bivalent copper ions, these forming metal complexes. contrary, in basic precipitate. Thecomplexes chitosan–copper in two configurations differingThe from each other by condition, these do notcomplexes form, andexist copper hydroxides precipitate. chitosan–copper the number of in amino that coordinate thefrom metallic ions. Both metallic complexes have complexes exist two groups configurations differing each other bythese the number of amino groups

that coordinate the metallic ions. Both these metallic complexes have optical absorption spectra

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characterized by the presence of a broad band, ranging from the red to the near infrared spectral interval, ascribable to d–d electron transitions of the Cu2+ ions. Such a band is pH sensitive, Sensors 2017, 17, 2198 4 of as 12 it is found to shift from the near infrared to the visible region, as the pH of the solution increases. Based on absorptionthe spectra characterized byabsorption the presenceband, of a broad band,atranging the in redthe to the these optical considerations, intense and broad centered about from 760 nm optical near infrared spectral interval, ascribable to d–d electron transitions of the Cu2+ ions. Such a band is absorption spectrum shown of Figure 1b, can be attributed to the copper ions, confirming that the pH sensitive, as it is found to shift from the near infrared to the visible region, as the pH of the solution green solution is a copper–chitosan complex. On the other side, the band at about 760 nm is not increases. Based on these considerations, the intense and broad absorption band, centered at about observed in the spectrum of the brown material, in Figure 1c.attributed According to Omar al. [35], 760 nm in the optical absorption spectrum shownshown of Figure 1b, can be to the copperet ions, the near infrared band ascribable toisthe complexation complex. of copper chitosan, disappears when a confirming that the green solution a copper–chitosan Onwith the other side, the band at about reducing agent is used to convert the chitosan-copper complexshown into chitosan, and copper oxide. 760 nm is not observed in the spectrum of the brown material, in Figure copper, 1c. According to Omar et al. [35], the near band ascribable the complexation of copper with chitosan, This consideration, andinfrared the presence of a singletoabsorption band positioned at about 445disappears nm, similar to when by a reducing agent et is al. used to and convert the chitosan-copper complex into chitosan, copper, and that found Basumallick [36] ascribed to chitosan coated copper oxides, suggest that the copper oxide. This consideration, and the presence of a single absorption band positioned at about electrochemically synthesized brown material can be identified as chitosan dispersion of copper oxides 445 nm, similar to that by Basumallickderived et al. [36]brown and ascribed to chitosan coated copper particles. In particular, thefound electrochemically suspension, which from nowoxides, on will be suggest that the electrochemically synthesized brown material can be identified as chitosan referred to as chitosan:CuOx, is likely to be a chitosan dispersion of the red cuprous oxide (Cu2 O) and dispersion of copper oxides particles. In particular, the electrochemically derived brown suspension, the black coupric oxide (CuO), mixed with a predominance of the former one, as the brownish color of which from now on will be referred to as chitosan:CuOx, is likely to be a chitosan dispersion of the the material suggests. red cuprous oxide (Cu2O) and the black coupric oxide (CuO), mixed with a predominance of the The results of the AFM morphological confirm the hypothesis on the nature of the former one, as brownish color of theinvestigations material suggests. electrochemically derived materials. The films deposited from the The results of AFM morphological investigations confirm thecopper/chitosan hypothesis on thecomplex nature ofsolutions the electrochemically materials. films deposited from the copper/chitosan complex solutionsfilms are quite smooth, as isderived evidenced in theThe upper-rightmost portion of Figure 2a. On the contrary, are quite as isliquid evidenced in the portionshaped of Figure 2a. On the contrary, films size obtained fromsmooth, the brown reveal theupper-rightmost presence of regularly particles, having average obtained from the brown liquid reveal the presence of regularly shaped particles, having average size smaller than 200 nm, as is evidenced in the AFM micrographs of Figure 2b. smaller than 200 nm, as is evidenced in the AFM micrographs of Figure 2b.

