Effect of Nanoparticles on Modified Screen Printed Inhibition Superoxide Dismutase Electrodes for Aluminum

sensors Article Effect of Nanoparticles on Modified Screen Printed Inhibition Superoxide Dismutase Electrodes for Aluminum Miriam Barquero-Quirós 1,* and María Julia Arcos-Martínez 2 1Department of Chemistry, University of Costa Rica, CELEQ, San Pedro de Montes de Oca, San José 11501-2060, Costa Rica 2Department of Chemistry, Faculty of Sciences, University of Burgos, Plaza Misael Bañuelos s/n, Burgos 09001, Spain; jar[email protected] *Correspondence: [email protected]; Tel.: +506-2511-2442; Fax: +506-2511-2446 Academic Editor: Roberto Pilloton Received: 26 July 2016; Accepted: 20 September 2016; Published: 26 September 2016 Abstract: A novel amperometric biosensor for the determination of Al(III) based on the inhibition of the enzyme superoxide dismutase has been developed. The oxidation signal of epinephrine substrate was affected by the presence of Al(III) ions leading to a decrease in its amperometric current. The immobilization of the enzyme was performed with glutaraldehyde on screen-printed carbon electrodes modifiedwith tetrathiofulvalene (TTF) and different types ofnanoparticles. Nanoparticles of gold, platinum, rhodium and palladium were deposited on screen printed carbon electrodes by means of two electrochemical procedures. Nanoparticles were characterized trough scanning electronic microscopy, X-rays fluorescence, and atomic force microscopy. Palladium nanoparticles showed lower atomic force microscopy parameters and higher slope of aluminum calibration curves and were selected to perform sensor validation. The developed biosensor has a detection limit of 2.0 ±0.2 µM for Al(III), with a reproducibility of 7.9% (n = 5). Recovery of standard reference material spiked to buffer solution was 103.8% with a relative standard deviation of 4.8% (n = 5). Recovery of tap water spiked with the standard reference material was 100.5 with a relative standard deviation of 3.4% (n = 3). The study of interfering ions has also been carried out. Keywords: superoxide dismutase biosensor; aluminum; tetrathiofulvalene; screen-printed electrodes; nanoparticles 1. Introduction Aluminum toxicity has been shown in vivo and in vitro , but complexity of its interactions with human organism makes it very difficult to assign the responsibility in Alzheimer’s disease. It can be considered as the combined effect of oxidant action, participation on amyloid cascade, neuronal degeneration [ 1 ] and accumulation in neurofibrillary tangles. Aluminum presence favors T protein link trough phosphate bridge [ 2 ] and alters homeostatic ion equilibrium [ 3 ], and it has shown a strong effect on reactive oxygen species (ROS) production on living organisms, due to iron accumulation in oxidative stress [ 4 ]. Markedly, ROS impaired enzymes such as superoxide dismutase (SOD) and catalase react with radical species such as O 2•− , OH •− , and ONNO •− . Although O 2•− radical is not so reactive itself, in the presence of Fe 2+ , Fenton reaction can turn it into hydroxyl radical, which is the most potent radical. These findings show that O 2•− is involved in cellular damage. Biosensors have employed superoxide dismutase (SOD), an enzyme that scavenges superoxide to measure superoxide anion accordingly with the reactions [5]: Cu2+ −SOD + O2•−→Cu1+ −SOD + O2 Sensors 2016,16, 1588; doi:10.3390/s16101588 www.mdpi.com/journal/sensors Sensors 2016,16, 1588 2 of 19 Cu1+ −SOD + O2•−+ 2H+→Cu2+ −SOD + H2O2 O 2•− affects cytochrome c (Cyt c) by oxidizing Fe 2+ to Fe 3+ and reducing itself to H 2 O 2 . O2•−radical levels were found to be elevated in homogenized cancerous brain tissue compared to normal human brain tissue [ 6 ]. O 2•− is formed in living biological systems by the donation of an electron to molecular oxygen, through oxidation of semiquinone-type radicals formed in the mitochondrial electron transport chain. In its presence, free radical scavengers, enzymes such as superoxide dismutase and glutathione peroxidase (GSH-Px), decrease their antioxidant status, and lipid peroxide levels are increased. Aluminum administration to laboratory animals induces SOD dysfunction and damage