Ca2+-Switchable Glucose Dehydrogenase Associated

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Ca -Switchable Glucose Dehydrogenase Associated with Electrochemical/Electronic Interfaces: Applications to SignalControlled Power Production and Biomolecular Release Ashkan Koushanpour, María Gamella, Zhong Guo, Elham Honarvarfard, Arshak Poghossian, Michael J. Schoening, Kirill Alexandrov, and Evgeny Katz J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b11151 • Publication Date (Web): 29 Nov 2017 Downloaded from http://pubs.acs.org on December 2, 2017

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The Journal of Physical Chemistry

Ca2+-Switchable Glucose Dehydrogenase Associated with Electrochemical/Electronic Interfaces: Applications to Signal-Controlled Power Production and Biomolecular Release Ashkan Koushanpour,† Maria Gamella,† Zhong Guo,◊ Elham Honarvarfard,† Arshak Poghossian,§‡ Michael J. Schöning,§‡ Kirill Alexandrov,◊* Evgeny Katz†* †

Department of Chemistry and Biomolecular Science, Clarkson University, Potsdam, NY 13699-5810,

USA ◊

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia

§

Institute of Nano- and Biotechnologies, FH Aachen, Aachen University of Applied Sciences, Campus

Jülich, Heinrich-Mußmann-Str. 1, D-52428 Jülich, Germany ‡

Institute of Complex Systems (ICS-8), Research Centre Jülich GmbH, D-52425 Jülich, Germany

Corresponding authors: * [email protected] (EK) * [email protected] (KA)

Supporting Information

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ABSTRACT: An artificial Ca2+-regulated PQQ glucose dehydrogenase (PQQ-GDH) enzyme was electrically connected to conducting electrodes and semiconductor interfaces. Direct electron transfer from the enzyme to the conducting electrode support was stimulated by the addition of Ca2+ cations resulting in reversible enzyme activation. A signal-switchable biofuel cell and biomolecular release have been realized using the Ca2+-activated enzyme immobilized on conducting electrodes. Interfacing the signalswitchable enzyme with a semiconductor chip allowed electronic read out of the enzyme ON-OFF states. The developed approach based on the signal-regulated PQQ-GDH enables numerous bioelectrochemical/bioelectronic applications of the developed systems in signal-activated biosensors and biofuel cells, as well as in biomolecular computing/logic systems.

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INTRODUCTION

Switchable, signal-controlled, features of biological systems are important for life and based on complex regulating mechanisms which are difficult to replicate in artificial biomolecular systems.1,2 Synthetic chemical/biochemical approaches have been applied to create artificial signal-switchable biomolecules and multi-component assemblies using methods and structures of limited complexity, much easier than the way used by Nature. Obviously, the artificial biomolecular systems perform functions much simpler than those of the natural biological systems. Biocatalytic systems with signal-switchable/tunable activity have been prepared by incorporation of enzymes in various stimuli-responsive materials changing their properties upon variation of pH, temperature or application of magnetic field.3,4 This approach is based on the variation of environment properties controlling diffusional processes for substrates or cofactors, rather than the catalytic activity of the enzymes themselves. A much more interesting approach to creation of switchable, signal-controlled, biocatalysts is based on control of the tertiary protein structure by various physical or chemical signals. These molecular changes actuated onto the enzyme’s active centers result in modulation of the enzyme’s biocatalytic activity. For example, light-signal switchable enzymes have been prepared by incorporation of photoisomerizable molecules into a protein structure. The photoisomerizable molecules could be randomly bound to functional groups of amino acid residues at various locations5 or specifically attached to the enzyme active centers resulting in much stronger effect on the enzyme activity.6 Site-specific modification of enzymes with oligonucleotides was used to prepare thermo-switchable enzymes.7 The signal-switchable part of the biocatalytic assembly could be represented by various biomolecular receptor units, including complex DNA “machines”,8 which interact with the biocatalytic units changing their conformations and catalytic activity. Another broadly used approach relies on construction of artificial chimeric enzymes that combine a reporter domain with a receptor unit capable of selective recognition of the desired substance. Typically the con3 ACS Paragon Plus Environment