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Figure 2. Atomic Force Microscopy (AFM) image close to the border of a thin film of chitosan-copper

Figure 2. Atomic Force Microscopy (AFM) image close to the border of a thin film of chitosan-copper complex deposited onto a silicon substrate (visible in the bottom left portion) (a); AFM image of the complex deposited onto a silicon substrate (visible in the bottom left portion) (a); AFM image of the chitosan:CuOx film(b). chitosan:CuOx film(b).

From the electrical point of view, depending on its average molecular weight, on its crystallinity, and on degree of acetilation, in the form behaves weight, as an ionic conductor. From theitselectrical point of view,chitosan depending onhydrated its average molecular on its crystallinity, Responsible for of charge transportchitosan are mobileinhydroxide ions that, in the presence water, originate and on its degree acetilation, the hydrated form behaves asofan ionic conductor. from the partial protonation of the amino groups bounded to the polymeric chain [36]. Figure 3 Responsible for charge transport are mobile hydroxide ions that, in the presence of water,shows originate the Cole plot of chitosan and of chitosan:CuOx. from the partial protonation of the amino groups bounded to the polymeric chain [36]. Figure 3 shows The opposite of the imaginary part of the impedance of chitosan, plotted versus the real one, the Cole plot of chitosan and of chitosan:CuOx. forms a wide semicircle, as happens in the case of samples having a parallel combination of resistance, The opposite ofWarburg the imaginary of the impedance chitosan, versus the real capacitance and elementspart as electrical equivalent. of Compared to plotted that of pure chitosan, the one, formschitosan:CuOx a wide semicircle, as happens in the case of samples having a parallel combination of resistance, Cole plot has a smaller semicircle, and therefore it is expected to have improved capacitance and Warburg elements as electrical equivalent. Compared to that of pure chitosan, the charge transport properties. chitosan:CuOx Cole plot has a smaller semicircle, and therefore it is expected to have improved charge transport properties.

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Figure 3. Opposite of the imaginary part of the impedance, plotted against the real part of the Figure 3. Opposite of the imaginary part of the impedance, plotted against the real part of the impedance, of chitosan and imaginary of chitosan:CuOx. Figure 3. of Opposite the part of the impedance, plotted against the real part of the impedance, chitosanofand of chitosan:CuOx. impedance, of chitosan and of chitosan:CuOx.

Figure 4a,b shows respectively the current–voltage plot of a couple of cells developed as Figure 4a,b shows current–voltage plot plot ofwith a couple of cells as sketched sketched in Figure 1c,respectively one having the the the goldcurrent–voltage electrode coated copper/chitosan complex and Figure 4a,b shows respectively ofthe a couple ofdeveloped cells developed as in the Figure 1c,having having the having gold electrode coated with the copper/chitosan complexcomplex and theand other other a chitosan:CuOx coated gold electrode. sketched inone Figure 1c, one the gold electrode coated with the copper/chitosan having a chitosan:CuOx coated gold electrode. the other having a chitosan:CuOx coated gold electrode.

(b) (a) (b) (a) Figure 4. (a) Current-voltage loop of a cell obtained using the electrochemically derived copperchitosan as the electrode; current-voltage plot of the Au/chitosan:CuOx/chitosan/Au cell (b). Figure 4.complex (a) Current-voltage loop cell obtained the electrochemically derived copperFigure 4. (a) Current-voltage loop of aof cella obtained usingusing the electrochemically derived copper-chitosan chitosan as the electrode; current-voltage plot of the Au/chitosan:CuOx/chitosan/Au cell (b). complex ascomplex the electrode; current-voltage plot of the Au/chitosan:CuOx/chitosan/Au cell (b).