on target organs [ 7 – 10 ]. It has been found that zinc [ 11 ], selenium [ 12 ] and therapies used against Alzheimer’s disease [ 13 – 16 ] have a protective role against aluminum induced toxicity, improving SOD function. Aluminum presence in humans is associated with oxidative stress [17,18]. Biosensors constructed with screen printed electrodes (SPCE) offer low detection limits, easier assembly of metallic nanoparticles (NPs), good reproducibility, low contamination and excellent biocompatibility with enzymes and antibodies [ 18 ]. The metal NPs’ small size, high mechanical strength and high chemical and thermal stability allow them, when acting as enzyme-carrier materials, to improve the efficiency of immobilized enzymes, facilitating reaction kinetics, and supplying a larger surface area, leading to higher enzyme loading per unit mass of particles. This fact allows achieving enhanced device sensitivity and reduced mass transfer resistance [ 19 ]. Besides, NPs increased electric conductivity and electron transfer between redox enzyme center and electrode [20,21]. Different types of NPs such as metal, metal oxides, semiconductors, polymers and composite-metal NPs have been used to assembly miniaturized electrochemical sensors and biosensors. Gold NPs (AuNPs), due to their unique properties, relatively low cost and ease of preparation, are the most used in many biochemical applications [ 22 , 23 ]. AuNPs can be synthesized by different chemical methods and applied to electrochemical detection of As(III) [ 24 ]. AuNPs use in amperometric biosensors and electrochemical techniques enhances detection sensitivity [25–27]. Due to their inertness, platinum NPs (PtNPs) are the principal metal NPs alternative for anodic current measurement and have been applied to formaldehyde [ 28 ], neurotransmitters [ 29 ], glucose [ 30 ], uric acid [31], and As(III) determination [32]. Glassy carbon electrodes modified with palladium nanoparticles (PdNPs) have been applied to catecholamines determination [ 33 ]. PtNPs and PdNPs/methylthiophene (PMT) sensors have been applied to dopamine (DA) and AA determination [34]. Rhodium NPs’ (RhNPs) main application has been as precursors for the preparation of catalytics [ 35 , 36 ] and for catalysis and sensing of cytochrome c [ 37 ] and H 2 O 2 [ 38 ], as well as for biosensing of α-ketoglutarate [39]. Due to sensitivity and specificity joint benefits produced by modified SPCEs with metallic NPs and enzymes, this research work was conducted with the goal to compare the effect of NPs of Au, Pt, Pd and Rh deposited by distinct electrochemical procedures on sensitivity of amperometric inhibition SOD by aluminum, with TTF as mediator using epinephrine (EPI) as substrate. Most developed biosensors that immobilize SOD enzyme in a carrageenan gel are based on H 2 O 2 amperometric detection [ 40 ] and were successfully applied as a tool for antioxidant capacity assessment to evaluate red and white wines [41], fresh herbs and fruits, olives, tea [42–44], algae [45], phytoterapeutic preparations [ 46 ], drugs containing salicylic and as corbic acid, and β -carotene [ 40 , 47 ]. This developed biosensor enables measurement of antioxidant capacity of healthy and diseased tissues in vitro [40], and was also applied to determination of total antioxidant capacity of berries [48]. The others SOD based biosensors that employ modified solid electrodes are shown in Table 1. Sensors 2016,16, 1588 3 of 19 Table 1. SOD based biosensors applied to samples of biological interest. Technique Electrode Potential Modification Range LOD Sample/Analite Reference CV 1SPCE −0.8–+0.8 V Pyrrole/SAMs 0.5 ×10−9–5 M 0.5 ×10−9Mcultured human keratinocytes NO2-[49] Amperometry CFME 2+0.25 V cysteine/AuNPs (13–104) ×10−9MO2•−[50] Amperometry SPCE −0.1 V porous Pt-Pd/nafion (16–1536) ×10−6M 0.13 ×10−6MCell culture medium/O2•−[51] CV 1Chronoamperometry Gold electrode −0.2–+0.5 V Au/Cys/SOD 9, Au/GNP/Cys/SOD 10 and Au/GNP/Cys/SOD/Chit 11 0.5–4 Gy 0.03 ×10−6Mthallium 201/water [52] Amperometry GC CFME 20.2 V NTA/HT 710−7–10−4M 21 ×10−9Mbrain tissue/O2•−[53] CV 1 Carbon paste electrode Electrochem 0–0.3 V cytochrome c in solution and Fe(III)-protoporphyrin immobilized (1–6) ×10−3M 0.3 ×10−3MXantine [54] System Carbon paste electrode Protoporphyrin and cytochrome both immobilized (1–8) ×10−3M 0.2 ×10−3M Cronoamperometry Composite electrode −0.3 V PtPd-PDARGO 6(0.016–0.24) ×10−3M 2 ×10−6M DMEM 5/O2•−[55] Amperometry GC 8−0.3 V MWCNT 4(0.01–0.3) ×10−3M1×10−6MWines, berry juice/O2•−[56] PEDOT 3 Amperometry SPCTTFE12 0.2 V SOD/PdNP 13 (1.0–60) ×10−5M2×10−6M Al(III) This article water samples 1 Cyclic voltammetry; 2 carbon fiber microelectrode; 3 poly(3,4-ethylenedioxythiophene); 4 multiwalled carbon nanotubes, 5 Dulbecco’s modified Eagle’s medium; 6 PtPd poly dopamine reduced graphene oxide; 7 Nitrilotriacetic acid/histidine-tag; 8 glassy carbon; 9 gold, cysteine, super oxido dismutase; 10 gold, gold nanoparticles cysteine, super oxido dismutase; 11 gold, gold nanoparticles cysteine, super oxido dismutase, chitosane; 12 screen printed carbon TTF5% electrode; 13 super oxide dismutase, palladium nanoparticles. Sensors 2016,16, 1588 4 of 19 Taking into account that TTF allows the rapid electron transfer between SOD and electrode surface can be carried out at lower potential [ 57 ], and that pro-oxidant activity of aluminum inhibits SOD activity [ 58 ], this study indicates that Al(III) SOD inhibition can be performed at lower potential compared to other aluminum enzymatic determinations. It was shown for the first time using SPC TTF Es that Al(III) inhibits SOD enzyme linked with Alzheimer’s disease at low concentrations. 2. Materials and Methods 2.1. Reagents All solutions were prepared with purified water supplied by TKA Gen Pure, inverse osmosis, with a UV lamp irradiation system. SOD enzyme (30 KU), EPI, bovine serum albumine (BSA), glutaraldehyde (GA) and hydrogen tetrachloroaurate (III) trihydrate (HAuCl 4 ) were obtained from Sigma-Aldrich (Sigma-Aldrich), Steinheim, Germany). Solutions of platinum, rhodium and palladium 0.1 mM were prepared from ICP solutions of 1000 mg/L (Merck, Darmstad, Germany). Titrisol solutions from (Merck, Darmstad, Germany)were used to prepare stock standard solutions of Al(III), Cu(II), Fe(III), Sn(II), Zn(II), Co(II), Ni(II), Se(IV) Cr(III), Cd(II), Pb(II), W(VI) and V(V). Mo(VI) and Ca(II) solutions were obtained from Inorganic Ventures (Lakewood, NJ, USA). As(V) and Hg(II) solutions were prepared from Atomic Spectroscopy Standards solutions (Perkin Elmer Co, Whaltham, MA, USA). Al(III) solutions used for spike were prepared from High Purity Standards (Charleston, SC, USA) confirmed against standard reference material SRM 3101. Britton Robinson (BR) supporting electrolyte solutions were prepared as usual with boric, phosphoric and acetic acids (Merck, Darmstadt, Germany), and the required pH was obtained by adjusting with NaOH solution (Suprapur, Merck, Darmstadt, Germany). Several inks were used in the fabrication of SPEs, namely Electrodag PF-407 A (carbon ink), Electrodag 6037 SS (silver/silver chloride ink) and Electrodag 452 SS (dielectric ink) supplied by Acheson Colloiden (Acheson Colloiden, Scheemda, The Netherlands). The working electrode ink was prepared by thoroughly mixing carbon ink with tetrathiofulvalene (CTTF) 5%. TTF was obtained from Acros Organics (Acros Organics, Geel, Belgium). 2.2. Equipment An electrochemical system Autolab PGSTAT Echo Chemie128 N with GPS software was used to record electrochemical measurements (Echo Chemie, Utrech, The Netherlands). All pH values were adjusted with a pHmeter (Mettler Toledo, Schwerzenbach, Switzerland). A S-3700 Hitachi was used to perform scanning electronic microscopy (SEM) of SPCEs. An IXRF Systems model 550iwas used to obtain spectra of elements on the SPCE. Atomic force microscopy (AFM) parameters and images were obtained with a NanoScopeQuadrex Digital Instruments Veeco Metrology Group. SPCTTFEs Construction SPC TTF Es were homemade built using a DEK 248 printing machine (DEK, Weymouth, UK) using polyester screens with appropriate stencil designs mounted at 45 ◦ to the printer stroke. These transducers consisted of three screen-printed electrodes deposited onto polyethylene terephthalate films (HiFi Industrial Film, Dardilly, France). The different inks were screen-printed and cured according to the manufacturer’s