Electrochemical and Optical Analysis of Various Compositions of Au and Ag Layers for Blood Cancer Prognosis

Abstract

The fabrication of silver (Ag) and gold (Au) thin film electrodes was successfully carried out using the DC sputtering deposition method. These thin film electrodes were able to detect the increase in serum albumin concentration that was used as a prognostic factor for leukemia. The simulation and the optical experimental analysis show that an increase in BSA concentration can increase the absorbance peak observed at a wavelength of 435 nm on hypoalbumin medium and 470 nm on normal concentration of serum albumin medium. The performance of the electrodes was electrochemically tested, in which it was shown that a decrease in oxidation and reduction peaks occurred with respect to an increase in BSA concentration. An oxidation peak was observed at a voltage of 0.5 V for the Ag thin film. For the Au, Au/Ag, and Ag/Au thin films, an oxidation peak was observed at a voltage of 1.0 V. The limits of detection (LODs) of the Ag, Ag/Au, Au, and Au/Ag thin films were 0.56, 0.24, 0.64, and 0.36 g/dL, respectively. Therefore, based on both the electrochemical and optical analysis, the Ag/Au thin film possessed the highest potential for prognosis monitoring of leukemia compared with the other Ag and Au thin films.

1. Introduction

Blood cancer is ranked eleventh among the types of cancer with the highest death rate, with a five-year relative survival rate of 65.7% [1,2]. Efforts can be made to increase the life expectancy of blood cancer patients by monitoring its prognosis factors. Serum albumin is the most abundant protein component of blood [3]. Serum albumin functions to maintain oncotic pressure, acid–base balance, and prevent platelet aggregation. Thus, serum albumin concentration is frequently used for monitoring the physical condition of patients [4]. A study conducted by Wang et al. in 2019 showed that there was an inverse correlation between serum albumin concentration in acute myeloid leukemia (AML) patients and their life expectancy [5]. Past studies have shown that AML patients with serum albumin <35 g/L require more intensive care and have lower survival rates. Consequently, prognosis monitoring of blood cancer can be carried out by measuring the concentration of serum albumin in the blood of patients [6,7]. Conventional methods to monitor the condition of blood cancer patients include bone marrow aspiration to evaluate blood cells and diagnose conditions related to the bone marrow of patients [8]. Spectroscopic and electrochemical methods have been widely developed for the early detection of blood cancer; however, the exploration of these methods for the monitoring of blood cancer patients is still rare [9,10,11].

Various combinations of biosensor electrodes have been developed, specifically for the detection of blood cancer [9]. Au nanoparticles have been widely used as a component in biosensors due to their great ability to provide a stable immobilization as a result of their high surface energy, to allow fast and direct electron transfer between a wide range of electroactive species and electrode materials, and also to be used as signal amplification tags due to their light-scattering properties and very large enhancement ability of the local electromagnetic field [12]. The combination of Au with other precious metals has also been studied to improve biosensor capabilities because the bimetallic structure offers greater stability and also higher electrical, magnetic, and optical properties [13]. Ag can also be explored as a material to be used as a component of biosensors due to their tunable plasmonic properties, higher thermal and electrical conductivities, and efficiency in electron transfer [14]. The combination of Ag and Au has been utilized for biosensors as a bioreceptor binder in electrochemical and optical experiments [15].

The development of biosensors is carried out by exploring the bond between biomarkers and transducers [16]. This bond can influence both optical and electrochemical properties in the form of alterations to electromagnetic wave propagation and the ability of electrodes to carry out redox reactions. Non-electrochemical molecules, such as cells and proteins, can decrease the ability to carry out reduction and oxidation because they inhibit the electron flow at the interface between an electrode and an electrolyte [11]. Meanwhile, the deposition of the biomarker on the transducer can produce a bond between them and change the refractive index value on the surface of the system [17].

