Article pubs.acs.org/cm
Redox Interfaces for Electrochemically Controlled Protein−Surface Interactions: Bioseparations and Heterogeneous Enzyme Catalysis Xiao Su,† Jonas Hübner,†,∥ Monique J. Kauke,† Luiza Dalbosco,† Jonathan Thomas,† Christopher C. Gonzalez,† Eric Zhu,† Matthias Franzreb,‡ Timothy F. Jamison,§ and T. Alan Hatton*,† †
Department of Chemical Engineering and §Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States ‡ Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces (IFG), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany S Supporting Information *
ABSTRACT: Redox-active materials are an attractive platform for engineering specific interactions with charged species by electrochemical control. We present nanostructured redoxelectrodes, functionalized with poly(vinyl)ferrocene embedded in a carbon nanotube matrix, for modulating the adsorption and release of proteins through electrochemical potential swings. The affinity of the interface toward proteins increased dramatically following oxidation of the ferrocenes, and, due to the Faradaic nature of the organometallic centers, the electrodes were maintained at sufficiently low overpotentials to ensure the preservation of both protein structure and catalytic activity. Our system was selective for various proteins based on size and charge distribution, and exhibited fast kinetics (200 mg/g) under moderate overpotentials (+0.4 V vs Ag/AgCl), as well as remarkable stability for binding under ferrocene oxidation conditions. The preservation of bioactivity and protein structure at the interface indicates the potential for these redoxmediated surfaces to be used as heterogeneous supports for enzyme catalysis. This work draws on the molecular selectivity of ferrocene-functionalized materials toward organic anion groups, and demonstrates that these smart redox-active materials can be used for modulation of the macroscopic affinity of surfaces for charged biomacromolecules to enhance processes such as bioseparations, electrochemically controlled protein purification, biocatalysis, and electrochemically mediated drug release.
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INTRODUCTION Proteins are amphiphilic biomacromolecules of great importance for biotechnology, therapeutics, and genetic engineering.1 The interaction of proteins with advanced material interfaces has been studied extensively in the context of self-assembly,2−5 bioadsorption, and permeation,6 with explored nanostructures ranging from organic and inorganic materials to composites.4,7,8 More recently, protein interactions with organic conducting polymers and under electrical stimuli have received increased attention for applications as varied as tissue engineering and biosensing.4,9 Stimuli-responsive interfaces represent a key direction in the development of nanostructured and robust materials for biological applications. Here, we explore chemically functionalized, nanoporous redox-active electrodes as a platform for modulation of the reversible electrosorption and release of proteins in bioseparations, and to serve as highly efficient, nondestructive catalyst immobilization supports. Studies of protein interactions with charged interfaces, both from an applied and a fundamental perspective, are of crucial importance in the design of more efficient electrochemically mediated systems for biological applications.9−12 To date, most investigations of electrochemical-type systems have been © 2017 American Chemical Society
limited to conducting electrodes that are not electroactive. Faradaic processes at the electrode interface, however, offer advantages over these earlier systems in that they allow for higher charge storage and the possibility of achieving molecular selectivity associated with a redox reaction, relying solely on modulation by electrochemical potential.13,14 Heterogeneous redox species have been used for electrocatalysis,15−17 analyte recognition,18,19 pseudocapacitive charge storage,20 and a range of other applications.21,22 Recently, we demonstrated that ferrocene, when oxidized to ferrocenium, has the ability to bind carboxylates reversibly from an over 30-fold excess of competing anions.23 This remarkable molecular selectivity was shown to rely on redox-activated hydrogen bonding of the organic functional groups with the cyclopentadienyl moiety.23 Proteins have a predominance of such carboxylic acid groups in their amino acid building blocks, and at the molecular level it is their distribution over the protein surfaces, and the resulting charge distribution, that dictates many of the Received: April 26, 2017 Revised: June 13, 2017 Published: June 13, 2017 5702
DOI: 10.1021/acs.chemmater.7b01699 Chem. Mater. 2017, 29, 5702−5712
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Chemistry of Materials
Figure 1. (a) Electrode preparation method for dip-coated PVF-CNT. (b) and (c) HR-SEM of dip-coated PVF/CNT electrode surface on carbon fibers at various magnifications. (d) Cathodic shift in oxidation peak from cyclic voltammetry at 0.05 V/s of PVF-CNT in 50 mM NaClO4 buffer with and without BSA.
