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

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.


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]:  This article water samples 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.
Solutions of platinum, rhodium and palladium 0.1 mM were prepared from ICP solutions of 1000 mg/L (Merck, Darmstad, Germany).
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).
The working electrode ink was prepared by thoroughly mixing carbon ink with tetrathiofulvalene (C TTF ) 5%. TTF was obtained from Acros Organics (Acros Organics, Geel, Belgium).

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 550i was 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. SPC TTF Es 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).

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.

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.

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.

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.

SOD Enzyme Immobilization onto AuNPs//SPC TTF 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 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.

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. The parameter Δ(I0-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(I0-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. 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/SPCTTFEs, SOD/PtNPs/SPCTTFEs, SOD/PdNPs/SPCTTFEs, and SOD/RhNPs/SPCTTFEs were obtained under the optimized conditions. 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. The parameter Δ(I0-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(I0-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. 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/SPCTTFEs, SOD/PtNPs/SPCTTFEs, SOD/PdNPs/SPCTTFEs, and SOD/RhNPs/SPCTTFEs were obtained under the optimized conditions. 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. 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.

XRF and SEM for NPs/SPC TTF E 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 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. 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. 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.

XRF and SEM for NPs/SPCTTFE 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 SPCTTFEs 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. The inhibition calibration curves for Al(III) are shown in Figure 3, where the lowest slope value corresponds to SPCTTFE without NPs deposited and the highest corresponds to SOD/AuNPs SPCTTFE. The other metal NPs modified SPCTTFE 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.

XRF and SEM for NPs/SPC TTFEs Study METHOD B
XRF percentagesforevery metal deposited with method B are shown in Table1. AuNPs, PtNPs, PdNPs and RhNPs were deposited on SPCTTFEs 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/SPCTTFEs (Figure 7). SEM image of PdNPs/SPCTTFEs at CV2 conditions is shown in Figure 8, where it is observed that PdNPs are deposited in a regular form on SPCTTFEs for the CV2 conditions.

XRF and SEM for NPs/SPC TTF Es 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 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.

XRF and SEM for NPs/SPC TTFEs Study METHOD B
XRF percentagesforevery metal deposited with method B are shown in Table1. AuNPs, PtNPs, PdNPs and RhNPs were deposited on SPCTTFEs 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/SPCTTFEs (Figure 7). SEM image of PdNPs/SPCTTFEs at CV2 conditions is shown in Figure 8, where it is observed that PdNPs are deposited in a regular form on SPCTTFEs for the CV2 conditions.

AFM Analysis of SPCTTFEs Prepared by Methods A and B
AuNPs deposit on SPCTTFE increases roughness of SPCTTFE compared with control electrode, as can be observed in AFM images of the surfaces of AuNPs/SPCTTFEs obtained by deposition of AuNPs at 0.18 V for15 s ( Figure 9). The highest slope presented for SOD/AuNPs/SPCTTFE using method A is afforded by lower AFM parameters of AuNPs at 0.18 V for15 s, than when the CV1or CV2 conditions are used. AFM images of PdNPs/SPCTTFEs obtained by deposition of PdNPs at CV1 and CV2 conditions are shown in Figure 10. Analysis of AFM parameters confirmed that metal deposited was nanometric size. Table 3 shows the most important parameters of NPs/SPCTTFEs and SPCTTFEs control electrode obtained through tapping mode and Roughness Kurtosis (RKu) and Skewness (RSk) coefficients. PdNPs/SPCTTFEs, obtained using CV2 conditions, showed lower Roughness Average (RA), Roughness Mean Square (RMS) and height of the highest peak above mean line in the profile (Rmax) than other SPCTTFEs modified with metallic NPs, indicating that PdNPs/SPCTTFEs prepared by CV2 condition present a more homogeneous surface [62]. AFM image of PdNPs prepared by CV2 method showed lower values of RA andRMS when compared tosurface prepared by CV1 method Table 3. AFM parameters of modified SPCTTFEs with metallic NPs.