The combination of Ag and Au has rarely been used as components of a modified biosensor electrode surface, especially for monitoring serum albumin concentration [18,19,20,21,22]. It is shown in this study that the Ag and Au thin film arrangements using a simple method of fabrication possess different optical and electrochemical characteristics when used in cancer prognosis monitoring. The optical characteristics of the Ag and Au thin film arrangements are investigated by means of experiments and electric field distribution simulations. The actualization of the Ag and Au thin film arrangements proves that it can be used to monitor cancer prognosis by monitoring the concentration of serum albumin using spectrophotometric and electrochemical methods.

2. Materials and Methods

2.1. Materials

Phosphate-buffered saline (PBS), potassium ferricyanide (K₃[Fe(CN)₆]·3H₂O), sodium chloride (NaCl), and bovine serum albumin (BSA) were bought from Sigma Aldrich (Singapore, Singapore). Ag (99.99%) and Au (99.99%) sputtering targets were obtained from Goodfellow (Huntingdon, UK). ITO glass with a transmittivity of 86%, a resistance of 30–40 Ω, and a thickness of 1.1 mm was bought from Ali Laboratory (Surabaya, Indonesia). Acetone and ethanol were obtained from Sigma Aldrich. Distilled water (DI-H₂O) was obtained from SIP (Surabaya, Indonesia).

2.2. Simulation Session

Electric field distribution simulations using the Finite-Difference Time-Domain (FDTD) method were carried out using the Comsol Multiphysics 5.4 software [11]. The geometric model used was the thin film arrangements consisting of ITO glass coated with Ag and Au. The upper part of the electrode geometric model was coated with varying concentrations of BSA. The inputs to the electric field distribution simulation were the refractive index of BSA and all the component materials of the electrode, and also the electromagnetic wavelength [23,24,25]. The electromagnetic wavelengths used were between 400 nm and 1000 nm. The outputs of the simulations using Comsol Multiphysics 5.4 were 3D images of the electric field distributions of the Ag and Au thin films, transmittance values, absorbance values, and the total electric and magnetic energy as a function of wavelength.

2.3. Ag and Au Deposition

The deposition of Ag and Au on ITO glass was performed using the direct current (DC) sputtering method and carried out in BATAN, Yogyakarta, Indonesia. For the DC sputtering process, the sputtering target, which acted as the cathode, was placed at a distance of 25 mm from the substrate, which acted as the anode. Furthermore, the base pressure for the sputtering chamber was set to 10⁻² Pa. Pure argon gas (99.99%) was inserted into the sputtering chamber through a valve with a constant pressure of 60 Pa. The voltage applied between the anode and cathode was 1 kV. The deposition by means of sputtering was carried out for 10 s.

2.4. Electrode Characterization

The microstructure analysis of the Ag and Au thin film electrodes was conducted based on X-ray Diffraction (XRD) using X’pert Pro (PANalytical, Almelo, The Netherlands) to find out the phase of the electrode. The morphological and elemental composition examinations of thin films were carried out using a Phenom scanning electron microscope (SEM) (Dynatech, Jakarta, Indonesia) equipped with an energy-dispersive X-ray spectroscopy (EDS) (Dynatech, Jakarta, Indonesia). Electrochemical performance experiments of the electrodes were carried out using a CorrTest CS2350 potentiostat (Wuhan, China). The experimental mediums were PBS (pH 7.4, 0.1 M), NaCl (0.3 M), and K₃Fe(CN)₆/K₄Fe(CN)₆ (0.01 M). The electrochemical experiments using quantitative analysis were carried out by means of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) with the use of a platinum sheet as the counter electrode, Ag/AgCl as the reference electrode, and the Ag and Au thin films as the working electrode. The concentration of the BSA solution was varied between 2.5 g/dL and 5 g/dL to be used as a prognostic parameter of blood cancer and dripped on top of the electrodes. The CV potential for Ag was −1 to 1 V vs. Ag/AgCl, while the Au, Ag/Au, and Au/Ag electrodes were tested with potentials between −0.5 to 1.5 V vs. Ag/AgCl. The reproducibility test was performed by measuring the CV for 10 cycles. The performance of the electrodes to detect BSA was also analyzed the optical properties qualitatively using Thermo Scientific (Waltham, MA, USA) Genesys 10S UV–Vis spectrophotometer. Hypoalbumin and normal serum albumin concentrations were carried out using serum albumin concentrations of 2.5 g/dL and 5 g/dL. The test was repeated 5 times.

