Development of a Novel Biosensor Based on Tyrosinase/Platinum Nanoparticles/Chitosan/Graphene Nanostructured Layer with Applicability in Bioanalysis

The present paper describes the preparation and characterization of a graphene, chitosan, platinum nanoparticles and tyrosinase-based bionanocomposite film deposited on the surface of a screen-printed carbon electrode for the detection of L-tyrosine by voltammetry. The redox process on the biosensor surface is associated with the enzymatic oxidation of L-tyrosine, which is favoured by graphene and platinum nanoparticles that increase electrical conductivity and the electron transfer rate. Chitosan ensures the biocompatibility between the tyrosinase enzyme and the solid matrix, as well as a series of complex interactions for an efficient immobilization of the biocatalyst. Experimental conditions were optimized so that the analytical performances of the biosensor were maximal for L-tyrosine detection. By using square wave voltammetry as the detection method, a very low detection limit (4.75 × 10−8 M), a vast linearity domain (0.1–100 μM) and a high affinity of the enzyme for the substrate (KMapp is 53.4 μM) were obtained. The repeatability of the voltammetric response, the stability, and the reduced interference of the chemical species present in the sample prove that this biosensor is an excellent tool to be used in bioanalysis. L-tyrosine detection in medical and pharmaceutical samples was performed with very good results, the analytical recovery values obtained being between 99.5% and 101%. The analytical method based on biosensor was validated by the standard method of analysis, the differences observed being statistically insignificant at the 99% confidence level.


Introduction
The recent development of sensors and biosensors based on nanomaterials for detecting amino acids which are vital for an organism, such as L-Tyrosine (Tyr), is of utmost interest in bioassay [1]. L-Tyrosine is one of the indispensable amino acids found in proteins, which is necessary in order to maintain a positive nitrogen balance in the body [2,3]. Tyr is the precursor of a series of compounds such as thyroxine, dopamine, epinephrine and norepinephrine, which condition the appropriate functioning of the body [4]. Tyr can be synthesized by the body if a sufficient amount of phenylalanine is given by exogenous intake [5]. The Tyr level in the body is correlated with the individual's health and its normal concentration level in blood plasma ranging between 30-120 µM [6]. nanoparticles film and the immobilization of tyrosinase enzyme for tyrosine and catechin biosensing. The polypyrrole acts as conducting matrix and gold nanoparticles play the role of electrocatalysts [25].
The graphene, platinum nanoparticles, chitosan were also used for the immobilization of other enzymes in the sensitive element of different biosensors with enhanced performance characteristics. For instance, a biosensor based on diamine oxidase/platinum nanoparticles/graphene/chitosan modified screen-printed carbon electrode showed good performance in the electrochemical detection of histamine. Enhanced sensitivity is related to the electrocatalytic synergetic effect of graphene and platinum nanoparticles on the electrochemical detection of H 2 O 2 [26]. Another biosensor based on platinum nanoparticles-reduced graphene oxide-laccase biocomposite for the determination of total polyphenolic content was developed. The combination of reduced graphene oxide and platinum nanoparticles leads to a synergistic effect, increasing the electroactive surface area of the electrode and enhancing electron transfer towards the electrode [27]. Therefore, the development of a composite nanomaterial based on graphene, platinum nanoparticles, chitosan and tyrosinase in order to build a new biosensor with superior characteristics is a complex and challenging research task.
The present paper describes the development of a new biosensor for detecting tyrosine, which has applicability in bioanalysis. The biosensor was fully characterized and experimental conditions were optimized so that sensitivity and selectivity were maximal. The analytical method based on the biosensor was validated in the laboratory by using the standard method for determining tyrosine.

Development of Platinum Nanoparticles/Chitosan/Graphene-Carbon Screen-Printed Electrode (PtNP/Chit/GPH-CSPE)
The dispersion of GPH was prepared by mixing 1 mg GPH with 1 mL chitosan solution (0.2% in acetic acid, pH = 5) followed by ultrasonication for 2 h. By this method a homogeneous dispersion of GPH in aqueous phase was obtained. CSPE was modified with 10 µL GPH dispersion by the casting method. The evaporation of the solvent was carried at room temperature in a desiccant.
After drying, on the surface of GPH/Chit-CSPE platinum nanoparticles (PtNP) were deposited by chronoamperometry at +0.4 V for 300 s from a 2 × 10 −3 M H 2 PtCl 6 and H 2 SO 4 aqueous solution.
The counter electrode was a Pt plate of 2 cm 2 and Ag/AgCl, KCl (3.5M) was the reference electrode. The PtNP/GPH/Chit/CSPE was rinsed with ultrapure water and dried in a desiccant [19,20].

