Power Generation by Selective Self-Assembly of Biocatalysts | ACS

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Power Generation by Selective Self-Assembly of Biocatalysts. Alexander Trifonov, Andreas Stemmer, and Ran Tel-Vered ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b03013 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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Power Generation by Selective Self-Assembly of Biocatalysts. Alexander Trifonov*, Andreas Stemmer, Ran Tel-Vered* ETH Zürich, Nanotechnology Group, Säumerstrasse 4, CH-8803 Rüschlikon, Switzerland. E-mail: [email protected], [email protected] ToC figure

Abstract Through a careful chemical and bioelectronic design we have created a system that uses selfassembly of enzyme-nanoparticle hybrids to yield bioelectrocatalytic functionality and to enable the harnessing of electrical power from biomass. Here we show that mixed populations of hybrids acting as catalyst carriers for clean energy production can be efficiently stored, selfassembled on functionalized stationary surfaces, and magnetically recollected to make the binding sites on the surfaces available again. The methodology is based on selective interactions occurring between chemically modified surfaces and ligand-functionalized hybrids. The design

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of a system with minimal cross-talk between the particles, outstanding selective binding of the hybrids at the electrode surfaces, and direct anodic and cathodic electron transfer pathways, leads to mediatorless bioelectrocatalytic transformations which are implemented in the construction of a fast self-assembling membrane-less fructose/O2 biofuel cell.

Keywords: self-assembly, biofuel cell, magnetic nanoparticles, fructose dehydrogenase, direct electron transfer.

Self-assembly is one of the foremost ways of nature to promote the ordering of chemical structures and the controlled growth of biological systems.1-4 Through a set of local interactions,5,6 structures of growing dimensions are assembled, showing in many cases an emergence of functionality.7,8 With being easier to control at the nano-scale,9 self-assembly has consequently become a prominent nanofabrication tool in many fields such as nucleic acidsdirected nano systems,10,11 in vivo molecular targeting,12,13 switchable catalytic surfaces,14,15 and synthesis of complex materials exhibiting improved electrical or structural properties.16,17 Frequently employed was the directed self-assembly of nanoparticles18,19 using topographic templates and force fields to energetically favor their aggregation as building blocks for a variety of processes. The application of dynamic external triggers, such as electric and magnetic fields,20-23 macroscopic viscous flows,24 and combinations of thereof,25 were reported. Furthermore, in recent years, several strategies to harness self-assembly for the generation and storage of energy were proposed. Theoretical considerations26,27 were accompanied by practical efforts mainly directed towards the construction of lithium battery28,29 and fuel cell


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components.30,31 Nevertheless, in none of these studies a fully functional energy generator has been directly formed through a self-assembly interaction of active components. Enzymatic biofuel cells are well recognized as clean energy biological generators,32,33 employing benign materials such as sugars, alcohols and oxygen, possessing intrinsic selectivity towards these substrates, and avoiding the use of toxic and/or expensive catalysts.34,35 In recent years, a new class of enzymatic biofuel cells was developed, enabling direct electron transfer (DET) between redox enzymes and their binding electrodes.36,37 and showing inherent advantages in terms of design and bioelectrocatalytic efficiencies.38 In this work, we designed and explored an all-DET, membrane-less fuel cell whose active components self-assemble following infusion of mixed catalysts-modified nanoparticles into the electrolyte.

Results The general concept behind the construction of the biofuel cell employing self-assembly is introduced in Figure 1. Two conductive surfaces were chemically modified with receptor units, R1 and R2, showing high affinity to ligands L1 and L2, respectively. The latter were individually functionalized on carbon coated magnetic nanoparticles (ccMNPs), co-modified with oxidation or reduction processes-catalyzing enzymes. Starting from a mixed suspension, the specific enzyme-coated ccMNP hybrids showed selective binding to the receptor-modified electrodes, thus self-assembling the anodic and cathodic elements required for the functioning of a biofuel cell. Our design followed six main requirements which were identified as important to the cells from a practical standpoint: (i) The self-assembly process should be selective, allowing only one specific population of the bioelectrocatalyst hybrids in the mixture to effectively functionalize


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the anode/cathode; (ii) The dynamics of the self-assembly process should be fast enough to allow activation of the biofuel cell within a few minutes;

Figure 1. Schematic presentation of the designed self-assembled/magnetic disassembled biofuel cell. The selfassembly processes are facilitated through receptor-ligand interactions between electrolyte-solubilized catalytic nanoparticles and stationary modified surfaces.