3. Results and Discussion

Figure 1a–d show the top and side views of the prepared Ag and Au thin films from the surface morphology analysis using SEM. A consistent and even surface morphology can be exhibited on all the electrodes, namely, Ag, Au, Ag/Au, and Au/Ag. It can be seen from Figure 1a–d that the particle arrangement that was deposited on top of the substrate possessed particles in the size of nanometer order. The layer that was formed had a thickness of 2.42, 1.83, 3.14, and 4.23 μm for the Ag, Au, Ag/Au, and Au/Ag thin films, respectively. The molecular weight composition of Au and Ag for the bimetallic thin films was 2.2% and 0.9%, respectively. This value is very small compared to the overall composition of the formed electrode. From the thickness and molecular weight composition, the layers that were formed can be categorized as a thin film [26]. The fabricated Ag and Au thin films were later used as the geometric model in the electric field distribution simulation.

Figure 1e shows the XRD pattern of each thin film. The phase that was formed on the thin films was the crystalline phase, indicated by the appearance of diffraction pattern peaks. The addition of thin film on ITO increased the level of crystallinity of the electrodes, which was proven by an increase in diffraction peaks. The highest level of crystallinity was observed on the Ag/Au electrode, which was shown by the highest diffraction peak. This shows that the Ag/Au electrode is sufficient to be used as a biosensor because a high crystalline structure can provide less scattering or diffusion paths for incident photons, which in turn increases the interaction between free electrons in metal and incident light and, as a result, increases the optical sensitivity of a biosensor [27]. The thin films were completely deposited on top of the ITO glass substrate, which resulted in diffraction peaks at 2θ = 30.203°, 49.738°, and 59.631°. These peaks are diffraction peaks on ITO glass with a hexagonal structure and side length of 9.559 Å [28]. The addition of Ag and Au thin layers caused a new phase to appear on the thin films. The crystal system that was formed on the Ag and Au thin films was the face-centered cubic structure with side lengths of 4.1480 Å and 4.0900 Å, respectively. Phase formation occurred at 2θ = 37.525° and 43.605°, which indicated the formation of an Ag crystalline phase with a Miller index of (111) and (002). Whereas, the deposition of Au shows that diffraction peaks were exhibited at 2θ = 38.078°, 44.256°, 64.376°, and 77.312° with Miller indexes of (111), (002), (022), and (113), respectively [29]. Figure 1f–i shows the results of the EDX test on each type of thin layer. The colors in the EDX figure show the components of the thin layer, where green is O, yellow is Si, dark blue is In, light blue is Au, and pink is Ag. The inset image on the EDX results shows the interaction of Ag and Au in the thin layer. This shows that the ITO glass substrate has been evenly deposited by Ag and Au.

Figure 2 shows the results of the simulation of electric field distribution on the Ag and Au thin films that were coated with BSA. Electromagnetic waves with wavelengths were perpendicularly shot on to the upper part of the thin films that were coated with BSA. The simulation of electric field distribution on the Au and Ag thin films was carried out using varying concentrations of BSA and varying wavelengths in order to observe the electromagnetic wave response as a result of changes in BSA concentration. The results of the simulation show that a slight difference in electric field distribution was observed between varying concentrations of BSA, in which the highest electric field distribution was observed at a normal BSA concentration. This was caused by the increase in refractive index, which in turn causes an increase in absorbance value [30].