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formational changes of the receptor domain modulate activity of that reporter domain. Chimeric switches with luminescent, fluorescent, proteolytic and various other enzymatic outputs have been reported.9,10 This strategy has been recently applied to PQQ-glucose dehydrogenase (PQQ-GDH)11 resulting in a PQQ-GDH-calmodulin chimer (PQQ-GDH-CaM) activated by Ca2+ cations.12

Integration of enzymes with various electrochemical and electronic interfaces is a centerpiece of various bioelectronic applications,13,14 including biosensors,15 biofuel cells,16 biocomputing logic systems,17,18 etc. Many different approaches have been used to design electrochemical systems with signalcontrolled switchable features, triggered by variation of pH,19 temperature,20 magnetic field,21 etc., mostly using stimuli-responsive materials (not necessarily containing enzymes) for modification of electrode surfaces. Construction of electrodes functionalized with signal-switchable redox enzymes would enable a range of novel bioelectronic applications. In particular the ability to engineer enzymes that are selectively modulated by the ligand of choice would enable creation of complex bioelectronic systems with adjustable input/output parameters. Here we report several novel bioelectrochemical systems controlled by Ca2+-regulated PQQ-GDH-CaM on a chemically modified electrode or at a semiconductor chip interface.

When switchable biocatalytic redox processes are studied by electrochemical means the best results could be observed if the enzyme active centers demonstrate direct electron transfer to/from the conducting electrode. This allows exclusion of the secondary effects related to mediated electron transport. Thus, while many different immobilization techniques are available for attaching enzymes to electrode surfaces,22 preferences should be given to the enzymes and immobilization methods providing direct electron transfer processes.23,24 The enzyme selection and the way how the enzyme is bound to the electrode surface are highly important (notably not all reported systems really provide direct electron trans4 ACS Paragon Plus Environment

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fer).25 Wild type PQQ-GDH (E.C. 1.1.5.2) has been used in many bioelectrocatalytic systems demonstrating direct electron transfer to electrode surfaces, particularly using electrodes composed of carbon nanotubes or graphene.26-32 We therefore assumed that Ca2+-switchable PQQ-GDH-CaM enzyme will behave in a similar way and have immobilized it on the electrode surface using the established protocols.29,32

EXPERIMENTAL SECTION 1. Chemicals and materials. Glucose oxidase (GOx; E.C.1.1.3.4, from Aspergirus niger sp), hemin, D-(+)-glucose, (1-ethyl-3[3(dimethylamino)propyl] carbodiimide (EDC), N-hydroxysuccimide (NHS), (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES-buffer), 2-(N-morpholino)ethanesulfonic acid (MES-buffer), 3(N-morpholino)propanesulfonic acid, (MOPS-buffer), calcium chloride and other standard organic and inorganic chemicals and solvents were purchased from Sigma-Aldrich and used as supplied without any further purification. 1-Pyrenebutanoic acid succinimidyl ester (PBSE) was purchased from AnaSpec Inc. Water used in all of the experiments was ultrapure (18.2 MΩ·cm) from a NANOpure Diamond (Barnstead) source. Ca2+-activated PQQ-dependent glucose dehydrogenase (PQQ-GDH-calmodulin chimer: PQQ-GDH-CaM) was prepared and characterized according to the procedures detailed in recently published report.12 Released DNA labelled with a fluorophore: 6-FAM-5’ TGC AGA CGT TGA AGG

ATC

CTC

(6-FAM

is

a

single

isomer

fluorescein

derivative;

https://www.idtdna.com/site/Catalog/Modifications/Product/1108) was purchased from Integrated DNA Technologies Inc. (San Diego, CA).

