UV-Vis-NIR and TEM characterization of gold nanotriangles
The synthesis of gold nanotriangle (AuNTr) using a phospholipid mixture as a reducing and stabilizing agent in water is relatively simple and reproducible. The mass ratio between the metal precursor and DMPG-Na/PC was kept constant (1:4) during the synthesis at 25 °C. Under such conditions, the solution turns deep red showing two UV-Vis absorption maxima at 523 nm and 870 nm, as reported in Fig. 1a (before purification), characteristic for the formation of spherical small dimension AuNPs and anisotropic NPs, respectively. The AuNTrs were characterized using transmission electron microscopy before (Fig. 1b) and after purification (Fig. 1d). By considering these TEM pictures, a shape yield higher than 95% was demonstrated, resulting in a green color solution due to the concentration of AuNTrs with sizes of about 50 nm and 200 nm after only one sedimentation and re-dispersion step. Consequently, a maximum at about 870 nm is observed in the UV-Vis-NIR spectrum (Fig. 1c, after purification) confirming that highly concentrated AuNTrs are present. Similar results have been observed by Liebig et al. [63] and Scarabelli et al. [64], using AOT and CTAC micelles, respectively, during the purification of AuNTr obtained by different approaches. As expected, the AuNTrs show a negative zeta potential of − 60 mV as a result of the coating with anionic surfactants (data not shown). Furthermore, AuNSphs were also characterized using transmission electron microscopy (TEM) as reported in Fig. S3 (see Electronic Supplementary Material, ESM), showing a good size distribution.
Electrochemical characterization of FDH-modified graphite electrode
CV experiments were performed with modified graphite electrodes in absence and in presence of substrate in order to assess the contribution of the electrode nanostructuration using differently shaped AuNPs (spherical and triangular) on the catalytic current related to the oxidation of d-(-)-fructose to 5-keto-d-(-)-fructose catalyzed by FDH.
Figure 2a shows the CVs for a FDH/G-modified electrode in 50 mM NaAc buffer pH 4.5 in the absence (black curve) and in the presence (red curve) of 1 mM d-(-)-fructose. From these CVs, it is clear that the non-turnover case reveals no apparent electroactivity of FDH, and in the presence of fructose, there is only a slight electrocatalytic wave with an onset potential, EONSET, at − 0.050 V vs. Ag|AgClsat rising up to maximum current of 2.5 μA at 0.2 V vs. Ag|AgClsat. The low catalytic current is probably due to a combination of the low roughness of the electrode surface and the random orientation of the enzyme onto the electrode surface.
Figure 2b depicts CVs for a FDH/AuNSphs/G-modified electrode in non-turnover conditions (black curve) (50 mM NaAc buffer pH 4.5), showing surprisingly no redox waves related to DET of CYTFDH. However, in turnover conditions (1 mM d-(-)-fructose, red curve), the modified electrode showed a higher electrocatalytical wave compared with FDH/G, starting at EONSET = − 0.070 V vs. Ag|AgClsat rising up to 7.6 μA at 0.2 V vs. Ag|AgClsat. In this case, the increase in the electrocatalytical wave is probably due to the enhanced real surface area of the modified electrode, which allows a higher enzyme loading. Nevertheless, it should be taken into account also the SDS layer onto the nanoparticles, which surprisingly creates a favorable environment for the immobilization of FDH.
Finally, Fig. 2c shows the CVs for the FDH/AuNTrs/G-modified electrode in 50 mM NaAc buffer pH 4.5; however, also here, there are no evident redox waves for the DET reaction of CYTFDH. The CV in the presence of substrate (red curve) showed the highest electrocatalytical current compared with the others with an EONSET starting at − 0.103 V vs. Ag|AgClsat rising up to 22.5 μA at 0.2 V vs. Ag|AgClsat. The result is probably ascribable to the efficient packing of AuNTrs onto the electrode surface, which would sensibly enhance the enzyme loading and also the ET rate constant.