Development of Tyrosinase (Ty)/PtNP/GPH/Chit-CSPE
The tyrosinase enzyme was immobilized on the surface of PtNP/GPH/Chit-CSPE by the casting method. Thus, 10 µL of 0.1 M phosphate-buffered saline (PBS) (pH = 7) containing 50 µg/µL of tyrosinase was added onto the surface of PtNP/GPH/Chit-CSPE. The biosensor was dried at room temperature in a desiccant overnight. The Ty/PtNP/GPH/Chit-CSPE biosensors were kept in a closed box at 4 • C in a fridge in order to prevent the denaturation of the sensitive element.

Apparatus
All the electrochemical results obtained from voltammetric experiments (cyclic voltammetry and square wave voltammetry) were recorded by a Biologic SP 150 potentiostat/galvanostat (Bio-Logic Science Instruments SAS, Claix, France) controlled by the EC-Lab Express software V5.52. All measurements were carried out at room temperature, ambient conditions and atmospheric pressure. The scanning electron microscope (SEM) images were captured by a FlexSEM 1000 (Hitachi, Tokyo, Japan) scanning electron microscope. A Cencom II centrifuge (JP SELECTA S.A., Barcelona, Spain) was used to centrifuging the sample solutions. An Inolab pH 7310 pH-meter (WTW, Weilheim, Germany) equipped with a combined glass electrode/Ag/AgCl was applied to pH buffer solutions adjustments. The ultraviolet (UV)spectrometric experiments were carried out with a Rayleigh UV-1601 spectrophotometer (Beijing Rayleigh Analytical Instrument Corporation, Beijing, China).

Biological and Pharmaceutical Samples
The determination of Tyr was carried out in heparinized blood plasma supplied by one hospital laboratory. The pharmaceutical samples were purchased from local pharmacies. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of "Dunarea de Jos" University of Galati (#3158/17). A recovery study was carried out by by spiking different known concentrations of the Tyr solution. Three replicate measurements (n = 3) were carried out for each measurement. The Tyr concentration was calculated from the calibration curve by employing the corresponding dilution factor. Figure 1 shows the SEM image of the Ty/PtNP/GPH/Chit-CSPE, which presents a typical 3D morphology, confirming the attachment of the GPH on the surface of CSPE, the successful PtNPs electrodeposition and the immobilization of tyrosinase enzyme.   The best results were obtained by the biosensor (Ty/PtNP/GPH/Chit-CSPE) (curve c), which has the highest peak current (74 μA) and the lowest potential corresponding to this peak (0.79 V).     The best results were obtained by the biosensor (Ty/PtNP/GPH/Chit-CSPE) (curve c), which has the highest peak current (74 μA) and the lowest potential corresponding to this peak (0.79 V). The best results were obtained by the biosensor (Ty/PtNP/GPH/Chit-CSPE) (curve c), which has the highest peak current (74 µA) and the lowest potential corresponding to this peak (0.79 V). The peak current identified for Ty/PtNP/GPH/Chit-CSPE is ≈4 times higher than that of CSPE (18.4 µA) and the anode peak potential is about 70 mV more reduced as compared to the positive potential observed for CSPE (0.86 V). The differences between the biosensor and the PtNP/GPH/Chit-CSPE electrode (curve b) are important, as well, the peak current being 2 times higher and the potential being shifted to the lower potential with 90 mV.