AFM Analysis of SPC TTF Es Prepared by Methods A and B
AuNPs deposit on SPC TTF E increases roughness of SPC TTF E compared with control electrode, as can be observed in AFM images of the surfaces of AuNPs/SPC TTF Es obtained by deposition of AuNPs at 0.18 V for 15 s (Figure 9). The highest slope presented for SOD/AuNPs/SPC TTF E using method A is afforded by lower AFM parameters of AuNPs at 0.18 V for 15 s, than when the CV1or CV2 conditions are used. AFM images of PdNPs/SPC TTF Es obtained by deposition of PdNPs at CV1 and CV2 conditions are shown in Figure 10. Analysis of AFM parameters confirmed that metal deposited was nanometric size. Table 3 shows the most important parameters of NPs/SPC TTF Es and SPC TTF Es control electrode obtained through tapping mode and Roughness Kurtosis (RKu) and Skewness (RSk) coefficients. PdNPs/SPC TTF Es, obtained using CV2 conditions, showed lower Roughness Average (RA), Roughness Mean Square (RMS) and height of the highest peak above mean line in the profile (Rmax) than other SPC TTF Es modified with metallic NPs, indicating that PdNPs/SPC TTF Es prepared by CV2 condition present a more homogeneous surface [62]. AFM image of PdNPs prepared by CV2 method showed lower values of RA andRMS when compared tosurface prepared by CV1 method.  It was observed that PdNPs/SPCTTFEs modified by means of method B and CV2 conditions showed lower AFM values than the other NPs/SPCTTFEs. Then,it was decided to analyze the response of EPI at SOD/NPs/SPCTTFEs in presence of aluminum.

Inhibition Behavior of Al(III) on SOD Enzyme
Michaelis Menten Km apparent constants were estimated by Lineweaver-Burk plot. It was obtained in presence and absence of Al(III) with SPCTTFEs modified with AuNPs, PtNPs, RhNPs and PdNPs. Modified electrodes were prepared under the best conditions for each NP deposition method used, namely 0.18 V for method A and CV2 conditions for method B. Figure 11 shows amperometric recording of SOD/PdNPs/SPCTTFEs (obtained by method B and CV2 conditions).  It was observed that PdNPs/SPCTTFEs modified by means of method B and CV2 conditions showed lower AFM values than the other NPs/SPCTTFEs. Then,it was decided to analyze the response of EPI at SOD/NPs/SPCTTFEs in presence of aluminum.

Inhibition Behavior of Al(III) on SOD Enzyme
Michaelis Menten Km apparent constants were estimated by Lineweaver-Burk plot. It was obtained in presence and absence of Al(III) with SPCTTFEs modified with AuNPs, PtNPs, RhNPs and PdNPs. Modified electrodes were prepared under the best conditions for each NP deposition method used, namely 0.18 V for method A and CV2 conditions for method B. Figure 11 shows amperometric recording of SOD/PdNPs/SPCTTFEs (obtained by method B and CV2 conditions). It was observed that PdNPs/SPC TTF Es modified by means of method B and CV2 conditions showed lower AFM values than the other NPs/SPC TTF Es. Then, it was decided to analyze the response of EPI at SOD/NPs/SPC TTF Es in presence of aluminum.

Inhibition Behavior of Al(III) on SOD Enzyme
Michaelis Menten Km apparent constants were estimated by Lineweaver-Burk plot. It was obtained in presence and absence of Al(III) with SPC TTF Es modified with AuNPs, PtNPs, RhNPs and PdNPs. Modified electrodes were prepared under the best conditions for each NP deposition method used, namely 0.18 V for method A and CV2 conditions for method B. Figure 11 shows amperometric recording of SOD/PdNPs/SPC TTF Es (obtained by method B and CV2 conditions). Km apparent values of modified electrodes are shown in Table 4.

Validation of SOD/PdNPs/TTF/SPCE Based Biosensor
SOD/PdNPs/SPCTTFEs were selected to perform validation of the developed biosensor trough estimation of their performance parameters.

