Development of electrode by using gold-platinum alloy nanoparticles for electrochemical detection of serum amyloid A protein

Gold-platinum alloy nanoparticles (AuPtNP) were electrochemically deposited on the surface of indium-tin-oxide (ITO) modified with (3-aminopropyl)triethoxysilane (APTES), after optimizing deposition conditions for the electroplating solution and number of deposition cycles. Different part ratios of Au/Pt used were 4/0, 3/1, 2/2, 1/3 and 0/4 (AuNP, Au 3 Pt 1 , Au 2 Pt 2 , Au 1 Pt 3 and PtNP, respectively) in preparation of 1 mM solutions. FE-SEM, EDAX and XRD surface characterization techniques were used to confirm the presence of deposited AuPt alloy nanoparticles on the modified ITO surface. Electrochemical methods (CV, DPV and EIS) were used to investigate the electrochemical properties of prepared electrodes in the presence of ferri/ferrocyanide redox couple, which indicated the following increasing order of electrocatalytic peak current: Au < Au 2 Pt 2 < Pt < Au 1 Pt 3 < Au 3 Pt 1 . The most active electrode, Au 3 Pt 1 NP/APTES/ITO (with Au/Pt part ratio 3:1), was further used to fabricate the immunoelectrode SAA-Ab/Au 3 Pt 1 NP/APTES/ITO. The prepared immunoelec-trode was tested for detection of serum amyloid A protein biomarker (APO-SAA) by immobilising SAA-specific antibodies (SAA ½ Ab) on its surface. Sensing studies on this immunoelectrode, performed by DPV technique, revealed the SAA biomarker detection in the linear range of 10 to 10 6 fg ml -1 . The limit of detection was calculated as 7.0 fg ml -1 .


Introduction
Bimetallic nanoparticles have gained great attention due to their unique catalytic efficiency, low cost, high stability, high electronic and optical properties.The presence of the second metal affects many parameters of prepared alloys like particle size, composition alterations, shape, surface morphologies, chemical and physical properties of materials, including catalytic activities and selectivity.Bimetallic nanoparticles of platinum and gold have proven their extraordinary electrocatalytic properties and are used for various applications, including fuel cells and biosensing [1][2][3][4].
Pt with outstanding electrocatalytic properties and Au with more electronegativity and less reactivity produce synergistic catalytic effects due to their interaction in their alloy [5][6][7].The greater availability of gold at a lower price than platinum favors this combination of metal nanoparticles to tap the benefits of both in one alloy besides reducing the cost of preparation of electrode [8][9][10][11].Electrochemical deposition of Au-Pt nanoparticles is a simple, easy, and direct method to fabricate electrodes with AuPtNP alloy film.
Indium tin oxide (ITO) has a wide range of applications and can be used in fields such as biosensors, transistors, electroluminescence devices, light-emitting diodes, solar cells and displays.Its surface can be modified in different ways to enhance its electrocatalytic activity [5,12,13].Due to high transparency, excellent electrical conductivity and large conducting surface area, ITO has wide potential to be used as a working electrode for electrochemical deposition of AuPtNP alloy film.As it can be easily etched and patterned, electrochemical biosensors can be fabricated using modified ITO surfaces to detect various biomolecules of interest [12].
Serum amyloid A (SAA) protein, an inflammatory biomarker, has recently gained attention as its concentration in human serum increases manifolds immediately after viral infection in the human body, which helps in the early detection of viral diseases.Its concentration may increase many times its original concentration and decrease at a faster rate during recovery from the infection [14][15][16].Fabricating an electrochemical biosensor to detect biomarkers is easy, cost-effective, less timeconsuming, and non-invasive as compared with the other techniques to detect SAA biomarker.These biosensors are sensitive, reproducible, reliable, selective, and stable towards target biomolecules and hence possess wide potential in clinical diagnosis [17,18].
Xia et al. reported SAA electrochemical immunoassay [19] to detect SAA as a chronic inflammatory biomarker, while Lee et al. reported the fabrication of anodic aluminum oxide (AAO) chip for the detection of SAA 1 as a lung cancer biomarker 20.Antibody array-based immunosensor was developed by Timucin et al. [21] to detect SAA as a cardiovascular disease biomarker, [11,22] presented the detection of SAA as a renal cell carcinoma biomarker in their report.SAA as an inflammatory rheumatic disease biomarker and for allograft rejection was reported by Sorić Hosman et al. [23], while SAA as a prominent acute phase reactant biomarker was reported by Malle et al. [16].
In the present manuscript, electrochemical deposition condition parameters were firstly optimized for the alloy of gold-platinum nanoparticles (AuPtNP) on the ITO surface.AuPtNP hybrids were electrochemically deposited on the ITO surface with different part ratios of Au and Pt salts in the electrolyte.Electrode with part ratio 3:1 for Au and Pt, respectively (Au3Pt1NP) was found to be of greater electrocatalytic activity and was further used to detect SAA biomarker by immobilizing antibodies on the surface of the optimized electrode (Scheme 1).