Since both kinds of spherical and triangular NPs were covered with a layer of SDS creating an unexpected favorable immobilization environment, we considered a possible influence of the shape of the NPs on the catalytic current caused by FDH [65]. In particular, Compton and his co-workers published a paper reporting on the diffusion-limited currents to NPs of various shapes supported on an electrode by means of a mathematical simulation on spherical and hemispherical shapes [66]. For this reason, we believe that the different behaviors between the FDH/AuNSphs/G and FDH/AuNTrs/G could be explained by the influence of the shape of the NPs on the limiting kinetic current part of the catalytic current. Therefore, in the section below, we reveal the results of our investigation of the influence of the shape of the NPs on the diffusion-limited current related to the catalytic oxidation of d-(-)-fructose by using two different approaches: the rotating disk electrode (RDE) and flow through amperometric wall jet cell [67,68,69].
The dependence of mass transfer–limited current on the shape of the NPs: rotating disk electrode and flow through amperometric wall jet cell studies
The reaction of FDH with fructose starts with the oxidation of d-(-)-fructose to form 5-keto-d-(-)-fructose, which corresponds to the 2e−/2H+ reduction of FAD to FADH2 followed by the first internal electron transfer through two cyt c moieties (one heme c is not involved at all) contained in subunit II for the partial regeneration of FADH· in its semi-oxidized state and the delivery of the 1st e−, as reported below (Eqs. (1–4)):
$$ \mathrm{D}-\left(-\right)-\mathrm{fructose}+\mathrm{FAD}\to 5-\mathrm{keto}-\mathrm{D}-\left(-\right)-\mathrm{fructose}+{\mathrm{FADH}}_2 $$
(1)
$$ {\mathrm{FADH}}_2+\mathrm{cyt}{c}_1-{\mathrm{Fe}}^{3+}\to {\mathrm{FADH}}^{\cdotp }+\mathrm{cyt}{c}_1-{\mathrm{Fe}}^{2+} $$
(2)
$$ \mathrm{cyt}{c}_1-{\mathrm{Fe}}^{2+}+\mathrm{cyt}{c}_2-{\mathrm{Fe}}^{3+}\to \mathrm{cyt}{c}_1-{\mathrm{Fe}}^{3+}+\mathrm{cyt}{c}_2-{\mathrm{Fe}}^{2+} $$
(3)
$$ \mathrm{cyt}{c}_2-{\mathrm{Fe}}^{2+}\to \mathrm{cyt}{c}_2-{\mathrm{Fe}}^{3+}+{\mathrm{e}}^{-} $$
(4)
In the last step, equation (4), cyt c2-Fe2+ is re-oxidised to cyt c2-Fe3+ at the electrode surface releasing the 1st e-. The FADH. radical formed in equation (2) is reoxidised to FAD by cyt c1-Fe3+ in equation (5) and the last two steps shown above (equations (3-4)) are repeated a second time for the regeneration of FAD and the protein in its native state, as follows (equations 5-7):
$$ {\mathrm{FADH}}^{\cdotp }+\mathrm{cyt}{c}_1-{\mathrm{Fe}}^{3+}\to \mathrm{FAD}+\mathrm{cyt}{c}_1-{\mathrm{Fe}}^{2+} $$
(5)
$$ \mathrm{cyt}{c}_1-{\mathrm{Fe}}^{2+}+\mathrm{cyt}{c}_2-{\mathrm{Fe}}^{3+}\to \mathrm{cyt}{c}_1-{\mathrm{Fe}}^{3+}+\mathrm{cyt}{c}_2-{\mathrm{Fe}}^{2+} $$
(6)
$$ \mathrm{cyt}{c}_2-{\mathrm{Fe}}^{2+}\to \mathrm{cyt}{c}_2-{\mathrm{Fe}}^{3+}+{\mathrm{e}}^{-} $$
(7)
where equations (6) and (7) are equal to equations (3) and (4), respectively. Equation (7) yields the 2nd e- to the electrode.