Limit of Detection
The limit of detection under the optimum working conditions (2.0 ± 0.2 μM) was calculated from the standard deviation (Sy/x) of five Al(III) inhibition calibration curves according to the criteria 3 Sy/x [63], and its RSD was 7.9%. Analogous to LOD, quantification limit (LOQ) was estimated under optimal conditions from the standard deviation of five Al(III)inhibition calibration curves using the criteria 10 Sy/x, and its value was 6.7 ± 0.5 μM, with a RSD of 7.9%.

Precision
This parameter is usually calculated in terms of reproducibility and repeatability. Repeatability was assessed using the same electrode surface. In this way, successive calibrations for Al(III) were tested with SOD/PdNPs/SPCTTFEs prepared under CV2 conditions. The electrodes were conditioned in a Britton Robinson buffer solution, pH 5.0, stirring for 5 min between experiments. The RSD obtained for the slopes of the first two graphs was 5.1%, but, in the third measurement, a decrease in Km apparent values of modified electrodes are shown in Table 4. Table 4. Km apparent values for SPC TTF Es modified with AuNPs, PtNPs, RhNPs, and PdNPs.

Validation of SOD/PdNPs/ TTF /SPCE Based Biosensor
SOD/PdNPs/SPC TTF Es were selected to perform validation of the developed biosensor trough estimation of their performance parameters.

Limit of Detection
The limit of detection under the optimum working conditions (2.0 ± 0.2 µM) was calculated from the standard deviation (Sy/x) of five Al(III) inhibition calibration curves according to the criteria 3 Sy/x [63], and its RSD was 7.9%. Analogous to LOD, quantification limit (LOQ) was estimated under optimal conditions from the standard deviation of five Al(III)inhibition calibration curves using the criteria 10 Sy/x, and its value was 6.7 ± 0.5 µM, with a RSD of 7.9%.

Precision
This parameter is usually calculated in terms of reproducibility and repeatability. Repeatability was assessed using the same electrode surface. In this way, successive calibrations for Al(III) were tested with SOD/PdNPs/SPC TTF Es prepared under CV2 conditions. The electrodes were conditioned in a Britton Robinson buffer solution, pH 5.0, stirring for 5 min between experiments. The RSD obtained for the slopes of the first two graphs was 5.1%, but, in the third measurement, a decrease in the biosensor sensitivity and a RSD increase, reaching 15%, were observed. Because the electrodes are disposables, the reproducibility is a better estimate of performance. Likewise, the reproducibility of the amperometric signal was checked using the slopes of five regression lines carried out with different electrode surfaces, RSD slope value estimated was 7.0%.

Accuracy
The accuracy of the developed method was tested by a recovery study in which a known amount of Al(III) standard reference material (SRM), SRM High Purity Standards solution (Lot Number 1121015, (1000 ± 3) mg·L −1 ) was spiked to a buffer solution.

Study of Interferences on SOD/PdNPs/SPC TTF E Biosensors
Interference study was performed comparing the percentage of inhibition showed by the developed SOD based biosensor in the presence of aluminum and other foreign ions. Three concentration levels of possible interfering ions, namely 1 × 10 −3 M, 1 × 10 −4 M, and 1 × 10 −6 M, were tested. Regarding Al(III), LOD value obtained for SOD/PdNPs/SPC TTF E is meaningful at 1 × 10 −4 M. As can be seen in Figure 12, the highest interference effect was found for Sn(II), Cd(II) and Mo(VI) for concentrations tested; however, these cations should usually not be present in water.