Scheme 1. Scheme for fabrication of SAA-Ab/AuPtNP/APTES/ITO immunoelectrode
The procedures for modification of ITO surface with APTES, preparation of electrolytic solutions and antibody immobilization have already been reported in our previous works 24,25.

Instrumentation
For electrochemical deposition and characterization, an advanced Metrohm potentiostat/galvanostat 204 was used with a three-electrode system [24,25] containing Ag/AgCl/3 M KCl reference electrode and Pt wire as a counter electrode.Electrochemical characterization techniques used were cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) in 100 mM PBS buffer solution with 0.9 % NaCl and 5 mM [Fe(CN)6] 3−/4− (pH 7.4).EIS measurements were performed using alternating signal amplitude of 10 mV, dc potential of 0.03 V and frequency range of 10 5 to 0.1 Hz.
For morphological characterization of AuPtNP-modified electrodes, FE-SEM (JEOL JSM-7610F Plus with attached EDX analysis tool) was Shimadzu UV2600sed.A 20 kV operation voltage was used over a collection period of 164 seconds to obtain the EDX spectrum.To determine the crystalline structures of the deposits, an X-ray diffraction (XRD) analyzer (Rigaku SmartLab StudioII,  = 0.1541 nm, Cu K radiation) was used.UV measurements were performed by Shimadzu UV2600 spectrophotometer.

Optimization of electrochemical deposition parameters for AuPtNP deposition on ITO surface
AuPtNP were deposited electrochemically from two different solutions (labelled as solution A and B, both containing platinum salts H2PtCl66H2O and HAuCl43H2O) on the surface of ITO with and without its modification with APTES.The CV technique was applied in the potential range of -0.6 to 1.0 V and a scan rate of 100 mV s -1 for 20 cycles.Only slight changes were observed in redox peak currents after depositing AuPtNP for more than 60 cycles on the electrode surface.
AuPtNP were first deposited on ITO surface (with/without modification with APTES) from solution A (0.01 M Na2SO4, 0.01 M H2SO4 and 1 mM total concentration of H2PtCl66H2O and HAuCl43H2O), and then from solution B (0.05 M PBS (pH 7.0) and 1 mM total concentration of H2PtCl66H2O and HAuCl43H2O) [26].The procedure for cleaning and modification of bare ITO electrodes of size 11 cm 2 with APTES was reported in previous works [24,25], along with a procedure for solution condition optimization.

