Biosensor for Aluminum(III) Based on α-Chymotrypsin Inhibition using a Disposable Screen-Printed Carbon Electrode and Acetyl-Tyrosine Ethyl Ester as Substrate

We report on a new amperometric assay for Al(III) ions based on the inhibition of α-chymotrypsin enzyme. The immobilization of the enzyme was performed on screen-printed carbon electrodes modified with gold nanoparticles and polyvinyl alcohol. N-acetyl-tyrosine ethyl ester, used as enzyme substrate, produces an oxidation amperometric signal, which at increasing aluminum concentration is diminished. The developed system has a detection limit of 1.5 ± 0.1 μM (n=7) for Al(III). The reproducibility of the method is 6.6% (n=5) and its repeatability is 8.2% (n=3). Main interferences at low concentration, include As(V), Cd(II), and Mo(VI) ions. The developed method was successfully applied to the determination of Al(III) in spiked white wine sample. Results agree with the certified value giving a value of percent recovery of 103 ± 4% (n=4).


Introduction
Aluminum is an element of ubiquitous presence being linked to several neurological disorders, such as Alzheimer's disease [1][2][3]. Moreover, increasing aluminum contamination due to industrial uses and clarification of tap water has augmented the interest on the development of methods for the analysis of this element at trace levels.
The determination of aluminum in aqueous solution has been traditionally performed by electrothermal atomic absorption spectrometry methods using complexing agents, long preconcentration time and sophisticated sample pretreatment. Another technique frequently used in the determination of Al(III) is fluorescence spectroscopy [4], which involves a high cost of analytical instrumentation. Electro analytical techniques allow to obtain low detection limits, however the high reduction potential of Al(III) limits their applicability. Ion complexation with chelating agents [5][6][7][8][9][10][11] has been presented as an interesting alternative to solve this problem. In this way, good results have been obtained with Hg electrodes, however their use is beginning to be reduced because of their toxicity and environmental issues. Amperometric biosensors constitute a good alternative based on their sensitivity, selectivity and environmental friendliness, accounting for their increasing analytical application in many fields [12,13]. The amperometric biosensor response is influenced by pH, applied potential and substrate concentration, thus the selection of proper conditions may considerably improves its performance and stability.
Enzymatic electrodes performance is linked to an immobilization enzyme method [14] that should not denature the protein nor modify its active site. An immobilization method that achieves this goal is polyvinyl alcohol (PVA) encapsulation-polymerization, which has led to the development of biosensors with high activity and stability [15][16][17]. On the other hand, the performance of enzymatic electrodes is improved with electrodeposited nanoparticles taking into account its increased use [18][19][20][21][22].
Aluminum accelerates proteolysis of a β-amyloid peptide that initiates neuritic plates in Alzheimer's disease [23]. The different effects of aluminum on the binding of synthetic substrates and macromolecular inhibitors to α-chymotrypsin suggest the occurrence of an aluminumlinked conformational change in the enzyme and pH dependence [24]. Other enzymes as calpains, which are Ca dependent, and cerebral cortex catepsins are also inhibited by Al(III) [25]. Aluminum induces Tau protein phosphorilation and aggregation through a phosphate bridge [26] and also promotes aggregation of β-amyloid protein both present in neuritic plates [27][28][29][30][31]. Their inhibitor effect over neurofilament proteins suggests accumulation inside neuronal space after aluminum administration [32]. Therefore, aluminum could affect proteases enzymatic activity, modifying their enzymatic structure, interfering in the recognition process of macromolecular inhibitors and altering the biochemical processes lead by proteases. Thus, the study Al(IIII) inhibitory effect on α-chymotrypsin using acetyl tyrosine ethyl ester (ATEE) as enzymatic substrate is of great importance. In this way, aluminum behaves as an inhibitor for chymotrypsin enzyme, which is a serine endopeptidase and hydrolyzes ATEE producing acetyl tyrosine which can be oxidized at an electrode surface [33].
Determination of Al(III) through this method offers several advantages, such as high sensitivity and low cost equipment. These advantages can be improved by using screen-printed electrodes (SPEs) due to their disposable character and great versatility. To the best of author's knowledge, there is no report in the literature of a screenprinted carbon electrodes (SPCEs) based biosensors for aluminum determination using ATEE as a substrate. This kind of biosensors offer low detection limits, narrower calibration range, and uses a lower previous works [36,37]. A picture of dimensions of the SPCE used is shown in Figure 1a.

Modification of SPCEs with gold nanoparticles
Metallic gold nanoparticles (AuNPs) deposits were obtained by direct electrochemical deposition on the SPCE surface using a 0.1 mM solution of HAuCl 4 in 0.5 M H 2 SO 4 . The deposition was performed by applying a potential of +0.18 V during 15 seconds under stirring conditions [38,39]. Figure 1b shows the described formation of AuNPs on SPCEs.
Signal stability of enzyme α-chymotrypsin electrode was improved with AuNPs. In this way, stability is better compared with enzyme electrode without gold nanoparticles as it is shown in Figure 2.