2. Preparation of bioelectrocatalytic electrodes. 2.1. Carbon fiber electrochemical in situ modification with graphene. 5 ACS Paragon Plus Environment


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Carbon paper composed of carbon fibers (SpectracarbTM 2050L-1050; Fuel Cell Store, TX) was used as the electrode material for formation of graphene nano-sheets. The electrode modification procedure falls in two successive phases. The first step, aiming at synthesis of graphene oxide nano-sheets (GONs), was done by cyclic voltammetry scanning potential from -0.5 to 3.0 V, with a potential sweep rate of 50 mV·s-1 for 15 successive cycles in phosphate buffer solution, 25 mM, pH 6.9, while the solution was stirred at 400 rpm. The second phase was set to reduce the GONs to graphene nano-sheets (GNs) in the same buffer solution by applying a step potential at -1.0 V for 120 s at the same swirling condition. The produced electrodes composed of carbon fibers functionalized with graphene nano-sheets are abbreviated as GNs-CF in the following text.

2.2. PQQ-GDH-CaM-modified electrode preparation. The GNs-CF electrodes were washed with isopropyl alcohol under moderate shaking for 15 min at room temperature (22 ± 2 °C) prior to their modification with the enzyme. Then, the electrodes were incubated with PBSE linker, 10 mM, in dimethyl sulfoxide (DMSO) solution with moderate shaking for 1 h at room temperature, subsequently rinsed with DMSO to remove excess of PBSE and then with HEPESbuffer (25 mM, pH 7.1) to remove DMSO. The PBSE-functionalized electrode was incubated for 1 h in a solution of PQQ-GDH-CaM (85 µg·mL−1) in HEPES-buffer (25 mM, pH 7.1) at ca. 4 °C in order to preserve the enzyme activity. Then, the enzyme-modified electrode was rinsed with HEPES-buffer (25 mM, pH 7.1) and stored (4 °C) in the same buffer solution. The electrode modification procedure is shown schematically in Figure 1A in the paper.

2.3. Preparation of the biocathode functionalized with hemin and GOx for assembling a biofuel cell. Hemin was adsorbed on the GNs-CF electrode due to its π-π stacking. Hemin (8 mg) was dissolved in 2 mL phosphate buffer solution, 0.1 M, pH 10.0, and the solution was sonicated for 1 minute. Then, the 6 ACS Paragon Plus Environment

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GNs-CF electrode was immersed in the hemin solution and left overnight at room temperature. The electrode with the adsorbed hemin was rinsed carefully with the phosphate buffer to remove the excess of the weakly bound hemin. The hemin-functionalized GNs-CF electrode was reacted at room temperature with 0.1 M EDC and 0.1 M NHS in MES-buffer (2 mL, 0.1 M, pH 5.0) for 1 hour. Then, the activated electrode was immediately immersed in a HEPES-buffer solution (2 mL, 25 mM, pH 7.1) containing 50 U·mL-1 of GOx, and was left to react at room temperature for 1.5 hours. The GOx-modified electrode was rinsed with HEPES-buffer (25 mM, pH 7.1). Note that the GOx molecules were covalently attached through amino groups of lysine residuals to the carboxylic groups of hemin; see Figure S1 for the illustration.

3. Electrochemical characterization of the PQQ-GDH-CaM modified electrode. Cyclic voltammetry (CV) measurements were performed using a conventional three-electrode cell at room temperature (22 ± 2 °C) with an ECO Chemie Autolab PASTAT 10 electrochemical analyzer using the GPES 4.9 (General Purpose Electrochemical System) software package. The working electrode was made of carbon fibers modified with graphene nano-sheets (GNs-CF) followed by the PQQ-GDHCaM enzyme immobilization as described above. The geometrical surface area exposed to the solution was ca. 2 cm2 accounting for both sides of the modified electrodes, but not taking into account the internal surface of the porous 3D-electrodes. A slab of glassy carbon (6.5 cm2 geometrical surface area) was used as a counter electrode and a Metrohm Ag|AgCl|KCl, 3 M, electrode served as a reference electrode. All potentials in the paper are reported vs. this reference electrode. The cyclic voltammograms were recorded at a scan rate of 2 mV·s−1 in a HEPES-buffer (0.025 M, pH 7.1) containing 0.1 M NaCl, in the presence, 20 mM, or absence of glucose and in the presence, 0.1 mM, or absence of Ca2+ cations (added in the form of soluble CaCl2 salt).