specifications. The working electrode ink was prepared by thoroughly mixing carbon ink with TTF (5% v/w) and immediately screen-printed. One electrode is shownin (Figure 1). Sensors 2016,16, 1588 5 of 19 Sensors 2016, 16, 1588 5 of 18 Figure 1. Screen printed electrodic system used. 2.3. Nanoparticles Electrodeposition Methods SPCTTFEs modification with nanoparticles (NPs/SPCTTFEs) was carried out by both controlled potentialand cyclic voltammetry scan methods. (A) Metal plating was carried at two different potentials namely +0.3 and +0.18 V, in a quartz cell containing Au(III), Pt(IV), Rh(IV) or Pd(IV) solutions (0.1 mM) in H2SO4 (0.5 M) [24]. Following electrodeposition process, the NPs/SPCTTFEs was removed from platting solution, rinsed with purified water and wiped carefully. (B) Cyclic voltammetry deposition was performed doing a set of seven successive voltammetric scans between +1.0 and −0.2 Vin a quartz cell containing Au(III), Pt(IV), Rh(IV) or Pd(IV) (0.1 mM) in H2SO4 (0.5 M) [59]. Electrodes were prepared by setting two cyclic voltammetric conditions namely CV1 and CV2. CV1: delay time 60 s, step potential 0.0150 V, scan rate 0.050 V/s CV2: delay time 120 s, step potential 0.025 V, scan rate 0.100 V/s. After nanoparticles deposition, the electrode was rinsed with purified water and wiped carefully. 2.4. SOD Enzyme Immobilization onto AuNPs//SPCTTF Es Enzyme was immobilized by crosslinking polymerization with glutaraldehyde [60] on the surfaces of AuNPs/SPCTTFEs, PtNPs/SPCTTFEs, PdNPs/SPCTTFEs, and RhNPs/SPCTTFEs. To carry out the immobilization procedure, superoxide dismutase enzyme solution was prepared by dissolving enzyme in Britton Robinson buffer at pH 7.0. To avoid loss of enzymatic activity, BSA was used in a mixture made of 20 μL of SOD (5.9 mg/mL), 10 μL of BSA (1.69% w/v) and 10 μL of GA (2.5% v/v) [61]. This mixture was dropped onto the surface electrode and stored at 4 °C before used and between measurements. The modified electrode was washed with purified water, before and after use. 3. Results 3.1. Optimization of Experimental Parameters EPI originates an amperometric signal at NPs/SPCTTFE with SOD enzyme immobilized (SOD/PdNPs/SPCTTFE), after which a steady-state current is reached. The presence of Al(III) ions produces SOD enzyme inhibition which causes a decrease in the EPI amperometric signal. Al(III) concentration influence inhibition process and can be quantitatively evaluated determining the difference between the steady state current in absence of Al(III), (I0), and the steady state current in the presence of Al(III), (I) namely Δ(I0-I).Accordingly, with the following working principle proposed in Scheme 1, a SOD based biosensor, with TTF incorporated in electrode ink, has been developed. Figure 1. Screen printed electrodic system used. 2.3. Nanoparticles Electrodeposition Methods SPC TTF Es modification with nanoparticles (NPs/SPC TTF Es) was carried out by both controlled potentialand cyclic voltammetry scan methods. (A) Metal plating was carried at two different potentials namely +0.3 and +0.18 V, in a quartz cell containing Au(III), Pt(IV), Rh(IV) or Pd(IV) solutions (0.1 mM) in H 2 SO 4 (0.5 M) [ 24 ]. Following electrodeposition process, the NPs/SPC TTF Es was removed from platting solution, rinsed with purified water and wiped carefully. (B) Cyclic voltammetry deposition was performed doing a set of seven successive voltammetric scans between +1.0 and − 0.2 Vin a quartz cell containing Au(III), Pt(IV), Rh(IV) or Pd(IV) (0.1 mM) in H 2 SO 4 (0.5 M) [ 59 ]. Electrodes were prepared by setting two cyclic voltammetric conditions namely CV1 and CV2. CV1: delay time 60 s, step potential 0.0150 V, scan rate 0.050 V/s. CV2: delay time 120 s, step potential 0.025 V, scan rate 0.100 V/s. After nanoparticles deposition, the electrode was rinsed with purified water and wiped carefully. 