Electrochemical characterization of variously deposited AuPtNP on ITO and APTES/ITO electrodes
AuPtNP (Au2Pt2) deposited for 20 cycles on bare ITO and APTES/ITO electrodes from solutions A and B were characterized by CVs recorded at 50 mV/s scan rate within -0.5 to 1.0 V potential range in 100 mM PBS buffer solution containing 5 mM [Fe(CN)6] 3−/4− (Figure 1a).The highest redox peak current was obtained for the AuPtNP/APTES/ITO electrode when AuPt nanoparticles were deposited from solution A. The reason for this could be easier electrochemical deposition in acidic conditions containing H2SO4.
Characterization of bare ITO and APTES/ITO modified with Au, Pt and AuPt metal nanoparticles was performed using CVs (at 50 mV/s scan rate and -0.6 V to 1.5 V potential range) in 0.05 M H2SO4 (Figure 1b) for electrodes AuNP/APTES/ITO, Au2Pt2NP/APTES/ITO and PtNP/APTES/ITO.The reduction peaks were observed at potentials 0.61 and 0.02 V for electrodes AuNP/APTES/ITO and PtNP/APTES/ITO, respectively.For electrode AuPtNP/APTES/ITO, two reduction peaks were observed at potentials 0.57 V and 0.07 V, corresponding to deposited AuNP and PtNP.These two peaks confirm the deposition of two noble metal alloy nanoparticles on ITO [27].Since the highest oxidation and reduction peak currents due to ferricyanide/ferrocyanide redox reaction were obtained for the APTES-modified ITO with AuPtNP deposited from solution A, solution A has been used throughout this study for deposition of AuPtNP.
EDX results for various AuPtNP hybrids deposited on APTES/ITO confirm the presence of deposited Au and Pt on the electrode surfaces.Further, the varying content of Au and Pt in different hybrids follows their varying composition in the salt solution.EDX of the electrode with an electrochemically deposited film of Au3Pt1NP shows a higher content of Au as compared to Pt, whereas EDX of Au1Pt3NP electrode shows less elemental presence of Au as compared to Pt.It is worth noting that in the case of the Au2Pt2NP electrode, where the same molar concentrations of Au and Pt in the salt solution were used, the EDX presents a content of Pt as compared to Au.This may be because Pt tends to get deposited on the electrode surface better than Au.The increased tendency of PtNP deposition compared to AuNP deposition is also reflected for Au1Pt3 and Au3Pt1 electrodes, where the content is not directly correlated with the parts of their respective salts in the solution.Rather, the weight of PtNP deposited is higher compared to its part ratio in the salt solution.

XRD analysis of deposited AuPt metal nanoparticles
X-ray diffraction spectra of different AuPtNP/APTES/ITO electrodes are presented in Figure 4. XRD diffraction patterns acquired for all prepared electrodes show different peaks, as listed in Table 2.In the 2θ range of 10 to 80°, different distinctive peaks have been obtained with (111), (200), ( 220) and (311) as their crystallographic planes, respectively.A fairly symmetric and single peak (111) face is shown by XRD profiles of the Au-Pt bimetallic system, which is intermediate of Au and Pt (111) peaks [24,25].Moreover, the values of Au-Pt hybrid nanoparticles lattice constant (d) are in between values of monometallic gold (0.407 nm) and platinum (0.392 nm) and increase linearly on increasing mole fraction of platinum in the electrolyte solution.Vegard's law was used to calculate lattice constant (d), which suggests an alloy feature in deposited Au-Pt hybrid where Au and Pt atoms are mixed tightly [28].

2 / °
Pt mole fraction in the electrolyte  Absorption spectra analysis of deposited AuPt nanoparticles UV absorption spectra for AuNP, PtNP and their alloys electrochemically deposited on APTES/ITO electrode surfaces in different ratios were compared in a range of 400 to 800 nm wavelength (Figure 5).No absorption peak was observed for PtNP (Figure 5a), while different broad peaks for AuNP, Au3Pt1NP, Au2Pt2NP and Au1Pt3NP were observed at maximum wavelengths of 567 nm (Figure 5b), 557 nm (Figure 5e), 551 nm (Figure 5c) and 515 nm (Figure 5d), respectively.A left shift of wavelength peaks in the obtained spectrum was observed with the increase of Pt content in the alloy [1].

Electrochemical characterization of different AuPtNP/APTES/ITO-modified electrodes
Further electrochemical investigations were done for all prepared AuPtNP-modified electrodes (Au3Pt1NP/APTES/ITO, Au2Pt2NP/APTES/ITO and Au1Pt3NP/APTES/ITO) in 100 mM PBS buffer solution containing 5 mM of [Fe(CN)6] 3−/4− redox couple.An increase in redox peak currents and a decrease in the separation of peak potential values were observed with the deposition of noble metal alloy nanoparticles on APTES/ITO (Figure 6a), which is reconfirmed by DPV peak current (Figure 6b) and a decrease in resistance of charge transfer (Figure 6c).The corresponding parameters for AuPtNP/APTES/ITO electrodes are summarized in Table 3 and compared with those already obtained for Au and Pt-modified APTES/ITO electrodes 24,25.Among AuPtNP-modified electrodes, an increase in anodic and cathodic current is maximal for Au3Pt1NP/APTES/ITO with ΔIp 1271.97 µA, for which the lowest charge transfer resistance (Rct) of 319.78  was obtained (Table 3).So, for the preparation of the biosensing electrode, Au3Pt1NP/APTES/ITO was used and modified by depositing SAA biomarker-specific antibodies.

