Mechanisms of Enhanced Hemoglobin Electroactivity on Carbon

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Mechanisms of Enhanced Hemoglobin Electroactivity on Carbon Electrodes upon Exposure to a Water-miscible Primary Alcohol Justin Tom, Philip J Jakubec, and Heather A. Andreas Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00117 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018

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Analytical Chemistry

Mechanisms of Enhanced Hemoglobin Electroactivity on Carbon Electrodes upon Exposure to a Water-miscible Primary Alcohol

Justin Tom, Philip J. Jakubec and Heather A. Andreas* Department of Chemistry, Dalhousie University, Halifax, NS, Canada B3H 4R2 *Corresponding author, email: [email protected]; Phone: 902-494-4505

Abstract Exposing a carbon electrode to hemoglobin (Hb) and alcoholic solvents, such as methanol, ethanol or 1-propanol, drastically changes Hb electroactivity, but until this work the important underlying mechanisms were unclear. For the first time, we show that these alcohols impact Hb electroactivity via three mechanisms: modification of the carbon surface oxides on the glassy carbon (GC) electrode, Hb film formation, and structural changes to Hb. C1s X-ray photoelectron spectroscopy provided evidence for significant alcohol-induced modification of the carbon surface oxides and differential pulse voltammetry showed links between these modifications and Hb electroactivity. Spectroscopic ellipsometry showed that Hb films formed during exposure to Hb- and alcohol-containing electrolytes increased in thickness with increasing alcohol content, although film thickness played only a minor role in Hb electroactivity. Alcohol-induced structural changes in Hb are confirmed with UV-visible absorption and fluorescence data, showing that Hb denaturation also was a significant factor in increasing Hb electroactivity. Carbon surface oxide modification and Hb denaturation worked in tandem to maximally increase the Hb electroactivity in 60% methanol. While in ethanol and 1-propanol, the significant increases in Hb electroactivity caused by Hb denaturation were offset by an increase in Hb-inhibiting carbon surface oxides. Knowledge of these mechanisms shows the impact of alcohols on both Hb and carbon electrodes, allows for thoughtful design of the Hb1

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sensing system, is vital for proper analysis of Hb electroactivity in the presence of these alcohols (e.g. when used as binder solvents for immobilizing Hb into films) and provides fundamental understanding of the Hb-carbon interactions.

Introduction Electrochemical biosensors are an area of active research with applications in biotechnology and diagnostics.1 For instance, detection of hemoglobin (Hb) is important due to its role in various diseases and potential cytotoxicity.2 Electrochemical biosensors generally provide inexpensive, portable, fast and sensitive analyte quantification1,3 and, conveniently, Hb is redox-active through the iron in its heme groups.4–6 However, the direct electron transfer between an electrode and Hb is difficult since the four redox-active heme groups are located in the interior, hydrophobic regions of the protein.7,8 While Hb can be detected in solution,5,9,10 electrochemical mediators,4,11 unfolding Hb,12 or immobilizing Hb directly onto the electrode4,13 are more commonly employed to observe its electroactivity. However, we recently recognized that simply exposing Hb and carbon to methanol (MeOH), ethanol (EtOH) or 1-propanol (1-PrOH) causes a marked increase in Hb electroactivity, resulting in the best detection limit for Hb in solution with a simpler and less expensive system (submitted for publication in Sensors and Actuators B). However, to further improve Hb electroactivity, it is vital to understand the mechanism by which the alcohol modifies the system, such that future development can be based on fundamental understandings of the alcohol-carbon and alcohol-Hb interactions. This work provides the necessary knowledge linking direct Hb electroactivity to alcohol-induced changes to the Hb protein and the carbon electrode.

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Herein, we provide a detailed analysis of three possible mechanisms: (1) the alcohol-induced modifications of the carbon surface oxides, since our previous work showed that ether and carbonyl surface groups inhibit Hb electroactivity;14 (2) alcohol-induced increases in the adsorbed Hb layer thickness, possibly due to increased Hb aggregation15 and (3) protein denaturation resulting in easier access to the electroactive heme group, since MeOH, EtOH, and 1-PrOH all change the Hb structure and denaturation of the protein’s conformational framework allows the hydrophobic Hb interior to be exposed to the solvent.15,16 This paper demonstrates that carbon-oxide modification and Hb denaturation, mechanisms (1) and (3) respectively, are the dominant mechanisms by which alcohols impact Hb electroactivity.