α-chymotrypsin immobilization in AuNPs/SPCEs
The enzyme was immobilized by entrapment with PVA on the surface of AuNPs/SPCEs. The optimum immobilization procedure was reached by mixing 40 μl of a 30 mg/ml of enzyme solution, 20 μl of 1.7% (w/v) BSA solution, 24 μl of a 5% (w/v) PVA and 20 μl Britton-Robinson buffer solution at pH 7.8 [35]. Then, 10 μl of this mixture were placed on the working electrode surface. Immediately afterwards 3 μl of enzyme solution were placed over the mixture. Once the aliquot of the mixture used to immobilize the enzyme was deposited on the electrode surface it was kept under laboratory ceiling fluorescent lamp (λ 350-750) nm at 9 × 10 2 lux for 4 h at 24°C in air conditioned system and then stored for 24 hours at 4°C before using and between measurements. These storage conditions allowed biosensors good performance.

Amperometric determination of aluminum
The α-chymotrypsin biosensor was placed in an electrochemical cell containing 5 mL of acetate buffer solution pH 7.8 with 40 mM NaCl and 10 mM CaCl 2 [40][41][42]. An adequate potential was then applied and once a steady-state current was established, a defined amount of ATEE substrate was added to the cell. A large oxidation current was observed applied potential [34,35], being their disposability character, low cost and environmental friendliness very significant. Moreover, this work reports for the first time the Km(app) (Michaelis Menten apparent constant) value of ATEE with and without Al(IIII) by means of SPCEs. Finally, the influence of NaCl concentration over noise and signal stability of biosensor electrode was determined.

Experimental Section Reagents
In order to prepare hand-made SPCE several inks were used including Electrodag PF-407 A (carbon ink), Electrodag 6037 SS (silver/ silver chloride ink) and Electrodag 452 SS (dielectric ink) from Acheson Colloiden (Scheemda, The Netherlands), in a DEK 248 printing machine (DEK, Weymouth, UK, http://www.dek.com) using polyester screens with appropriate stencil designs mounted at 45° to the printer stroke.

Screen printed electrodes preparation
Inks used to construct hand-made SPCEs used in the determination of aluminum were placed as successive layers on a polyester substrate. Four different screens with appropriate stencils were used to transfer the required design following the printing procedure described in   due to the oxidation of the enzymatic reaction product after the addition of ATEE substrate. Once a steady-state current was set again, a selected volume of aluminum stock solution was consecutively added and a calibration curve was constructed. The addition of aluminum resulted in a decrease of the amperometric response proportional to the amount of metal added. Enzyme electrodes were conditioned in the acetate buffer solution pH 7.8 between each calibration set.

Results and Discussion
The α-chymotrypsin/AuNPs/SPCE biosensor produces an amperometric signal, which is quantitatively related to the concentration of ATEE as shown in Figure 3. The ATEE oxidation signal is considerably  affected by addition of Al(III) producing a decrease in its amperometric response which relates to Al(III) concentration. In this way, Al(III) inhibition action was quantitatively evaluated by determining the difference between the steady-stated current obtained for ATEE in absence of Al(III) (I 0 ) and the steady-state current in the presence of Al(III) (I). The parameter ΔI (I 0 -I) presents a linear dependence in the Al(III) concentration range from 3.6 μM to 36 μM.

Selection of experimental conditions
Aluminum inhibition effect was quantitatively evaluated by determining the difference between the ATEE steady-state current in the absence of aluminum (I 0 ) and the steady-state current in the presence of aluminum (I). The parameter ΔI (I 0 -I) depends on ATEE concentration, applied potential (Eap) and pH of the buffer solution. Different experiments were carried out in order to optimize these variables.
First, the effect of applied potential in the chronoamperometric response of the developed biosensor was studied. The inhibitive signal of Al(III) was analyzed using operational potentials ranging from +0.3 to +0.8 V at pH 6.8 and 7.8, recommended for the α-chymotrypsin enzyme, as it is shown in Figure 4, parts a and b. A high quality amperometric signal at +0.7 V was obtained, thus, this potential was selected as the best value for the determination of aluminum.
The influence of pH was also studied in the range from 5 to 8 values. The results obtained are shown in Figure 4, part c, obtaining a value of 7.8 as the most adequate for Al(III) determination, taking into account the better stability conditions for the enzymatic electrode. Higher pH values produce enzyme instability and aluminum precipitation.
Finally, the influence of the concentration of ATEE was also studied in the range from 0.10 mM up to 0.30 mM. As it is shown in Figure 4 part d, the current increases in linear dependence with ATEE concentration showing a tendency to substrate saturation. A concentration of 0.20 mM ATEE in cell was then found to give a high inhibition response of aluminum, while upper concentrations were noisy.

