A Surface Network Based on Polytyramine/ Gold Nanoparticles: Characterization, Kinetics, Thermodynamics and Selective Determination of Norepinephrine

: A solid-state sensor was fabricated by a spontaneous electrochemical deposition of polytyramine (Ptyr) film onto a glassy carbon electrode (GCE) which was further peripherally supported by gold nanoparticles (AuNPs). The surface materials of the developed sensor (AuNPs.Ptyr-GCE) were characterized by X-ray photoelectron spectroscopy (XPS), electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The rate constant of charge transfers ( k ct ) of the as-prepared sensor (8.77 × 10 -4 cm/s) was evaluated by fitting the charge transfer resistance ( R ct ) data in the presence of ferric-ferrous hexacyanide redox couple solution, [Fe(CN) 6 ] 3-/4- . The voltammetric behavior of norepinephrine (NOR) was confirmed to follow an irreversible reaction mechanism at which the estimated diffusion coefficient value was 7.39 × 10 -5 cm 2 /s. The sensor showed a large enhancement on NOR oxidation and comparatively lowered its detection limit (DL 3  ) to 0.130  M (22 ppb). It was also applied for selective determination of NOR in the presence of high concentrations of ascorbic acid (AA) and uric acid (UA). The interference study highlighted the great stability of the proposed sensor by generating a similar sensitivity as in the pure NOR solution. The analytical performance of the proposed system was validated successfully for pharmaceutical and biological samples with tolerable

dipotassium phosphate (K 2 HPO 4 ) were obtained from BDH, UK. Gold nanoparticles (AuNPs) of 30 nm size come with a citrate ligand (Nanopartz Inc., USA). A moderately alkaline background electrolyte (pH = 7.4) made of 0.1 M phosphate buffer solution (PBS) was used for selective determination of NOR in the electrochemical cell (size = 20 mL) which was degassed for 10 min prior to measurement with oxygen-free nitrogen gas, and which was blanketed throughout the experiment.

Fabrication of the surface network
The proposed AuNPs.Ptyr-GCE surface network was fabricated by deposition of Ptyr onto the clean GCE via reversible voltammetric cycling followed by a drop casting of 20 L AuNPs. The electrochemical deposition of the Ptyr was achieved by continuous scanning of the WE potential between 0 mV and 1400 mV vs. Ag/ AgCl at 50 mV/s for 30 scans (15 cycles) in 0.1 M of H 2 SO 4 containing 20 mM tyr monomer. The deposition time limit for controlling the thickness of the Ptyr film is a crucial factor to build up a sensitive surface material possessing a good conductivity. Therefore, lowering the number of repetitive cycles to 15 was sufficient to fabricate a thin and stable platform for doping electroactive materials such as AuNPs and presenting an active surface for electron shuttling with the electrode substrate. The electrode sensitivity decreases dramatically with a larger deposition time limit of Ptyr (above 20 cycles), in agreement with previous experimental data describing the surface resistivity, passivation and limitation of active area due to increasing film thickness [30].

Characterization of the surface network
The surface materials of the modified electrode were essentially identified (structure, composition and reactivity) using various analytical tools. An X-ray Photoelectron Spectroscopy (XPS) machine (Omicron Nanotechnology XPS system, Germany) was employed to determine the composition of materials deposited onto the GCE. The obtained XPS spectra of individual components were de-convoluted using a Gaussian Lorentzian function after background subtraction with a Shirley function in Casa XPS software (Casa Software Ltd, UK). The binding energies were calibrated with respect to a adventitious C 1s feature at 284.6 eV.
Electrochemical impedance spectroscopy (EIS) experiments using a Bi-Potentiostat PC-14 (Gamry, USA) were conducted to identify the resistivity of the deposited materials on the electron transfer processes. The EIS data were recorded in a three-electrode cell containing 5 mM of [Fe(CN) 6 ] 3-/4solution within a frequency range of between 100 kHz to 1 Hz at an amplitude of 5 mV. Randle's equivalent circuit was used to demonstrate the electrochemical properties at the solid/electrolyte interface at open circuit potential.
Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) techniques (BAS Software, USA) were used for electrochemical deposition of the modified electrode, reactivity and selectivity tests, and analytical performance for sensitivity of determination of NOR in artificial and real samples.

Preparation of real samples
A noradrenaline ampoule (1 mg/mL) and plasma blood samples were obtained from SQU hospital, Muscat, Oman. The drug solution was diluted 10 times with PBS (0.1 M of pH = 7.4) to prepare a 0.591 mM stock solution which was applied directly for DPV measurements of NOR. On the other hand, the plasma blood was diluted 3 times with PBS and the standard addition method for as-prepared 0.5 mM of NOR was applied. The recovery percentage of NOR in both real samples was computed to evaluate the sensitivity and reliability of the proposed sensor.