physicochemical properties of these biomacromolecules.10 Thus, it is anticipated that binding of these biomacromolecules with redox-active interfaces can also be modulated through changes in the potential applied to the electrodes; studies on these interactions would provide a natural and valuable extension of the fundamental understanding and practical applications of redox-active materials for the processing of a range of charged targets, through small organic ions to macromolecular species. Protein separation has been a primary challenge in biotechnology for many decades,1 and with the advances of both functional materials and increasing demands in selectivity, there is a strong incentive to seek alternative concepts for their purification.24,25 Electrochemically based separations can have significant advantages over traditional ion-exchange methods26,27 as they have dramatically faster kinetics for adsorption of charged species,28 reusability by reversible adsorption, and, most importantly, do not need chemical regenerants or changes in pH to release the adsorbed proteins, which often account for much of the chemical costs of separation.29,30 All these advantages also lead to modularity and ease of scale-up. Conventional electrosorption-based methods are limited in their ion adsorption capacity, however, which is often quite low (in the 10 mg/g range at most for carbon-based electrodes)28 and, most importantly, when utilized for bioseparations, can affect the integrity of the proteins based on the degree of overpotential applied.31−34 The structure of proteins is determined by a delicate folding process driven by a range of attractive forces,35,36 and direct electrical currents associated with high voltages can have damaging effects on their chemical structure and bioactivity.32,37 In contrast, the redox-based materials proposed in the current work offer a solution to circumvent both these challengesthrough their high pseudocapacitance and charge storage properties, we can increase the ion capacity of our systems beyond that of
capacitive-type electrosorption processes, and, most importantly, we can maintain overpotentials at the low to moderate redox potentials of the immobilized Faradaic species. Furthermore, if the selectivity of the redox surface is enhanced to a sufficient extent for a native protein, it may obviate the need for specific affinity tags as used in some processes, and thereby eliminate the need for post-translational modifications.38 The preservation of protein integrity at the interface of our functionalized surfaces without degradation of their structure represents a significant step for enzymatic catalysis. Heterogeneous enzyme catalysis is important for bioprocessing and the chemical industry,39,40 as it allows for ready recovery and reuse of the catalysts, applications in fixed-bed reactor configurations, and optimal performance in nonaqueous media. A variety of porous materials have been investigated as potential immobilization substrates,41,42 with attachment of the enzymes by covalent methods that can affect the protein structure and decrease its activity, or with confinement of the protein within the pores of the substrate, where they are often susceptible to leaching.39 Electrostatics combined with surface affinity, however, can provide a strong immobilization of the proteins without actually forming damaging chemical bonds. When the supporting surface is maintained at a moderate potential, high enough to oxidize the redox-charged sites but low enough to prevent peptide degradation, we can achieve a heterogeneous surface that has minimal effect on enzyme activity, and due to sorption reversibility by electrical potential swings, may allow for the possible advantages of multiple enzymatic sites for cascade reactions43 or boomerang catalysis.44 In this work, we investigate the use of nanostructured ferrocene-functionalized redox electrodes for electrochemical modulation of interfacial interactions with proteins, and test our redox-based systems as the basis for reversible, potentialmodulated electrosorption processes and immobilized enzyme 5703
DOI: 10.1021/acs.chemmater.7b01699 Chem. Mater. 2017, 29, 5702−5712
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Chemistry of Materials
Figure 2. (a) α-Chymotrypsin (α-CHY) uptake (mg proteins/g adsorbent) at pH = 11 (50 mM carbonate), desorption at pH = 7. (b) Bovine serum albumin (BSA) adsorbed at pH = 11 (50 mM phosphate) and desorbed at pH = 7 (50 mM phosphate). (c) Effect of solution pH on α-CHY adsorption. (d) Adsorption capacity of PVF-CNT redox-electrode toward various proteins at pH = 7 (LYS: lysozyme; MYO: myoglobin; HRP: horseradish peroxidase). (e) Charge electrostatic mapping in various proteins at pH = 7 by accounting for protons, 50 mM ionic strength calculated based on an adaptive Poisson−Boltzmann solver (see details in SI).