The curves are obtained in deionized water, by measuring the current in response to zero average voltage inputs 40 s period voltagethe ratecurrent of change with time). Thetriangular curves are obtained in with deionized water,(±50 by mV/s measuring in response to After zero The curves are obtained in deionized water, by measuring the current in response to zero average aaverage transient phase involtage which inputs the current initially grows increasing current triangular with 40 s period (±50with mV/sthe voltage rate ofvoltage, changethe with time). starts After triangular voltage inputs with 40 sinperiod (± mV/s voltage rate of change with time). After a to cycle, forming loops shown Figure 4. 50 Current loops whenever the current is affected a transient phase the in which the current initially grows with theform increasing voltage, the current starts transient which the shown current initially grows with the increasing voltage, the current starts to not onlyphase by theinvoltage, but also byin the time rate of the voltage change: such a situation is commonly to cycle, forming the loops Figure 4. Current loops form whenever the current is affected cycle, forming thevoltage, loops shown inby Figure 4. Current loops form whenever the current affected observed when dealing with capacitive-resistive systems. This may be the case in the chitosan basednot not only by the but also the time rate of the voltage change: such a situation isiscommonly cells, double layer capacitance forms at the interface between electrode conducting only bywhere thewhen voltage, but also by the time rate ofsystems. the voltage change: such a situation is commonly observed dealing with capacitive-resistive This may the be the case inand the the chitosan based polyelectrolyte. In addition, wheneverforms the electrode contains electroactive undergoing redox observed when dealing with capacitive-resistive This maythe beelectrode thespecies case in the based cells, where double layer capacitance at thesystems. interface between and thechitosan conducting processes over the swept voltage range, current peaks arise. the polyelectrolyte. Inlayer addition, whenever the characteristic electrode contains electroactive species undergoing redox cells, where double capacitance forms at the interface between theusually electrode andObserving the conducting current–voltage plot of Figure 4a, a range, weak peak (marked by an arrow) can be undergoing noticed at about processes overInthe swept voltage characteristic current peaks usually arise. Observing the polyelectrolyte. addition, whenever theforward electrode contains electroactive species redox −45 mV and a weaker reverse peak is observed at about −450 mV: such a couple of peaks may current–voltage plot of Figure 4a, a weak forward peak (marked by an arrow) can be noticed at about processes over the swept voltage range, characteristic current peaks usually arise. Observingbethe 2+ consistent with the hypothesis of processes involving the Cu Compared that ofmay Figure −45 mV and a plot weaker reverse peak is observed about −450 mV: such a couple oftonoticed peaks be current–voltage of Figure 4a, aredox weak forwardat peak (marked by ions. an arrow) can be at about 2+ 4a, the I–V loop measured using the chitosan:CuOx modified electrode, shown in Figure 4b, has a consistent with the hypothesis of redox processes involving the Cu ions. Compared to that of Figure −45 mV and a weaker reverse peak is observed at about −450 mV: such a couple of peaks may five times larger current intensity, and exhibits a forward current peak positioned at 4b, 600has mV, 4a, the I–V loop measured using the chitosan:CuOx modified electrode, shown in Figure 2+ be consistent with the hypothesis of redox processes involving the Cu ions. Compared to thata of approximately. A shoulder, marked by arrow in 4b,current indicates thatpositioned a reverse current peak five times current intensity, andthe exhibits a Figure forward mV,4b, Figure 4a, thelarger I–V loop measured using the chitosan:CuOx modified peak electrode, shownatin600 Figure is likely positioned between marked 500 mV by andthe 600arrow mV. The separation of the forward and reverse current approximately. A shoulder, in Figure 4b, indicates that a reverse current peak has a five times larger current intensity, and exhibits a forward current peak positioned at 600 mV, peaks, their intensity ratiomV (considerably unity), indicate that the processes is likelyand positioned between 500 and 600 mV.below The separation of the forward andredox reverse current approximately. A shoulder, marked by the arrow in Figure 4b, indicates that a reverse current peak is responsible the intensity peaks are ratio irreversible. To investigate natureindicate of such processes, current–voltage peaks, and for their (considerably belowthe unity), that the redox processes likely positioned between 500 mV and 600 mV. The separation of the forward and reverse current peaks, measurements beenare performed using V amplitude voltage inputs, current–voltage with a different responsible for have the peaks irreversible. To1investigate thetriangular nature of such processes, and their (considerably below unity), indicate that the redox processes rate of intensity change have ofratio voltage with time.using Figure collects the results. Starting from with theresponsible inner loopfor measurements been performed 1 V5a amplitude triangular voltage inputs, a different themeasured peaks are irreversible. To investigate the nature of such processes, current–voltage measurements at theof lowest timewith rate time. of change (±0.6 outward (i.e.,the as the voltage rate of change voltage Figure 5a mV/s), collectsand theproceeding results. Starting from inner loop have been performed using 1 V amplitude triangular voltage inputs, with a different rate of change measured at the lowest time rate of change (±0.6 mV/s), and proceeding outward (i.e., as the voltage

