DNA Redox Hydrogels: Improving Mediated Enzymatic

Mar 10, 2016 - We present the preparation of a redox DNA hydrogel for mediated bioelectrocatalysis of oxidoreductase enzymes for biosensor and biofuel...
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DNA Redox Hydrogels: Improving Mediated Enzymatic Bioelectrocatalysis Khiem Van Nguyen,†,‡ Yaovi Holade,† and Shelley D. Minteer*,† †

Departments of Chemistry and Materials Science and Engineering, University of Utah, 315 S 1400 E Room 2020, Salt Lake City, Utah 84112, United States ‡ Institute of Research and Development, Duy Tan University, Da Nan, Vietnam S Supporting Information *

ABSTRACT: We present the preparation of a redox DNA hydrogel for mediated bioelectrocatalysis of oxidoreductase enzymes for biosensor and biofuel cell applications. The noncovalent functionalization of DNA with redox molecules is achieved by intercalation of aromatic redox probes into the DNA double helix or electrostatic binding of redox-active tetraalkylammonium ions to phosphate groups on DNA. Prepared DNA redox hydrogels demonstrate the capability of mediating bioelectrocatalytic glucose oxidation by oxidoreductase enzymes. This is the first evidence that redox DNA hydrogels can replace redox polymer hydrogels for self-exchangebased mediation for bioelectrocatalytic applications. This study contributes toward advances in the use of DNA, an emerging biomaterial, in enzymatic bioelectrocatalysis-based applications.

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INTRODUCTION Immobilizing enzymes on the electrode surface while retaining their activity and allowing good electrical connection to the electrode is critical for the development of bioelectrocatalysisbased devices such as biofuel cells and biosensors.1 Various strategies have been investigated for enzyme immobilization, ranging from physical adsorption2 to covalent binding to the electrode3 and encapsulation or entrapment of enzymes in polymers.4−6 Among them, enzyme immobilization within hydrogels is probably the most widely used strategy. The hydrogel state keeps enzymes from leaching off the electrode while still allowing free diffusion of small molecules such as substrates, products, and enzyme cofactors.7 In addition, the polymer backbone can be modified with redox molecules capable of mediating electron transfer between the enzyme active site and the electrode.7 This helps to significantly increase the performance of enzymatic biofuel cells and biosensors. Common hydrogel polymers used for enzyme immobilization include poly(ethylenimine) (PEI) and poly(4vinyl)pyridine (PVP) with redox moieties (i.e., osmium complexes and ferrocene) covalently bound to the backbone polymer to allow self-exchange-based conduction of electrons to/from the electrode.7,8 We recently introduced self-assembled DNA hydrogels as a new class of biomaterial for enzyme immobilization.9 DNA hydrogels are formed under physiological conditions, thus allowing in situ encapsulation of enzymes. In our previous study, a redox mediator in solution was used to mediate electron transfer between an enzyme and an electrode. However, immobilized mediators are more desirable in terms of practical applications. In this study, we investigate the possibility of modifying DNA with small redox © 2016 American Chemical Society

molecules for mediated electron transfer (MET) in bioelectrocatalytic systems. Since most high current density mediated electron transfer (MET) processes rely on facile self-exchange between redox centers along the polymer backbone, the polymer needs to be functionalized with a high density of redox moieties for efficient MET. This is normally achieved by covalent attachment of redox molecules to the hydrogel polymer backbone; for example, PEI that has nucleophilic amine groups can be functionalized with electrophilic halogen derivatives of ferrocene.8 Although the covalent modification of DNA polymers with redox probes at the 5′- or 3′-ends is possible, the redox centers are obviously not dense enough for efficient self-exchange processes. High-density covalent modification of DNA that involves internal bases will likely lead to disruption of the double-helical structure. Thus, the development of alternative strategies for redox functionalization of DNA is necessary. Herein, we present two noncovalent methods for high-density functionalization of DNA with redox moieties to form redox polymers that can be used for MET between oxidoreductase enzymes and the electrode. In the first method, a redox probe with an aromatic structure was intercalated into the double-stranded DNA. The second method utilized a tertiary ammonium salt containing a redox center, whereby the redox center is immobilized onto DNA through electrostatic interactions with the phosphate backbone of DNA. Received: November 27, 2015 Revised: March 3, 2016 Published: March 10, 2016 2603