Discussion
SOD biosensor was developed looking for effect of metallic NPs on sensibility of slopes of Al(III) calibration curves. One initial hypothesis was that NPs generation methodology influence sensitivity of biosensor. For these reasons, two different methodologies to deposit NPs were tested, direct deposit at 0.18 V produced the highest slope for AuNPs, CV2 deposit methodology produced the highest slope for PdNPs, followed for RhNPs andCV1 deposit methodology produced the highest slope for PtNPs. Criteria used for slope selection were linearity and sensibility. Regarding all optimum slopes values obtained with SOD/NPs/SPCTTFEs biosensors under the two methodologies, the highest slope value was obtained for SOD/PdNPs/SPCTTFEs based biosensor. NPs' physical characteristics are also modified by deposit methodologies.Although SEM were performed on every NPs/SPCTTFEs prepared by means of the above-mentioned methodologies and NPs were visualized, AFM is a more appropriate instrument for NPs/SPCTTFEs surface characterization. AuNPs/SPCTTFEs prepared by method A showed lower AFM parameters, namely RA, RMS and Rmax, than CV deposition methodologies. PdNPs/SPCTTFEs prepared by CV2 showed lower AFM parameters than PdNPs/SPCTTFEs prepared by CV1. Although PtNPs/SPCTTFEs prepared by CV2/CV1 showed similar AFM values and slopes, best linearity was obtained for CV2 condition. RhNPs/SPCTTFEs prepared under CV2 condition showed much better linearity than CV1. All RKu values are near 3, providing evidence that the obtained values are closer to a normal distribution, and the surface is named Mesokurtic, for kurtosis minor 3, surface is flat and called Platykurtic. When kurtosis is greater than 3, surface owns more peaks than valleys. RSk measures the profile of symmetry about mean line. If the height distribution is asymmetrical and the surface has more peaks than valleys, skewness is positive, while in the opposite case, skewness is negative. SOD/NPs/SPCTTFEs based biosensor was based on Al(III) inhibition of SOD, and Km inhibition were estimated for SOD/NPs/SPCTTFEs under method A andCV2 condition. It was established that Al(III) exerts its inhibitory action at low concentration. Inhibitory effect for SOD/NPs/SPCTTFEs was confirmed by means of Km app values with Al(III) increasing concentrations. This fact is in accordance with theoretical considerations regarding enzymatic behavior in presence of inhibitors. However, the last measurement for SOD/RhNPs/SPCTTFEs, displayed an unusual behavior.
SOD/PdNPs/SPCTTFEs were selected to perform validation of the developed biosensor. The selection was based on the Al(III) inhibition calibration curves of SOD enzyme, that clearly showed higher sensibility by modifying SPCTTFEs with PdNPs prepared under method B and CV2 conditions than the other metallic NPs. Al(III) inhibition on SOD/NPs/SPCTTFEs could be used with analytical purposes, but at first it is necessary to perform developed biosensor validation. This goal was achievedby performance parameters estimation. Precision was established through reproducibility