2.4. SOD Enzyme Immobilization onto AuNPs//SPCTTF Es Enzyme was immobilized by crosslinking polymerization with glutaraldehyde [ 60 ] on the surfaces of AuNPs/SPC TTF Es, PtNPs/SPC TTF Es, PdNPs/SPC TTF Es, and RhNPs/SPC TTF Es. To carry out the immobilization procedure, superoxide dismutase enzyme solution was prepared by dissolving enzyme in Britton Robinson buffer at pH 7.0. To avoid loss of enzymatic activity, BSA was used in a mixture made of 20 µ L of SOD (5.9 mg/mL), 10 µ L of BSA (1.69% w/v) and 10 µ L of GA (2.5% v/v) [ 61 ]. This mixture was dropped onto the surface electrode and stored at 4 ◦ C before used and between measurements. The modified electrode was washed with purified water, before and after use. 3. Results 3.1. Optimization of Experimental Parameters EPI originates an amperometric signal at NPs/SPC TTF E with SOD enzyme immobilized (SOD/PdNPs/SPC TTF E), after which a steady-state current is reached. The presence of Al(III) ions produces SOD enzyme inhibition which causes a decrease in the EPI amperometric signal. Al(III) concentration influence inhibition process and can be quantitatively evaluated determining the difference between the steady state current in absence of Al(III), (I 0 ), and the steady state current in the presence of Al(III), (I) namely ∆ (I 0 -I).Accordingly, with the following working principle proposed in Scheme 1, a SOD based biosensor, with TTF incorporated in electrode ink, has been developed. Sensors 2016,16, 1588 6 of 19 Sensors 2016, 16, 1588 6 of 18 Scheme 1. Oxidation of EPI on SOD/SPC TTF E. The parameter Δ(I 0 -I) depends on EPI concentration, applied potential (Eap) and pH solution. Therefore, an optimization of these variables was performed in order to ensure the quality of the results. Because the dependence between Δ(I 0 -I) and Al(III) concentration is linear, substrate response was obtained from pH 5.0 to pH 8.0, and a pH of 5.0 was selected regarding substrate stability to autoxidation. In the same way, substrate response was obtained from +0.20 V to +0.60 V, and a potential of 0.2 V was selected driving substrate oxidation to epinephrinequinone [57]. Then, several aluminum inhibition calibration curves were performed at different potential and pH values and their slopes were compared, in order to obtain Al(III) inhibition effect with pH and Eap. Slope calibration curve with pH was calculated from pH 5.0 to pH 8.0. In the same way, slope calibration curve with potential was calculate from +0.20 V to +0.60 V. Higher slope values were obtained at pH 5.0 and Eap of +0.2 V, so these conditions were chosen to perform Al(III) inhibition calibration curves. Slopes of calibration curves with potential and pH are shown in Figure 2. Figure 2. (a) Slope values of Al(III) calibration curves with pH; and (b) Slope value of Al(III) calibration curves with applied potential. [EPI] = 1.6 × 10 −4 M; Britton Robinson buffer pH 5.0, Eap = +0.2 V vs. Ag/AgCl. Findings indicated that substrate stability improved at low values of pH and potential; furthermore, if applied potentials were higher than +0.6 V, the electrodes showed erratic behavior. Since substrate response increases with concentration, a value of 1.6 × 10 −4 M for EPI was chosen, as this concentration gives a proper sensibility, and a very stable signal with very low noise. Upper concentrations produced higher noise on amperometric recording of calibration curves. Under the selected conditions, the electrodes showed good performance. Calibration curves of Al(III) using SOD/AuNPs/SPC TTF Es, SOD/PtNPs/SPC TTF Es, SOD/PdNPs/SPC TTF Es, and SOD/RhNPs/SPC TTF Es were obtained under the optimized conditions. Scheme 1. Oxidation of EPI on SOD/SPCTTFE. The parameter ∆ (I 0 -I) depends on EPI concentration, applied potential (Eap) and pH solution. Therefore, an optimization of these variables was performed in order to ensure the quality of the results. Because the dependence between ∆ (I 0 -I) and Al(III) concentration is linear, substrate response was obtained from pH 5.0 to pH 8.0, and a pH of 5.0 was selected regarding substrate stability to autoxidation. In the same way, substrate response was obtained from +0.20 V to +0.60 V, and a potential of 0.2 V was selected driving substrate oxidation to epinephrinequinone [ 57 ]. Then, several aluminum inhibition calibration curves were performed at different potential and pH values and their slopes were compared, in order to obtain Al(III) inhibition effect with pH and Eap. Slope calibration curve with pH was calculated from pH 5.0 to pH 8.0. In the