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4. Biofuel cell polarization curve measurements. The performance of the biofuel cell was analyzed in a HEPES-buffered (0.025 M, pH 7.1) electrolyte solution containing 0.1 M NaCl. The biocatalytic anode-cathode pair (each electrode with the geometrical surface area of ca. 2 cm2 accounting for both electrode sides) was inserted into the working solution with a distance between the electrodes of ca. 1 cm. A polarization function was obtained when the cell was connected to an external variable resistance load (varied from 0 to 1.0 MΩ) and the voltage/current produced on the load was measured by a high-impedance multimeter (Meterman 37XR). The biofuel cell was characterized in the presence of glucose, 20 mM, and in the presence, 0.1 mM, or absence of Ca2+ cations (added in the form of soluble CaCl2 salt).

5. The PQQ-GDH-CaM enzyme immobilization and operation on the semiconductor device. Electrolyte-insulator-semiconductor (EIS) chips (with sizes of 10 mm × 10 mm) consisting of an Al (300 nm)–p-Si–SiO2 (30 nm)–Ta2O5 (60 nm) structure were prepared from a p-doped Si wafer (thickness: ca. 400 µm, resistivity of 1–10 Ωcm, Si-Mat, Germany; the authors thank H. Iken for preparation of the EIS chips). The PQQ-GDH-CaM immobilization on the gate surface was performed by mixing the enzyme with Nafion and then depositing on the surface of the EIS gate insulator. The enzyme membrane was prepared by mixing 1.8 µL of PQQ-GDH-CaM (300 nM) in HEPES-buffer (1 mM, pH 7.0) with 4 µL of a Nafion 117 solution (~5% in a mixture of lower aliphatic alcohols and water; SigmaAldrich). The membrane was deposited by drop-casting 5 µL of the mixture of the enzyme/Nafion solution on the Ta2O5 surface. After drying the semiconductor chip in air at room temperature (22 ± 2 °C), it was immersed in a HEPES-buffer solution (1 mM, pH 7.0) for 30 minutes for enzyme membrane equilibration. The enzyme-membrane-EIS sensor was assembled in a homemade electrochemical cell. The sidewalls and backside contacts of the EIS sensor chip were protected from the electrolyte solution by means of a sealing O-ring, thus excluding the need for a complex encapsulation process. The contact 8 ACS Paragon Plus Environment

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area of the EIS sensor was determined by the O-ring diameter, about 0.5 cm2. An Ag/AgCl/KCl, 3 M, electrode was used as a reference electrode, while the rear-side aluminum electrical contact of the chip was wired through a gold pin. Capacitance-voltage (C-E) and impedance spectroscopy methods were applied to measure electrochemical parameters of the EIS chips, using an ECO Chemie Autolab PSTAT-10 electrochemical instrument equipped with a frequency response analyzer. A variable DCbiasing voltage (from -1 to +1 V) was applied between the conducting rear side of the chip and the reference electrode to set the working potential of the EIS sensor chip, and a small alternating voltage (20 mV) was applied to the system in order to measure the capacitance. All measurements were performed in a dark Faraday cage at room temperature in 1 mM phosphate buffer solution, pH 7.0, containing 2 mM KCl. Capacitance-voltage curves were obtained for the Ta2O5-gate EIS sensor modified with the PQQ-GDH-CaM upon adding 20 mM glucose and 0.1 mM Ca2+ cations (added in the form of soluble CaCl2 salt). Capacitance measurements were performed at a frequency of 100 Hz in a HEPES-buffer solution (1 mM, pH 7.5) containing 2 mM KCl at room temperature. Mott-Schottky plots derived from the impedance spectra were obtained for the Ta2O5-gate EIS sensor modified with the PQQ-GDH-CaM upon adding 20 mM glucose and 0.1 mM Ca2+ cations. The measurements were performed in the frequency range of 1 mHz to 100 kHz.