(iii) The two populations of the enzyme hybrids should not interact in the mixed solubilized state prior to the assembly in a way that leads to loss of activity and self-discharge; (iv) A membrane-


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less design is required for separating and targeting the hybrids in the cell; (v) The use of relay molecules to mediate the bioelectrocatalytic transformations should be avoided, and thus only enzymatic configurations supporting a genuine direct electron transfer (DET) should be considered; and (vi) The use of magnetic nanoparticles as bioelectrocatalyst carriers will allow the application of external magnetic field gradients to disassemble the catalytic layers on the electrode surfaces while regenerating the availability of receptors for binding. Aiming for a design meeting all requirements above indicated the possible use of stationary glassy carbon slides functionalized with either R1=pyrene-linked thiolated -cyclodextrin, acting as the receptor unit participating in the anodic bioelectrocatalyst binding, and R2=multi-walled carbon nanotubes (CNTs)-modified polyethyleneimine (PEI), acting as a stabilized receptive element for the cathodic assemblies. The self-assembled elements were composed of fructose dehydrogenase (FDH) or bilirubin oxidase (BOD)-adsorbed ccMNPs, respectively acting as bioelectrocatalytic anodic and cathodic hybrid carriers. Prior to the enzymatic functionalization, the “anodic” and “cathodic” nanoparticles were respectively adsorbed with L1=adamantanelinked pyrene and L2=pyrene tetrasulfonate (PTS) ligands. The explicit chemical structures of the designed components are elaborated in Figures 2a and 3a. The self-assembling anodic layer relies on the strong host-guest complexation between the R1 and L1 termini, Figure 2a, which was experimentally estimated to be ca. Kc=6.8.104 M-1, ca. 6.6 kcal.mole-1, Figure S1. The value of association constant found for monolayer configuration is slightly higher, than the reported Kc in solution, in agreement with the previous study.39 The assembly of the FDH carrier particles to the surface is expected to increase the loading of the redox enzyme on the electrode and to provide a means for the anodic bioelectrocatalytic transformation. To follow the dynamics of the assembly process, we


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measured the buildup of both the bioelectrocatalytic currents and the open circuit voltage (OCV) on the R1-modified electrode surface in the presence of fructose and L1-modified FDH/ccMNPs hybrids. Figure S2, Supporting Information depicts a series of consecutive cyclic voltammetry scans performed parallel to the assembly process. A time-dependent increase in bioelectrocatalytic anodic currents, starting at ~50 mV vs. Ag/AgCl, is clearly evident, with the bioelectrocatalytic responses at 0.5V presented in Figure 2b. The results indicate that the current responses begin to level off after ~300 seconds from the infusion time. A similar time-evolution pattern was detected while monitoring the OCV changes during the assembly, showing a gradual shift from ~25 to ~44 mV vs. Ag/AgCl within a few minutes, with a further modest 5% increase to 46 mV after 2 hours of continuous recording. The steady state OCV was correlated to the onset of the direct electron transfer (DET) bioelectrocatalysis potential in Figure S2, Supporting Information. The good agreement between the measurements implies that the majority of the self-assembly process occurs within the first 5 minutes of interaction, and thus to ensure a good coverage of the electrode surface, a 10 minutes interval was used for the rest of the anodic experiments. Complementary evidence for the complexation-driven assembly was obtained both by scanning electron microscopy imaging as well as by spectroscopically assaying the FDH content on the electrode surface, see Supporting Information. The micrographs in Figure S3, Supporting Information imply that following interaction with the ligand-bound ccMNPs the modified electrode was covered by a layer of nanoparticles. Under these conditions the surface loading of the FDH was estimated to be 1.5.10-12 mole.cm-2 with a relative activity of 91% compared to the solubilized state, Figure S4b, Supporting Information. Figure 2c depicts the amperometric responses obtained for the self-assembled electrode in the presence of variable concentrations of fructose. The currents, gradually increasing towards


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saturation, Figure 2c inset, indicate that fructose oxidation is conducted through a DET mechanism, with an onset potential typical to FDH on carbonaceous nanostructures.40 These findings comply with the strict requirements reported in several studies, defining a genuine DET path.41,42 Notably, the DET was assisted both by direct interactions between the carbonaceous coating of the particles and the FDH, facilitating contacting in the adsorbed configurations,43,44 and by the coating’s high electronic conductivity which contributes in relaying the electrons between the heme c moieties of FDH and the glassy carbon (GC) collector. Using the saturation current and the enzymatic loading, the turnover rate of FDH in the assembly was calculated to be ket=260 s-1. At this stage we were interested in understanding the correlation between several structural effects related to the assembly of the active layer and the observed emerging bioelectrocatalysis. To this end we have used the amperometric responses as a means to transduce the effects. The calibration curve associated with the self-assembly of the L1-modified FDH/ccMNPs hybrids on the R1-modified surface yielded current densities in excess of 60 A.cm-2 and is presented in Figure 2d curve (i). A similar configuration, equilibrating in the presence of 1-adamantaneacetic acid, showed a 3-fold lower response due to competition on the available receptor sites by both the hybrids and the freely soluble adamantane molecules, curve (ii). In two further experiments either the R1 receptor, curve (iii), or the L1 ligand units, curve (iv), were excluded from the system. Evidently, in both cases sharp decreases of ~75% in the electrocatalytic signals were recorded, implying that the R1-L1 complexation is essential for achieving the high amperometric responses desired for a self-assembled biofuel cell. The origin of the observed remaining catalytic currents is rationalized on the grounds of a non-specific adsorption, evidenced by SEM images probing the configurations, Figure S3c and S3d,