DOI: 10.1021/acscatal.5b02699 ACS Catal. 2016, 6, 2603−2607

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ACS Catalysis

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RESULTS AND DISCUSSION DNA Modification through Intercalation. Small redox molecules with aromatic structures can intercalate into the DNA double helix via π−π stacking with DNA bases.10 This phenomenon has been widely utilized for studying DNA hybridization and developing DNA electrochemical sensors.11,12 However, investigating the possibility of using this redox probe intercalated DNA as a mediator in MET processes is still relatively unexplored. There is only a single report on the use of toluidine blue O (TBO) intercalated DNA for MET between peroxidase and an electrode, but this system was using the DNA for MET, not for immobilization, and so it did not provide the normal properties of a redox hydrogel based bioelectrode.13 TBO is a phenothiazine dye that can intercalate into double-stranded DNA with high affinity.14 Thus, TBO can be used for DNA staining and as a redox-active probe to detect DNA hybridization.15,16 In addition, TBO has been known to undergo MET between various enzymes and the electrode,17−19 although it should be noted that even though TBO has been shown to mediate NAD-dependent dehydrogenases, it is not the best mediator in the literature, but it is the only mediator that has also been shown to be an intercalator. For these reasons, TBO was also chosen to prepare redox DNA hydrogels via intercalation in this study. Self-assembled DNA hydrogels were prepared from Y-DNA and L-DNA scaffolds as described in our previous study.9 The diluted DNA polymer solution (50 μM) was then dropcast onto the surface of a Toray carbon paper electrode (TP) and dried at room temperature for 2 h. The DNA-modified electrode was then immersed into a solution of TBO (0.1 mM in PBS buffer at pH 7) for 5 min to allow TBO intercalation into the DNA double helix (Figure 1A). Finally, the electrode was rinsed with PBS for 30 min to remove weakly adsorbed TBO. Figure 1B presents cyclic voltammetry (CV) of the TBO-

intercalated DNA (TBO/DNA) hydrogel. The redox peaks of immobilized TBO are negatively shifted in comparison with those of TBO in solution, indicating that TBO is intercalated into the DNA double helix, as described in previous studies.13,20 After incorporating TBO into DNA to form a redox hydrogel (TBO/DNA), we then investigated the possibility of using this material for mediated bioelectrocatalysis. We hypothesized that TBO/DNA might not communicate efficiently with the active site of cofactor-bound enzymes due to steric hindrance. Our preliminary experiment showed that TBO/DNA was not able to mediate electron transfer from FAD-dependent glucose dehydrogenase to the electrode. Thus, the following studies focused on enzymes utilizing diffusional cofactors such as NAD-dependent dehydrogenases. Efficient bioelectrocatalysis of NAD-dependent dehydrogenases normally requires electrode materials capable of oxidizing NADH at low potentials. TBO can oxidize NADH at ∼0 V vs SCE and has been used to modify electrodes for bioelectrocatalytic devices using NAD-dependent dehydrogenase enzymes.18 However, the ability of intercalated TBO to oxidize NADH has not been explored. Therefore, we next investigated the electrooxidation of NADH by TBO/DNA hydrogel immobilized on a Toray carbon paper electrode (TP) by CV over a larger potential range, from −0.4 to +0.3 V vs SCE. Figure 2A displays typical cyclic voltammograms of TBO on a bare Toray carbon paper (TBO/TP) electrode (black line) and TBO/DNA hydrogel immobilized on a Toray carbon paper (TBO/DNA/TP) electrode (red line) in the absence of NADH (dashed curves) and in the presence of 4 mM NADH (solid curves). The TBO/TP electrode serves as a control to ensure that the electrocatalytic oxidation of NADH on a TBO/ DNA/TP electrode is not solely from physically adsorbed TBO on the surface of TP. We observed the NADH oxidation starting at about −0.2 V vs SCE for the TBO/DNA/TP electrode and at about −0.1 V vs SCE for the TBO/TP electrode. This is in good agreement with the above observation that the redox peaks of intercalated TBO are negatively shifted in comparison with those of TBO physically adsorbed on the electrode surface (Figure 1B). The current density from the electrocatalytic oxidation of NADH at the TBO/DNA/TP electrode is 65 μA/cm2, which is 4-fold higher than the value of 16 μA/cm2 obtained for the TBO/TP electrode. This clearly indicates the capability of intercalated TBO to mediate NADH oxidation at low potential. To further ensure the observed electrocatalytic activity of NADH oxidation at 0 V vs SCE for the TBO/DNA/TP electrode derived from TBO mediator but not DNA, we performed additional control experiments to study the direct oxidation of NADH at the unmodified Toray carbon paper (TP) and the DNA hydrogel modified Toray carbon paper (DNA/TP) electrodes (Figure S1 in the Supporting Information). As expected, neither TP nor DNA/TP electrodes show significant electrocatalytic oxidation of NADH at 0 V vs SCE. It is known that carbon electrodes are capable of electrooxidizing NADH at higher potential. This is confirmed in Figure S1, in which the oxidation occurs at potentials higher than +0.1 V vs SCE. Interestingly, the presence of DNA at the electrode also improves substantially the NADH electrooxidation at potentials higher than 0.2 V vs SCE. This result suggests that the interaction between DNA and NADH might also affect the redox property of NADH. Nevertheless, all cyclic voltammetric data clearly demonstrate that the observed electrocatalytic oxidation of NADH at a TBO/DNA/TP