Fabrication of the surface network materials
Electrochemical deposition of Ptyr film on the GCE surface was obtained by repetitive voltammetric scanning of 30 segments (15 cycles) using the cyclic voltammetry (CV) method. The electrode potential was reversibly stepped between 0 and 1400 mV vs Ag/AgCl at a rate of 50 mV/s in an electrochemical cell containing 20 mM of tyr monomer dissolved in 0.1 M H 2 SO 4 . Figure 1 shows an irreversible anodic peak for tyr oxidation at 1175 mV (for the first cycle) which decreases significantly at the preliminary four successive cycles. Moreover, the anodic peak potential is shifted negatively to 1125 mV in the fourth cycle. The above trend suggests a surface passivation due to the generation of low conductivity short oligomers. The growth of the polymeric film in the subsequent cycles from cycle 5 (dotted black) to cycle 15 (solid black) as shown in the inset is associated with reduction of the surface resistivity, apparent by a spontaneous increase of the anodic peak current at 1135 mV. The propagation of the polymer (Ptyr) is predominantly carried out by the formation of cationic radical intermediates due to an oxidation reaction of the phenolic group present in the tyr monomer [31]. The phenoxy cationic radical produced in the initiation step undergoes a dimerization reaction by attacking a new tyr molecule (linkage via the ortho position in the aromatic ring). With subsequent cycling, the oligomerization process is continued to form an insoluble propagated polymer that is deposited onto the electrode surface in a mechanism consistent with those demonstrated for Ptyr deposition at sputtered [32] and graphite [33] electrodes. In the present experimental conditions, the 15 repetitive cycles were appraised to build a thin polymeric film on the electrode surface. It was observed that the anodic peak current adopted nearly a constant value above 15 cycles, indicating the growth of a thicker film. The thickness and the amount of Ptyr deposited on the electrode surface are directly affected by the nature of the electrode substrate, the concentration of tyr monomer and electrolyte composition (mostly the pH), the number of potential cycles and scan rates. It should be noted that the oxidation of tyr in an acidic medium results in the formation of Ptyr with a rich number of free cationic amino groups (one per a chain). Accordingly, the experimental conditions and data presented in Figure 1 were selected to generate a thin Ptyr platform film for doping active surface materials such as gold nanoparticles (AuNPs). The above pattern of fabrication increases the electrode surface area and enhances the electron transfer kinetics with the targeted electroactive species. For this task, a drop of 20 L AuNPs was mechanically added onto the moiety of the fabricated Ptyr modified surface.
The proposed sensor (AuNPs.Ptyr-GCE) was characterized using various analytical tools and finally applied for the selective determination of NOR in artificial and real samples.

XPS studies
The XPS core level spectra of C 1s peaks obtained from different samples are shown in Figure 2. The XPS spectra of a bare GCE surface shown in Figure 2 (A) produces approximately an equal percentage of (C-C)/ (C-H) sp 2 hybridization obtained at (284.5 eV) and sp 3 hybridization at (285.0 eV), respectively. The structure and percentage of (C-C)/(C-H) function are changed upon deposition of the polymer and came as a single peak at its preference position of 284.6 eV with 93.6% as shown in Figure 2 (B). Two extra convoluted peaks were obtained at 285.7 eV (21.3%) and 287.1 eV (12.6%) characterized for functional groups of (C-N)/(C-O) and (C=O), respectively. Upon casting of the AuNPs, the C 1s spectra for the fabricated (AuNPs.Ptyr-GCE) shows some distinctive features on the nature of surface functionalities in terms of binding energies and percent concentrations.   [34] were obtained with a spinorbit separation of 3.7 eV confirming the attachment of Au species onto the surface moiety [35]. The experimental data and all features presented in Figure 2 are evidence for successful surface modification and the final preparation of the proposed sensor.

