Development of a Novel Nanoarchitecture of the Robust Photosystem I from a Volcanic Microalga Cyanidioschyzon merolae on Single Layer Graphene for Improved Photocurrent Generation

Abstract

Here, we report the development of a novel photoactive biomolecular nanoarchitecture based on the genetically engineered extremophilic photosystem I (PSI) biophotocatalyst interfaced with a single layer graphene via pyrene-nitrilotriacetic acid self-assembled monolayer (SAM). For the oriented and stable immobilization of the PSI biophotocatalyst, an His₆-tag was genetically engineered at the N-terminus of the stromal PsaD subunit of PSI, allowing for the preferential binding of this photoactive complex with its reducing side towards the graphene monolayer. This approach yielded a novel robust and ordered nanoarchitecture designed to generate an efficient direct electron transfer pathway between graphene, the metal redox center in the organic SAM and the photo-oxidized PSI biocatalyst. The nanosystem yielded an overall current output of 16.5 µA·cm^-2 for the nickel- and 17.3 µA·cm^-2 for the cobalt-based nanoassemblies, and was stable for at least 1 h of continuous standard illumination. The novel green nanosystem described in this work carries the high potential for future applications due to its robustness, highly ordered and simple architecture characterized by the high biophotocatalyst loading as well as simplicity of manufacturing.

Keywords: Cyanidioschyzon merolae; biohybrid nanodevices; biophotovoltaics; direct electron transfer; photosystem I; single layer graphene.

Scheme 1

Diagrammatic representation of the full nanoarchitecture of the SLG/pyr-NTA-M²⁺/His₆-PsaD-PSI nanoconstruct. M²⁺ redox center in the organic interface corresponds to Co²⁺ or Ni²⁺ cations used in this study.

Scheme 2

Graphic representation of the modified psaD (CMV144CT) gene inserted by homologous recombination in the upstream region of the URA locus. The first construct indicates introduced linear DNA, the second construct shows the genomic structure of the C. merolae uracil-auxotrophic mutant M4 and the third construct depicts the genomic structure of the C. merolae transformant expressing His₆-PsaD protein. The asterisk shows the position of a frameshift mutation in the URA gene resulting in truncation of the C-terminal half of the URA protein in the M4 mutant. To express the his-psaD gene, the promoter and nucleotide sequence encoding the chloroplast-targeting peptide (Chl-TP) of APCC and β-tubulin terminator were connected to the upstream and downstream regions of the nucleotide sequence encoding the His₆-PsaD protein. The arrows indicate the position of the primers used to confirm the integration of the construct into the chromosome.

Figure 1

Characterization of the C. merolae recombinant cell lines expressing the nuclear encoded His₆-PsaD protein. (A) PCR analysis of the three independent transformants (7, 12, 16). WT strain was used as a negative control. The position of the primers is shown in Scheme 1. (B) Western blot analysis of the total cell lysates of the two transformant lines (12 and 16) and the WT-negative control. The His₆-tag was detected with anti-His₆ monoclonal antibody, as described in Materials and Methods. The specific signals corresponding to the His₆-PsaD bands are marked with a black box. Non-specific signals (marked with an asterisk) were used as a protein loading control.

Figure 2

Biochemical characterization of the native PSI and His₆-PsaD-PSI from C. merolae. (A) RT Absorption spectra of the native (black) and His₆-PsaD-PSI (blue) complexes. (B) SDS-PAGE analysis of the native (thylakoids and WT PSI) and modified (thylakoids and His₆-PsaD-PSI) proteins. The bands corresponding to the His₆-tagged PsaD and native PsaD proteins are marked with a white box.

Figure 3

Confocal imaging of the PSI-biofunctionalized SLG/FTO electrodes. (A) FTO/SLG/pyr-NTA-Ni device used as a negative control. (B) FTO/SLG/pyr-NTA-Ni/PSI device. (C), FTO/SLG/pyr-NTA-Ni/cyt c₅₅₃/PSI device, as analyzed in [37]. (D), FTO/SLG/pyr-NTA-Ni/His₆-PsaD-PSI sample. The visualized surface area was ~0.41 mm². Excitation was at 639 nm. Scale bar is 100 µm.

Figure 4

Cross-sectional imaging using scanning electron microscopy of glass/FTO/SLG electrodes functionalized with: (A) pyr-NTA-Ni SAM (d = 15 nm); (B) pyr-NTA-Ni/PSI (d = 97.5 nm); (C) pyr-NTA-Ni/cyt c₅₅₃/PSI (d = 101 nm); and (D) pyr-NTA-Ni/His₆-PsaD-PSI (d = 83 nm) nanoconstructs. d, cross-sectional thickness.

Figure 5

Photochronoamperometric analysis of the biohybrid His₆-PsaD-PSI based nanoassemblies. (A) Photocurrent densities obtained from the freshly prepared FTO/SLG/pyr-NTA-Ni/His₆-PsaD-PSI and FTO/SLG/pyr-NTA-Co/His₆-PsaD-PSI samples under 30 s ‘light ON/OFF’ cycles vs. FTO/SLG control. These data were obtained for 2 independent samples (n = 2). (B) Photocurrent curves recorded for the representative FTO/SLG/pyr-NTA-Ni/His₆-PsaD-PSI and FTO/SLG/pyr-NTA-Co/His₆-PsaD-PSI samples vs. FTO/SLG/pyr-NTA control. Photocurrents were recorded at −0.3 V during 1 h continuous illumination (100 mW·cm⁻²).

Figure 6

Photochronoamperometric analysis of the biohybrid FTO/SLG/pyr-NTA-Co²⁺/His₆-PsaD-PSI nanoassemblies. (A) Photocurrent densities obtained from the freshly prepared samples in the presence (black) or absence (red) of oxygen under 30 s ‘light ON/OFF’ cycles. These data were obtained for 2 independent samples (n = 2). (B) Photochronoamperometric curves recorded for the representative FTO/SLG/pyr-NTA-Co/His₆-PsaD-PSI sample at −0.3V during 1 h continuous illumination (100 mW·cm⁻²).