Experimental Characterization Electrochemical measurements of Hb electroactivity were conducted by collecting differential pulse voltammograms (DPVs) between 300 and -900 mV using a 20 mV s-1 scan, 50 mV pulse height, 250 ms pulse width, -25 mV step height, and 1250 ms step time. The initial potential was held for 2 seconds. The 0.1 M phosphate buffer (PB) electrolyte (pH 7.08) was a 64.5% to 35.5% mole ratio of K2HPO4 (ACS Reagent, Sigma Aldrich, ≥98%) and KH2PO4 (Sigma Life Science, ≥99.0%) in 18.2 MΩ·cm Millipore water. For some experiments, the electrolyte contained 0.2 g L-1 Hb (Sigma Life Science, lyophilized powder, hemoglobin from bovine blood) and/or various concentrations of MeOH (Fisher Scientific, 99.9%), EtOH (anhydrous, 100%), or 1-PrOH (Caledon Laboratory Chemicals, 99.5%). Electrolytes containing alcohol were used immediately after preparation to mitigate alcohol evaporation and ensure accurate alcohol concentrations. The glassy carbon working electrode (GC, CHI 104, 3.0 mm 3



carbon diameter sealed in Kel-F) was connected to a Bio-Logic VMP3 multipotentiostat along with a platinum mesh counter electrode and a Hg/Hg2SO4 reference electrode (saturated K2SO4, measured as 0.692 V versus the standard hydrogen electrode, used to avoid the allosteric chloride effect on Hb17). The three electrodes were examined in an all-glass one-compartment electrochemical cell. All experiments were performed in triplicate and graphical error bars indicate one standard deviation. Prior to testing, the electrochemical cell, Pt and GC electrodes were cleaned by wiping with a Kimwipe, submersed in 1 M NaOH for five minutes to remove residual Hb, followed by rinsing with 18.2 MΩ·cm water. Subsequently, the GC was polished on nylon pads (BASi PK-4 MF-2060 polishing kit) using 3 and 1 µm diamond polishes and rinsed with copious amounts of 18.2 MΩ·cm water between polishing steps. X-Ray photoelectron spectroscopy (XPS) and spectroscopic ellipsometry samples were prepared on GC plates (SPI Supplies, 2.5 mm thick). The GC plates were cleaned by wiping with a Kimwipe, washing with distilled water, followed by a 10 minute soak in 1 M NaOH; this procedure was repeated total three times. After the cleaning procedure, the GC plates were ultrasonicated in 18.2 MΩ·cm water for 10 minutes, polished to a mirror-like finish using 1 micron diamond polish, and again ultrasonicated in 18.2 MΩ·cm water for 10 minutes followed by a final rinse with 18.2 MΩ·cm water to remove residual diamond polish. C1s XPS data were collected at room temperature and at a pressure of 1 x 10-9 Torr using a Thermo VG Scientific Multilab ESCA 2000 spectrometer equipped with a Mg Kα X-ray source (1253.6 eV, 0.6 mm diameter spot size) and a CLAM4 MCD electron energy analyzer with a pass energy of 30 eV. A Shirley background and Gaussian-Lorentzian functions were used when fitting XPS data with the CasaXPS version 2.3.17PR1.1 software. The counts per second of all spectra were normalized relative to the graphitic peak; when necessary the graphitic peak was 4


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shifted to 284.3 eV. C1s XPS peak assignments were based on known literature values: C=C at 284.3 eV,18–21 C-O at 285.5 eV,20–24 C=O from 286.8 to 287.1 eV,19,20,23,25–27 COOR at 288.8 eV,19,20,25–28 π-π* shakeup from 290.3 to 290.4 eV,18,25,28,29 and plasmon processes at 292.6 eV.25,29 Error estimates representing one standard deviation were provided from Monte Carlo simulations using CasaXPS software. Spectroscopic ellipsometry data were collected for wavelengths from 210 to 1000 nm using a M-2000F J.A. Woollam spectroscopic ellipsometer in reflectance mode with a 65° angle of incidence relative to the vertical normal for all measurements. To ensure reproducibility, scans were performed on nine different areas of each sample. CompleteEASE version 4.24 software was used for all ellipsometry data processing. A two-layer model was used where a B-spline model described the GC substrate and a Cauchy model was used for Hb. The transparency assumption for the Cauchy model was tested and described in the supporting information. After surface modification (described in Experimental Surface Modification Section below), the samples were air-dried for approximately one hour prior to taking measurements. Ultraviolet-Visible (UV-Vis) absorption spectroscopy was conducted using a Cary 5000 spectrophotometer (Varian, Inc.) in double beam split mode scanning between 200 - 800 nm at a rate of 600 nm min-1. Fluorescence was recorded on a Cary Eclipse instrument using a 280 nm excitation wavelength and emission wavelengths from 290 - 420 nm with a measurement rate of 120 nm min-1 and excitation and emission slits of 5 nm. UV-Vis and fluorescence spectra were collected for electrolytes containing 0.2 g L-1 Hb in PB and 0 to 60% by volume of alcohol (MeOH, EtOH, or 1-PrOH) in a 10 mm quartz cuvette.

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Hb-free solutions were recorded as background scans. For double-beam UV-Vis measurements, the reference compartment contained 0.1 M PB in an identical quartz cuvette.

Surface Modification Adsorbed Hb films were formed by submerging the GC in 0.1 M PB (pH 7.08) with or without MeOH, EtOH or 1-PrOH. Hb dissolved in 0.1 M PB was then pipetted into the mixture, attaining a final Hb concentration of 0.2 g L-1 and alcohol content of 0%, 30%, or 60% by volume. GC was incubated in the mixture for 30 minutes, followed by a 10 minute submersion in 18.2 MΩ·cm water and a final rinse with 18.2 MΩ·cm water to remove any excess Hb before electrochemical or ellipsometric testing. Alcohol-induced modification of the GC surface oxides was effected by incubating the GC in various concentrations of one of MeOH, EtOH, or 1-PrOH for 30 minutes. For electrochemical measurements, the GC electrode was thoroughly rinsed using 18.2 MΩ·cm water prior to experiments. For XPS analysis, GC plates were submersed in 18.2 MΩ·cm water for 10 minutes and then rinsed to avoid the crystallization of phosphates from the PB. The treated GC plate samples were kept dry in a desiccator until XPS analysis.