formal potential of ferrocene, indicating a favorable binding interaction between the redox species and the proteins in solution (Figure 1d), similar to the potential shift seen in smallmolecule studies with carboxylates.23 In the case of proteins, however, broader voltammograms were obtained due to the heterogeneous nature of the interactions and greater mass transfer limitations than occur in small molecule sensing. Bovine serum albumin (BSA) and alpha-chymotrypsin (αCHY) were chosen as the model proteins for the proof-ofconcept electro-swing adsorption studies. In the first set of electrosorption tests, adsorption of BSA was performed at pH = 7, 50 mM phosphate buffer, and of α-CHY at pH = 11, 50 mM carbonate, to make sure that all proteins were significantly negatively charged (zeta potential of −25.55 mV for BSA and −32.44 mV for α-CHY). To reach equilibrium uptake capacities, the electrosorption was carried out at a constant current of +100 μA (∼1 mA/cm2 current density) to the PVF/ CNT electrode for 10 min. For recovery of the proteins, the loaded PVF/CNT electrode was immersed in a clean buffer solution at the same pH and ionic strength as used in the adsorption step, and a current of −100 μA was applied to drive the reduction of the ferrocenes and release the adsorbed proteins. Between the adsorption and desorption experiments, the platinum counter-electrode was switched and cleaned extensively to ensure only the effect of the PVF-CNT electrode was observed. The electrical potential of the system stabilized at
catalysis, showing that the noncovalent chemical interactions preserve bioactivity in both the supported and released states. We explore a range of proteins with sizes from ∼10 to ∼70 kDa, and include a series of enzymes to be tested for catalytic activity when immobilized. In addition to the aforementioned applications, the system shows promise also as a platform for sensing and even for potential-controlled therapeutic release.
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RESULTS AND DISCUSSION Redox Electrodes. The redox electrodes were prepared by dip-coating of conductive carbon fibers (Electrochem Inc.) in a 1:1 suspension of poly(vinyl)ferrocene (PVF) and carbon nanotubes (CNT);23 the carbon fibers provided a stable support for the functional material (Figure 1a; for details see Experimental Section). The PVF/CNT coating formed a porous conductive layer around the carbon fibers (Figure 1b and 1c) with nanoscale pores. Cyclic voltammetry at 50 mV/s clearly showed a linear dependence of charge on the number of dip layers coating the electrode. Three layers were found to be optimal both for film morphology and for maximization of protein adsorption per adsorbent massan increase in protein uptake was not observed with additional layers due to reduced accessibility of the protein to the inner redox sites (see SI). In the presence of proteins (e.g., at 1 mg/mL BSA and 50 mM phosphate buffer at pH = 7), we observed a cathodic shift in the 5704
DOI: 10.1021/acs.chemmater.7b01699 Chem. Mater. 2017, 29, 5702−5712
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themselves could act as the counterions to bind with the redoxelectrodes during oxidation. Multiple Proteins. A range of proteins with various isoelectric points, sizes, and charge distribution were tested to investigate the generality of our system, ranging from small proteins at ∼14 kDa to larger macromolecules at ∼66 kDa, and with a pI range from 4 to 11 (Table 1). As shown in Figure 2d,
between 0.3 and 0.4 V during oxidation, which corresponded to the peak oxidation of the ferrocene−ferrocenium couple, and reflected the Faradaic nature of the system. The protein uptake observed with chronoamperometry (+0.4 V) was similar to that noted in chronopotentiometry runs with current densities of ∼0.25 A/m2, but in this case less than 60 s was needed to reach equilibrium redox potential, demonstrating the remarkable kinetics of these redox-mediated systems. Protein Electrosorption. The uptake of proteins on the redox-electrodes was quantified via a bicinchoninic acid (BCA) assay of the supernatant. Bare conductive carbon fibers (CF) and fibers coated solely with carbon nanotubes (CNT) were used as controls. Negligible passive adsorption was observed when the various electrodes were soaked in the protein solutions for up to 8 h. There was some limited protein uptake with positively polarized CNT electrodes, which was probably due to electrical double-layer effects (Figure 2a). The oxidized PVF/CNT anode adsorbed almost 3-fold more α-CHY than