SAA ½ antibodies immobilization on Au3Pt1NP/APTES/ITO
Solution of SAA ½ antibodies of concentration 10 μg ml -1 was prepared as reported in our previous work [24,25], and 20 μl were poured on the surface of Au3Pt1NP/APTES/ITO electrode and left for the whole day for immobilizing antibodies by self-assembled monolayer (SAM).Such prepared immunoelectrode (SAA-Ab/Au3Pt1NP/APTES/ITO) was further used for biosensing of biomarker SAA by various electrochemical techniques [24,25].
Electrochemical properties of SAA-Ab/Au3Pt1NP/APTES/ITO electrode The electrochemical properties of bare Au3Pt1NP/APTES/ITO and SAA-Ab/Au3Pt1NP/APTES/ITO electrodes in the presence of the [Fe(CN)6] 3−/4− redox couple were defined by different electrochemical techniques, i.e.CV, differential pulse voltammetry (DPV) and EIS (Figure 7).The observed variations in peak potential difference (ΔEp) and peak redox currents (ΔIp) for recorded CVs profiles in 100 mM PBS buffer solution containing 5 mM [Fe(CN)6] 3−/4− in -0.5 to 0.7 V potential range and at 50 mV/s scan rate, have indicated variations in current values and other parameters, not only between two electrodes, but for ITO and APTES/ITO electrodes [24,25] too.CV of bare ITO showed ΔEp of 0.27 V, oxidation and reduction peak potentials at 0.26 and -0.02 V, respectively, and ΔIp of 1078.68 μA [24,25].Decreased ΔIp value (775.21μA) was obtained when ITO was modified with APTES with an increased (ΔEp) value of 0.59 V.This decrease in ΔIp value could be due to inhibition in electron transfer between the redox probe and the surface of the electrode, which also confirms the modification with APTES [24,25].An increased ΔIp value (1271.97μA) was seen for Au3Pt1NPs modified APTES/ITO electrode with ΔEp 0.31 V, which can be due to the large surface area and increased conductivity provided by electrochemical deposition of AuPtNPs.This led to faster electron transfer between the redox probe and AuPtNPs modified ITO electrode.with specific antibodies (SAA-Ab/Au3Pt1NP/APTES/ITO).This could be ascribed to the insulating layer formation after antibodies immobilization on the electrode surface, which hinders electron transfer between immunoelectrode and redox probe.The pattern of variations obtained for redox peak currents was similar to the pattern of current variations in DPV scans (Figure 7b) of all these modifications of ITO (in -0.3 to 0.7 V potential range) [1,27].Further, the electrochemical impedance spectroscopy (EIS) technique was used to compare the electrochemical properties of Au3Pt1NP/APTES/ITO and SAA-Ab/Au3Pt1NP/APTES/ITO electrodes.The Nyquist plots obtained after EIS measurements (Figure 7c) indicated variations in impedance spectra for each modification of ITO [29].In Nyquist plots, a semicircle diameter represents the electron transfer resistance (Rct), which helps to investigate the interfacial properties of the modified electrode [30,31].The corresponding Rct values for bare ITO, APTES/ITO [24,25], and two ITO modifications presented in Figure 7c are given in Table 4.It was already shown that bare ITO and APTES/ITO showed Rct values of 677.14 and 1728.64Ω, respectively [24,25].However, the smallest Rct value (319.78 ) was obtained for APTES/ITO modified with Au3Pt1NPs, indicating excellent conductivity of AuPt alloy nanoparticles.After immobilization of SAA ½ antibodies on the surface of the Au3Pt1NP/APTES/ITO electrode, the Rct value was increased (568.77Ω), which refers to the decreased conductivity of electrode SAA-Ab/Au3Pt1NP/APTES/ITO due to the insulating layer formation after antibodies immobilization.These resulted in higher impedance of charge exchange due to the insulation property of protein molecules.Variations in Rct values and obtained impedance spectra with different sizes of semicircle region in Nyquist plots confirm different modifications of ITO and are also in conformity with obtained ΔIp values of CV measurements (Table 4).