Results and Discussion The impact of alcohol exposure on Hb electroactivity can be clearly seen in the DPVs of Hb in various alcohol-containing PB electrolytes (Fig. 1). In the absence of alcohol, the GC electrode exhibited a small Hb cathodic peak near -0.675 V. Large Increases in Hb-reduction current are seen with alcohol contents of > 30% MeOH (Fig. 1a), ≥ 20% EtOH (Fig. 1b) or ≥ 10% 1-PrOH (Fig. 1c). At high alcohol contents, a negative shift in the Hb peak potential is 6


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seen, consistent with previous peak shifts identified as either Hb unfolding exposing the redoxactive heme30,31 or the incorporation of Hb into a film,32 suggesting the first two of the three mechanisms under study in this work. Considering protein denaturation first, unfolding of the protein may grant greater access to the buried redox-active heme groups leading to the enhanced Hb electrochemical response (proposed mechanism 1, see section below). Figure 1 shows that the onset of Hb reduction occurs at increasingly negative potentials in the order of 1-PrOH, 0.425 V < EtOH, -0.450 V < MeOH, -0.475 V at 60% alcohol concentration. This is consistent with our later observations (Mechanism 1) that 1-PrOH denatures Hb the most, increasing the ease of access to the redox-active heme groups (lowering the onset of Hb reduction potential), followed by EtOH and MeOH when comparing similar alcohol concentrations. The denaturation mechanism is consistent with the findings that glycerol, which stabilizes the protein structure, shows no increased electroactivity,33 while dimethyl sulfoxide denatures Hb34 and evidences increased Hb electroactivity.32 Likewise, MeOH,15,16,35 EtOH,15,16,35 and 1-PrOH15,16 all denature Hb and Fig. 1 shows increasing Hb electroactivity upon exposure to these alcohols. Additionally, the denaturation of the Hb may lead to a decrease in Hb solubility, which may result in precipitation of some Hb as a surface film (proposed mechanism 2, see section below). Enhanced Hb adsorption leads to an effective Hb-concentration increase at the electrode surface, improving Hb electroactivity. The negative potential shift at high alcohol concentrations (see Fig. 1 insets, peak potentials based on three replicates) would then be a result of slow electron transfer through the film. Our previous work also showed that significant changes to Hb electrochemistry can arise from changes in the carbon oxides on the carbon electrode surface (proposed mechanism 3, see section below); specifically, ethers and carbonyls are known to inhibit Hb electroactivity on 7



carbon surfaces.14 While it is known that exposing a carbon material to MeOH decreases the amounts of surface oxygen,36 likely also changing the carbon-oxygen surface groups, it is unclear exactly what effect that change has on Hb electroactivity. The impact of EtOH and 1PrOH on the Hb-inhibiting surface functionalities is entirely unknown. The reduction charge (used because of peak broadening in Fig. 1a-c likely due to slow electron transfer, due to Hb rearrangement and re-orientation as it is incorporated into increasingly thick films) reveals that the maximal Hb reaction occurs upon exposure to 60% MeOH. Conversely, the electroactivity improvements are slowed or reversed at the highest concentrations of EtOH and 1-PrOH, suggesting an inhibiting mechanism (such as increased ether or carbonyl surface groups) is introduced in these highly-alcoholic electrolytes. This work examines these complex relationships between the enhancing and inhibiting mechanisms.

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Figure 1. DPVs and Hb-reduction peak potential (insets) of GC in 0.2 g L-1 Hb + 0.1 M PB electrolyte with various amounts of (a) MeOH, (b) EtOH, or (c) 1-PrOH. Hb-reduction charge as a function of alcohol concentration are shown (d) for MeOH (circles, solid line), EtOH (squares, dashed line), and 1-PrOH (triangles, dotted line), where the blank DPVs of GC in PB were used for background subtraction. These data were also submitted to Sensors and Actuators B, Dec 2017 (Tom, Jakubec, Andreas).

