Margot Jacquet‡
a,
Małgorzata Kiliszek‡a,
Silvio Osella‡b,
Miriam Izzoa,
Jarosław Sara,
Ersan Harputluc,
C. Gokhan Unlud,
Bartosz Trzaskowskib,
Kasim Ocakogluc and
Joanna Kargul*a
aSolar Fuels Laboratory, Centre of New Technologies, University of Warsaw, Banacha 2C, 02-097 Warsaw, Poland. E-mail: j.kargul@cent.uw.edu.pl
bChemical and Biological Systems Simulation Lab, Centre of New Technologies, University of Warsaw, Banacha 2C, 02-097 Warsaw, Poland
cDepartment of Engineering Fundamental Sciences, Faculty of Engineering, Tarsus University, 33400, Tarsus, Turkey
dDepartment of Biomedical Engineering, Pamukkale University, TR-20070 Denizli, Turkey
First published on 25th May 2021
Construction of green nanodevices characterised by excellent long-term performance remains high priority in biotechnology and medicine. Tight electronic coupling of proteins to electrodes is essential for efficient direct electron transfer (DET) across the bio-organic interface. Rational modulation of this coupling depends on in-depth understanding of the intricate properties of interfacial DET. Here, we dissect the molecular mechanism of DET in a hybrid nanodevice in which a model electroactive protein, cytochrome c553 (cyt c553), naturally interacting with photosystem I, was interfaced with single layer graphene (SLG) via the conductive self-assembled monolayer (SAM) formed by pyrene–nitrilotriacetic acid (pyr–NTA) molecules chelated to transition metal redox centers. We demonstrate that efficient DET occurs between graphene and cyt c553 whose kinetics and directionality depends on the metal incorporated into the bio-organic interface: Co enhances the cathodic current from SLG to haem, whereas Ni exerts the opposite effect. QM/MM simulations yield the mechanistic model of interfacial DET based on either tunnelling or hopping of electrons between graphene, pyr–NTA–M2+ SAM and cyt c553 depending on the metal in SAM. Considerably different electronic configurations were identified for the interfacial metal redox centers: a closed-shell system for Ni and a radical system for the Co with altered occupancy of HOMO/LUMO levels. The feasibility of fine-tuning the electronic properties of the bio-molecular SAM upon incorporation of various metal centers paves the way for the rational design of the optimal molecular interface between abiotic and biotic components of the viable green hybrid devices, e.g. solar cells, optoelectronic nanosystems and solar-to-fuel assemblies.
Recent works showed that non-covalent modification of graphene with pyrene (pyr) and its derivatives provides a promising route for immobilisation of a wide range of enzymes while retaining their activity.17,18 Notably, the pyr molecules forming the self-assembled monolayer (SAM) on graphene via π–π stacking, allowed for stabilisation and controlled oriented immobilisation of electroactive and catalytic light harvesting proteins, such as photosystem I (PSI), while maintaining high conductivity and structural integrity of the graphene monolayer.19,20 Moreover, the electronic properties of the pyr SAM itself can be tuned by the introduction of specific divalent transition metal redox centres, in order to improve the kinetics and directionality of direct electron transfer (DET) between graphene and pyr SAM.20,21
Cytochrome c553 (cyt c553) is a model electroactive protein involved in a fundamental process of natural oxygenic photosynthesis. The homologues of this protein are involved in mitochondrial respiration and apoptosis.22 In photosynthesis, this protein functions in the early events of solar light conversion, as it mediates ET between cytochrome b6f complex and the photo-oxidised P700+ chlorophylls forming the reaction centre of PSI.23 The X-ray analysis of the red algal cyt c553 protein, which is the robust electroactive protein used in this study, revealed that it is a Class I c-type cytochrome in which the redox-active prosthetic group is formed by haem covalently bound to Cys34 and Cys37 residues. The central Fe atom of the haem group displays octahedral coordination with His18 and Met58 axial ligands.24 The cyt c553 protein has been successfully applied in various types of photoactive nanodevices, in which the domain-specific molecular recognition between cyt c553 and PSI, that occurs in vivo, has been utilised for the specific orientation of PSI (reaction centre side toward the electrode surface) ensuring the preferred direction of ET through the system and formation of the oriented PSI photoactive monolayer on the electrode surface.20