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Sensors 2017, 17, 2198 12the of voltage with time. Figure 5a collects the results. Starting from the inner loop measured6 ofat Sensors 2017, 17, 2198 6 of 12 lowest time rate of change (±0.6 mV/s), and proceeding outward (i.e., as the voltage time rate of time rate of change increases), the and loops enlarge the increase current peaks intensity increase their move intensity and a change increases), theincreases), loops enlarge the currentand peaks towards time rate of change the loops enlarge and the current their peaks increaseand their intensity and move towards a larger voltage. Figure 5b shows the intensity of the forward current peak of Figure larger voltage. Figure 5b shows the intensity of the forward current peak of Figure 5a, plotted against move towards a larger voltage. Figure 5b shows the intensity of the forward current peak of Figure 5a, plotted against the root square of the modulus of the voltage rate of change with time. The the root square of the of the rate of of change with time. experimental dataThe are 5a,experimental plotted against themodulus root square ofvoltage the modulus the voltage rate The of change with time. data are distributed along a straight line, as is evidenced by the best fitting curve, distributed along a are straight line, as along is evidenced by the best fitting curve, indicating that a diffusion experimental data distributed a straight line, is evidenced byforward the best fitting curve, indicating that a diffusion limited electrochemical process isasresponsible for the current peak. limited electrochemical process is responsible for the forward current peak. According to the results indicating that a diffusion limited electrochemical process is responsible for the forward current peak. According to the results shown in Figures 4 and 5, both the chitosan complex and the chitosan:CuOx shown in Figures 4 and 5, both the chitosan complex and the chitosan:CuOx dispersions seem to be According to the results shown in Figures 4 and 5, both the chitosan complex and the chitosan:CuOx dispersions seem to be electroactive in the voltage window between ±1 V. We believe it could be of electroactive in thetovoltage window between ±1 V.the We believebetween it could be of We interest to investigate the dispersions be electroactive in the voltage window V. it could be interest toseem investigate the possibility to exploit solid state cells±1 based onbelieve such materials as of possibility to exploit the solid state cells based on such materials as electrochemical sensing systems. interest to investigate the possibility to exploit the solid state cells based on such materials as electrochemical sensing systems.

electrochemical sensing systems.

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Figure 5. (a) Current-voltage loop of the Au/chitosan:CuOx/chitosan/Au cell, (b) measured at different (a) Figure 5. (a) Current-voltage loop of the Au/chitosan:CuOx/chitosan/Au cell, measured at different scan speeds; (b) Intensity of the forward current peak of Figure 5a, plotted against the square root of scan speeds; (b) Intensity of the forward current peak of Figure 5a, plotted against the square root of Figure 5. (a) Current-voltage loop of the Au/chitosan:CuOx/chitosan/Au cell, measured at different the scan speed. the scan speed. scan speeds; (b) Intensity of the forward current peak of Figure 5a, plotted against the square root of