The distribution of electric field intensity was caused by a change in electromagnetic wave source. This change in electromagnetic wave source was caused by a change in refractive index. Furthermore, electric field intensity is also influenced by changes in the wavelength value, as shown in the following equation:

(1)$\mathrm{E}\left(\mathrm{x},\mathrm{t}\right)={ \mathrm{E}}_0\exp \left(-\frac{\mathsf{\omega }}{\mathrm{c}}\mathrm{nkx}\right)\exp \mathrm{i}\mathsf{\omega }\left(\frac{\mathrm{n}}{\mathrm{c}}\mathrm{x}-\mathrm{t}\right)$

where E₀ is the initial electric field on the medium, ω is the wave frequency, n is the real part of the medium’s refractive index, k is the absorption coefficient of the medium, and c is the speed of light [31].

The analysis of absorbance value from the results of the experiment are shown in Figure 3a. Hypoalbumin was tested with an albumin concentration of 2.5 g/dL, while normal blood samples were tested with an albumin concentration of 5 g/dL. Based on the experimental results, the absorbance peak was exhibited on the Au/Ag electrode at a wavelength of 435 nm on hypoalbumin medium and 470 nm on normal concentration of serum albumin medium. It is caused by optimal energy absorption of the electromagnetic wave at this particular wavelength. Figure 3a shows a higher absorbance value of a thin layer on normal albumin concentration medium compared with hypoalbumin. This is caused by a larger energy of electromagnetic waves absorbed in higher concentrations of serum albumin molecules [32]. The change in absorbance value due to the addition of serum albumin was also caused by the change in refractive index of the electrodes as a result of the bond between the electrodes and serum albumin. Generally, the absorbance curve from the results of the experiment and simulation, as shown in Figure 3b, exhibit similar patterns; therefore, optical analysis to compare biomolecule concentration can be carried out by identifying the absorbance value of the thin films.

An increase in absorbance was exhibited, which indicates an increase in the absorption of electromagnetic wave energy by the electrons of the component materials; as a result, the electrons experience transition in their molecular orbitals [32]. The resulting energy from the wavelength of the absorbed photon can be calculated using the band gap energy value. Figure 3c shows the calculation of the bandgap energy values for Ag/Au thin films on serum albumin with normal concentrations, while Figure 3c insets the bandgap energy values for all types of electrodes with variations in serum albumin concentrations calculated using the same method. The Ag, Ag/Au, Au, and Au/Ag thin layer electrodes on ITO glass substrates had values of 1.636, 1.445, 1.573, and 1.598 eV, respectively. The value of the band gap energy for each type of electrode has a value that is not much different because the band gap energy of pure Ag and Au also has almost the same value [33]. This indicates that all the formed thin films can be classified as narrow band gap semiconductors. The addition of serum albumin on top of the thin films caused an increase in the band gap energy of the thin films. The increase in band gap energy may be due to the optical scattering effect and electronic absorption that occurred on the thin-film serum albumin system [34]. The largest increase in band gap energy values along with the addition of serum albumin concentration was found in the Ag/Au thin layer. So, Ag/Au is the best type of thin layer arrangement for optically monitoring serum albumin concentrations because Au bonds with biomolecules can cause high optical scattering effects and electronic absorption as well as large signal amplification due to the high conductivity of Ag.

The performance of the various types of Ag and Au thin films in detecting serum albumin concentration was electrochemically tested in [Fe (CN)₆]^3−/4− media that was dripped with serum albumin with varying concentrations and varying scan rates. The CV graph that was formed for each electrode possessed dissimilar patterns, depending on their reduction–oxidation characteristics. Figure 4a shows the results of the CV measurements of each electrode. The coating of Au and Ag on ITO glass was proven to increase the reduction and oxidation ability of the electrodes as a result of the increase in the density of the current that was formed during the reduction and oxidation process. The process of the reduction and oxidation reaction of Au that occurred is as follows:

Au* + H₂O → Au^δ+.OH^δ− + H⁺ + e⁻(2)

Au^{δ+ ·} OH^δ− + H⁺ + e⁻ → Au(OH)₃(3)