same way, slope calibration curve with potential was calculate from +0.20 V to +0.60 V. Higher slope values were obtained at pH 5.0 and Eap of +0.2 V, so these conditions were chosen to perform Al(III) inhibition calibration curves. Slopes of calibration curves with potential and pH are shown in Figure 2. Sensors 2016, 16, 1588 6 of 18 Scheme 1. Oxidation of EPI on SOD/SPC TTF E. The parameter Δ(I 0 -I) depends on EPI concentration, applied potential (Eap) and pH solution. Therefore, an optimization of these variables was performed in order to ensure the quality of the results. Because the dependence between Δ(I 0 -I) and Al(III) concentration is linear, substrate response was obtained from pH 5.0 to pH 8.0, and a pH of 5.0 was selected regarding substrate stability to autoxidation. In the same way, substrate response was obtained from +0.20 V to +0.60 V, and a potential of 0.2 V was selected driving substrate oxidation to epinephrinequinone [57]. Then, several aluminum inhibition calibration curves were performed at different potential and pH values and their slopes were compared, in order to obtain Al(III) inhibition effect with pH and Eap. Slope calibration curve with pH was calculated from pH 5.0 to pH 8.0. In the same way, slope calibration curve with potential was calculate from +0.20 V to +0.60 V. Higher slope values were obtained at pH 5.0 and Eap of +0.2 V, so these conditions were chosen to perform Al(III) inhibition calibration curves. Slopes of calibration curves with potential and pH are shown in Figure 2. Figure 2. (a) Slope values of Al(III) calibration curves with pH; and (b) Slope value of Al(III) calibration curves with applied potential. [EPI] = 1.6 × 10 −4 M; Britton Robinson buffer pH 5.0, Eap = +0.2 V vs. Ag/AgCl. Findings indicated that substrate stability improved at low values of pH and potential; furthermore, if applied potentials were higher than +0.6 V, the electrodes showed erratic behavior. Since substrate response increases with concentration, a value of 1.6 × 10 −4 M for EPI was chosen, as this concentration gives a proper sensibility, and a very stable signal with very low noise. Upper concentrations produced higher noise on amperometric recording of calibration curves. Under the selected conditions, the electrodes showed good performance. Calibration curves of Al(III) using SOD/AuNPs/SPC TTF Es, SOD/PtNPs/SPC TTF Es, SOD/PdNPs/SPC TTF Es, and SOD/RhNPs/SPC TTF Es were obtained under the optimized conditions. Figure 2. ( a ) Slope values of Al(III) calibration curves with pH; and ( b ) Slope value of Al(III) calibration curves with applied potential. [EPI] = 1.6 × 10 −4 M; Britton Robinson buffer pH 5.0, Eap = +0.2 V vs. Ag/AgCl. Findings indicated that substrate stability improved at low values of pH and potential; furthermore, if applied potentials were higher than +0.6 V, the electrodes showed erratic behavior. Since substrate response increases with concentration, a value of 1.6 × 10 −4 M for EPI was chosen, as this concentration gives a proper sensibility, and a very stable signal with very low noise. Upper concentrations produced higher noise on amperometric recording of calibration curves. Under the selected conditions, the electrodes showed good performance. Calibration curves of Al(III) using SOD/AuNPs/SPC TTF Es, SOD/PtNPs/SPC TTF Es, SOD/PdNPs/SPC TTF Es, and SOD/RhNPs/SPCTTFEs were obtained under the optimized conditions. Sensors 2016,16, 1588 7 of 19 Preliminary experiments showed that modification of electrode surface with NPs increased the sensitivity of the biosensor; therefore, a thorough study of conditions of NPs deposition was carried out. AuNPs, PtNPs, PdNPs and RhNPs were deposited on electrodes surfaces according to methods described in the Experimental Section. 