6. Deposition of alginate on the graphite electrode. Sodium alginate (1.5% w/v) was dissolved in 100 mM Na2SO4 (pH 6.0) and stirred overnight at 45 °C. The solution was cooled to room temperature (22±2 ºC) and FeSO4 (35 mM) and 6-FAM-labeled DNA (1 µM) were added and mixed well. The mixture was deposited on a graphite rod electrode (geometrical area of ca. 0.57 cm2 exposed to the solution) upon oxidation of Fe2+ cations by applying a potential of +0.85 V (vs Ag/AgCl) for 180 sec resulting in an alginate cross-linked film on the electrode surface. The electrode was washed and allowed to incubate for 3 minutes in 1.0% (w/v) of poly(allylamine hy9 ACS Paragon Plus Environment


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drochloride) (PAH) prepared in water. The resulting electrode was washed thoroughly in water, stayed in MOPS-buffer solution (50 mM, pH 7.4) for 2 hours, and then used in the release experiments.

7. DNA release from the alginate film triggered by Ca2+ signal. The alginate-modified electrode was electrically connected with a wire to the PQQ-GDH-CaMmodified electrode. Both electrodes were located in different test tubes with the electrolyte solutions connected with a salt bridge (NaCl in water, 0.5 M), Figure S2. The PQQ-GDH-CaM-modified sensing electrode was immersed in a HEPES-buffer solution (25 mM, pH 7.1) containing 0.1 M NaCl in the presence (20 mM) or absence of glucose and in the presence (0.1 mM) or absence of Ca2+ cations, while the alginate-electrode was in a MOPS-buffer solution (50 mM, pH 7.4) containing 0.1 M NaCl. The reason to separate the electrolyte solutions was to exclude the added Ca2+ cations from their contact with the alginate film (note that Ca2+ cations are well known cross-linkers for alginate, thus they can prevent the alginate dissolution and inhibit the DNA release). The fluorescence, λmax = 520 nm, corresponding to the FAM fluorescent label on the DNA was monitored in the solution before and after the addition of Ca2+, corresponding to the DNA leakage and signal-triggered release, respectively. Fluorescent measurements were performed using a fluorescent spectrophotometer (Varian, Cary Eclipse).

RESULTS AND DISCUSSION

Figure 1A schematically shows immobilization of the PQQ-GDH-CaM on a graphene-functionalized carbon fiber electrode using 1-pyrenebutanoic acid succinimidyl ester (PBSE) as a hetero-bifunctional linker. The graphene nano-sheets (Figure 1B) were produced on the electrode surface (carbon paper, SpectracarbTM 2050L-1050; Fuel Cell Store, TX) by oxidative-reductive cyclic treatment resulting in peeling off graphene layers from the carbon fibers (see the experimental details in the Supporting In10 ACS Paragon Plus Environment

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formation, SI).33 Then, the PBSE linker was adsorbed on the electrode surface due to π-π staking of the pyrene units on the graphene nano-sheets. Finally, the active ester groups of the PBSE linker reacted with the amino groups of the enzyme lysine residues, resulting in the enzyme covalent immobilization. The PQQ-GDH-CaM-functionalized electrode was studied in the absence and presence of Ca2+ cations in glucose-containing buffer. The bioelectrocatalytic electrode demonstrated activity only in the presence of Ca2+ cations, which produced conformational changes in the protein resulting in the enzyme activation. Figure 2 shows cyclic voltammograms measured with the modified electrode in the absence and presence (20 mM) of glucose, both obtained in the absence of Ca2+ cations (curves a and b, respectively). As expected for the PQQ-GDH-CaM,12 both experiments result in almost identical cyclic voltammograms showing no electrocatalytic activity for glucose oxidation. It should be noted, just for comparison, that the electrode modified with the wild-type PQQ-GDH shows well-defined anodic electrocatalytic current in the presence of glucose.32 Addition of Ca2+ cations (0.1 mM) to the solution resulted in activation of the PQQ-GDH-CaM for glucose oxidation, thus producing the anodic electrocatalytic current (Figure 2, curve c). The observed electrocatalytic current is measured as the increase of the Faradaic anodic current in the presence of the activating Ca2+ cations comparing with the cyclic voltammogram obtained in the presence of glucose but in the absence of Ca2+ cations. It should be noted that Ca2+ cations operated as the activating switch for the bioelectrocatalytic process, while glucose was a “fuel” being a source of the electrons. Obviously, in the absence of glucose the electrocatalytic current was not observed regardless of the presence or absence of Ca2+ cations. Cyclic addition-removal of Ca2+ cations by changing the solution in the electrochemical cell resulted in ON-OFF switching of the bioelectrocatalytic activity of the modified electrode (Figure 2, inset). The reversible activation-inactivation of the bioelectrocatalytic process could be performed many times without decreasing the switchable enzyme activity.