theRecently, scan speed. Geng et al. [33] have shown that the presence of hydrogen peroxide in water can be Recently, Gengelectrode et al. [33]measurements have shown that the of buffer, hydrogen peroxide in water can be detected by three in 0.1 Mpresence phosphate by using a copper complex Recently, Geng et al. [33] have shown that presence of hydrogen in can detected bychitosan three electrode measurements in 0.1 phosphate buffer, by peroxide using a copper complex based on applied on a titanium plate asthe aM sensitive electrode. Figure 6 shows thewater results of be detected three electrode in current-voltage 0.1aM phosphate buffer, by using a copper complex based on by chitosan appliedby onmeasurements a titanium plate as sensitive electrode. Figure 6 shows the results of sensing tests performed us, measuring the loop of a typical Au/chitosan–copper based on chitosan applied on a titanium plate as a sensitive electrode. Figure 6 shows the results of complex/chitosan/Au cell into deionized water, with and without 1 mM of hydrogen peroxide. It can sensing tests performed by us, measuring the current-voltage loop of a typical Au/chitosan–copper be noticed in response to H 2O2, the loop modifies: in particular, forward current peak (marked sensing teststhat performed byinto us, measuring the current-voltage loop of a of typical Au/chitosan–copper complex/chitosan/Au cell deionized water, with and without 1amM hydrogen peroxide. It can by an arrow 6), into arises at about 500 mV. In addition, it is found that the current–voltage loop complex/chitosan/Au cell water, with and without 1 mM of hydrogen peroxide. It can be noticed thatin inFigure response to Hdeionized O , the loop modifies: in particular, a forward current peak (marked by 2 2 turns back to its original shape (black line in Figure 6), when the solution containing hydrogen be noticed that in response to H 2 O 2 , the loop modifies: in particular, a forward current peak (marked an arrow in Figure 6), arises at about 500 mV. In addition, it is found that the current–voltage loop turns isin replaced with deionized water. byperoxide antoarrow Figure 6), arises atline about 500 mV. addition, it is found that thehydrogen current–voltage loop back its original shape (black in Figure 6), In when the solution containing peroxide is turns back to deionized its original shape (black line in Figure 6), when the solution containing hydrogen replaced with water. peroxide is replaced with deionized water.

Figure 6. Current-voltage loop of the Au/chitosan–Cu-complex/chitosan/Au cell, measured in pure water, and in the presence of 0.5 mM hydrogen peroxide.

It can6.be inferred that the solid state cell schematized in Figure 1d, having electrochemically Figure Current-voltage loop of the Au/chitosan–Cu-complex/chitosan/Au cell,the measured in pure Figure 6. Current-voltage loop of the Au/chitosan–Cu-complex/chitosan/Au cell, measured in pure derived chitosan-copper complex coated on the top of one of the gold electrodes, is able to detect water, and and in in the the presence presence of of 0.5 0.5mM mMhydrogen hydrogenperoxide. peroxide. water, H2O2 in water without the need of any supporting electrolyte, when the analyte’s concentration is in theItrange 10−3 M. Measurements performed exposing the cells to 1d, different concentrations of the can beofinferred that the solid state cell schematized in Figure having the electrochemically

derived chitosan-copper complex coated on the top of one of the gold electrodes, is able to detect H2O2 in water without the need of any supporting electrolyte, when the analyte’s concentration is in the range of 10−3 M. Measurements performed exposing the cells to different concentrations of the