The oxidation current for d-(-)-fructose at a FDH-modified electrode can be limited by the mass transfer of d-(-)-fructose to the electrode and/or by the kinetics of the enzymatic reaction. The measured current, I, is a combination of both the mass transfer–limited current, Ilim, the kinetically limited current, Ikin, and the current related to the interfacial electron transfer, IE, according to Eq. (8):
$$ \frac{1}{I}=\frac{1}{I_{\mathrm{lim}}}+\frac{1}{I_{\mathrm{kin}}}+\frac{1}{I_{\mathrm{E}}} $$
(8)
The mass transfer–limited current consists of the current observed when the d-(-)-fructose is consumed by the enzyme reaction much faster than d-(-)-fructose which is transported to the electrode surface. For a rotating disk electrode (RDE), the mass transfer–limited current depends on the angular velocity (ω) and the bulk concentration of d-(-)-fructose (c*) according to the Levich equation [70], as follows in Eq. (9a):
$$ {I_{\mathrm{lim}}}^{\mathrm{planar}}=0.620 nFc\ast {D}^{2/3}{A}_{\mathrm{geo}}{v}^{-1/6}\ {\omega}^{1/2} $$
(9a)
where n and F have their usual meanings, D is the diffusion coefficient for d-(-)-fructose (7 × 10−6 cm2 s−1 [71]), A is the geometrical area of the electrode (0.073 cm2), and v is the kinematic viscosity of water (0.01 cm2 s−1).
Moreover, the mass transfer–limited current was evaluated also by using flow-through amperometry in a wall jet cell, for which the equation derived by Yamada and Matsuda can be applied (Eq. (9b)) [72]:
$$ {I_{\mathrm{lim}}}^{\mathrm{planar}}=0.898 nFc\ast {D}^{2/3}{A_{\mathrm{geo}}}^{3/8}{v}^{-5/12}{V}^{3/4}{a}^{-1/2} $$
(9b)
where V is the volumetric flow rate and a is the radius of the capillary nozzle.
In this regard, we assumed that the AuNTr and the AuNSphs have a different self-packing pattern onto the electrode surface resulting in a different real surface area [73]. This can be determined by scanning the electrodes in H2SO4 and integrating the area under the wave for formation of gold oxide (data not shown). The real surface area (Areal) resulted to be 4.6 ± 0.3 cm2 and 1.1 ± 0.2 cm2 for the AuNTr and the AuNSph modified electrodes, respectively. After this theoretical consideration, both Eqs. (9a) and (9b) were re-formulated as follows:
$$ \frac{I_{\mathrm{lim}}^{\mathrm{real}}}{I_{\mathrm{lim}}^{\mathrm{planar}}}=\frac{0.620 nF{c}^{\ast }{D}^{2/3}{A}_{\mathrm{real}}{v}^{-1/6}{\omega}^{1/2}}{0.620 nF{c}^{\ast }{D}^{2/3}{A}_{\mathrm{geo}}{v}^{-1/6}{\omega}^{1/2}} $$
(10a)
$$ \frac{I_{\mathrm{lim}}^{\mathrm{real}}}{I_{\mathrm{lim}}^{\mathrm{planar}}}=\frac{0.898 nF{c}^{\ast }{D}^{2/3}{A}_{\mathrm{real}}^{3/8}{v}^{-5/12}{V}^{3/4}{a}^{-1/2}}{0.898 nF{c}^{\ast }{D}^{2/3}{A}_{\mathrm{geo}}^{3/8}{v}^{-5/12}{V}^{3/4}{a}^{-1/2}} $$
(10b)
where n, F, c*, D, v, V, a, and ω have their usual meanings while Areal/Ageo is the roughness factor calculated for the two different modified electrodes.