Here, we report the comprehensive electrochemical and quantum mechanical characterisation of the DET processes in a cyt-functionalised single layer graphene (SLG) nanodevice in which the His6-tagged cyt c553 monolayer is used as the electro-responsive biotic component. The conductive interface between cyt c553 and the graphene monolayer is formed by an organic SAM composed of π–π-stacked pyr molecules functionalised with nitrilotriacetic acid (NTA) chelated to various transition metals (Co2+ or Ni2+). We demonstrate by three independent electrochemical approaches (cyclic voltammetry, photochronoamperometry and impedance spectroscopy) that efficient DET occurs between graphene and cyt c553 molecules and whose kinetics, directionality and stability depend on the metal redox centre incorporated into the bio-organic interface. The electrochemical data in conjunction with quantum mechanical simulations yielded the mechanistic models of DET occurring between graphene, pyr–NTA–M2+ SAM and cyt c553. Finally, we demonstrate the remarkable long-term stability of the cyt-based graphene nanodevice over the period of up to 5 months of interim illumination at ambient conditions.
(1) |
The ET constant rate (k0et) values were extracted using the following equation:28
(2) |
To assess the orientation and position of the cyt c553 on the SLG/SAM interface, classical MD simulations were carried out with the GROMACS 2018 program29 using the CHARMM36 force-field,30 with the time-step of 2 fs and the total simulation time of 300 ns at 300 K, in the NVT ensemble. Different geometrical analyses, carried out to assess the equilibration of the system, revealed the interface equilibration after 200 ns (see ESI†). Next, 56 snapshots have been extracted from the last 10 ns of MD and used for Quantum Mechanics/Molecular Mechanics (QM/MM) calculations (see ESI† for more details). Since the geometry of the assembly is only weakly affected by the nature of the metal centre considered, we replaced the metal cation (Ni2+ to Co2+) in the extracted frames for the QM/MM analysis, without performing additional MD simulations for the Co2+ system. Within the QM/MM method, the system was split into two parts: the haem/NTA pair was described at the DFT level of theory, while the cytochrome was described using the electrostatic embedding scheme, to assess the effect of an anisotropic environment (as the final device is in an all-solid state, water molecules and ions were not considered in this part of the computational study). The CAM-B3LYP functional31 and the LACV3P** basis set32 were used, as implemented in the Jaguar v.9.5 program.33 For each MD snapshot extracted, a single point calculation was performed, and electronic properties were analysed. The details of the computational methodology, which is similar to our previous work,34 are described in the ESI.†
Fig. 1 (A) A representative Raman spectrum of the SLG layer on FTO substrate. (B) SEM images of the FTO/SLG surface visualised at two different magnifications. |
The FTO/SLG surfaces were then functionalised with a pyrene–nitrilotriacetic acid self-assembled monolayer (pyr–NTA SAM) due to the π–π staking interactions between the sp2 lattice of the SLG and the pyrene moiety. The free NTA moiety was chelated with two different metals, cobalt and nickel, by immersing the surfaces in aqueous solutions of NiSO4 or Co(NO3)2. The obtained functionalised electrodes containing either cobalt (FTO/SLG/pyr–NTA–Co) or nickel (FTO/SLG/pyr–NTA–Ni) were characterised by X-ray Photoelectron Spectroscopy (Fig. S1, ESI†), SEM (Fig. 2A and C and S2, ESI†) and Energy-Dispersive X-ray spectroscopy (EDX) analyses (Fig. 2E and G).