Alcohol-induced Hb Structural Changes and Denaturation (Proposed Mechanism 1) The redox-active heme groups are buried within the hydrophobic regions of Hb, which are difficult to access electrochemically; therefore, alcohol-induced changes to the Hb conformation15,16,37 may lead to the increased Hb-reduction seen in Fig. 1. MeOH, EtOH, and 1-PrOH are all known to change the Hb conformation,15 and Hb may have specific binding sites

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for MeOH and EtOH.35 Jun et al. previously used circular dichroism to show significant changes in Hb structure (specifically decreased alpha helix content) upon exposure to alcohol.15 The structure of the redox-active heme groups can be probed through UV-Vis absorption spectroscopy, specifically using the Soret peak, generally found between 405 - 430 nm for Hb.38,39 A red-shifted Soret peak indicates stronger interactions between the Fe and an axial ligand (i.e. histidine, His) within a heme group, while blue shifts indicate decreased Fe-His interactions and greater steric hindrance.39 UV-Vis absorption data (Fig. 2a, derived from spectra provided in Fig. S.5) show an initial small blue-shift when adding small amounts of alcohol (10% MeOH or EtOH), and red shifts for solutions containing 30 – 40% MeOH, 10 – 40% EtOH or 0 – 20% 1-PrOH, with the Soret peak shifting blue again at higher alcohol contents. These results indicate that alcohols have a significant impact on the Hb structure: low concentrations of MeOH and EtOH cause a slight relaxing of the Fe-His interaction (no similar relaxation is seen for 1-PrOH); low 1-PrOH contents and mid-range MeOH and EtOH contents cause tightening of the interaction; and, for high alcohol concentrations, the Fe-His interaction relaxes, coupled with an increase in steric hindrance.39 Clearly, these data also show that less 1-PrOH was required to initiate the changes, implying that 1-PrOH denatures Hb more than EtOH and MeOH, likely due to 1-PrOH being a less polar alcohol than EtOH and MeOH. Because of the absorption cutoff by the aliphatic alcohols near 210 nm,40 the interpretation of the peptide bond absorption (210 – 220 nm) is unreliable here. Still, previous circular dichroism studies of Hb in aqueous-alcohol solutions demonstrate a decreased alpha helix content in Hb upon alcohol exposure, suggesting a more loose protein conformation.15 The UV-Vis data (Fig. 2b) exhibit a change in absorption near 280 nm, often attributed to tryptophan (Trp) and tyrosine (Tyr) residues, where an increase in absorption suggests the 10


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residues are moving into a less polar environment,41 such as being buried in a more hydrophobic region of the protein. Trp residues are well-known to have a higher absorptivity than Tyr residues, but Tyr residues are more sensitive to changes in polarity than Trp residues;41 thus, the absorption changes near 280 nm were mainly attributed to Tyr. The absorbance increase near 280 nm between 20 – 50% MeOH, 10 – 30% EtOH, or 0 – 10% 1-PrOH (Fig. 2b) suggests that the environment near Tyr residues is becoming less polar, meaning the residues are likely moving into a more hydrophobic region of Hb. The absorption peak decreases when exposed to > 50% MeOH, > 30% EtOH, or > 10% 1-PrOH (Fig. 2b), suggesting that the polarity of the Tyr environments increased,41 which may be interpreted as increased solvent exposure relative to their previously hydrophobic surroundings and an unravelling of the protein. Consistent with the Fe-His results, these data show that 1-PrOH denatures Hb at a lower concentration than EtOH, which in turn denatures at a lower concentration than MeOH.15 (b) 1.6

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Figure 2. Soret peak wavelength (a) and Trp and Tyr peak absorbance at 280 nm (b) as a function of MeOH (circles, solid line), EtOH (squares, dashed line), and 1-PrOH (triangles, dotted line) concentrations in 0.2 g L-1 Hb solutions. Error bars indicate one standard deviation from three replicates. UV-Vis spectra are provided in the supporting information (Fig. S.5). Fluorescence was also used to track polarity changes for Trp and Tyr residues; these changes are most strongly associated with Trp since it has the highest quantum yield.42,43 A 11



polarity increase near Trp typically red-shifts the fluorescence peak42,43 and is coupled with a fluorescence intensity decrease where solvent reaching the Trp residue disperses more energy from the excited fluorescing Trp.42 However, Trp fluorescence intensity may also be influenced by Förster resonance energy transfer (FRET),44–46 where the energy of an excited chromophore (e.g. Trp) is transferred to another chromophore within 10 nm (e.g. heme group),42 resulting in a lower fluorescence intensity.44–46 Hb has a low Trp fluorescence intensity46 because the six Trp residues in Hb are each approximately 1.4 to 1.6 nm from the nearest heme.17,47 The quantum efficiency of Trp fluorescence increases ~40% if all heme groups were removed from Hb, with no shift in the peak emission wavelength.46 Consequently, it is expected that any alcoholinduced conformational changes resulting in increased Trp-heme distance will decrease the energy transfer efficiency and increase the fluorescence intensity. The fluorescence results (Fig. 3, derived from spectra provided in Fig. S.6) confirm the existence of alcohol-induced changes to the Hb structure. As with the UV-Vis results, similar protein changes occur for the three alcohols, but at different concentrations, with 1-PrOH causing significant changes at the lowest concentrations. The high errors in peak position for MeOH (Fig 3a) make assigning trends difficult, but we believe MeOH mirrors the changes seen with EtOH and this is supported by the UV-Vis data for Fe-His and Tyr. At 0 – 20% MeOH and 0 – 10% EtOH, the Trp peak red shifts (Fig. 3a), suggesting a more polar Trp environment due to the Trp residues being slightly exposed to the solvent. While the expected intensity decrease is seen for MeOH (Fig. 3b), the EtOH exhibits a peak intensity increase (Fig. 3b) which suggests that the protein denatured slightly and caused an increase in the Trp-heme distance (i.e. less FRET). At higher MeOH and EtOH contents (20 – 40% and 10 – 20%, respectively) the peak is blue shifted (Fig. 3a), suggesting the protein has contracted, pulling the Trp into a hydrophobic 12