Rotating linear sweep voltammograms (RLSVs) for all the modified electrodes (viz. FDH/G, FDH/AuNSphs/G, and FDH/AuNTrs/G), obtained in presence of 1 mM d-(-)-fructose, are reported in Fig. 3a–c. As can be seen in Fig. 3, d-(-)-fructose oxidation at all modified electrodes resulted in a mass transfer–limited reaction (highly dependent from rotation speed). For a more deeper evaluation of the limiting steps of the performance of the RDE, the currents measured at different rotation speeds were as usually plotted in Koutecky-Levich coordinates (1/1 vs. ω−1/2) [74]. The d-(-)-fructose oxidation currents obtained with one FDH/G, FDH/AuNTrs/G, and FDH/AuNSphs/G at different [d-(-)-fructose] and ω are presented as Koutecky-Levich plots in Fig. 4a–c, for FDH/G, FDH/AuNTrs/G, and FDH/AuNSphs/G, respectively. Nevertheless, we applied the following Koutecky-Levich equation (Eq. (11)):
$$ \frac{1}{I}=\frac{1}{0.620 nF{c}^{\ast }{D}^{2/3}\left({A}_{\mathrm{geo}}\right){v}^{-1/6}{\omega}^{1/2}}+\frac{1}{nF\left(\raisebox{1ex}{${A}_{\mathrm{real}}$}\!\left/ \!\raisebox{-1ex}{${A}_{\mathrm{geo}}$}\right.\right)\Gamma {k}_{\mathrm{cat}}{c}^{\ast }}+\frac{1}{nF\left(\raisebox{1ex}{${A}_{\mathrm{real}}$}\!\left/ \!\raisebox{-1ex}{${A}_{\mathrm{geo}}$}\right.\right)\Gamma \left({k}_{s1}+{k}_{s2}\right)} $$
(11)
It can be seen that the electrode current depends on the ω (which is the criterion for diffusion limitation in the bioelectrocatalytic oxidation of d-(-)-fructose) in the range 50–400 μM. However, the data obtained for the FDH/G were fitted according to Eq. (10a) considering the graph 1/1lim vs. [d-(-)-fructose] (data not shown), while the data for FDH/AuNTrs/G and FDH/AuNSphs/G well fitted (Eq. (10a)) taking into account the same graph. In this graph, it should be considered that the slope is proportional to the number of electrons transferred per molecule of d-(-)-fructose oxidized at the modified electrode, which was found to be 1.86 ± 0.02 for FDH/G, 1.93 ± 0.14 for FDH/AuNSphs/G, and 1.89 ± 0.20 for FDH/AuNTrs/G, values actually close to the theoretical value of 2, while kcat (s−1) can be calculated from the intercept. The mass transfer–limited currents were also evaluated by considering Eq. (10b) valid for flow-through setup obtaining similar results. The equivalent Koutecky-Levich plots obtained by the flow-through setup for FDH/G, FDH/AuNSphs/G, and FDH/AuNTrs/G, respectively, are reported in Figs. 5a–c. These results were in great agreement with those reported for RDE as confirmed from the correlation factor R2 = 0.98, as shown in the correlation graph reported in Fig. 5d.
The dependence of kinetically limited current on the nanoparticle shape: rotating disk electrode and flow through amperometry studies
Before discussing the data on the kinetically limited current, we should consider the equation for the kinetically limited current, as follows (Eq. (12)):
$$ \frac{1}{I_{\mathrm{kin}}}+\frac{1}{I_{\mathrm{E}}}=\frac{1}{nFA\Gamma}\frac{1}{\left({k}_{\mathrm{cat}}{c}^{\ast }+{k}_{s1}+{k}_{s2}\right)} $$
(12)
The equation above, Eq. (12), is valid for the FDH/G electrode, while for FDH/AuNTrs/G and FDH/AuNSphs/G, a contribution on the enhancement of the electrode area should be considered; therefore, Eq. (12) can be rearranged as follows (Eq. (13)):
$$ \frac{1}{I_{\mathrm{kin}}}+\frac{1}{I_{\mathrm{E}}}=\frac{1}{nF\left(\raisebox{1ex}{${A}_{\mathrm{real}}$}\!\left/ \!\raisebox{-1ex}{${A}_{\mathrm{geo}}$}\right.\right)\Gamma}\frac{1}{\left({k}_{\mathrm{cat}}{c}^{\ast }+{k}_{s1}+{k}_{s2}\right)} $$
(13)
At this stage, we need to further approximate the system in order to determine the catalytic constant, kcat, and the heterogeneous electron transfer rate constant, kSt, considering that the internal electron transfer is not the rate-limiting step in the overall electron transfer mechanism [75]. Thus, we would consider reactions (1), (5), and (9) in order to deeply evaluate the effect of shape of the nanoparticles on kcat and kSt. Finally, Eq. (13) was rearranged as follows (Eq. (14)):