Aluminum inhibition behavior
Experiments performed with this biosensor showed that addition of CaCl 2 and NaCl improved stability of calibration curve. Therefore, their effect on the stability of the biosensor was investigated following the method of Lineweaver-Burk. Measurements were performed using NaCl concentrations ranging from 10 mM to 80 mM in CaCl 2 10 mM. Considering apparent Michaelis Menten (Km app) values, signal stability, linearity and sensitivity of plots, concentrations of 10 mM CaCl 2 and 40 mM NaCl were selected as the most suitable for the inhibitive determination of Al(III). Figure 5 shows Lineweaver-Burk plots for NaCl increasing concentrations. The results obtained indicate that slope and therefore Km app values increase with NaCl concentration.
Once NaCl concentration was selected, at 40 mM, the inhibitory effect of Al(III) ions on the response of the α-chymotrypsin biosensor was investigated following the method of Lineweaver-Burk in presence of increasing amounts of inhibitor. The Km value (1.17 ± 0.06) ×10 -4 found without Al(III) was lower than the one obtained in presence of 100 μl of aluminum 3.70×10 -4 M (2.41 ± 0.07)×10 -4 and 400 μl of aluminum 3.70×10 -4 M (4.52 ± 0.31)×10 -4 . Al(III) presence increases slope and Km app value and diminishes enzyme-substrate affinity. As it is shown in Figure 6, the studied process resembles a competitive inhibition.

Calibration and detection limit
As it has been mentioned above, Figure 3 showed a linear   Figures of merit such as precision and detection limit were evaluated by means of calibration curves obtained at optimum conditions. Standard deviation (Sy/x) of seven calibration curves was used to calculate limit of detection, giving a value of (1.5 ± 0.1) μM for this parameter under the optimum working conditions, using 3Sy/x criteria.

Precision
This parameter was calculated in terms of repeatability and reproducibility. Repeatability was carried out using the same electrode surface. In this way, several successive calibrations for Al(III) were tested. The electrodes were conditioned for 5 min in a buffer solution, pH 7.8, between experiments. The relative standard deviation (RSD) obtained for the slopes of the three curves with the same electrode was 8.2%. Likewise, the reproducibility of the amperometric signal was checked using the slopes of five regressions carried out with different electrode surfaces. The RSD reproducibility value obtained was 6.6%. These results suggest that the fabrication procedure of the chymotrypsin/AuNPs/SPCEs biosensors is reliable and allows reproducible electroanalytical responses to be obtained with different electrodes constructed using the method described in this work as it is shown in Table 1.

Accuracy
The accuracy of the developed method was evaluated by means of the analysis of a standard reference material (SRM) High Purity Standards solution (Lot Number 1121015, (1000 ± 3) mg/L, confirmed against reference material SRM 3101 (lot number 060502), spiked in white wine sample, using the standard addition method. Results are shown in Table 2. The aluminum average concentration quantified by the developed procedure, (1025 ± 30) mg/L (n=4; α=0.05), matches the certified value of the standard reference material in agreement with aluminum stock concentration, and mean deviation. These results suggest that the developed biosensor method with α-chymotrypsin enzyme is accurate and reliable for aluminum determination in white wine sample.

Interferences
Interference study was performed comparing the percentage of inhibition showed by the developed α-chymotripsine based biosensor in the presence of aluminum and other foreign ions. Three concentration levels were tested, namely 1 mM; 0.1 mM and 1 µM. As it can be seen in (Figure 7), the highest interference effect was found for As(V); Cd(II) and Mo(VI) for the lowest level of concentration tested, but toxic ions should not be naturally present in wine. Due to some proteases enzymes are activated by Ca(II), this one is not a real interference, neither common ions as Mg(II) and Fe(III).

Conclusions
The development of a novel biosensor based on the inhibition of α-chymotripsin enzyme using AuNPs/SPCEs allows the amperometric determination of aluminum. The biosensor precision was studied in terms of the RSD of the slopes of several calibrations resulting in a reproducibility value of 6.6%. The method developed in this work presents several advantages, including lower detection limit, 1.5 μM, than other previous described ones [34,35].
The cross linking and entrapment method with PVA allowed immobilizing the enzyme over the AuNPsSPCE electrode surface. The optimum conditions for the best amperometric response of ATEE were at working potential of +0.7 V and a pH value of 7.8. In addition, the presence of Ca(II) and NaCl produce a stabilizing effect over the amperometric signal; in this way concentrations of 10 mM of Ca(II) and 40 mM of NaCl resulted to be optimal considering sensitivity, stability and Km(app) values. The Lineweaver-Burk plots obtained in the presence of Al(III) showed competitive inhibition and the performance of the developed biosensor was evaluated through its figures of merit that were adequate to quantify Al(III) at low concentration level. Moreover, the analytical performance of the developed biosensor was demonstrated by its application to the analysis of spiked white wine samples with a High-Purity Standards stock solution. The recovery value (1025 ± 30) mg/L obtained agreed with the certified reported aluminum SRM value of 1000 mg/L ± 3 mg/L. Finally, the main interferences at low level concentration included As(V), Cd(II) and Mo(VI).
Overall, the ATEE substrate was suitable for quantitative determination of aluminum using a SPE modified with AuNPs and α-chymotripsin enzyme. The developed method presents a lower detection limit and uses a lower potential than other enzymatic methods for the analysis of Al(III).