Exploratory Studies for the Detection of Tyr by Biosensor
These results prove that Ty immobilized in the matrix of nanostructured materials is viable and acts efficiently on the oxidation of the substrate.
The mechanism of the biosensor detection is illustrated in Figure 3.
Materials 2019, 12, x FOR PEER REVIEW 6 of 14 The peak current identified for Ty/PtNP/GPH/Chit-CSPE is ≈4 times higher than that of CSPE (18.4 μA) and the anode peak potential is about 70 mV more reduced as compared to the positive potential observed for CSPE (0.86 V). The differences between the biosensor and the PtNP/GPH/Chit-CSPE electrode (curve b) are important, as well, the peak current being 2 times higher and the potential being shifted to the lower potential with 90 mV. These results prove that Ty immobilized in the matrix of nanostructured materials is viable and acts efficiently on the oxidation of the substrate.
The mechanism of the biosensor detection is illustrated in Figure 3. Tyrosinase catalyses the Tyr hydroxylation reaction in the ortho position relative to the hydroxyl group (cathecholase) by transforming it into the L-3,4-dihydroxyphenylalanine (levodopa, L-DOPA) amino acid. The subsequent stage is represented by the oxidizing of the ortho-quinonic derivative (a-dopaquinone) [13,28].
The biosensor detection mechanism described proves that the enzymatic reaction is dependent on the concentration of O2 in the solution and on the pH of the medium. Therefore, biosensor measurements should be carried out under ambient conditions in the presence of air and in buffer solution with the pH suitable for the optimal functioning of the enzyme [13,28].
By comparing the cyclic voltammograms of electrodes which were modified successively with nanomaterials or biomaterials, their influence and role in the biosensor detector element may be determined.
Furthermore, by comparing the cyclic voltammograms of PtNP/GPH/Chit-CSPE and Ty/PtNP/GPH/Chit-CSPE, the increased sensitivity of the electrode due to incorporating the enzyme into the solid matrix is obvious. The decrease of the anodic peak potential related to Tyr oxidation process in the presence of the enzyme may be noticed, as well.
When the cyclic voltammograms of CSPE, Chit/GPH-CSPE and PtNP/GPH/Chit-CSPE are compared, an increase in the anodic peak current may be observed, aspect which indicates that both GPH and PtNP facilitate the reaction from the biosensor surface. The differences regarding the mechanisms of action are significant. Thus, GPH through the bidimensional special nanostructure increases the electrical conductivity of the sensitive layer, while PtNPs facilitate the transfer of electrons to the biosensor surface [18,20]. The corresponding effects are synergistic when both nanomaterials are components of a nanocomposite material. Tyrosinase catalyses the Tyr hydroxylation reaction in the ortho position relative to the hydroxyl group (cathecholase) by transforming it into the L-3,4-dihydroxyphenylalanine (levodopa, L-DOPA) amino acid. The subsequent stage is represented by the oxidizing of the ortho-quinonic derivative (a-dopaquinone) [13,28].
The biosensor detection mechanism described proves that the enzymatic reaction is dependent on the concentration of O 2 in the solution and on the pH of the medium. Therefore, biosensor measurements should be carried out under ambient conditions in the presence of air and in buffer solution with the pH suitable for the optimal functioning of the enzyme [13,28].
By comparing the cyclic voltammograms of electrodes which were modified successively with nanomaterials or biomaterials, their influence and role in the biosensor detector element may be determined.
Furthermore, by comparing the cyclic voltammograms of PtNP/GPH/Chit-CSPE and Ty/ PtNP/GPH/Chit-CSPE, the increased sensitivity of the electrode due to incorporating the enzyme into the solid matrix is obvious. The decrease of the anodic peak potential related to Tyr oxidation process in the presence of the enzyme may be noticed, as well.
When the cyclic voltammograms of CSPE, Chit/GPH-CSPE and PtNP/GPH/Chit-CSPE are compared, an increase in the anodic peak current may be observed, aspect which indicates that both GPH and PtNP facilitate the reaction from the biosensor surface. The differences regarding the mechanisms of action are significant. Thus, GPH through the bidimensional special nanostructure increases the electrical conductivity of the sensitive layer, while PtNPs facilitate the transfer of electrons to the biosensor surface [18,20]. The corresponding effects are synergistic when both nanomaterials are components of a nanocomposite material.  As it may be noticed, an increased scanning rate leads to an increase in the anodic peak current and to a slight shift of the peak potential to more positive values. Anodic peak current associated with Tyr oxidation at the surface of the biosensor varies linearly with the scanning rate (I = 288.79v + 45.667; R² = 0.9994), proving that the electrochemical process is controlled by the adsorption process (Figure 4b) [29].

Studies for the Optimization of the Experimental Parameters
In order to obtain the optimal value of the applied potential, the amperometric signal of Ty/PtNP/GPH/Chit-CSPE towards Tyr was determined under continuous and constant stirring of the sample. The applied potential is one of the most relevant parameters, which significantly influences the electrochemical signal of a biosensor, affecting both sensitivity and selectivity. The most intense response of the biosensor was obtained at +0.8 V vs. Ag (Figure 5a). Therefore, the sensitivity of the sensor is maximum for Tyr detection when this value of the potential is applied. As it may be noticed, an increased scanning rate leads to an increase in the anodic peak current and to a slight shift of the peak potential to more positive values. Anodic peak current associated with Tyr oxidation at the surface of the biosensor varies linearly with the scanning rate (I = 288.79v + 45.667; R 2 = 0.9994), proving that the electrochemical process is controlled by the adsorption process ( Figure 4b) [29].