Scan rate studies of fabricated electrodes
Scan rate studies were also performed for Au3Pt1NP/APTES/ITO and SAA-Ab/Au3Pt1NP/APTES/ITO modified electrodes in 100 mM PBS buffer solution containing 5 mM [Fe(CN)6] 3−/4− in the scan rates range of 10 to 200 mV/s (Figure 8).As seen in Figures 8a and c, with every increase in the scan rate, an increase in redox peak current is observed [7].The ratio of redox peak currents (Ipc and Ipa) was near one.The quasi-reversible nature of CV and electron transfer kinetics were confirmed by this ratio, which is supported by an increase in peak potential difference, ΔEp, with an increase in the scan rate.Linear plots of redox peak currents (Ipa and Ipc) and square root of scan rate () were obtained for all examined modified electrodes in the scan rates range of 10 to 200 mV/s (Figures 8b  and 8d).

Electrochemical response to SAA biomarker
A stock solution of concentration 100 μg ml -1 of SAA biomarker was prepared as was reported in our previous work [24,25].DPV technique was used to investigate the electrochemical current in a potential range of -0.3 to 0.7 V.As seen in Figure 9a, the oxidation peak current response of immunoelectrode SAA-Ab/Au3Pt1NP/APTES/ITO as a function of concentration of SAA biomarker revealed linear decrease in recorded DPVs, when 10 μl of SAA biomarker was poured from concentrations range of 10, 10 2 , 10 3 , 10 4 , 10 5 and 10 6 fg ml -1 , and kept on the immunoelectrode for 10 minutes every time.The current decrease could be due to the formed SAA/SAA-Ab, which hinders the electron transfer.The linear relationship between DPV peak current values (with negative slope) and the concentration range of the SAA biomarkers can be seen in the obtained calibration plot (Figure 9b) [32].The linearity is defined by equation ( 1): I / μA = -24.08703(μA (log (fg ml -1 )) -1 [log (cSAA / fg ml -1 )] + 263.91593 μA (1) The regression coefficient for the linearity equation (R 2 ) is 0.94.Using equation 3σ/sensitivity (where σ is the standard deviation, which indicates a "typical" deviation from the mean), the limit of detection (LOD) was calculated as 7.0 fg ml -1 .The sensitivity of fabricated bioelectrodes was determined by linearity curve slope [33][34][35].

Conclusion
Parameters for electrochemical deposition of AuPt nanoparticles (AuPtNP) alloy from solutions containing different molar fractions of Au and Pt salts on ITO modified by 3-aminopropyltriethoxysilane (APTES) surfaces were optimized.The prepared AuPtNPs/APTES/ITO electrodes, having different part ratios of Au and Pt, were characterized by XRD, FESEM, EDAX and UV absorption techniques, confirming different morphologies and the presence of two metals in various modifications at electrode surfaces.Electrochemical testing of electrodes performed by CV and EIS techniques using a redox probe showed that Au/Pt alloy with the part ratio of 3:1, i.e., Au3Pt1NP/APTES/ITO electrode displayed excellent electrocatalytic activity.This electrode was used for SAA-specific antibody immobilization to fabricate the immunoelectrode SAA-Ab/Au3Pt1NP/APTES/ITO, successfully detecting SAA biomarkers in the 10 to 106 fg ml -1 concentration range.A linear relationship was obtained between increasing SAA concentrations and decreasing peak current values of recorded DPVs, with a calculated LOD of 7.0 fg ml -1 .
concentration of metallic salt (out of 1 mM concentration) in the electrolyte solution (Au salt + Pt salt) Concentrations of metallic salts in 20 ml solution (Au salt + Pt salt)

Figure 8 .Figure 9 .
Figure 8. CV plots at scan rates from 10 mV to 200 mV (a and c) and linear plots of reduction and oxidation peak currents against square root of scan rate, with indicated intercepts, slopes and R 2 values (b abd d) for: (a and b) Au3Pt1NP/APTES/ITO; (c and d) SAA-Ab/Au3Pt1NP/APTES/ITO electrodes in 100 mM PBS buffer solution containing 5 mM [Fe(CN)6] 3−/4− .Au3Pt1NP were deposited for 60 cycles from solution A on electrode

Table 2 .
Peak positions and d values for various prepared electrodes

Table 4 .
CV oxidation and reduction peak potentials, their difference (ΔEp), peak current difference (ΔIp) and charge transfer resistance (Rct) for ITO modifications