The comparison of the XPS results between the bare FTO/SLG and the functionalised electrodes confirms the construction of the two organic interfaces (Fig. S1A†). The signals corresponding to Sn3d5/2, O1s, and C1s were found as expected in all the samples at 485.4 eV, 531.6 eV and 282.9 eV, respectively. For the functionalised surfaces, specific additional signals of the nitrogen from the pyr–NTA (Fig. S1E†) and from the different metals Ni2p3/2 (Fig. S1B†) and Co2p3/2 (Fig. S1C†) were found at 397.86 eV, 853.20 eV and 780.17 eV, respectively.
The EDX-SEM analyses (Fig. 2) allowed for the precise elemental mapping of the metalorganic interfaces, with the specific detection of nitrogen and nickel atoms for the FTO/SLG/pyr–NTA–Ni SAM (Fig. 2E) and the presence of nitrogen and cobalt atoms for the FTO/SLG/pyr–NTA–Co counterpart (Fig. 2G). Moreover, the obtained EDX maps and the corresponding quantitative elemental analyses (Fig. 2F and H) clearly confirm high homogeneity and high surface coverage of both types of samples.
Finally, the cyt c553 biocomponent was immobilised within the different devices following the binding of its C-terminal His6-tag to the metal–NTA SAM on the electrode surface.
To ensure efficient DET between SLG and cyt c553 and to obtain a stable monolayer of this electroactive protein, we used a conductive organic SAM composed of pyr–NTA molecules. A similar strategy was used before to anchor cyt c553 via its C-terminal His6-tag on various types of electrode materials.25,27,37,38 Previous studies demonstrated that incorporation of additional redox-active metal centres into the pyr–NTA SAM on graphene allows for manipulation of the output and directionality of the resultant photocurrents.25 In this study, we focused on the dissection of the precise molecular mechanism of DET within a much more complex molecular system comprising a graphene monolayer, pyr–NTA–M2+ SAM and the haem group of cyt c553 to determine the optimal supramolecular architecture of such nanoassembly for efficient DET. As a first step to assess the influence of each metal centre on DET, the redox behaviour of the cyt c553 protein, either suspended in the water-based electrolyte or captured on the SLG surface, was analysed by cyclic voltammetry (CV) using the FTO/SLG assembly as a working electrode (WE) (see Fig. 3).
Fig. 3A shows that the FTO/SLG electrode is redox inactive in the potential range from 0.5 V to −0.05 V vs. Ag/AgCl with only a small capacitive current observed. After the addition of cyt c553 in solution, a clear redox signature is observed between 0 V and 0.35 V, confirming the presence of DET between graphene and the haem group of cyt c. A broad oxidation peak is detected in the potential range of 0.15–0.33 V, while the reductive peak is more defined and observed around 0.02 V. We then proceeded to the electrochemical characterisation of the full FTO/SLG/pyr–NTA–M2+/cyt c553 bionanoassembly containing two distinct transition metal cations (Co2+ or Ni2+), since the previous study demonstrated the importance of these metallic redox centres for improvement of the kinetics and directionality of ET between graphene and the pyr-based SAM.21
Fig. 3B and C show cyclic voltammograms for the SLG/pyr–NTA–Co and SLG/pyr–NTA–Ni assemblies in the presence or absence of the cyt c553 thin layer. As expected, both types of samples show a higher capacitive current compared to the FTO/SLG sample due to the presence of the metallo-organic monolayer on the highly conductive SLG. In the applied potential range, the NTA–M2+ SAM is redox inactive (see blue and red traces in Fig. 3B and C). Binding of cyt c553 through the His6-tag, genetically introduced into the structure of this protein at the C-terminus, onto the pyr–NTA–M2+ SAM results in a subtle change of the redox behaviour between 0.05 V and 0.4 V. For the pyr–NTA–Co/cyt configuration (Fig. 3B), a slightly higher capacitive current is recorded, and the small redox peaks are present at 0.35 V and 0.14 V attributed to the FeIII/FeII couple in the haem group. Concerning the pyr–NTA–Ni/cyt assembly (Fig. 3C), the electrochemical signature of cyt is more visible with an oxidative peak at 0.28 V and a reductive peak at 0.1 V. Surprisingly, for both bio-functionalised electrodes, the redox peaks of cyt are positively shifted in comparison to those found for cyt c553 in the electrolyte solution. This observation could be explained by a stabilisation of the cyt electrochemical behaviour after its immobilisation on SLG via the pyr–NTA–M2+ SAM. Nevertheless, the electrochemical detection of the cyt redox peaks confirms tight electronic communication between the haem group and SLG via the pyr–NTA–M2+ metallo-organic interface.