did the conductive CNT electrode with similar results seen for BSA (Figure 2b). In sum, in terms of protein uptake, the redoxelectrodes showed much higher protein capacities than did the nonelectroactive conductive surfaces. Elemental analysis by ICP indicated that each 3-layer electrode contained an average of 0.11 wt % iron, which translates to 84.8 μg of PVF and 169.6 μg total adsorbent mass per electrode (PVF + CNT). Specific uptake capacities in the current work are based on protein successfully released after sorption divided by this average adsorbent mass, which in the case of α-CHY, was ∼200 mg/g. The redox-electrode mass-based specific uptake values are comparable with and often superior to those of high-capacity porous adsorbents reported in the literature, such as silica beads, porous polymers, and magnetic particles.6,46−48 Also, based on the specific geometric surface area, the equilibrium adsorption capacities at pH = 7 for the proteins were 18.7 μg/ cm2 for α-CHY and 13.4 μg/cm2 for BSA, significantly higher than those observed with various polymeric and inorganic materials.6,49,50 Most importantly, in addition to the high capacities, the interaction of the proteins with the redoxelectrodes was shown to be tunable by electrochemical potential due to the redox-reversibility of the ferrocene units. There is a significant effect of pH on the protein charge distribution, which in turn is known to affect adsorption performanceas expected, lower adsorption was observed at lower solution pH owing to the more positive protein surface charge (Figure 2c). On the other hand, the pH of the release buffer did not play a major role in desorption (whether at pH = 7 or 11), as ∼80% of the protein was able to be desorbed at the same pH at which the adsorption step was carried out. Moreover, the adsorption capacity was found not to be affected significantly by the ionic strength of the phosphate solution (0, 25, 50, and 100 mM), which was remarkable as polymeric charged and zwitterionic systems suffer from lower capacity at low ionic strengths.50 This independence of adsorption capacity on ionic strength can be attributed to the more ion-selective nature of the receptor ferrocene units, which previously have been shown to present a 1:1 stoichiometry toward carboxylates, and thus are primarily selective toward the anionic groups on the proteins over any competing electrolyte tested. For example, BSA adsorption was significant (>200 mg/g) under 50 mM of phosphate, carbonate, or perchlorate as competing electrolyte (Figures S8 and S9). In fact, it was noted that adsorption capacity for the various proteins was unchanged even when no competing salt was used, as the proteins
Table 1. Size and Charge Properties of Proteins Tested in This Work
protein name
approximate size (Da)a
isoelectric point (PI)a
lysozyme (LYS) myoglobin (MYO) horseradish peroxidase (HRP) ribonuclease-A (R-A) α-chymotrypsin (α-CHY) bovine serum albumin (BSA)
14 000 16 900 44 000 14 700 25 000 66 500
11.3 7.2 7.2 7.8 8.6 4.9
internal dipole moment (Debye)b 216 239 188 819 659 579
a
Values obtained from common literature sources; isoelectric points are subject to significant deviations depending on buffer conditions. b Dipole moments were calculated from the Protein Dipole Moments Server (PDMS) based on each protein’s PDB file, taking only the peptide sequence.45
under chronopotentiometry the redox-mediated interaction with the proteins depended primarily on their isoelectric points in the more extreme casese.g., lysozyme (pI ∼ 11), is significantly positively charged and thus has significantly lower uptake (200 mg/g). For proteins with closer isoelectric points, possible differences in uptake can be attributed to effects such as hydrophobic or hydrophilic interactions, and protein size and charge distribution over the protein surface. The relatively higher uptake capacity for smaller proteins such as myoglobin and ribonuclease (size ∼16.9 and 14.7 kDa) when compared to BSA (∼66 kDa), which has a much lower pI, could be due to their more concentrated surface charge distributions and better geometrical stacking on the surfaces of the porous electrodes.51 Extensive spectroscopic studies have shown that charge, shape, hydrophobic properties, and peptide chemical structure play a crucial role in these surface stacking interactions.51 The spatial distribution of the protein charges on a given protein was determined with a Poisson−Boltzmann solver (APBS and PDB 2PQR) that accounts for all side-chain pKas using PROPKA. Simulations conducted for the different proteins