Figure 1. (A) TBO intercalated into the DNA double helix. (B) Representative cyclic voltammograms of adsorbed TBO on a bare Toray paper electrode (black line) and immobilized TBO on a DNA hydrogel-modified Toray paper electrode (red line) in PBS buffer (50 mM phosphate, 100 mM NaCl, pH 7.5). The scan rate was 20 mV/s. 2604

DOI: 10.1021/acscatal.5b02699 ACS Catal. 2016, 6, 2603−2607

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more, the stability of the TBO/DNA/TP electrode was also investigated by chronoamperometry at 0 V vs SCE (Figure S2 in the Supporting Information). The results indicate that the designed electrode has good stability. Overall, these results constitute evidence that the intercalated TBO in DNA is the mediator for NADH electrooxidation. The ability of TBO/DNA to mediate bioelectrocatalysis of NAD-dependent dehydrogenase enzymes was studied next. We chose glucose dehydrogenase (GDH) as a model enzyme, because the enzyme can be used to construct either a glucose biosensor or a glucose biofuel cell (Figure 3A). The GDH/

Figure 2. (A) Representative cyclic voltammograms of TBO on a bare Toray paper (TP) electrode (black line) and immobilized TBO on a DNA hydrogel modified Toray paper electrode (red line) in PBS buffer (50 mM phosphate, 100 mM NaCl, pH 7.5) in the absence of NADH (dashed curves) and in the presence of 4 mM NADH (solid curves). The scan rate was 20 mV/s. (B) Amperometric response of the DNA hydrogel modified electrode (black) and TBO/DNA hydrogel modified electrode (red) upon the addition of NADH in PBS buffer (50 mM phosphate, 100 mM NaCl, pH 7.5). The inset shows a representative i−t curve with the applied potential at 0 V vs SCE.

Figure 3. (A) Scheme describing the mediated bioelectrocatalytic oxidation of glucose by NAD-dependent glucose dehydrogenase (GDH) within the TBO/DNA modified electrode. (B) Amperometric response of the modified electrode upon the addition of glucose in PBS buffer (50 mM phosphate, 100 mM NaCl, pH 7.5). Error bars represent 1 standard deviation (n = 3). The inset shows a representative i−t curve with an applied potential of 0 V vs SCE.