Studies for the Optimization of the Experimental Parameters
In order to obtain the optimal value of the applied potential, the amperometric signal of Ty/PtNP/GPH/Chit-CSPE towards Tyr was determined under continuous and constant stirring of the sample. The applied potential is one of the most relevant parameters, which significantly influences the electrochemical signal of a biosensor, affecting both sensitivity and selectivity. The most intense response of the biosensor was obtained at +0.8 V vs. Ag (Figure 5a). Therefore, the sensitivity of the sensor is maximum for Tyr detection when this value of the potential is applied. The decreased biosensor response may be related to the enzyme denaturation, this leading to the loss of biocatalytic activity. Based on the results of this present study, it was decided that the electrochemical determination of Tyr should be performed at the optimal pH value of 7.

Analytical Performance Characteristics of the Ty/PtNP/GPH/Chit-CSPE Biosensor
In order to achieve superior analytical performance, the square wave voltammetry (SWV) electroanalytical detection technique was used. This method has a much greater sensitivity as compared to cyclic voltammetry or amperometry [33].
The optimal parameters of the SWV method were: pulse amplitude 0.090 V, increment 5 mV, frequency 15 Hz. The potential range used was 0.2 to 1.2 V. The voltammograms obtained in Tyr solutions of variable concentrations are shown in Figure 6.  The decreased biosensor response may be related to the enzyme denaturation, this leading to the loss of biocatalytic activity. Based on the results of this present study, it was decided that the electrochemical determination of Tyr should be performed at the optimal pH value of 7.

Analytical Performance Characteristics of the Ty/PtNP/GPH/Chit-CSPE Biosensor
In order to achieve superior analytical performance, the square wave voltammetry (SWV) electroanalytical detection technique was used. This method has a much greater sensitivity as compared to cyclic voltammetry or amperometry [33].
The optimal parameters of the SWV method were: pulse amplitude 0.090 V, increment 5 mV, frequency 15 Hz. The potential range used was 0.2 to 1.2 V. The voltammograms obtained in Tyr solutions of variable concentrations are shown in Figure 6. pH optimization is necessary in order for the enzyme to have the best biocatalytic activity and to avoid the denaturation of the enzyme immobilized in the detector element of the biosensor. The pH influence on the electrochemical determination of L-Tyrosine with Ty/PtNP/GPH/Chit-CSPE was studied by amperometry in Tyr 10 −4 M solutions in 0.1 M PBS with different pHs in the range from 4 to 10. The response of the Ty/PtNP/GPH/Chit-CSPE biosensor to Tyr 10 −4 M increased when the pH was modified from 4 to 7, and decreased when the pH reached values from 7 to 10 ( Figure  5b).
The decreased biosensor response may be related to the enzyme denaturation, this leading to the loss of biocatalytic activity. Based on the results of this present study, it was decided that the electrochemical determination of Tyr should be performed at the optimal pH value of 7.

Analytical Performance Characteristics of the Ty/PtNP/GPH/Chit-CSPE Biosensor
In order to achieve superior analytical performance, the square wave voltammetry (SWV) electroanalytical detection technique was used. This method has a much greater sensitivity as compared to cyclic voltammetry or amperometry [33].
The optimal parameters of the SWV method were: pulse amplitude 0.090 V, increment 5 mV, frequency 15 Hz. The potential range used was 0.2 to 1.2 V. The voltammograms obtained in Tyr solutions of variable concentrations are shown in Figure 6.  Analysing the voltammograms obtained by using the SWV technique, the fact may be noticed that the anodic peak of Tyr is better defined as compared to that observed in the cyclic voltammograms. This result is due to the application of potential in the form of a pulse, which reduces the consumption of the electroactive species and the background current.
The relationship between the SWV responses and the concentrations of Tyr solutions was determined by using the regression method (Figure 6b).
The dependence obtained is typical for a biosensor, a linearity range and a plateau, where the current of the anodic peak does not increase when the concentration is increased.
The linearity range between the anodic peak current and the Tyr concentration of the solutions analysed was 0.1-100 µM, as it may be noticed in Figure 6b (insert).
The limit of detection (LOD) of the biosensor developed in this study was calculated in accordance with the IUPAC recommendations (3 S/m, where S is the standard deviation of the blank, and m is the slope of the calibration curve) [34]. The replicates number of the blank measurements for the S calculation was 7. The LOD of the biosensor for L-tyrosine in the range of 0.1-100 µM was found to be 4.75 × 10 −8 M.
The Hill coefficient was determined from the dependence equation between log[I/(I max − I)] vs. log [Tyr], which is the slope of this dependence. The value of the Hill coefficient was 1.02, a value very close to the ideal value, which suggests that the enzymatic reaction follows a type of Michaelis-Menten mechanism [35].
Moreover, the biosensor calibration curve and the steady-state current mathematical expression of the Lineweaver-Burk equation for an electrochemical system (Equation (2)) were used to calculate the enzyme kinetics parameters [36].
where: I is the anodic current, I max is the steady-state current, [Tyr] is the molar concentration of tyrosine, and K app M is the apparent Michaelis-Menten constant. The apparent Michaelis-Menten constant calculated for the immobilized tyrosinase was 53.4 µM, a value lower than other values reported in the literature, indicating the efficient immobilization of the enzyme in the bionanocomposite material [26,29,37].
The performance characteristics of the biosensor recommend it for the applications in the analysis of Tyr in real samples.