Au(OH)₃ + 3H⁺ + 3e⁻ → Au** + 3H₂O(4)

Au* depicts the atoms on the surface of the electrode that are more active than the atoms of the other constituent elements of the electrode. The ^δ+ and ^δ− indices represent the surface dipole conversion that is able to form more organized oxide molecules. Furthermore, Au** are Au atoms on the surface that are more active than Au*, which are activated due to the low coordination number of the lattice. The oxidation reaction on Au occurs when Au reacts with water, enabling the release of electrons to the electrolyte. The reduction and oxidation reaction on Ag occurs through the following process:

Ag + OH⁻ ↔ ½ Ag₂O + ½ H₂O + e⁻(5)

½ Ag₂O + OH⁻ ↔ AgO + ½ H₂O + e⁻(6)

The oxidation reaction on Ag occurs when Ag reacts with water, enabling the release of electrons to the electrolyte and the formation of silver oxide. Meanwhile, the reduction reaction occurs when silver oxide captures electrons and forms Ag atoms. The process of reduction and the oxidation reaction on Au and Ag are shown in Figure 4b.

From the experimental results, oxidation and reduction peak potentials of Ag were exhibited at 0.5 and −0.5 V vs. Ag/AgCl, respectively. Meanwhile, oxidation and reduction peak potentials of Au were exhibited at 0.9 and −0.2 V vs. Ag/AgCl, respectively. This is in accordance with the studies conducted by Luo et al. [27] and Burke et al. [28] that identified the reduction potential and oxidation potential of Ag and Au, respectively. The combination of Au and Ag caused a shift in oxidation and reduction peaks, as shown in Figure 4a. This is due to the occurrence of two reactions, namely, the reaction of Au and Ag on the electrode. On the Au and Ag bimetallic electrode, the value of reduction and oxidation peak potentials shifted between the oxidation and reduction peaks due to the different diffusion-controlled process on the electrode [35]. For the electrode with an uppermost layer of Au, the oxidation and reduction peaks exhibited a slight shift from the oxidation and reduction peak potentials of Au; meanwhile, the electrode with an uppermost layer of Ag possessed an oxidation and reduction peak potential close to that of Au. This is due to the redox reaction that dominantly occurred on the outermost layer of the electrode. The CV measurements made with 10 cycles show that each electrode has a fairly high suitability of the current and voltage chart. Its reproducibility value reaches 0.99 as shown in the Figure S1.

The reaction kinetic study on the various types of Au and Ag thin films is shown in Figure 5a. The CV measurements using varying scan rates indicate an increase in the current density of reduction and oxidation peaks with respect to an increase in scan rate. This is in accordance with the theory on reduction and oxidation reactions proposed by Randles–Sevcik, in which it is stated that the oxidation and reduction peak current values are proportional to the square root of the scan rate value; therefore, a larger scan rate will result in a larger reduction and oxidation current [36]. The linear increase in current can be seen in the graph in Figure 5b, which shows that all the electrodes possess an R square value larger than 0.99 and the resulting graphs of all the electrodes are straight line graphs. This indicates that the oxidation and reduction reactions possess a stable pattern, and also the diffusion process that occurred during the electrochemical reaction was adequately controlled by the electrodes.

The type of reaction that occurred in the CV test can be determined by calculating the ratio between the oxidation peak current (i_ox) and the reduction peak current (i_red). The formed reaction is reversible if the ratio value of the peak currents is 1. On the other hand, the formed reaction is irreversible if the ratio value of the peak currents is far from a value of 1. The Ag, Ag/Au, Au, and Au/Ag electrodes possess an i_ox/i_red ratio value of 1.00, 1.47, 1.85, and 1.74, respectively. Therefore, the chemical reactions that occurred on all the different types of Au and Ag thin film electrodes were quasi-reversible reactions because the potentials of the reduction and oxidation peaks shift slightly apart as a function of scan rate and as the current ratio of their oxidation and reduction peaks approaches 1 [37].