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It can be inferred that the solid state cell schematized in Figure 1d, having the electrochemically derived chitosan-copper complex coated on the top of one of the gold electrodes, is able to detect Sensors 2017, 17, 2198 7 of 12 H 2 O2 in water without the need of any supporting electrolyte, when the analyte’s concentration is in − 3 the range of 10 M. Measurements performed exposing the cells to different concentrations of the analyte show show that that the the solid solid state state cells cells based based on on the the chitosan-copper chitosan-copper complex complex reversibly reversibly respond respond in in aa analyte reproducible way way to to hydrogen reproducible hydrogen peroxide peroxide in in the the millimolar millimolar range, range, with with aa limit limit of of detection detection of of 0.2 0.2 mM. mM. Sensing tests performed on solid state cells having chitosan:CuOx as the active electrode, reveal that Sensing tests performed on solid state cells having chitosan:CuOx as the active electrode, reveal that the copper copper oxide oxide dispersions dispersions have have higher higher sensitivity sensitivity towards towards H H2O O2 compared to the copper–chitosan the 2 2 compared to the copper–chitosan complex. The set of measurements of Figure 7a are performed by exposing aa solid solid state state cell, cell, having having complex. The set of measurements of Figure 7a are performed by exposing chitosan:CuOx as the active electrode, to hydrogen peroxide at a concentration that increases in steps chitosan:CuOx as the active electrode, to hydrogen peroxide at a concentration that increases in steps of 0.2 0.2 µM. μM. Unexpectedly, Unexpectedly,however, however,starting startingfrom from current-voltage loop measured in pure water, of thethe current-voltage loop measured in pure water, the the reverse current progressively decreases and the original forward current peak rapidly weakens reverse current progressively decreases and the original forward current peak rapidly weakens as the as the hydrogen peroxide concentration increases. In particular, the intensity of the forward hydrogen peroxide concentration increases. In particular, the intensity of the forward currentcurrent peaks, peaks, plotted against the hydrogen peroxide concentration, as shown in Figure 7b, decreases in a plotted against the hydrogen peroxide concentration, as shown in Figure 7b, decreases in a linear way linear way with a slope of about 1 μA per 0.1 μM of H 2O2. On the contrary, according to the scientific with a slope of about 1 µA per 0.1 µM of H2 O2 . On the contrary, according to the scientific literature, literature, electrochemical measurements out in the of electrolytes supporting show electrolytes show electrochemical measurements carried out carried in the presence of presence supporting that sensors that sensors based graphite and glassy modified carbon electrodes modified with copper[37–39], oxide based on graphite andonglassy carbon electrodes with copper oxide nanoparticles nanoparticles [37–39], and with nanosized copper oxide dispersed into mixtures of multiwalled and with nanosized copper oxide dispersed into mixtures of multiwalled carbon nanotubes and carbon nanotubes and chitosan [40], doamounts respondoftohydrogen increasing amounts of hydrogen peroxide with chitosan [40], do respond to increasing peroxide with an increasing current. an increasing current.

(a)

(b)

Figure 7. 7. (a) loop of of the theAu/chitosan:CuOx/chitosan/Au Au/chitosan:CuOx/chitosan/Au cell, Figure (a) Current-voltage Current-voltage loop cell,measured measured in in water, water, and and in the the presence presence of of hydrogen hydrogen peroxide peroxide at at different different concentrations; concentrations; (b) (b) Intensity Intensity of of the the forward forward current current in peak intensities intensities of of Figure Figure 7a, 7a, plotted plotted against against the the H H2O O2 concentration. peak concentration. 2