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Figure 1. (A) Schematics of the PQQ-GDH-CaM covalent immobilization on the graphenefunctionalized carbon fiber electrode and its reversible switching ON-OFF by the additionremoval of Ca2+ cations. Glc = glucose; GlcA = gluconic acid. (B) Scanning electron microscopy image of the graphene nano-sheets on the carbon fiber electrode surface.


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Figure 2. Cyclic voltammograms obtained with the PQQ-GDH-CaM-modified electrode: (a) in the background solution: 25 mM HEPES-buffer, pH 7.1, 0.1 M NaCl, (b) in the presence of 20 mM glucose, (c) in the presence of 20 mM glucose and 0.1 mM Ca2+. Potential scan rate, 2 mV/s. Inset: Reversible activation (ON) and inhibition (OFF) of the bioelectrocatalytic process by cyclic adding-removing Ca2+ cations. The electrocatalytic current, Icat, was measured at 0.5 V.

Some bioelectronic applications are expected to benefit from functional integration of signalswitchable enzymes with semiconductor electronic devices.34-36 To test the suitability of the switchable PQQ-GDH-CaM for such applications it was immobilized in the form of a membrane (see SI) on the Ta2O5 gate surface of the electrolyte-insulator-semiconductor (EIS) chip (Figure 3A). The Ta2O5 is one of the best pH-sensitive materials with nearly-Nernstian pH sensitivity of 56-59 mV/pH.37,38 When the PQQ-GDH-CaM becomes active for oxidation of glucose in the presence of Ca2+ cations and phenazine methosulfate (PMS) operating as an electron acceptor, the local pH in the proximity to the Ta2O5 surface is decreasing due to the biocatalytically produced gluconic acid (the product of glucose oxidation). The 13 ACS Paragon Plus Environment


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pH changes produced by the enzyme in its active state were analyzed by impedance spectroscopy and represented as the EIS interfacial capacitance changes and Mott-Schottky plots measured at different bias potentials. The biocatalytically produced acidic pH results in the shifts of the capacitance-voltage curve in the depletion region along the capacitance axis (∆C, at a constant bias voltage) or along the voltage axis (∆E, at a constant capacitance) (Figures 3B and 3C, respectively). In the absence of Ca2+ cations the enzyme was in the mute (non-active) state resulting in the measured capacitance and MottSchottky plot indistinguishable from the background measurements in the absence of glucose (see almost identical curves a and b in Figures 3B and 3C). Cyclic addition-removal of Ca2+ cations (changing the solution at the gate) resulted in the reversible activation-inhibition of the PQQ-GDH-CaM, thus resulting in the reversible capacitance changes measured on the EIS chip.


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Figure 3. (A) Schematics of the electrolyte-insulator-semiconductor (EIS) device interfaced with the PQQ-GDH-CaM-containing membrane reversibly switchable by the Ca2+ signals between the ON-OFF states. (B) Capacitance-voltage curve of the EIS chip. (C) Mott-Schottky plots derived from the capacitance measurements. The curves were obtained: (a) in the background solution of 1 mM HEPES-buffer, pH 7.5, and 2 mM KCl, (b) in the presence of 20 mM glucose, (c) in the presence of 20 mM glucose and 0.1 mM Ca2+.