TBO/DNA-modified electrode was fabricated as described above for the TBO/DNA electrode, except that GDH was added to the DNA hydrogel solution before casting on the electrode surface. The amperometric response of the modified electrode upon the addition of glucose was investigated at an applied potential of 0 V vs SCE (Figure 3B). By assuming Michaelis−Menten kinetics, an apparent Km value of 5.1 ± 0.5 mM and an associated Jmax value of 8.3 ± 0.4 μA/cm2 were determined. These values are also comparable to those produced by a TBO/PEI modified electrode as studied previously,18 which shows that the DNA based redox hydrogel has electrochemical properties similar to those of a polymerbased redox hydrogel. The stability was investigated as well and is shown in Figure S3 in the Supporting Information. DNA Modification through Electrostatic Interaction. We have demonstrated that it is possible to prepare DNA redox hydrogels by intercalating a small redox molecule within the DNA double helix. The newly formed material can be used to immobilize NAD-dependent GDH and mediate the bioelec-

electrode at low potential (0 V vs SCE) is due to the mediation of intercalated TBO. We next investigate the kinetic of NADH oxidation mediated by intercalated TBO by an amperometric method. Although the NADH oxidation can be triggered at about −0.2 V vs SCE, indicated by the CV experiments above, we used an applied potential of 0 V vs SCE to better monitor the catalytic current. By fitting of the data to the Michaelis−Menten model, an apparent Km value of 2.4 ± 0.2 mM and a Jmax value of 82.4 ± 3.3 μA/cm2 were obtained for the TBO/DNA/TP electrode. Consistent with the above CV data, these results clearly demonstrate the capability of TBO/DNA to efficiently oxidize NADH at a low potential (0 V vs SCE), while there is no significant NADH oxidation at a DNA modified Toray carbon paper electrode in the absence of TBO at this low potential, as shown in Figure 2B. The catalytic current for NADH oxidation by the TBO/DNA hydrogel is comparable to that achieved from TBO-modified PEI on a graphite electrode.18 Further2605

DOI: 10.1021/acscatal.5b02699 ACS Catal. 2016, 6, 2603−2607

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ACS Catalysis trocatalysis of this enzyme to the electrode surface. However, this strategy is only applicable for aromatic redox mediators capable of intercalating into DNA with high affinity. In addition, the mediator is buried inside the DNA double helix; thus, the use of this material to mediate bioelectrocatalysis might be limited to enzymes with diffusional cofactors. Although the electrochemical properties of the TBO mediator can be tuned by modifying its structure, for example the 3amine group, it is still necessary to utilize an alternative strategy for immobilizing redox mediators for enzymes with associated and/or deeply buried cofactors. Functionalization of DNA by tetraalkylammonium salts through electrostatic interaction with the phosphate backbone of DNA has been routinely used in applications that require more hydrophobic DNA, such as gene delivery.21 This is achieved by ion exchange between a tetraalkylammonium ion and a metal ion present in DNA. Recently, this strategy was used to attach a pyrene functional group to DNA for designing a light-harvesting system.22 We postulate that DNA can also be modified with redox-active tetraalkylammonium salts in the same fashion for MET. The redox centers of this functionalized DNA are likely oriented facing outward from the DNA double helix, thus providing an opportunity for improved interactions with enzymes in comparison to intercalated probes and a chemical environment that would be more like a polymer backbone with redox moieties bound on side chains off the backbone polymer. As a proof of concept, our initial experiment was carried out on DNA isolated from salmon testes (stDNA) to examine if the functionalized DNA is able to communicate with enzymes on the electrode surface. Ferrocene, a common redox mediator for enzymatic bioelectrocatalysis, was chosen for DNA modification (Figure 4A). Ferrocene-containing tetraalkylammonium complexed with DNA and RNA has been used to electrochemically deliver DNA or siRNA through cell membranes by controlling the redox states of the ferrocene moiety.23,24 In our study, we first prepared a tetraalkylammonium-containing ferrocene (TEAF) from 6-bromohexylferrocene and triethylamine, following a literature procedure.25 After aqueous solutions of TEAF and stDNA were mixed, the stDNA-TEAF salt was precipitated and collected by centrifugation. The modified DNA was washed five times with water to remove excess TEAF and resuspended in an appropriate amount of ethanol to obtain a concentration of 10 mg/mL. The capability of TEAF-modified stDNA (TEAF/stDNA) to undergo MET with glucose oxidase (GOx) was then investigated (Figure 4B). For this purpose, TEAF/stDNA was coimmobilized with GOx in a tetrabutylammonium bromide modified Nafion (TBAB/ Nafion) film onto the surface of a glassy-carbon electrode (GCE). The bioelectrocatalysis of GOx was studied by cyclic voltammetry (Figure 4C). The increase in the anodic current in the presence of glucose clearly indicates that TEAF/DNA is able to undergo MET from the active site of GOx to the electrode. Therefore, unlike the redox probe intercalated DNA, functionalization of DNA with redox mediators facing outward from the double helix allows good electrical communication with cofactor-bound enzymes, such as GOx. We then investigated the possibility of incorporating TEAF into a GOx-encapsulated DNA hydrogel for MET. In this case, the mediator density on the electrode surface would be much higher than that of the previous design, in which TEAF/stDNA was coimmobilized with GOx in a TBAB/Nafion polymer. Thus, we expected to observe enhanced bioelectrocatalysis. The