2

The experimentally observed behavior of Figure 7 suggests that somehow the electrochemically The experimentally observed behavior of Figure 7 suggests that somehow the electrochemically derived chitosan:CuOx, applied to the top of the gold electrode, as schematized in Figure 1d, does derived chitosan:CuOx, applied to the top of the gold electrode, as schematized in Figure 1d, does interact with H2O2, but the way in which this interaction manifests itself is a sort of reversible interact with H2 O2 , but the way in which this interaction manifests itself is a sort of reversible quenching of the electroactivity of chitosan:CuOx. quenching of the electroactivity of chitosan:CuOx. Aimed at evaluating the behavior of the Au/chitosan:CuOx/chitosan/Au cells towards analytes Aimed at evaluating the behavior of the Au/chitosan:CuOx/chitosan/Au cells towards analytes other than H2O2, sensing tests have been carried out in the presence of a base and of a weak acid, other than H2 O2 , sensing tests have been carried out in the presence of a base and of a weak acid, namely ammonia and acetic acid. The results are shown in Figures 8 and 9 respectively. It can be namely ammonia and acetic acid. The results are shown in Figures 8 and 9 respectively. It can be noticed that in both cases the forward current peak found at about 600 mV in the current–voltage noticed that in both cases the forward current peak found at about 600 mV in the current–voltage loop of Au/chitosan:CuOx/chitosan/Au in water disappears and new current peaks, the positions of loop of Au/chitosan:CuOx/chitosan/Au in water disappears and new current peaks, the positions of which depend on the analyte, arise. In particular, Figure 8a shows that when exposed to ammonia in which depend on the analyte, arise. In particular, Figure 8a shows that when exposed to ammonia the micromolar range, the current–voltage loops of the Au/chitosan:CuOx/chitosan/Au cells enlarge, in the micromolar range, the current–voltage loops of the Au/chitosan:CuOx/chitosan/Au cells and a forward current peak arises, the intensity of which is linearly related to the ammonia enlarge, and a forward current peak arises, the intensity of which is linearly related to the ammonia concentration, as the results of Figure 8b suggest. In the presence of acetic acid, as is illustrated in concentration, as the results of Figure 8b suggest. In the presence of acetic acid, as is illustrated in Figure 9a, a couple of forward and reverse current peaks arise, and again, according to the results Figure 9a, a couple of forward and reverse current peaks arise, and again, according to the results shown in Figure 9b, the intensity of the forward current peak is found to be linearly related to the acetic acid concentration. In both the examined cases, the observed modification is reversible, as after a transient time the current-voltage loops turn back to their original shape and intensity by immersing the cells in deionized water. These latter results, being the investigated analytes a base and a weak acid, could be explained in terms of the pH sensitivity of nanosized copper oxide [41], which may

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Sensors 2017, 17, 2198 11 persist when the material is dispersed in the chitosan host and used in the solid state8 of cell persist when the material is dispersed in the chitosan host and used in the solid state cell configuration of Figure 1d. configuration of Figure 1d. Thein fact that9b,inthe the case of of H2O2 forward the amplitude the ispeaks increasing shown Figure currentofpeak foundistodecreasing be linearlyfor related to the The fact that in theintensity case of Hthe 2O2 the amplitude of the peaks is decreasing for increasing concentration while the opposite occurs in the case of a weak acid and a base, can be used to acetic acid concentration. In both the examined cases, the observed modification is reversible, as after concentration while the opposite occurs in the case of a weak acid and a base, can be used to discriminate thethe presence of hydrogen peroxide with respect to other species. With by regard to the adiscriminate transient time current-voltage loops turn back to their original shape and intensity immersing the presence of hydrogen peroxide with respect to other species. With regard to the feasibility employing the investigated structure for the the realization ofanalytes an H2O2 sensor, we must the cells inof deionized water. latter results, being and a weak feasibility of employing the These investigated structure for the investigated realization of an H2Oa2 base sensor, we must observe that,beclearly, the fact that the decreases of with increasing concentration sets an upper acid, could explained in terms of response the pH sensitivity nanosized copper oxide [41], which may observe that, clearly, the fact that the response decreases with increasing concentration sets an upper limit to the maximum detectable concentration and, hence, to the dynamic range. This possible persist materialdetectable is dispersed in the chitosan hosthence, and used in the solid state cell This configuration limit towhen the the maximum concentration and, to the dynamic range. possible disadvantage is partially compensated by the high sensitivity that is observed at lower of Figure 1d. disadvantage is partially compensated by the high sensitivity that is observed at lower concentrations, as summarized in Table 1. concentrations, as summarized in Table 1.