The two experiments described above clearly demonstrated that the Ca2+-activated PQQ-GDH-CaM enzyme can be interfaced with conducting electrodes and semiconductor devices resulting in their signal-switchable electrochemical/electronic features. The next step in our research was aimed at demonstrating some practical applications based on the switchable functions of the enzyme-modified interfaces. To demonstrate electrochemical applications of the PQQ-GDH-based switchable enzymes we used the developed Ca2+-activated electrodes to construct signal-activated biofuel cells16 and molecularrelease systems.39



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The Ca2+-activated PQQ-GDH-CaM-modified glucose oxidizing electrode was used as an anode in a biofuel cell. The cathode was prepared using graphene-functionalized carbon fibers with the adsorbed hemin and covalently immobilized glucose oxidase (GOx; E.C. 1.1.3.4) (see SI for the experimental details and Figure S1 for the cathode assembly). It should be noted that the cathode in the biofuel cell could be organized in a different way, for example, using laccase for the bioelectrocatalytic reduction of oxygen.32 Both biocatalytic electrodes operated in the presence of glucose and O2, however, the anode also required Ca2+ cations for its function. The PQQ-GDH-CaM-anode was oxidizing glucose and producing an anodic current while being in the active state in the presence of Ca2+ cations. GOx immobilized on the cathode produced H2O2 as the concomitant product of glucose oxidation by O2. Then, the in situ generated H2O2 was catalytically reduced by hemin, thus generating a cathodic current on the electrode. Overall, the biofuel cell produced voltage and current, while being in the active state (Figure 4A). Removing Ca2+ cations from the solution resulted in the inhibition of the anodic process because of switching off the PQQ-GDH-CaM. This resulted in the dramatic decrease of the current and power generated by the biofuel cell (Figures 4B and 4C, respectively). The reversible activation-inhibition of the power generation can be achieved by cyclic addition-removal of Ca2+ cations. It should be noted that Ca2+ cations operated as the switch for activating the anodic process in the biofuel cell, while glucose was the “fuel” providing electrons for the anodic process. The biofuel operation was not possible in the absence of the glucose “fuel” regardless of the presence or absence of Ca2+ cations. In other words, the open circuit voltage and short circuit current measured in the absence of glucose were negligible, not different from the background values obtained for bare electrodes.

While this is not the first example of a signal-switchable biofuel cell,40-42 the present system is the very first realization of a switchable biofuel cell with the use of a signal-activated enzyme (all previous


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switchable biofuel cells were based on signal-responsive polymers and mediators controlling the electron transport).

Figure 4. (A) Schematics of the biofuel cell operation. Polarization function (B) and power release on external variable resistance of the biofuel cell (C) in the active (in the presence of 0.1 mM Ca2+) and inactive (in the absence of Ca2+) states, curves a and b, respectively. The solution included 25 mM HEPES-buffer, pH 7.1, 0.1 M NaCl and 20 mM glucose.

Signal-triggered release of biomolecules (e.g., insulin, DNA) entrapped in Fe3+-cross-linked alginate hydrogels has been recently extensively studied.39 This process is based on different propensity of Fe2+ and Fe3+ cations to serve as a cross-linker of the alginate polymer. While Fe3+ cations effectively crosslink alginate yielding a hydrogel, Fe2+ cations do not cross-link alginate resulting in its soluble state (see 17 ACS Paragon Plus Environment


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Figure S3 in the SI for illustration).43 Electrochemical oxidation of Fe2+ to Fe3+ on the electrode surfaces results in gelation of the soluble alginate and entrapment of dissolved biomolecules. These can be released in controlled fashion by electrochemical reduction of Fe3+ to Fe2+ resulting in the alginate hydrogel dissolution. The reductive dissolution of the alginate film can be achieved by electrochemical means applying negative potential from an electrochemical instrument43 or by connecting the alginatemodified electrode to a biosensing electrode generating negative potential in the presence of specific chemical signals.39