EIS and electrochemical studies
The structure of the solid/liquid interface is subject to some limiting factors that affect the reactivity of the fabricated sensors. Restriction factors are centralized by the internal resistance generated by surface modification and can be represented by the impedance of the electrodes under investigation. The internal resistance is composed of three components: charge transfer resistance (R ct ) which arises by surface activation, the omhic component which is described by the solution resistance (R s ) due to solution resistivity and the nature of surface materials, and finally the concentration or mass transport resistance (generally considered as a pure diffusion). These important features can be studied efficiently using the electrochemical impedance spectroscopy (EIS) under experimental conditions of a narrow potential window and a wide frequency range.
The Nyquist plot presented in Figure 3(A) demonstrates the applied potential at the open circuit value of a redox probe (5 mM of [Fe(CN) 6 ] 3-/4-) which is characterized by both faradaic and non-faradaic processes. The faradaic process is initiated by the surface activation process and defined by the electrode kinetics (fast or slow). Therefore, it is a direct measurement of (R ct ) and describes the extent of impedance (inhibition) of the electron transfer process at the modified surfaces.
Mathematically, it is inversely proportional to the rate constant of the electron transfer process (k ct ) induced by surface materials and simply given by (R ct * k ct = K) [36], where K is a constant equal to 0.749 cm.ohm/s for the present experimental conditions, which involve the transfer of a single electron (n = 1) at ambient temperature (T = 298 K). The non-faradaic process arises due to charging of the double layer capacitance (C dl ) via electrostatic attraction of ions at the interface satisfied by the value of potential across it. In addition, a typical semicircular curve with resistance zone combines both (R ct ) and the uncompensated solution resistance (R s ) imposed by the working electrolyte, both obtained at high frequency. The mass transport process (diffusion) is depicted as a straight line obtained at low frequencies.  Figure 3(B) depicts the electrochemical behavior of bare GCE (solid black) and as-prepared modified electrodes Ptyr-GCE (solid red) and AuNPs.Ptyr-GCE (dotted blue), respectively, in the presence of a redox probe (5 mM of [Fe(CN) 6 ] 3-/4-) using the CV method. Both bare GCE and AuNPs.Ptyr-GCE produce very well-defined and comparable reversible redox waves with peak potential separation (E p = E p,a  E p,c ) of 106 mV and 188 mV, respectively. In the case of Ptyr-GCE, the calculated (E p = 282 mV) value becomes much larger due to the evident potential shift (far positive and negative) of both anodic and cathodic peaks, suggesting slow electrode kinetics. The presence of AuNPs onto the Ptyr moiety improves the electron transfer process by lowering the electrode over-potential by approximately 100 mV. Moreover, the response currents (anodic and cathodic) of the AuNPs.Ptyr modified surface are much greater than that recorded for the virgin Ptyr surface, consisting with the experimental data of EIS presented in Figure 3(A). The above characteristic mapping confirms a successful construction of modified electrodes and the importance of AuNPs as a reactive dopant. A further investigation on the behavior of NOR oxidation at various electrodes was conducted to characterize the significance of AuNP doped surface materials. Figure 4 depicts the electrochemical oxidation of 50 M NOR at various electrodes in 0.1 M of PBS (pH = 7.4) and (v = 50 mV/s) using the DPV technique. The electrochemical measurements were expressed by the change in the current response of NOR oxidation (I) after eliminating the current response of the background electrolyte generated by each electrode. Obviously, the current response at the bare GCE is the lowest compared to the as-prepared modified electrodes which can be attributed to weak adsorption of NOR. In the intermediate (Ptyr-GCE) surface, the current response is little higher than the bare substrate due to the presence of more active surface sites to attract NOR. The proposed sensor (AuNPs.Ptyr-GCE) shows a prominent current response approaching 1.75 times greater than that of the (Ptyr-GCE) modified electrode. The presence of AuNPs has an evident synergism action for accelerating the electron transfer process with NOR and also offers a wide adsorption assembled surface for stronger bonding. It should be noted that the peak potential of NOR oxidation is shifted in a more negative direction by 25 mV compared to the modified Ptyr-GCE. It is good evidence for a rapid electrochemical reaction that is explicitly pertinent to the surface materials of the proposed sensor.

The behavior of NOR oxidation at AuNPs.Ptyr-GCE
The electrochemical behavior of NOR at the developed AuNPs.Ptyr-GCE was monitored using the cyclic voltammetry (CV) technique as shown in Figure 5 (A). Apparently, the reaction mechanism of NOR is following an irreversible mode concluded by the growth of an anodic peak at 240 mV in the forward scan corresponding to NOR oxidation, which increases linearly with increasing [NOR]. In the reverse cathodic scan, only a peak shoulder of constant current response is collected at 130 mV for the entire [NOR] range. This trend can be attributed to the reaction mechanism of NOR oxidation which involves a cyclization step [37]. It is important to note that some literature reports that NOR experiences a reversible reaction by varying the recorded anodic and cathodic peaks with electrode scan rates without considering the size (ratio) of both obtained peaks. Figure 5 (B) depicts the linear relationship of the anodic peak current (I p ) as a function of [NOR] collected from the basic experimental data of Figure 5 (A). The resulting slope of (0.0577 A/M) is important to calculate the diffusion coefficient of NOR applying the Randles-Sevcik equation for irreversible processes [36]. The calculated value is 7.39 × 10 -5 cm 2 /s assuming a transfer of two electrons (n = 2) by NOR oxidation. The obtained value for the present system is approximately two times greater than (3.30 × 10 -5 cm 2 /s) evaluated at a ZnO/CNTs modified electrode [38]. The minimum and maximum boundaries of NOR diffusion coefficient values were not identified since this thermodynamic parameter is affected by the electrolyte composition, the nature of surface materials, the ionic or molecular size, and the temperature.
The reactivity of the proposed sensor (AuNPs.Ptyr-GCE) was tested under experimental conditions given in Figure 5 (A) for 10 cycles (20 segments) in the presence of 50 M of NOR. The anodic peak current decreased spontaneously with subsequent cycles to fall to 86.0% (in the last three cycles) of its ideal initial value as shown in Figure 6. This marks a change in the nature of the surface due to the accumulation of inactive oxidation byproducts. Moreover, it describes the robustness and stability of the developed sensor against surface poisoning. It is worth noting that the oxidative pathway of NOR is initiated by the transfer of two-electrons and two-protons, forming an unstable oquinone derivative that rapidly cyclized via intermolecular attraction to produce a leucochrome derivative. The reaction is propagated to the formation of an electrochemically inactive melanin polymeric species [37,39].