Discussion
SOD biosensor was developed looking for effect of metallic NPs on sensibility of slopes of Al(III) calibration curves. One initial hypothesis was that NPs generation methodology influence sensitivity of biosensor. For these reasons, two different methodologies to deposit NPs were tested, direct deposit at 0.18 V produced the highest slope for AuNPs, CV2 deposit methodology produced the highest slope for PdNPs, followed for RhNPs andCV1 deposit methodology produced the highest slope for PtNPs. Criteria used for slope selection were linearity and sensibility. Regarding all optimum slopes values obtained with SOD/NPs/SPC TTF Es biosensors under the two methodologies, the highest slope value was obtained for SOD/PdNPs/SPC TTF Es based biosensor. NPs' physical characteristics are also modified by deposit methodologies.Although SEM were performed on every NPs/SPC TTF Es prepared by means of the above-mentioned methodologies and NPs were visualized, AFM is a more appropriate instrument for NPs/SPC TTF Es surface characterization. AuNPs/SPCTTFEs prepared by method A showed lower AFM parameters, namely RA, RMS and Rmax, than CV deposition methodologies. PdNPs/SPC TTF Es prepared by CV2 showed lower AFM parameters than PdNPs/SPC TTF Es prepared by CV1. Although PtNPs/SPC TTF Es prepared by CV2/CV1 showed similar AFM values and slopes, best linearity was obtained for CV2 condition. RhNPs/SPC TTF Es prepared under CV2 condition showed much better linearity than CV1. All RKu values are near 3, providing evidence that the obtained values are closer to a normal distribution, and the surface is named Mesokurtic, for kurtosis minor 3, surface is flat and called Platykurtic. When kurtosis is greater than 3, surface owns more peaks than valleys. RSk measures the profile of symmetry about mean line. If the height distribution is asymmetrical and the surface has more peaks than valleys, skewness is positive, while in the opposite case, skewness is negative.
SOD/NPs/SPC TTF Es based biosensor was based on Al(III) inhibition of SOD, and Km inhibition were estimated for SOD/NPs/SPC TTF Es under method A andCV2 condition. It was established that Al(III) exerts its inhibitory action at low concentration. Inhibitory effect for SOD/NPs/SPC TTF Es was confirmed by means of Km app values with Al(III) increasing concentrations. This fact is in accordance with theoretical considerations regarding enzymatic behavior in presence of inhibitors. However, the last measurement for SOD/RhNPs/SPC TTF Es, displayed an unusual behavior.
SOD/PdNPs/SPC TTF Es were selected to perform validation of the developed biosensor. The selection was based on the Al(III) inhibition calibration curves of SOD enzyme, that clearly showed higher sensibility by modifying SPC TTF Es with PdNPs prepared under method B and CV2 conditions than the other metallic NPs. Al(III) inhibition on SOD/NPs/SPC TTF Es could be used with analytical purposes, but at first it is necessary to perform developed biosensor validation. This goal was achievedby performance parameters estimation. Precision was established through reproducibility of calibration curves slope, as SPC TTF Es are disposable, and this reproducibility is a good precision estimate. LOD and LOQ values allowed quantification of low Al(III) concentrations. Recovery percentage of certified SRM afforded accuracy of SOD/NPs/SPC TTF Es based biosensor and indicated that developed SOD/PdNPs/SPC TT Es biosensor can be applied to Al(III) determination in aqueous solutions.Validation results suggest that the fabrication procedure of the SOD/PdNPs/SPC TTF Es based biosensor is reliable and allows reproducible amperometric responses to be obtained with different electrodes constructed using the method described in this work.
A weakness of biosensor SOD/PdNPs/SPC TTF Es is its response to interfering ions, but these toxic ions should not be naturally present in water. Ca(II) and Fe(III) usually found in water do not interfere. Al(III) showed inhibition on SOD enzyme at all tested concentrations.

Conclusions
A novel amperometric biosensor based on SOD/PdNPs/C TTF /SPCEs was developed, validated and applied to Al(III) determination in aqueous matrixes. The biosensor was based on inhibitory effect of Al(III) on SOD enzyme and presents fast response, very good reproducibility, stability, and low LOD. Michaelis Menten constants were calculated from Lineweaver-Burk plots and showed increasing values with Al(III) concentration in accordance with theory of enzymatic inhibition.
SOD enzyme immobilization was easily and rapidly achieved by crosslinking using glutaraldehyde and allowed obtaining a good reproducibility value of biosensor.
Modification of SPC TTF Es with different types of NPs improves biosensor performance. A study of electrolytic generation conditions of NPs of Au, Pt, Rh and Pd onto SPC TTF E surface was carried out and results showed that SPC TTF Es modified with PdNPs by means of cyclic voltammetry under method B and CV2 conditions (delay time 120 s, step potential, 0.025 V, scan rate 0.1 V/s) gave a higher sensibility on amperometric inhibition of Al(III) calibration curves.
SEM images showed presence of the metallic NPs deposited on SPC TTF Es. XRF study was conducted to evaluate percentages of every metal deposited on SPC TTF Es. In addition, AFM study showed roughness, characteristic of SPC TTF Es and NPs/SPC TTF Es surfaces and provided useful information about morphology and surface homogeneity. It was also found that PdNPs/SPC TTF Es deposited by method B and CV2 conditions had lower RA, RSM and Rmax than the others metallic NPs.
Biosensor validation was performed under optimized conditions: pH 5.0, applied potential of 0.2 V and a concentration of EPI of 1.6 × 10 −4 M. The recovery value obtained using certified material, supported the feasibility of SOD/PdNPs/SPC TTF Es based biosensor for Al(III) determination.
Developed biosensor presents LOD similar to other developed Al(III) biosensors, but it has the advantage of using a lower applied potential of only +0.2 V. The possibility of using this biosensor at low potentials results in a muchhigher selectivity compared with the others Al(III) biosensors.