(a) (a)

(b) (b)

Figure 8. (a) loop of of the Au/chitosan:CuOx/chitosan/Au cell, measured in pureinwater, Figure (a)Current-voltage Current-voltage loop Au/chitosan:CuOx/chitosan/Au cell, measured pure Figure 8. 8. (a) Current-voltage loop of thethe Au/chitosan:CuOx/chitosan/Au cell, measured in pure water, and in and the presence of ammonia at different concentrations; (b) Intensity of the of forward current peak water, in the presence of ammonia at different concentrations; (b) Intensity the forward current and in the presence of ammonia at different concentrations; (b) Intensity of the forward current peak intensities of Figure (a), plotted against the NH peak intensities of Figure (a), plotted against the3 concentration. NH3 concentration. intensities of Figure (a), plotted against the NH3 concentration.

(a) (a)

(b) (b)

Figure 9. (a) Current-voltage loop of the Au/chitosan:CuOx/chitosan/Au cell, measured in pure water, Figure 9. 9. (a) Current-voltage loop ofof thethe Au/chitosan:CuOx/chitosan/Au cell, measured in pureinwater, Figure Current-voltage loop Au/chitosan:CuOx/chitosan/Au measured pure and in the (a) presence of acetic acid at different concentrations; (b) Intensity of thecell, forward current peak and inand the presence of acetic acid atacid different concentrations; (b) Intensity of theof forward current peak water, in the presence of acetic at different concentrations; (b) Intensity the forward current intensities of Figure 9a, plotted against the acetic acid concentration. intensities of Figure 9a, plotted against the acetic acid concentration. peak intensities of Figure 9a, plotted against the acetic acid concentration.

The fact that in the case of H2 O2 the amplitude of the peaks is decreasing for increasing concentration while the opposite occurs in the case of a weak acid and a base, can be used to discriminate the presence of hydrogen peroxide with respect to other species. With regard to the feasibility of employing the investigated structure for the realization of an H2 O2 sensor, we must observe that, clearly, the fact that the response decreases with increasing concentration sets an upper limit to the maximum detectable concentration and, hence, to the dynamic range. This possible disadvantage is partially compensated by the high sensitivity that is observed at lower concentrations, as summarized in Table 1.

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Table 1. Sensitivity of the Au/chitosan:CuOx/chitosan/Au cell towards H2 O2 , compared to that of other electrochemical sensors. Sensor Au/chitosan:CuOx/chitosan/Au Glassy carbon electrode modified with copper nanoparticles decorated silver nanoleaves Glassy carbon electrode modified with FeS nanosheets Carbon paste electrode modified Ni-Al/layered double hydroxide/Ag nanoparticles Glassy carbon electrode modified with Ag@C core-shell nanomaterials Graphene oxide electrode modified with Binary Mn-Co Oxides

Sensitivity (µA·mM−1 cm−2 )

Ref.

10,000

This work

6190

[42]

36.4

[43]

1.836

[44]

22.94 53.65

[45] [46]

4. Conclusions It is demonstrated that solid state cells with simple design, having electrochemically derived materials based on copper and chitosan applied to the top of one gold electrode, can be successfully used to detect the presence of hydrogen peroxide in water, at concentrations below 1 µM, without the need of any supporting electrolyte. While characterized by high sensitivity, the cells using chitosan:CuOx dispersions are found to respond to the presence of H2 O2 with a decreasing current, unlike other conventional electrochemical sensors based on nanosized copper oxides. The difference between the two kinds of behavior can be likely ascribed to the fact that copper oxide still mantaints its electroactivity towards H2 O2 but the way in which the material responds to the analyte is affected by the absence of a supporting electrolyte. Author Contributions: This paper is the result of a strong collaborative effort among all authors. Antonella Arena and Graziella Scandurra designed the study and performed most of the experimental work. Carmine Ciofi contributed in the discussion of the experimental data. All authors were involved in the writing and argumentation of the paper and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflict of interests.

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