$$ \frac{1}{I_{\mathrm{kin}}}+\frac{1}{I_{\mathrm{E}}}=\frac{1}{nF\left(\raisebox{1ex}{${A}_{\mathrm{real}}$}\!\left/ \!\raisebox{-1ex}{${A}_{\mathrm{geo}}$}\right.\right)\Gamma}\frac{1}{\left({k}_{\mathrm{cat}}{c}^{\ast }+{k}_{\mathrm{St}}\right)} $$
(14)
At limiting step, by considering 1/IE = 0. The experimental conditions at which 1/IE = 0 are low substrate concentration (C), rotation speed (ω) of the electrode, and applying sufficiently large an electrochemical driving force |E-E0′|. In this way, it was possible to increase the influence of the Levich and the enzymatic component in Eqs. (8) and (14); therefore, the kinetics contribution (1/IE) would be negligible (1/IE = 0). Therefore, we can simplify Eq. (14) as follows (Eq. (15)):
$$ \frac{1}{I_{\mathrm{kin}}}=\frac{1}{nF\left(\raisebox{1ex}{${A}_{\mathrm{real}}$}\!\left/ \!\raisebox{-1ex}{${A}_{\mathrm{geo}}$}\right.\right)\Gamma}\frac{1}{k_{\mathrm{cat}}{c}^{\ast }} $$
(15)
Kinetically limited currents of the oxidation of d-(-)-fructose can be evaluated from the intercepts of the Koutecky-Levich plots. According to the mathematical expression for Ikin (Eq. (14)), the slope of this plot is proportional to the rate of the reaction between d-(-)-fructose and FDH (constant kcat in reaction (1)), while the intercept is proportional to the heterogeneous electron transfer (constant kSt in reactions (2–4) and (5–7)) between reduced FDH and the graphite modified surface (AuNTrs and AuNSphs). To evaluate the rates of these reactions, the surface concentration of FDH on the graphite modified electrode must be known. The theoretical surface coverage resulted in 0.80 nmol cm−2 was considered in this paper for all the modified electrodes, namely FDH/G, FDH/AuNSphs/G, and FDH/AuNTrs/G. Therefore, it was possible to estimate k1, the kinetic constant for reaction (1), and the heterogeneous electron transfer (constant kSt in reactions (2–4) and (5–7)). The results calculated for FDH/G, FDH/AuNSphs/G, and FDH/AuNTrs/G are summarized in Table 1. From these results, it is possible to see that the shape of the NPs had no effect on the catalytic constant (kcat), while the kSt for FDH/AuNTrs/G calculated as 3.8 ± 0.3 s−1 resulted in a 5 times higher value compared with both the NPless graphite electrode 0.7 ± 0.1 s−1 and the AuNSphs/G modified electrode 0.9 ± 0.1 s−1. These results are probably related to the shape of the NPs because the AuNTrs due to their triangular geometry have different self-packing mechanism compared with the spherical ones ensuring a higher real surface area. Nevertheless, it should be taken into account also the interaction between the enzyme molecules and the NPs highlighting that the interaction enzyme-NPs can occur on the edge of the triangle while the spherical shape is limiting the number of enzyme molecules interacting with each NP.
Table 1 Kinetic parameters calculated from the RDE data for FDH/G, FDH/AuNSphs/G, and FDH/AuNTrs/G. n is equal to the number of electrons participating in the reaction As further investigations, we studied also the storage and operational stability of the proposed modified electrodes, namely FDH/G, FDH/AuNSphs/G, and FDH/AuNTrs/G, and the results are reported in Fig. S2A and B (see ESM), showing quite a stable signal for 24 h of continuous injections of substrate into the flow system, while in the storage stability test, it was possible to observe a significant drop in the retained current values of approximately 65% for FDH/AuNTrs/G, 72% for FDH/AuNSphs/G, and 70% for FDH/G (compared with the initial current value) achieved after 20 days.