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region, and resulting in a decreased Trp-heme distance, which has resulted in more FRET and an offset of the expected intensity increase (resulting in a decrease intensity for MeOH (Fig. 3b) and a nearly constant peak intensity for EtOH (Fig. 3b). When the solvent polarity is sufficiently low (>40% MeOH, 20 – 40% EtOH and 0 – 20% 1-PrOH), the protein unravelling has caused the Trp to be more exposed to the polar solvent (red-shifted peak), but also caused an increased Trpheme distance (decreasing FRET and increasing peak intensity). Finally, for the highest concentrations of EtOH and 1-PrOH (40 – 60% EtOH, 20 – 60% 1-PrOH) the blue-shifting peak, with increasing intensity is consistent with the Trp being more exposed to the solvent and the decreasing polarity is a result of the changes (increases) in the alcohol to water ratio. (a) 346

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Figure 3. The Trp peak emission wavelength (a) and intensity (b) as a function of alcohol content for MeOH (circles, solid line), EtOH (squares, dashed line), and 1-PrOH (triangles, dotted line) with one standard deviation from three replicates. Spectra are provided in the supporting information (Fig. S.6). Taken together, the UV-Vis and fluorescence data paint a cohesive picture of the impact of alcohols on the Hb protein structure. The changes to the protein structure appear to proceed in several stages, depending on alcohol content. At low alcohol content for MeOH and EtOH, the Trp appears to be slightly exposed to solvent and the Fe-His interaction lengthens, suggesting some small loosening of the protein structure; although this small change does not result in any

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change in the environment around the Tyr residues. With 20-40% MeOH or > 10% EtOH, the protein structure contracts (tightening the Fe-His and heme-Trp interactions, and pulling Tyr and Trpresidues into hydrophobic pockets). At higher alcohol contents, some of the protein starts to unravel, the Trp residues are exposed to the solvent and move away from the heme (while Fe-His is tightening and Tyr residues continue to move into a more hydrophobic environment). At high alcohol content, the protein structure loosens (Fe-His and Trp-heme lengthen, both Trp and Tyr are more exposed to the solvent) where the Trp residues show the earliest signs of denaturation by becoming more exposed to the solvent. The spectroscopic results show significant modification to the Hb structure upon exposure to alcohols; and, a change in Hb structure is known to impact Hb electroactivity,12 though alcohol-induced changes have not been related to Hb electroactivity until this work. Reedy et al. suggested that a solvent-exposed heme could result in a negative heme reduction potential shift,31 which is in evidence in the DPV data (Fig. 1) supporting that the improved Hb electroactivity derives, at least in part, from alcohol-induced Hb structure modification. Unravelling the globin structure likely increases accessibility to the redox-active hemes, increasing the measured Hb-reduction. The degree of protein structural changes appears to correlate with significant changes in Hb electroactivity for EtOH and 1-PrOH; large changes in Hb current at > 10% EtOH and > 0% 1-PrOH correlate with large structural Hb changes evidenced near these concentrations in the UV-Vis and fluorescence spectra (Fig. 2 and 3). Therefore, unravelling of the Hb structure appears to be an important factor involved in increasing Hb electroactivity. However, protein denaturation cannot be used alone to reliably predict Hb electroactivity, since this mechanism cannot explain the plateau and decrease in Hb

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electroactivity for high EtOH and 1-PrOH concentrations, nor can it explain why MeOH, which has the least significant protein unfolding, exhibits the highest Hb reduction charge (Fig. 1d).

Alcohol-induced Changes to Adsorbed Hb Film Thickness (Proposed Mechanism 2) The negative peak potential shift and increasing peak breadth in Fig. 1 is consistent with the incorporation of Hb into a film,32 leading to an increase in Hb electroactivity since the film effectively increases the Hb concentration at the electrode surface. The formation of an Hb film is related to denaturation of the Hb by alcohols (Mechanism 1) resulting in increased aggregation15 likely enhancing adsorption. To test for an adsorbed Hb film, the GC was incubated in a Hb-containing solution (with and without alcohol), then soaked in water, rinsed, and then tested in a Hb- and alcohol-free electrolyte; the presence of a Hb-signal reveals that Hb was transferred as an adsorbed film. GC incubated in an alcohol-free, Hb-containing solution evidenced a small Hb-reduction peak (Fig. 4a-c), suggesting a small amount of Hb adsorbed during incubation even in the absence of alcohol. When an alcohol was present during incubation, larger Hb-reduction peaks were observed, suggesting more adsorbed Hb on the GC; 1-PrOH exhibited more Hb electroactivity than EtOH or MeOH, suggesting that it formed the thickest film. 0

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Figure 4. Representative DPVs of GC in 0.1 M PB (pH 7.08) (dotted) after incubation in 0.2 g L-1 Hb in PB with (a) MeOH, (b) EtOH, or (c) 1-PrOH containing 0% (dot-dash), 30% (dash) and 60% (solid) alcohol content in the incubation solution. 15