A Comparative Approach to the (Bio)Sensors Reported in the Literature
The results obtained by using the biosensor developed in this research study (linear range, LOD) were compared to a series of results reported in literature and the results obtained are shown in Table 1. As may be noticed, the Ty/PtNP/GPH/Chit-CSPE biosensor has features similar or superior to various (bio)sensors used for Tyr detection.

Interference Study
To assess biosensor selectivity for Tyr detection in multicomponent samples, the influence of several chemical species, which might interfere with bioassay such as Ca 2+ , Mg 2+ in the determination of 5 × 10 −5 M Tyr under optimal experimental conditions was studied. The tolerance limit was calculated as being the maximum concentration of the interfering chemical species, which causes a relative error of ±5% for the quantitative determination of Tyr. The results obtained are shown in Table 2. Tyr determination is influenced more strongly by the presence of ascorbic acid and uric acid, while Ca 2+ , Mg 2+ , Na + have a reduced influence in quantitative determination. Considering these results, it may be argued that the biosensor developed in this study is selective for Tyr determination in multicomponent samples.

Repeatability and Stability of the Ty/PtNP/GPH/Chit-CSPE Biosensor
In order to analyse the repeatability values of Ty/PtNP/GPH/Chit-CSPE, the Tyr values were recorded by using the same biosensor and the square wave voltammetry in a 10 −5 M Tyr solution. In between the measurements, the biosensor was removed from the assay solution, rinsed with 0.1 M PBS pH 7.0 and then stored for 5 min in PBS. The relative standard deviation for 10 successive measurements was 1.4%, which demonstrates that the biosensor may be used repeatedly for quantitative determinations.
The biosensor stability was analysed by recording the SWV response in the 10 −5 M Tyr solution weekly for a four-week period. In between the measurements, the biosensor was stored in a refrigerator at 4 • C in a sealed closed box. Voltammetric results quantified as the anodic peak intensity proved that the biosensor retained 95.8% of the initial response, thus being almost unchanged four weeks after its manufacture. This very good stability may be related to the protective effect of the bionanocomposite matrix including graphene, chitosan and platinum nanoparticles, which prevents enzyme denaturation and loss of biocatalytic properties.

Use of the Biosensor in Bioassay and Validation of the Quantitative Determination Method
The final step in this study consisted in determining Tyr in biological (3 heparinized blood plasma samples-HBPS) and pharmaceutical samples by using the biosensor developed in this research study and in comparing the results obtained when using the standard determination method (absorbance at 275 nm). The results obtained are shown in Table 3. As can be noticed, the differences between the values obtained for the analytical recovery are lower than 2% for all the samples analysed, which indicates that the biosensor developed in this research study is useful and may be used in bioassay. Analysis of variance (ANOVA) showed that there was no statistically significant difference at the 99% confidence level between the results obtained by using the two methods, i.e., the biosensor and the ultraviolet (UV) spectrometry at 275 nm [42]. In addition, the results obtained in the case of pharmaceutical samples were compared with the values of the active compound concentration mentioned on the label of commercial products, the almost identical values obtained demonstrating the accuracy of the biosensor measurements developed in this research study.

Conclusions
A new nanostructured enzyme biosensor was developed and characterized for the qualitative and quantitative determination of L-tyrosine from standard solutions and real samples. Tyr electro-oxidation at the biosensor detector element is favoured by using a biocompatible matrix to immobilize the enzyme and the nanomaterials, which provide a rapid transfer of electrons from the redox reaction. The biosensor has high sensitivity, low detection limit, and the interferences caused by other analytes are minimal. Repeatability and stability in Tyr determination are appropriate and recommend this biosensor for routine determinations. The analytical method involving the use of the biosensor and the SWV detection techniques was validated by comparing the results obtained when using this method with those obtained when using UV spectroscopy. The results confirmed that the method is reliable, accurate and applicable in the Tyr bioassay.