In order to quantify the density of immobilised cyt c553, the surface coverage value (Γ) was calculated from the linear dependency (Fig. S3, ESI†) between the scan rate and current intensity of the cyt redox peaks obtained from the SLG/pyr–NTA–Ni/cyt and SLG/pyr–NTA–Co/cyt samples using eqn (1) (see Materials and methods). The surface coverage values for the cyt protein were estimated as 2.07 × 10−11 mol cm−2 and 2.08 × 10−11 mol cm−2, which corresponds to an ultrathin layer of this redox active protein. Notably, similar Γ values were reported for other types of (bio)organic and inorganic monolayers assembled on various types of electrode materials including graphene.39–42
To obtain a better insight into the ET process occurring between SLG, pyr–NTA SAM and cyt c553, electrochemical impedance spectroscopy (EIS) measurements were performed at the E1/2 recorded for immobilised cytochrome (Fig. 4). Table 1 presents fitted values of the EIS spectra to the equivalent circuit model (see Fig. 4, inset) represented by the electrolyte resistance (R1), the charge transfer resistance (R2) and the Warburg resistance associated with ions diffusion (Ws) in parallel to the double layer capacitance expressed as the Constant Phase Element (CPE1) parameter (see Table S1 for additional parameters, ESI†). The comparison of Nyquist plots between both types of bio-electrodes shows a higher resistance for Co-based configuration regardless of the presence or absence of illumination. This observation is in accordance with the CV analysis (Fig. 3B and C), whereby the electrochemical detection of cyt was higher in the case of Ni-based SAM reflecting a smaller resistance from this type of interface. Upon illumination, both bio-functionalised electrodes reveal a decrease of their respective resistance due to the photoelectrochemical activity of both nanoassemblies.
Sample | Illumination | R1 (kΩ) | R2 (kΩ) | k0et (10−2 s−1) |
---|---|---|---|---|
Pyr–NTA–Ni/cyt | OFF | 3.04 | 643 | 1.99 |
Pyr–NTA–Ni/cyt | ON | 2.92 | 569 | 2.26 |
Pyr–NTA–Co/cyt | OFF | 3.04 | 727 | 1.76 |
Pyr–NTA–Co/cyt | ON | 2.91 | 642 | 1.99 |
The EIS and surface coverage data for the biofunctionalised electrodes were employed to calculate the k0et kinetic constants of DET using the eqn (2) (see Materials and methods). The k0et values (Table 1) are estimated to be 1.99 × 10−2 s−1 for SLG/pyr–NTA–Ni/cyt and 1.76 × 10−2 s−1 for SLG/pyr–NTA–Co/cyt systems under dark conditions and respectively 2.26 × 10−2 s−1 and 1.99 × 10−2 s−1 under illumination. These values are similar to the previously reported data.43–45 In line with the EIS results, the values of k0et constant are smaller for the Co-based system. These observations are in accordance with quantum mechanical analyses, pointing on one hand towards a better capability of cathodic current generation in the case of Co redox centre, and on the other hand to anodic photocurrent generation in the presence of Ni-SAM.