3.2. XRF and SEM for NPs/SPCTTFE Study METHOD A Two different controlled potentials, +0.18 V and +0.3 V, were applied for 15 s to SPCEs in order to deposit NPs of every metal. X-ray fluorescence emission (XRF) spectra were obtained from surfaces of SPC TTF Es modified with the different type of NPs. Table 2shows XRF percentage of elements deposited using indicated potentials. The plating of metals at +0.18 V for 15 s produced a higher percentage of Au, Pd and Rh. Pt deposited percentage was higher at +0.30 V. Since the Eap of +0.18 V applied for 15 s produced a higher percentage for Pd, Rh and Au, and the application of a deposition potential of +0.3 V did not deposit Pd or Au, conditions of Eap of +0.18 V and 15 s of method A were selected to deposit NPs of metals. Table 2. XRF percentage of element deposited on SPCTTFEs by method A and B. Method A Method B Element XRF% XRF% XRF% XRF% (+0.18 V, 15 s) (+0.30 V, 15 s) CV1 CV2 Pd 0.136 0.00 0.557 0.632 Pt 0.223 1.48 2.74 2.71 Rh 0.693 0.380 4.49 2.95 Au 1.42 - 1.87 2.23 The inhibition calibration curves for Al(III) are shown in Figure 3, where the lowest slope value corresponds to SPC TTF E without NPs deposited and the highest corresponds to SOD/AuNPs SPC TTF E. The other metal NPs modified SPC TTF E tested showed lower linear adjustment than SOD/AuNPs SPCTTFE. SEM images of AuNPs obtained by method A deposited on SPCTTFE are presented in Figure 4. Sensors 2016, 16, 1588 7 of 18 Preliminary experiments showed that modification of electrode surface with NPs increased the sensitivity of the biosensor; therefore, a thorough study of conditions of NPs deposition was carried out. AuNPs, PtNPs, PdNPs and RhNPs were deposited on electrodes surfaces according to methods described in the Experimental Section. 3.2. XRF and SEM for NPs/SPC TTF E Study METHOD A Two different controlled potentials, +0.18 V and +0.3 V, were applied for15 s to SPCEs in order to deposit NPs of every metal. X-ray fluorescence emission (XRF) spectra were obtained from surfaces of SPC TTF Es modified with the different type of NPs. Table 2 shows XRF percentage of elements deposited using indicated potentials. The plating of metals at +0.18 V for 15 s produced a higher percentage of Au, Pd and Rh. Pt deposited percentage was higher at +0.30 V. Since the Eap of +0.18 V applied for 15 s produced a higher percentage for Pd, Rh and Au, and the application of a deposition potential of +0.3 V did not deposit Pd or Au,conditions of Eap of +0.18 V and 15 s of method A were selected to deposit NPs of metals. Table 2. XRF percentage of element deposited on SPC TTF Es by method A and B. Method A Method B Element XRF% XRF% XRF% XRF% (+0.18 V, 15 s) (+0.30 V, 15 s) CV1 CV2 Pd 0.136 0.00 0.557 0.632 Pt 0.223 1.48 2.74 2.71 Rh 0.693 0.380 4.49 2.95 Au 1.42 - 1.87 2.23 The inhibition calibration curves for Al(III) are shown in Figure 3, where the lowest slope value corresponds to SPC TTF E without NPs deposited and the highest corresponds to SOD/AuNPs SPC TTF E. The other metal NPs modified SPC TTF E tested showed lower linear adjustment than SOD/AuNPs SPC TTF E. SEM images of AuNPs obtained by method A deposited on SPC TTF E are presented in Figure 4. Figure 3. Calibration plots using: ( ) SOD/AuNPs/SPC TTF Es; (■) SOD/PdNPs/SPC TTF Es; (▲) SOD/RhNPs/SPC TTF Es; (  ) SOD/PtNPs/SPC TTF Es and (  ) SPC TTF Es performed with NPs/SPC TTF Es prepared by method A, [EPI] 1.6 × 10 −4 M, Britton Robinson buffer pH 5.0, Eap = +0.2 V vs. Ag/AgCl. Figure 3. Calibration plots using: ( • ) SOD/AuNPs/SPC TTF Es; (  ) SOD/PdNPs/SPC TTF Es; ( K ) SOD/RhNPs/SPC TTF Es; (  ) SOD/PtNPs/SPC TTF Es and ( • ) SPC TTF Es performed with NPs/SPC TTF Es prepared by method A, [EPI] 1.6 × 10 −4 M, Britton Robinson buffer pH 5.0, Eap = +0.2 V vs. Ag/AgCl. Sensors 2016,16, 1588 8 of 19 Sensors 2016, 16, 1588 8 of 18 Figure 4. SEM image of AuNPs/SPCTTFE deposited at 0.18 V for15 s. 3.3. XRF and SEM for NPs/SPC TTF Es Study METHOD B XRF percentagesforevery metal deposited with method B are shown in Table1. AuNPs, PtNPs, PdNPs and RhNPs were deposited on SPC TTF Es according to method B and modified with immobilized SOD. SOD/SPCEs modified with metallic NPs showed the best linear adjustedAl(III) calibration curve at CV1 conditions for PtNPs and at CV2 conditions for PdNPs (Figures 5 and 6). Regressions with the best linear fit performed by methods A and B showed that the highest slope corresponds to SOD/PdNPs/SPC TTF Es (Figure 7). SEM image of PdNPs/SPC TTF Es at CV2 conditions is shown in Figure 8, where it is observed that PdNPs are deposited in a regular form on SPC TTF Es for the CV2 conditions. Figure 