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Figure 5. (A) Schematics of the DNA release from the Fe3+-cross-linked alginate hydrogel stimulated by the Ca2+-signal applied on the PQQ-GDH-CaM-modified electrode. Note that the releasing and sensing electrodes operate in different solutions connected through a salt bridge (see details in the SI). (B) Fluorescence increase in the solution upon DNA leakage and Ca2+-stimulated release: (a) the alginate-electrode is not connected to the PQQ-GDH-CaM-modified electrode, (b) both electrodes are electrically connected and the PQQ-GDH-CaM-modified electrode is reacted with 20 mM glucose, (c) both electrodes are electrically connected and the PQQ-GDH-CaM-modified electrode is reacted with 20 mM glucose and 0.1 mM Ca2+ cations.

We decided to test if the Ca2+-regulated PQQ-GDH-CaM can be used as a trigger for the alginate phase transition. The alginate hydrogel film was deposited electrochemically on a graphite electrode including DNA molecules labelled with a fluorescent dye entrapped in the film (see experimental details in the SI). The alginate-modified electrode was connected electrically to the PQQ-GDH-CaM-electrode 19 ACS Paragon Plus Environment


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described above. This electrode was inactive in the presence of glucose (20 mM) unless Ca2+ cations were added. In the presence of Ca2+ cations (0.1 mM) the PQQ-GDH-CaM-electrode was activated generating ca. -70 mV potential (vs. Ag/AgCl reference electrode). This produced the reducing current passing from the switchable enzyme electrode to the alginate electrode resulting in the Fe3+ reduction and alginate film dissolution, thus releasing the entrapped DNA molecules. The released DNA was detected in the solution by measuring fluorescence related to the fluorescent label associated with the DNA. A similar release process has been demonstrated using the wild-type GDH enzyme producing the reductive potential in the presence of glucose,44 however, the present system was activated by the presence of Ca2+ cations, while glucose was always present in the solution. It should be noted that the present bioelectronic system was not reversible, thus being different from the previous examples, because of irreversible degradation of the alginate film and concomitant process of the DNA release. However, this irreversibility reflects the features of the alginate releasing system, rather than switchable enzyme.

Overall, the designed bioelectronics systems based on the Ca2+-activated PQQ-GDH-CaM enzyme demonstrated good stability (operating a few hours under lab conditions), reproducibility (providing the measurable results within ±10% distribution), and re-usability (being active if measured after a few days of storage in a fridge). These features are typical for enzyme-based bioelectrochemical biosensor systems.

CONCLUSIONS

The present study provides the first example of direct electrical coupling of the engineered ligandmodulated PQQ-GDH-CaM with conducting electrodes and semiconductor interface. PQQ-GDH has recently been demonstrated to be an excellent platform for protein engineering and was used to construct electrochemical receptors of ions, small molecules, proteins and biochemical activities.11,12 Practi20 ACS Paragon Plus Environment

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cal applications in the signal-controlled electrical power generation and biomolecular release have been demonstrated. The developed approach is straightforward and can be easily adapted to numerous bioelectrochemical and bioelectronic systems switchable by external molecular signals. The possible applications range from various biosensors/biofuel cells to biomolecular computing/logic systems.

Supporting Information

Additional figures illustrating the experiments.

AUTHOR INFORMATION Corresponding Authors Evgeny Katz: [email protected], Kirill Alexandrov: [email protected]

Notes KA is a shareholder of Molecular Warehouse Ltd that holds license to PQQ-GDH based biosensor used in this study.

ACKNOWLEDGMENT This work at Clarkson University (EK) was supported by the NSF awards CBET-1403208 and the work at The University of Queensland (KA) was supported by ARC DP grants DP150100936, DP160100973, and NHMRC Development grant APP1113262.



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Ca2+-Switchable Glucose Dehydrogenase Associated

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