The viability of the biohybrid devices depends to a large extent on their long-term stability. To this end, the short- and long-term stability of the cyt-based FTO/SLG/SAM assemblies was studied by photochronoamperometry, an approach that permits to record the photocurrent output (Fig. 5). The electrodes were initially subjected to continuous standard light illumination (1 sun) for up to 1 hour for the concomitant photocurrent measurement (short-term stability assessment, see Fig. 5A). The stability assessment revealed a similar electrochemical behaviour of the FTO/SLG/pyr–NTA–Ni and FTO/SLG/pyr–NTA–Co control electrodes, while the presence of cyt c553 on the FTO/SLG/pyr–NTA–Ni SAM results in a 2-fold higher photocurrent output compared to the control sample devoid of this protein (see Fig. 5A). On the other hand, the FTO/SLG/pyr–NTA–Co/cyt system produces the highest current value which is 5.5-times higher after 1 hour of constant illumination compared to the control devoid of cyt. In fact, the FTO/SLG/pyr–NTA–Co/cyt system showed the highest short-term stability compared to the other samples analysed in this study.
For the long-term stability assessment of the cyt-based nanoassemblies, the photocurrent output, produced within 30 s of ON/OFF illumination, was compared for the same samples after 5 months of storage under ambient conditions (Fig. 5B). For the freshly prepared biophotoelectrodes, the FTO/SLG/pyr–NTA–Co/cyt sample showed 22% higher current output (100 nA cm−2) compared to the FTO/SLG/pyr–NTA–Ni/cyt electrode (81.8 nA cm−2). After 5 months of storage, the respective current densities decreased to 97.7 nA cm−2 and 71.5 nA cm−2, which represents a mere 4.5% and 16.7% decrease of the photocurrent output for the Co- and Ni-containing biohybrid electrodes, respectively (Fig. 5B). These data clearly show a remarkable long-term stability of the Co-containing samples as well as the higher power output of the FTO/SLG/pyr–NTA–Co/cyt nanoassembly compared to the Ni-containing counterpart.
Within the 200–300 ns time frame, we extracted the minimum distance between the Fe2+ ion of the haem group and the Ni2+ cation of the SAM molecules, as well as the tilt angle between the haem group and the SLG, as they are the key parameters responsible for the DET efficiency of the whole conductive interface.34 The average minimum distance is 0.5 nm (Fig. S6, ESI†), while the tilt angle distribution peaks around 81 degrees, with a very narrow spread indicating the limited degrees of freedom for the movement of cyt c due to the presence of the anchoring peptide linker (Fig. S7, ESI†).
The extracted snapshots from the MD simulation are used to calculate the electronic properties of the pyr–NTA–M/haem interface. Among the 56 extracted snapshots, we observe two different sets; a major one composed of 48 frames with haem connected to one particular pyr–NTA–M molecule and a minor one of 8 frames with haem connected to a different pyr–NTA–M system (Fig. S8, ESI†). The difference between these distributions is the orientation of the pyr–NTA–M system with respect to the haem group. Importantly, the probability of occurrence of the two sets varies, with 86% of configurations belonging to the major set and only 14% representing the minor counterpart. Thus, we expect the first set to be more representative of the experimental nanodevice examined electrochemically in our study, although both should be considered to draw accurate conclusions.
Due to the nature of the coordinating metal centres considered, two different electronic configurations were obtained: a closed-shell system for Ni and a radical system for the Co-containing nanoassembly. This, in turn, leads to very different frontier molecular orbitals (FMOs) localisation, as depicted in Fig. 6A and S9 (ESI).† From the pyr–NTA–Ni/haem FMO distribution, obtained from the different MD snapshots, we observe an average value of −3.74 ± 0.27 eV for the HOMO and −3.22 ± 0.17 eV for the LUMO, leading to an energy gap of 0.52 ± 0.26 eV (Fig. 6A). The rather high values for the standard deviations are due to the presence of thermal fluctuations arising from MD simulations. The analysis of the orbitals reveals that the HOMO is mainly localised over the haem group (with some occasional, small degrees of delocalisation over the NTA moiety), while the LUMO is always localised over the NTA–Ni moiety of the SAM molecule, thus explaining the small energy gap value obtained. This localisation of the FMOs indicates a DET from haem to pyr–NTA–Ni molecule, which should be enhanced by an external anodic bias. The same behaviour and DET direction have been found for the minor set (Fig. S9, details in ESI†). Our QM/MM findings are in the full agreement with the electrochemical results; in fact, when applying a cathodic bias, the generated current for this interface is lower, due to the counteraction of the external electric field and the internal DET.