5. Calibration curves of Al(III) inhibition for (▲) SOD/RhNPs/SPC TTF Es; (■) SOD/PdNPs/SPC TTF Es; () SOD/PtNPs/SPC TTF Es and (  ) SOD/AuNPs/SPC TTF Es performed with NPs/SPC TTF Es prepared under CV1 condition, [EPI] 1.6 × 10 −4 M, Britton Robinson buffer pH 5.0, Eap = +0.2 V vs. Ag/AgCl. Figure 4. SEM image of AuNPs/SPCTTFE deposited at 0.18 V for 15 s. 3.3. XRF and SEM for NPs/SPC TTFEs Study METHOD B XRF percentagesforevery metal deposited with method B are shown in Table 1. AuNPs, PtNPs, PdNPs and RhNPs were deposited on SPC TTF Es according to method B and modified with immobilized SOD. SOD/SPCEs modified with metallic NPs showed the best linear adjustedAl(III) calibration curve at CV1 conditions for PtNPs and at CV2 conditions for PdNPs (Figures 5and 6). Regressions with the best linear fit performed by methods A and B showed that the highest slope corresponds to SOD/PdNPs/SPC TTF Es (Figure 7). SEM image of PdNPs/SPC TTF Es at CV2 conditions is shown in Figure 8, where it is observed that PdNPs are deposited in a regular form on SPC TTF Es for the CV2 conditions. Sensors 2016, 16, 1588 8 of 18 Figure 4. SEM image of AuNPs/SPCTTFE deposited at 0.18 V for15 s. 3.3. XRF and SEM for NPs/SPC TTF Es Study METHOD B XRF percentagesforevery metal deposited with method B are shown in Table1. AuNPs, PtNPs, PdNPs and RhNPs were deposited on SPC TTF Es according to method B and modified with immobilized SOD. SOD/SPCEs modified with metallic NPs showed the best linear adjustedAl(III) calibration curve at CV1 conditions for PtNPs and at CV2 conditions for PdNPs (Figures 5 and 6). Regressions with the best linear fit performed by methods A and B showed that the highest slope corresponds to SOD/PdNPs/SPC TTF Es (Figure 7). SEM image of PdNPs/SPC TTF Es at CV2 conditions is shown in Figure 8, where it is observed that PdNPs are deposited in a regular form on SPC TTF Es for the CV2 conditions. Figure 5. Calibration curves of Al(III) inhibition for (▲) SOD/RhNPs/SPC TTF Es; (■) SOD/PdNPs/SPC TTF Es; () SOD/PtNPs/SPC TTF Es and (  ) SOD/AuNPs/SPC TTF Es performed with NPs/SPC TTF Es prepared under CV1 condition, [EPI] 1.6 × 10 −4 M, Britton Robinson buffer pH 5.0, Eap = +0.2 V vs. Ag/AgCl. Figure 5. Calibration curves of Al(III) inhibition for ( K ) SOD/RhNPs/SPC TTF Es; (  ) SOD/PdNPs/SPC TTF Es; (  ) SOD/PtNPs/SPC TTF Es and ( • ) SOD/AuNPs/SPC TTF Es performed with NPs/SPC TTF Es prepared under CV1 condition, [EPI] 1.6 × 10 −4 M, Britton Robinson buffer pH 5.0, Eap = +0.2 V vs. Ag/AgCl. Sensors 2016,16, 1588 9 of 19 Sensors 2016, 16, 1588 9 of 18 Figure 6. Calibration curves of Al(III) inhibition for (■) SOD/PdNPs/SPC TTF Es; () SOD/PtNPs/SPC TTF Es; (▲) SOD/RhNPs/SPC TTF Es and (  ) SOD/AuNPs/SPC TTF Es performed with NPs/SPC TTF Es prepared under CV2 conditions, [EPI] 1.6 × 10 −4 M, Britton Robinson buffer pH 5.0;Eap = 0.2 V vs. Ag/AgCl. Figure 7. Calibration curves of Al(III) for (■) SOD/PdNPs/SPC TTF Es; (▲) SOD/RhNPs/SPC TTF Es prepared method B; CV2 conditions; () SOD/PtNPs/SPC TTF Es, prepared by method B and CV1 conditions and () SOD/AuNPs/SPC TTF Es, prepared by method A, [EPI] = 1.6 × 10 −4 M, Britton Robinson buffer pH 5.0, Eap = +0.2 V vs. Ag/AgCl. Figure 6. Calibration curves of Al(III) inhibition for (  ) SOD/PdNPs/SPC TTF Es; () SOD/PtNPs/SPCTTFEs ; ( K ) SOD/RhNPs/SPC TTF Es and ( • ) SOD/AuNPs/SPC TTF Es performed with NPs/SPC TTF Es prepared under CV2 conditions, [EPI] 1.6 × 10 −4 M, Britton Robinson buffer pH 5.0; Eap = 0.2 V vs. Ag/AgCl. Sensors 2016, 16, 1588 9 of 18 Figure 6. Calibration curves of Al(III) inhibition for (■) SOD/PdNPs/SPC TTF Es; () SOD/PtNPs/SPC TTF Es; (▲) SOD/RhNPs/SPC TTF Es and (  ) SOD/AuNPs/SPC TTF Es performed with NPs/SPC TTF Es prepared under CV2 conditions, [EPI] 1.6 × 10 −4 M, Britton Robinson buffer pH 5.0;Eap = 0.2 V vs. Ag/AgCl. Figure 7. Calibration curves of Al(III) for (■) SOD/PdNPs/SPC TTF Es; (▲) SOD/RhNPs/SPC TTF Es prepared method B; CV2 conditions; () SOD/PtNPs/SPC TTF Es, prepared by method B and CV1 conditions and () SOD/AuNPs/SPC TTF Es, prepared by method A, [EPI] = 1.6 × 10 −4 M, Britton Robinson buffer pH 5.0, Eap = +0.2 V vs. Ag/AgCl. Figure 7. 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