The performance of the electrodes in detecting serum albumin concentration was tested by dripping serum albumin on top of the thin films and varying the concentration of serum albumin between 2.5 g/dL and 5 g/dL with a scan rate of 100 mV/s. A scan rate of 100 mV/s is used to measure electrode performance because the scan rate is not too fast; so, it does not cause imperfect reactions because the analyte contact time is too fast and does not cause other reactions that will interfere with the analyte reduction process due to the scan rate being too slow. A decrease in reduction and oxidation peak currents occurred with respect to an increase in serum albumin concentration, as shown in Figure 6a. This is caused by an increase in the bond formed between the electrode and serum albumin, and as a result, diminishes the ability of the electrode to transfer charges to the electrolyte [38].

Electrochemical tests using CV were carried out to determine which thin film performed the best in detecting biomolecules—in this case, serum albumin—for biosensor applications. Figure 6b shows the correlation between serum albumin concentration and oxidation peak current, which is the slope of the graph and can be called the sensitivity of the electrode. As the serum albumin concentration increased from 2.5 g/dL up to 5 g/dL, the oxidation peak current decreased because serum albumin is a non-electroactive substance that can inhibit the flow of electrons [11]. A linear correlation was observed between serum albumin concentration and oxidation peak current with a correlation coefficient of 0.96. The Ag, Ag/Au, Au, and Au/Ag thin films achieved a sensitivity value towards the decrease in current with respect to the increase in serum albumin of 4.03 × 10⁻⁵, 196.19 × 10⁻⁵, 94.41 × 10⁻⁵, and 87.36 × 10⁻⁵ A/(g/dL), respectively. Furthermore, the Ag, Ag/Au, Au, and Au/Ag thin film electrode achieved limits of detection (LODs) of 0.56 g/dL, 0.24 g/dL, 0.64 g/dL, and 0.36 g/dL, respectively. The Ag/Au thin film exhibited the best sensitivity in detecting serum albumin because it had the best response to the change in oxidation peak as the concentration of serum albumin increased compared with the other Au and Ag thin films. This is due to the great ability of Au to bond with biomolecules with the presence of the Au–S bond as electrostatic interactions [39]. The addition of an Ag layer underneath the Au layer in the thin film arrangement can increase the conductivity of the thin film; therefore, the measured electrochemical signal possesses an adequately large current value [40].

The EIS tests were carried out on various Au and Ag thin film arrangements using the [Fe (CN)₆]^3−/4− medium and without the addition of serum albumin. Figure S2 shows the results of the EIS tests on various types of electrodes. It can be seen from Figure S2 that the ITO thin film achieved the largest impedance value. With the addition of an Au and Ag thin layer, the conductivity of the electrode increased and, as a result, decreased its impedance value. With the addition of Au and Ag, which increase the conductivity, the performance of the biosensor will be higher and it also can amplify the electrochemical signal [16]. Due to the ability of Au and Ag to bind organic molecules, it can be seen from Figure S3a that there is an increase in the impedance value of the electrode, which is indicated by an increase in the diameter of the Nyquist plot. This is caused by the increasing number of organic molecules that bind to the surface of the electrode and, as a result, hinder the electron flow on the electrolyte–electrode system [41]. Figure S3b shows the response of the electrodes, which is indicated by the increase in the resistance values of the electrodes with respect to an increase in the concentration of the serum albumin that is deposited on top of the electrodes. This is caused by an increase in the number of molecules that bind to the biosensor resulting in a greater resistance between the electrolyte and the electrode. Besides being affected by the interfacial bond between Au, Ag, and serum albumin, the R_ct value is also affected by the interfacial bond between ITO and Ag or Au. Ag/Au has greater interfacial stability on the substrate when compared with Au/Ag [42]. In addition, repeated electrochemical tests may result in damage to the interface between the electrode and the electrolyte. Ag in the outer layer can no longer play a role in the electrochemical process so that it can cause a significant increase in R_ct in the Au/Ag electrode arrangement [43].