Figure 4. (A) Modification of salmon testes DNA (stDNA) with ferrocene-containing tetraalkylammonium salt (TEAF). (B) Mediated bioelectrocatalysis oxidation of glucose by glucose oxidase enzyme on the TEAF/DNA modified electode. (C) Cyclic voltammograms of the glassy-carbon electrodes modified with TEAF/stDNA and GOx coimmobilized in a TBAB/Nafion film in the absence (black line) and presence (red line) of glucose in PBS buffer (50 mM phosphate, 100 mM NaCl, pH 7.5). The scan rate was 10 mV/s.

Toray carbon paper electrode was modified with the GOxencapsulated DNA hydrogel as described in our previous study.9 After that, the electrode was immersed into an aqueous solution of TEAF for 30 min for the salt exchange process. The ferrocene-containing tetraalkylammonium ion will replace sodium ion as a counterion for phosphate groups of DNA. The electrode was then incubated with water for 30 min to remove weakly adsorbed TEAF on the film. The electrochemical behavior of the modified electrode was first studied by cyclic voltammetry in the presence and absence of glucose substrate (Figure 5). Initial evaluation of the bioelectrode demonstrates the mediated bioelectrocatalytic oxidation of glucose by the GOx-encapsulated TEAF/DNA hydrogel. As expected, the catalytic current achieved for this electrode is higher than that produced by the TEAF/DNA/GOx/TBAB/ Nafion-modified electrode described above. The amperometric response of the modified electrode following the addition of glucose is shown in Figure 5B, indicating a maximum current density (Jmax) of 60 μA/cm2 within 20 mM glucose.

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CONCLUSION In conclusion, we have presented two strategies for the noncovalent modification of DNA with redox-active molecules for the preparation of a redox DNA hydrogel. In the previous study, we introduced a self-assembled DNA hydrogel as a new biomaterial for enzyme immobilization in bioelectrocatalysisbased applications.9 Herein, we have demonstrated the 2606

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ACKNOWLEDGMENTS Financial support from the Air Force Office of Scientific Research is greatly acknowledged.

Figure 5. (A) Cyclic voltammograms of the Toray paper electrodes modified with GOx-encapsulated TEAF/DNA hydrogel in the absence (black line) and presence (red line) of glucose in PBS buffer (50 mM phosphate, 100 mM NaCl, pH 7.5). (B) Amperometric response of the modified electrode upon the addition of glucose. Error bars represent 1 standard deviation (n = 3). The applied potential was 0.25 V vs SCE.

possibility of the coimmobilization of redox mediators and glucose oxidoreductase enzymes in DNA hydrogels. These modified materials are capable of mediating the bioelectrocatalytic oxidation of glucose by GDH using a diffusional cofactor (NAD) or an FAD cofactor bound GOx. Due to the simplicity of the DNA modification procedures, which do not require cross-linking or polymer synthesis, our strategies can be easily generalized for other oxidoreductase enzymes and mediators and allow for the immobilization of high loadings of oxidoreductase enzymes without decreases in activity due to the addition of cross-linking agents. With its unique properties, DNA is an emerging biomaterial that has attracted significant attention in recent years. Our study contributes to advancing the use of this material to improve enzymatic bioelectrocatalysis for biosensor and biofuel cell applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.5b02699. Experimental procedures and additional data, as well as a figure detailing the electrochemical stability of the system (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail for S.D.M.: [email protected]. Notes

The authors declare no competing financial interest. 2607

DOI: 10.1021/acscatal.5b02699 ACS Catal. 2016, 6, 2603−2607