A different scenario arises when the pyr–NTA–Co/haem interface is considered. Since it is a formally radical system, it is necessary to consider the presence of a semi-occupied molecular orbital (SOMO) in addition to HOMO and LUMO. From the FMO distribution, we observe that both HOMO and SOMO are very close in energy, with values of −3.83 ± 0.33 eV and −3.75 ± 0.29 eV, respectively, while the LUMO is found at −2.86 ± 0.33 eV, leading to an energy gap of 0.88 ± 0.34 eV (Fig. 6A). Interestingly, the presence of Co destabilises the LUMO with respect to the Ni system, while the HOMO is only slightly affected by the presence of the different cations, leading to an energy gap more than 1.5 times larger for Co than Ni, which might have dramatic consequences for the DET mechanism (see Discussion). For the second minor set of snapshots, the same trend has been found, although the difference in energy gap between the two interfaces is smaller, 3.43 and 3.11 eV for Co and Ni, respectively (see Fig. S9, ESI†). This is somehow unexpected, considering the different localisation of the FMOs when Co is present as the coordinating metal. In fact, the additional electron does not only affect the localisation of the orbitals but also the electron flow at the interface. In particular, for the pyr–NTA–Co/haem interface two different pictures, stemming from two different sets of FMOs, emerge: (i) both HOMO and SOMO are localised over the NTA–Co moiety of the SAM molecule while the LUMO is localised over the porphyrin fragment of the haem, and (ii) the occupied orbitals are localised over the porphyrin fragment and the LUMO is localised over the NTA–Co moiety. These two different configurations arise from the subtle changes in the conformation, orientation and distance of the two components of the interface due to thermal fluctuation from the MD simulation. Our calculations suggest a prevalence of the first case (up to 65%) in which the DET occurs from the pyr–NTA–Co to the haem, thus explaining the enhancement of generated current when an external cathodic bias is applied.
Interestingly, for the minor set of snapshots of the pyr–NTA–Co/haem interface only one distribution is obtained, in which the occupied orbitals are localised over the porphyrin fragment and the LUMO is localised over the pyr moiety of the SAM molecule (Fig. S9, ESI†), explaining the higher energy LUMO value obtained and, in turn, the bigger energy gap computed. As a result, for this complex interface we can consider the final DET flowing from the pyr–NTA–Co to the haem, in agreement with the experimental data. When a cathodic bias is applied, the external electric field and the DET flow along the same direction (as opposite to the case of the pyr–NTA–Ni/haem interface), strongly increasing the photogenerated current.
In summary, our data demonstrate the successful application of Co as the redox metal centre in the organic interface based on pyr–NTA SAM for enhancement of the cathodic current from SLG to the haem group of cyt c. Importantly, the bionanoassemblies described here are characterised by the excellent long-term stability, with only a minor decrease of the power output over a period of up to 5 months of interim standard illumination at ambient conditions. The QM/MM-driven identification of the two distinct molecular mechanisms of DET occurring in the bio-organometallic interface with two different metallic centres paves the way for the rational design of the optimal molecular interface between abiotic and biotic components of the high-performance green hybrid devices ranging from solar cells, optoelectronic nanosystems and solar-to-fuel electrochemical cells.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/d1ra02419a |
‡ These authors contributed equally. |
This journal is © The Royal Society of Chemistry 2021 |