at an ionic strength of 50 mM yielded the charge distributions shown to scale in Figure 2e, with negative charges in red and positive charges in blue. On the basis of these results, we can infer that the relatively high adsorption of ribonuclease and α-CHY could be due in part to their internal dipole moment, with their anionic and cationic domains being more segregated over the particle surfaces than is the case for the other proteins (Figure 2e), and thus providing concentrated pockets of interaction with the uniformly distributed oxidized ferrocenium groups on the surface. The internal dipole moment for each protein was estimated (Table 1) with the Weizmann Institute of Science dipole moment server based on the protein PDB files.45 As can be seen both in the table and in Figure 2e, the charged groups (positive and negative) of both chymotrypsin and ribonuclease 5705
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the system due to the surface Faradaic process, and indicating that these types of pseudocapacitive systems can be a promising platform for fast-throughput separations and analysis. From the single-electrode adsorption studies, BSA and MYO were shown to have similar uptakes, and yet these proteins have significantly different properties (Table 1). Most evidently, BSA is the much larger of the two proteins and thus the binary mixture separation between the two species is an interesting case to study. A size-exclusion column in fast protein liquidchromatography (FPLC) was used to quantify the relative protein concentrations in solution following the desorption step. Significantly more myoglobin than BSA was desorbed (Figure 3b), indicating that in the case of proteins of different size, the electrode has preferential selectivity toward the smaller macromolecules even when pI effects are discounted. Upon integration of the FPLC peaks and determination of concentrations from standard calibration curves, the separation factor between myoglobin and BSA for an equimolar adsorption feed mixture was found to be 3.9 (see Experimental Section). Electrode Reusability and Protein Binding Stability. The stability of the electrodes under cycling was tested and they were found to be effective for at least five consecutive adsorption and desorption cycles (Figure 4a and 4b). A high adsorption capacity (∼200 mg/g) was maintained, with both electrochemical stability and regenerability for bioseparations. Adsorption isotherms (Figure 4c) determined by chronopotentiometry with the PVF/CNT electrode at various protein concentrations (+100 μA, equilibrium potential ∼0.4 V) showed a significantly higher adsorption capacity for α-CHY at low to moderate concentrations than for BSA, with both proteins displaying significant uptake across a range of concentrations down to 100 μg/mL. These isotherms are consistent with the expected adsorption behavior on porous surfaces, where the relative protein amounts adsorbed increase proportionally with protein solution concentration,51 and the changing adsorption amounts between various proteins at different concentrations depend on a series of factors including molecular packing, size, charge, chemical affinity, and hydrophobicity. The immobilization efficiency of the protein on the electrodes was studied carefully with assay of the liquid phase by BCA and characterization of the surface chemistry by XPS following the adsorption and desorption processes (Figure 5a). The degree of “leaching” of the enzyme was estimated at the end of an adsorption−desorption cycle (adsorption at pH = 11 and desorption at pH = 7 of α-CHY). In the first step, the protein was adsorbed from a 1 mg/mL pH = 11 solution. Following oxidation at an applied potential of +0.4 V, the electrode was transferred to a fresh 50 mM solution at pH = 7. Under oxidation, the surface-bound α-CHY was highly stable, with less than 1 μg of detectable enzyme present in solution when assayed by BCA, indicating that no protein desorbed owing to pH effects, and that the ferrocenium−anionic complexes were highly stable. After the potential was switched off after 10 min of oxidation, at pH = 7, ∼15 μg of protein desorbed through possibly the self-discharge of poly(vinyl)ferrocenium, while upon application of a forced 0 or negative potential at −0.2 V (again for 10 min), all the remaining protein desorbed (Figure 5b). XPS analysis of the fiber surfaces indicated that most of the protein adsorbed was effectively desorbed (>95%) following electrochemical reduction (Figure 5c), as can be seen by the ratio of the N 1s and F 1s peak areas.