Selective determination of NOR at AuNPs.Ptyr-GCE
The practical application of AuNPs.Ptyr-GCE for NOR quantification is presented in Figure 7 employing DPV in 0.1 M of PBS (pH = 7.4) at (v = 50 mV/s). A well-defined anodic peak current was obtained at 192 mV vs Ag/AgCl corresponding to NOR oxidation, which directly correlated to successive increments of [NOR]. The stability and reactivity of the sensor is diagnosed by bringing the anodic peak to a constant potential value for the entire applied [NOR] range. The linear plot of (I p ) as a function of [NOR] shown in the inset of Figure 7 produces a slope of (0.1189 A/M), by which the calculated detection limit (DL 3 ) was 0.130 M (22 ppb).  Table 1 lists the analytical performance of various modified electrodes [40][41][42][43][44][45][46] for NOR quantification in terms of detection limit, sensitivity and applied electrochemical method. The present electrochemical system is highly competent and exhibits superior performance to some relevant polymeric constituent surfaces.
Inspection of the competency of the proposed sensor was performed by selective determination of NOR in the presence of AA (250 M) and UA (50 M) as electroactive interference species. Figure 8(A) shows the DPV anodic peak for NOR oxidation obtained at 192 mV which increases gradually with subsequent addition of NOR for a concentration range between 0.5 M and 25 M. The inset of Figure 8(A) shows that the DPV anodic peaks for AA and UA oxidation (in the absence of NOR) occurred at 2 mV and 266 mV, respectively. A potential distance of 190 mV from AA and 74 mV from UA is adequate to overcome any possible overlap of anodic peaks of the three species. Moreover, the adsorption capacity of the electrode surface materials is capable of assembling all molecules and their byproducts indicating a robust and reactive sensor. A close scrutiny of the linear plot, (I p ) vs [NOR], given in Figure 8(B), demonstrates that the slope of (0.1015 A/M) is consistent with that in the absence of AA and UA (shown above in the insert of Figure 7) which is evidence of good reproducibility and the stability of the present sensor. Moreover, the calculated detection limit (DL 3 ) was 0.142 M (24 ppb). The reliability and sensitivity of the present electrochemical system were tested for the determination of NOR in drug and blood plasma samples using a DPV method as described above in section 2.4. The average recovery percentage (n = 3) of NOR listed in Table 2 was obtained by conversion the DPV peak current into [NOR] using the regression data illustrated in Figure 8(B). The minimum (95%) and maximum (101%) of reported percentages under the present experimental conditions describe the extent of reactivity and analytical performance of the sensor developed for sensitive NOR determination.  The electrochemical stability of the developed sensor in the last injection of NOR (25 M) NOR in the presence of (250M AA) and (50 M UA) was verified by DPV repetitive scanning for 12 runs with 5 min intervals. The anodic peak potential of NOR oxidation was consistently obtained at 192 mV and decreased negligibly with subsequent runs to approach 2.6% of its maxima in the last run, suggesting a reliable and stable sensor for selective NOR quantification in real samples.

Conclusion
This paper describes an approach for the fabrication of a solid-state sensor based on electrochemical polymerization of tyramine (Ptyr) film onto the surface of a glassy carbon electrode (GCE) followed by a drop casting of gold nanoparticles (AuNPs). The as-prepared surface materials (AuNPs, Ptyr-GCE) were characterized using surface image spectroscopy and electrochemical methods. These comprehensive investigative methods explore properties of the built up surface materials such as composition and structure, resistivity and reactivity, and analytical performance. The proposed sensor was tested for norepinephrine (NOR) determination in an artificial sample at which the limit of detection was brought down to 0.130 M. The sensor excellently passed the selectivity test in the presence of excess concentrations of electro-active biological interference agents such as ascorbic acid (AA) and uric acid (UA). Moreover, the sensor's performance was substantiated practically for NOR determination in a drug formulation and in plasma blood samples with satisfactory recovery percentages.