4. Conclusions

The fabrication of Ag and Au electrodes in the form of Ag, Ag/Au, Au, and Au/Ag electrodes was successfully carried out using the DC sputtering deposition method. The electrical and optical characteristics of the thin films were systematically investigated using various concentrations of serum albumin as a prognostic factor in blood cancer. The results of the experiments and simulations show that the largest shift of absorbance peak was observed on the Ag/Au electrode with a shift of 35 nm as the concentration of BSA was increased to 2.5 g/dL. The electrochemical tests show a decrease in anodic and cathodic peak currents with respect to an increase in BSA concentration in the electrochemical analysis. The Ag/Au thin film electrode is more promising than the other electrodes to be used in biosensor applications for the monitoring of serum albumin level as a prognostic biomarker of blood cancer.

Author Contributions

Conceptualization, U.F., N.N., A.S.M., and A.R.; methodology, U.F., V.Z.Z., and A.S.M.; software, U.F.; validation, U.F., N.N., and I.A.; formal analysis, I.A. and D.D.; investigation, U.F., N.N., and U.F.; resources, H.S. and D.D.; data curation, U.F.; writing—original draft preparation, U.F. and N.N.; writing—review and editing, A.S.M. and A.R.; visualization, U.F., H.S., and M.F.; supervision, A.S.M., A.R., and V.Z.Z.; project administration, A.R.; funding acquisition, N.N. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors gratefully acknowledge financial support from the Institut Teknologi Sepuluh Nopember and BRIN-RISTEK DIKTI for this work, under the project scheme of the Publication Writing and IPR Incentive Program (PPHKI). The researcher would also like to express gratitude to Bernard in PT Dynatech International for SEM EDS analysis.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures

Figure 1. The results of the SEM analysis of the (a) Ag, (b) Au, (c) Ag/Au, and (d) Au/Ag thin films. (e) The results of the XRD analysis of the thin films. The results of the EDX analysis of the (f) Ag, (g) Au, (h) Ag/Au, and (i) Au/Ag thin films (inset: composition of all parts of the film). * is ITO, ° is Ag, and x is Au.

Figure 2. The top view and side view of the electric field distribution simulation on the (a) Ag, (b) Ag/Au, (c) Au, and (d) Au/Ag electrodes using the (1) normal and (2) hypoalbumin mediums.

Figure 3. Results of the testing on the optical characteristics of the thin films using UV–Vis Spectroscopy: (a) experimental results; (b) simulation results; (c) the calculation of band gap energy values of Ag/Au thin film on serum albumin with normal concentration (inset: band gap energy value of Ag, Ag/Au, Au, and Au/Ag thin films using different concentration of serum albumin).

Figure 4. (a) The results of the CV test of the various thin films without the deposition of BSA with a scan rate of 100 mV/s (small picture is CV test of Ag thin film); (b) the oxidation and reduction reactions on the thin films.

Figure 5. (a) The result of the CV test using varying scan rates on the Ag/Au thin film; (b) oxidation peak (represented in squares) and reduction peak (represented in circles) of the Ag, Ag/Au, Au, and Au/Ag thin films.

Figure 6. (a) The results of the CV tests with varying BSA concentrations on the Au/Ag thin film. (b) oxidation peaks of the Ag, Ag/Au, Au, and Au/Ag thin film electrodes with varying BSA concentrations.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/coatings13010186/s1. Figure S1: The result of 10 cycles of the CV test using the (a) Ag, (b) Ag/Au, (c) Au, and (d) Au/Ag thin films; Figure S2: The results of the EIS tests on the various types of electrodes without the deposition of BSA; Figure S3: (a) The results of the EIS test with varying concentrations on the Au/Ag thin film. (b) The R_ct values of the Ag, Au, Ag/Au, and Au/Ag thin films with varying concentrations of BSA.

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