are more polarized than are those for the other proteins, as reflected in their higher dipole moments (819 and 659 D, respectively). The higher uptake of these two proteins relative to the others studied suggests a preferential affinity of the redox-interfaces for more polarized proteins. Further studies are necessary to unravel in detail the actual mechanism, as the interaction of charged proteins with charged interfaces can be extremely complex.10 Indeed, the region of the protein that is contact with the surface material does not need to bear the same sign as the entire protein itself, and the adsorption process is hypothesized, to a large extent, to be dictated by the local electrostatic environment. Under this scenario, the protein uptake capacity would not be expected to correlate directly with the isoelectric point of a protein, which reflects the overall protein charge. In fact, surface charges and charge distribution, based on the so-called charge regulation mechanism,10,52 can play a crucial role in determining preferential affinity of proteins of similar size with an interface, especially one that is chemically selective and redox-functionalized. The selectivity of the redox-electrode for different proteins was investigated by electrosorption and release from a binary mixture (+0.4 V adsorption, and 0 V desorption) in 50 mM phosphate (1 mg/mL BSA + 1 mg/mL MYO, pH = 7 in water, 50 mM phosphate buffer). Figure 3a shows that for a potential swing of 0.4 V, a time of 60 s was more than sufficient for the current to reach equilibrium during both charging and discharge, highlighting the fast electron-transfer kinetics of
Figure 3. Selective separation in binary protein mixture (MYO/BSA, 1 mg/mL each in pH = 7, 50 mM). (a) Chronoamperometry voltage and current profiles with 80 s, +0.4 V adsorption, fixed 0 V for desorption. (b) FPLC profiles for BSA and MYO of adsorption stock (1 mg/mL BSA, MYO each) and desorption supernatant (after electrosorption and release using pVF/CNT) analyzed through a size exclusion column, normalized based on MYO peak. 5706
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Enzyme Kinetics. Enzymatic assays are considered to be the prime method for testing preservation of the chemical and conformational integrity of proteins.31 The enzymatic activity of lysozyme, HRP, and α-CHY was thus evaluated to probe the effect of surface charge and other properties on the structure and biological activity of the proteins. Differences in activity under homogeneous solution conditions between the original, untreated enzyme and the released enzyme after an adsorption/ desorption cycle were used to quantify any degradation in enzyme properties. As shown in Figure 7a, over 80% of enzymatic activity was retained for all three proteins upon release, indicating that the electrochemical process, in general, did not have an impact on protein stability and that the chemical structure remained intact, with retention of >90% of the original catalytic activity by HRP and lysozyme. The minor losses in activity, in the case of α-CHY, were found not to be dependent on the duration of the oxidation or reduction steps (under conditions of both constant current at 1 mA/cm2 and constant potential at