The thickness of the adsorbed Hb film was estimated using spectroscopic ellipsometry data (Table 1, derived from data in Figure S.4). The film formed by incubation in an alcoholfree Hb-containing solution has a thickness 49 Å (Table 1), which is larger than the 40 Å thickness on a gold substrate,48 but is similar to one of the known dimensions of Hb, which are 50 x 55 x 65 Å.17,47,49 The layer is likely thinner on gold due to strong binding between Au and sulfur-containing amino acid residues50,51 in Hb, such as methionine and cysteine.17,47,49 Table 1 shows that generally the adsorbed Hb films are thicker for alcohol-containing incubations, consistent with the improved Hb-electroactivity in the DPVs (Fig. 4) and supporting the theory that some Hb-electroactivity improvement is derived from the formation of Hb films. Table 1. Hb film thickness of Hb layers on GC obtained by spectroscopic ellipsometry using a Cauchy and B-spline two-layer model. Errors in the thickness were estimated based on a 90% confidence interval.* GC incubated in Thickness / Å Hb + no alcohol 49.3 ± 0.3 Hb + 30% MeOH 63 ± 2 Hb + 60% MeOH 80 ± 20 Hb + 30% EtOH 48 ± 2 Hb + 60% EtOH 66.4 ± 0.9 Hb + 30% 1-PrOH 170.3 ± 0.7 Hb + 60% 1-PrOH 101.1 ± 0.5 * Very little light reflection was detected for the 60% MeOH-formed film, despite repeated trials and realignments; the thickness estimate for this film should be interpreted cautiously. Figure 5 shows the Hb reduction charge as a function of the film thickness. All Hb films formed with co-exposure to alcohols evidenced both higher thickness and electroactivity than the film formed without alcohol, with the exception of 30% EtOH. These results confirm the presence of an electroactive adsorbed Hb film. Further, they show that the presence of an alcohol causes a thicker and more electroactive Hb film to form. A similar adsorbed Hb film

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will form in experiments which contain both Hb and alcohol in the electrolyte (Fig. 1), and these results suggest some of the enhanced Hb activity in Fig. 1 arises from the adsorbed Hb film. For the incubated films (Fig. 5), MeOH shows a clear correlation between film thickness and Hb activity; however, for EtOH and 1-PrOH the relationship is not perfect. A 30% EtOH incubation solution resulted in increased Hb electroactivity but no increase in film thickness, possibly due to protein denaturation or dehydration52,53 leading to a more densely covered surface, orientating the Hb more favorably on the electrode surface, or modifying the protein structure to facilitate electron transfer while insufficiently unravelling the protein to increase film thickness. Similarly, 60% 1-PrOH produced a thinner, more electroactive film than that formed with 30% 1-PrOH, again, likely due to protein denaturation (examined in Mechanism section above). While the film formation mechanism clearly improves Hb electroactivity, the Hbreduction charge for incubated films (Fig. 5) was much smaller than the reduction evidenced for electrolytes containing both Hb and alcohol (Fig. 1d) suggesting the adsorbed Hb film contributes only part of the Hb electroactivity, implying that other mechanisms also play a significant role. 70 Charge from Film DPV / µC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60% 1-PrOH 30% 1-PrOH

60 50

60% EtOH

40 30% EtOH

30

60% MeOH

20 30% MeOH 10 no alcohol 0 0

50

100 150 Film Thickness / nm

200

Figure 5. Background-subtracted Hb reduction charge as a function of film thickness on GC for Hb films formed by incubation in aqueous-alcohol with Hb. 17



Alcohol-induced Changes to Carbon Surface Oxides (Proposed Mechanism 3) To effect possible alcohol-induced modifications to the GC surface oxides without impacting the Hb structure or film adsorption, the GC was incubated in MeOH, EtOH, or 1-PrOH and then tested electrochemically in Hb-free and Hb-containing electrolytes. The DPVs in Hb-free electrolyte (Fig. 6a) showed an increase in cathodic current between -0.5 and -0.9 V for all three alcohols used, suggesting that the alcohols are modifying the carbon surface, likely through changes to the carbon surface oxides which are then reduced in this potential window. (a)

(b) 0

0

-1

-1

-2

I / µA

-2 I / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 36

-3

-3

-4

-4

-5

-5

-6

-6 -1


0.4

-1


0.4

Figure 6. DPVs of untreated GC electrode (dotted line) and pretreated with MeOH (solid), EtOH (long dash) or 1-PrOH (short dash) in (a) 0.1 M PB and (b) 0.1 M PB + 0.2 g L-1 Hb. Figure 6b shows a significant increase in Hb-reduction current for the MeOH-modified carbon, while both the EtOH- and 1-PrOH-modified carbons exhibit decreases. Clearly, MeOH modifies the carbon surface to enhance Hb reduction, likely by removing Hb-inhibiting carbon surface oxides, such as quinones, carbonyls, or ethers.14 Likely the MeOH-induced removal of these oxides is one reason for the increased Hb electroactivity in Fig. 1 for MeOH-containing Hb electrolytes. However, the Hb reduction for MeOH-incubated carbon (Fig. 6) is significantly smaller than that exhibited when MeOH is included directly in the electrolyte (Fig. 1), consistent with the discussion in the mechanism section that MeOH also modifies the Hb protein to 18


Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


enhance Hb reduction. The decrease in Hb-electroactivity with 1-PrOH and EtOH-modified carbons suggests that exposure to these alcohols creates Hb-inhibiting surface oxides, such as C=O or C-O. These new inhibiting surface oxides are likely responsible for the plateau and decrease in Hb-reactivity seen at high EtOH and 1-PrOH concentrations in Fig. 1d. Carbon-oxygen groups were examined by C1s XPS for GC incubated in mixtures of PB and alcohols (at various concentrations) to examine whether alcohol exposure is changing the Hb-inhibiting ether or carbonyl surface groups. Fitting of the XPS data (Table 2 from data in Fig. S.7) shows two ranges of alcohol concentration (0 – 30% MeOH and 0 – 40% EtOH) where the C-O increases while the C=O decreases, making it difficult to draw a relationship between surface group and Hb-electroactivity since the expected Hb-activity improvements from removing C=O would be offset by the increased Hb-inhibiting C-O groups. However, while the Hb electroactivity is relatively constant through the 0 – 30% MeOH range in Fig. 1d, consistent with this offset, the 0 – 40% EtOH range evidences a large increase in electroactivity (Fig. 1d), suggesting that another enhancement mechanism dominates in these EtOH concentrations. Similarly, 0 – 30% 1-PrOH shows an increase in C-O and C=O that is expected to result in a decrease in Hb electroactivity, yet an increase is seen; again, indicating another mechanism dominates. Nevertheless, the changes in carbon surface oxides correlate with Hb electroactivity through much of the alcohol concentration ranges. For instance, increases in inhibiting C=O for ≥ 50% EtOH and C-O for ≥ 50% 1-PrOH predict a decrease in Hb activity, and indeed the Hb activity plateaus for EtOH and decreases for 1-PrOH, consistent with these surface groups inhibiting Hb electroactivity. Additionally, increasing the MeOH above 30% resulted in significant decreases in C-O and C=O surface groups (Table 2) that likely contributed to the large increase in Hb-reduction seen for these MeOH concentrations (Fig 1a). Overall, the data 19



Page 20 of 36

show that carbon oxides play a significant role in enhancing or inhibiting Hb electroactivity but that the relation is imperfect, consistent with the existence of multiple mechanisms, such as Hb denaturation and Hb film formation (discussed above). Indeed, as will be discussed below, the 60% MeOH-containing electrolyte results in the highest Hb activity of the three tested aliphatic alcohols (Fig. 1d), due to the carbon surface modification working in tandem with protein denaturation. Table 2. A summary of fitted peaks representing different surface groups from C1s XPS data for each alcohol treatment used on GC. Each entry listed in the table contains the relative surface composition based on integration under the fitted peak, the full width at half maximum, and the centre position of the fitted peak. The associated fitting errors for the carbon surface oxides were estimated using Monte Carlo simulations. Polished GC Exposed to no alcohol 30% MeOH + PB 40% MeOH + PB 60% MeOH + PB 40% EtOH + PB 50% EtOH + PB 60% EtOH + PB 30% 1-PrOH + PB 50% 1-PrOH + PB 60% 1-PrOH + PB

C=C

C-O

C=O

COOR

73.5% 1.26 eV 284.3 eV 71.6% 1.23 eV 284.3 eV 70.5% 1.18 eV 284.3 eV 75.8% 1.16 eV 284.3 eV 73.1% 1.18 eV 284.3 eV 73.6% 1.20 eV 284.3 eV 71.0% 1.36 eV 284.3 eV 66.9% 1.34 eV 284.3 eV 69.1% 1.25 eV 284.3 eV 65.1% 1.34 eV 284.3 eV

13.2 ± 0.5% 1.50 eV 285.5 eV 17.1 ± 0.6% 1.50 eV 285.5 eV 15.6 ± 0.3% 1.50 eV 285.5 eV 15.3 ± 0.3% 1.50 eV 285.5 eV 15.5 ± 0.8% 1.50 eV 285.5 eV 15.7 ± 0.8% 1.50 eV 285.5 eV 16.0 ± 0.5% 1.50 eV 285.5 eV 16.1 ± 0.4% 1.50 eV 285.5 eV 15.9 ± 0.3% 1.50 eV 285.5 eV 16.9 ± 0.6% 1.50 eV 285.5 eV

6.2 ± 0.3% 1.50 eV 286.8 eV 5.3 ± 0.3% 1.50 eV 286.9 eV 5.0 ± 0.3% 1.50 eV 286.9 eV 4.0 ± 0.2% 1.50 eV 287.0 eV 5.0 ± 0.6% 1.50 eV 286.8 eV 4.6 ± 0.6% 1.50 eV 286.9 eV 7.4 ± 0.3% 1.50 eV 286.8 eV 7.0 ± 0.3% 1.50 eV 286.9 eV 6.5 ± 0.4% 1.50 eV 286.9 eV 7.0 ± 0.3% 1.50 eV 286.9 eV