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Supporting Information. These images indicated a lower coverage matching the amperometric differences between the R1-L1-bound and the non-specifically adsorbed surface states, Figure S5, Supporting Information. A similar behavior is demonstrated when the L1 ligands on the FDH/ccMNP hybrids are replaced with L2, namely the PTS ligands associated with the other half cell configuration, curve (v). These results indicate that self-assembly can be both efficient and ligand-selective for potential use in modular biofuel cells. Another experiment shown in Figure 2d is an attempt to generate currents from a L1-R1-bound system in which the FDH/ccMNPs were exposed to a heat pretreatment, curve (vi). Evidently, the exposure affected the tertiary structure of the enzyme, inflicting denaturation and impotency to convert fructose, as reflected by the diminished catalytic currents. The use of magnetic nanoparticles as selectively binding bioelectrocatalyst carriers also allowed us to disassemble the self-organized functional structure using an external magnetic force. Figure 2e shows a dramatic decrease in the catalytic current upon removal of the R1-L1 attached FDH-ccMNPs particles from the surface by a magnetic field. The 8-fold drop in the icat net values was also accompanied by a lower capacitance due to the consequential decrease in the surface area. Evidently, the SEM micrographs of the surface prior and after the removal, Figure S6, Supporting Information, support the amperometric responses, indicating a major release of the hybrids from the surface. Furthermore, complementary spectroscopic analysis detected a 84% decrease in the FDH loading, as compared to the bound state. A series of selfassembly/magnetic disassembly cycles using fresh supply of dispersed hybrids is shown in Figure 2e inset. As can be seen, in both the assembled and detached states the amperometric responses appear stable, leading us to assume that the surface-bound hybrids not removed by the


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Figure 2. Design and characteristics of the self-assembled anode. (a) Molecular design of the assembling elements. (b) Open circuit voltage and catalytic current (at Ecat=0.5V) evolution during the self-assembly. (c) Electrocatalyzed fructose oxidation upon self-assembly for 10 minutes and in the presence of variable fructose concentrations (i-ix: 080 mM, at a 10 mM interval), with the corresponding calibration curve in the inset. (d) Calibration curves for fructose oxidation on the full anode construction, curve i, vs. control experiments: ii - in the presence of adamantane acetic acid, 1 µM, iii - lacking R1, iv - lacking L1, v - using L2 instead of L1, and vi - following a heat pretreatment for 20 minutes at 80oC. The inset compares the catalytic responses at 60mM fructose. (e) Magnet-assisted disassembly of the electrocatalytic ccMNPs. Curves i and ii correspond to the self-assembled system in the absence, and presence of 60 mM fructose. Curve iii depicts the response following the magnetic removal of the nanoparticles. The inset demonstrates the cyclic amperometric responses to infusion and removal of the particles on the modified stationary surface. Voltammograms were taken at 10 mV.s-1. The Icat values include subtraction of the amperometric response in the absence of fructose. Error bars correspond to a set of N=4 experiments.


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magnetic field are persistently trapped in energy favorable defective sites prevalent on the GC surface.45 Notably, the reproducible pattern is also in agreement with the stronger interaction expected between the - bridges46 holding together the receptor on the surface of the GC collector and the ligands on the ccMNPs, as compared to the binding energy of the cyclodextrin/adamantane complex.47 Aiming to design an analogous self-assembled cathodic system which could be coupled to our anode, we have chosen bilirubin oxidase (BOD) as the biocatalyst enzyme due to the longrecognized compatibility of its oxygen-reducing T1 copper center to directly wire to various forms of carbon upon adsorption.48,49 The binding between the BOD/ccMNP hybrids and the stationary GC surface was designed to rely on electrostatic interactions between the pH=5.5 positively charged R2=CNTs-modified polyethyleneimine (PEI)50 and the negatively charged L2=PTS ligand, Figure 3a. As was evident from preliminary impedance spectroscopy measurements, Figure S7, Supporting Information, the incorporation of the CNTs to the PEI increased the stability of the receptor unit on the GC, and further enhanced the electrical conductivity at the electrode/solution interface. The R2-L2-driven self-assembly of the cathodic system was directly monitored by following the OCV response, Figure 3b. Evidently, a near steady state value was achieved after ~200 seconds, with

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