3. Results and Discussions
3.2. Characterization of GCE/AuNPs/PG-DAN-Naf nanobiosensor probe
Using the synthesized PG-DAN nanocomposite a nanobiosensor probe was fabricated as described in Scheme 4.1(B). The modified probe surfaces were characterized using AFM, where we recorded the surface topologies and z-deflection profiles (Figure 4.3) after every step of modification.
Figure 4.3: The AFM micrographs (3D and 2D) and z-deflection profile of different probe surfaces (A) bare electrode, (B) AuNPs deposited electrode, and (C) PG-DAN-Naf coated surfaces.
Firstly, the bare electrode surface was captured, where smooth morphology was observed with the z-deflection of 2.10 nm. After electro-deposition, change in the surface topology with clearly visible spherical nanostructures were observed due to the formation of AuNPs.
The z-deflection in this case was increased and was found to be 7.42 nm. When PG-DAN- Naf was coated at the AuNPs deposited electrode surface, distinct morphological changes and a z-deflection of 150.30 nm were observed due to the presence of PG-DAN nanocomposite matrix. Thereafter, to assess the charge transfer properties of fabricated GCE/AuNPs/PG-DAN-Naf probe, we characterized the fabrication process using electrochemical methods. For that, the formation of AuNPs was characterized by LSV, where a clear reduction peak at 0.095 (V) vs. Ag/AgCl was observed which was due to the reduction of Au3+ to Au0 on electrode surface, forming AuNPs (Figure not shown).
Interestingly, the peak current at 0.095 V increased with three consecutive LSV sweeps, indicating the formation of highly conducting electrode surface. In the next step, modified electrode was characterized by recording CV in the potential window of -0.8 V to 0.8 V at a scan rate of 50 mV/s in 5mM Zobell’s solution. Figure 4.4(A) shows the CV responses for bare GCE (black), GCE/AuNPs (red), GCE/PG-DAN-Naf (blue), and GCE/AuNPs/PG-DAN-Naf (green) surfaces. Representative voltammogram due to the redox process of [Fe(CN)6]3−/4− was observed at bare GCE, while anodic (Ipa) and cathodic (Ipc) peak currents increased when GCE/AuNPs surface was examined. This was due to presence of deposited AuNPs on the GCE surface, which increases the conductivity and surface area of electrode. Further, bare GCE was modified with synthesized nanocomposite (PG-DAN-Naf) and CV was recorded to analyze the electrochemical compatibility of PG-DAN. Interestingly, we observed the increased Ipa and Ipc compare to the bare GCE and GCE/AuNPs surfaces, indicating that the nanocomposite is conducting and capable of giving amplified signal. It is interesting to note that, a new redox peak at -
0.03 / -0.21 V was observed for PG-DAN-Naf coated surface in CV, which was not present when bare GCE and GCE/AuNPs electrode was analyzed in the similar experimental conditions. This redox peak was most likely due to the electrochemical behavior of DAN present in sensing matrix (Jin et al.,1995). In final step, when we fabricated GCE/AuNPs/PG-DAN-Naf probe, maximum Ipa and Ipc were observed which was due to the synchronous effects of AuNPs and PG-DAN. Based on the results, GCE/AuNPs/PG- DAN-Naf sensing probe was applied for the analytical applications.
Figure 4.4: (A) CV and (B) EIS responses at different surfaces of bare GCE(black), GCE/AuNPs(red), GCE/PG-DAN-Naf(blue), and GCE/AuNPs/PG-DAN-Naf (green) in PBS buffer containing potassium ferro/ferri cyanide (5mM; pH- 7.0; Scan rate: 50mV/S ); histogram showing Rct values in the different surfaces (in inset).
The charge transfer through modified electrode is a complex phenomenon. In order to investigate the charge transfer behavior and stability of final sensing probe for electrochemical analysis, we have performed scan rate studies at bare GCE, GCE/AuNPs, GCE/PG-DAN-Naf, and GCE/AuNPs/PG-DAN-Naf. Here, CV responses were recorded at different scan rates (i.e.10-100 mV/s) in 5mM Zobell’s solution. The peak currents in CV responses were obtained and plotted against the square root of scan rates for bare GCE,
GCE/AuNPs (figure not shown), GCE/PG-DAN-Naf (figure not shown), and GCE/AuNPs/PG-DAN-Naf (Figure 4.5). The Ipa and Ipc were found to be directly proportional to the square root of scan rates in all cases with the correlation coefficient between 0.997 and 0.999; indicating the stability and diffusion-controlled charge transfer processes at electrode surface.
Figure 4.5: (A) CV responses of GCE/AuNPs/PG-DAN-Naf sensor probe at different scan rates (10-100 mV/s) in PBS buffer containing potassium ferro/ferri cyanide (5mM; pH- 7.0; Scan rate: 50mV/S ), (B) Plot obtained using the Ipa and Ipc of scan rate dependent CV responses.
To assess the importance of modified surface towards electrochemical sensing, we have calculated the diffusion coefficients (D) of bare GCE and each layer of the modified electrode using Randles - Sevcik’s model (equation 4.1).
(
5)
3/2 1/2 1/22.69 10
Ip = n ACD v ……….. Equation 4.1
Where, Ip is the peak current (in ampere), n is the number of electron transferred in redox process (here n=1), A is the electrode surface area (in cm2: here A = 0.01 cm2 ), C is the concentration of electroactive species (in mole cm−3), D is the diffusion coefficient (in cm2 s-1), and v is the scan rate (in V s−1).
The D values for bare GCE, GCE/AuNPs, GCE/PG-DAN-Naf, and GCE/AuNPs/PG- DAN-Naf modified surfaces were found to be 6.47 x 10-4, 9.54 x 10-4,1.61 x 10-3,and 2.66 x 10-3 cm2s-1, respectively, which clearly indicates the 4.12 fold higher transfer of charged species through the GCE/AuNPs/PG-DAN-Naf modified electrode surface than the bare GCE. The findings of Randles-Sevick’s model clearly suggests that the developed sensing surface is highly conducting. The results obtained by CV were also validated using the EIS, where spectra in the Nyquist plot were recorded for bare GCE, GCE/AuNPs, GCE/PG-DAN-Naf, and GCE/AuNPs/PG-DAN-Naf (Figure 4.4(B)) surfaces to obtain the Rct. The Rct values obtained were 3441± 68.832 Ω, 2120 ± 42.41 Ω, 298.5 ± 17.97 Ω, and 50 ± 4.10 Ω for the bare GCE, GCE/AuNPs, GCE/PG-DAN-Naf, and GCE/AuNPs/PG- DAN-Naf surfaces, respectively. It is interesting to note that the lowest Rct was obtained for GCE/AuNPs/PG-DAN-Naf surface compared to other tested surfaces. In this case, this was due to the fastest electron transfer kinetics at electrode/electrolyte interface. The results obtained in EIS also corroborates with the results of CV analysis. These results confirm that the developed GCE/AuNPs/PG-DAN-Naf probe is highly stable, conducting, and sensitive; hence, it is suitable for electrochemical analysis.
3.3. Analytical performance of GCE/AuNPs/PG-DAN-Naf nanobiosensor