3.7 ± 0.3% 2.30 eV 288.8 eV 3.1 ± 0.3% 2.00 eV 288.8 eV 3.1 ± 0.5% 2.25 eV 288.8 eV 1.8 ± 0.6% 2.40 eV 288.8 eV 4.0 ± 0.8% 2.10 eV 288.9 eV 3.4 ± 0.7% 2.20 eV 288.8 eV 5.2 ± 0.4% 2.21 eV 288.8 eV 5.2 ± 0.6% 2.40 eV 288.8 eV 4.1 ± 0.5% 2.30 eV 288.8 eV 6.1 ± 1% 2.40 eV 288.8 eV

20


π-π* shakeup 3.4% 3.47 eV 290.4 eV 2.9% 3.83 eV 290.4 eV 3.7% 3.00 eV 290.4 eV 3.1% 3.37 eV 290.3 eV 2.4% 2.00 eV 290.4 eV 2.7% 3.00 eV 290.4 eV 0.4% 5.00 eV 290.3 eV 4.8% 4.87 eV 290.3 eV 4.4% 4.00 eV 290.4 eV 3.5% 5.00 eV 290.3 eV

Plasmon Negligible

Negligible 2.1% 3.48 eV 292.6 eV Negligible

Negligible

Negligible

Negligible

Negligible

Negligible 1.4% 2.07 eV 292.6 eV

Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Dominant Mechanisms Impacting Hb Electroactivity in Alcoholic Electrolytes As shown above, multiple factors affect the electrochemical reduction of Hb, including: structural changes in Hb, incorporation of Hb into a film, and changing the carbon-oxygen surface groups. For each alcohol, different mechanisms appear to be dominant and the dominant mechanism can change depending on the alcohol concentration. Herein we elucidate the dominant mechanisms for each alcohol and concentrations in terms of increasing Hb electroactivity. This knowledge will be useful for identifying and evaluating the impact of other alcohols or organic solvents on not only Hb electroactivity but perhaps other similar redox-active proteins such as myoglobin or their synthetic analogs. The Hb electrochemical response (charge) in Fig. 1d shows that MeOH contents up to 30% resulted in very little improvement in Hb reduction, but higher concentrations significantly enhanced Hb electroactivity, with 60% MeOH exhibiting the largest Hb response across all three alcohols and concentrations used in this research. For MeOH, film thickness is not a significant mechanism since the small increase in film thickness for MeOH-contents of 30% (resulting in significant activity). Protein denaturation only plays an important role at high MeOH content, since significant protein changes (especially for the Fe-His in the redox active heme groups) are only seen at >50% MeOH. Thus, carbon-surface-oxide modification is the dominant mechanism, and indeed there is a good correlation between the MeOH concentration where inhibiting oxides are removed (≥ 30%) and the enhanced Hb electroactivity in the same range. It is interesting to note that the high Hb electroactivity of 60% MeOH (highest across all alcohols and concentrations tested) is due to protein denaturation and carbon oxide removal working in tandem to improve 21



Hb activity; as will be shown below, for the high concentrations of EtOH and 1-PrOH, these mechanisms counteract, resulting in lower Hb electroactivities. Thus, carbon-oxide modification dominates at mid-range MeOH contents, but at high MeOH-content there is also enhancement due to protein denaturation. In the case of EtOH, Fig. 1d shows a gradual increase in Hb electroactivity for EtOHcontents ≥ 10%, with a plateau ≥ 50% EtOH. As seen with MeOH, film thickness is not a good indicator of Hb electroactivity in EtOH-containing electrolytes. However, unlike MeOH, protein denaturation appears to play a significant role over most of the concentration range, with all three measures of protein denaturation (Fe-His, Tyr and Trp) showing significant changes at ≥ 20% EtOH, mirroring the increase of Hb electroactivity at these concentrations. The carbon oxidation modification is a significant factor only at ≥ 50% EtOH, where the increase in inhibiting C=O groups offsets the Hb enhancement due to protein denaturation, resulting in the Hb activity plateau in these high EtOH-containing solutions (Fig. 1d). These interpretations suggest that denaturation of the protein structure is the dominant mechanism for improving Hb reduction in EtOH containing solutions. Of the three alcohols tested in this research, 1-PrOH denatured Hb the most, producing the thickest film on the carbon electrode. Yet, 1-PrOH also increased the surface concentration of carbon oxides, which would inhibit Hb reduction; but an increase in Hb reduction was still observed, suggesting that the carbon-oxide mechanism was more than offset by the significant Hb denaturation. Protein denaturation was most significant for 1-PrOH concentrations up to 40% and the biggest increases in Hb electroactivity were observed for those concentrations. However, increasing 1-PrOH concentrations beyond 40% resulted in a plateau (50% 1-PrOH) and a decrease in Hb electroactivity (60% 1-PrOH) that may have been caused by the formation 22


Page 22 of 36

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of a thinner film and more inhibiting C-O surface groups, suggesting that the film thickness may also be an important factor in Hb-reduction for these high 1-PrOH concentrations. These results and interpretations suggest that the enhanced Hb-reduction due to 1-PrOH were likely overall dominated by the denaturation of Hb, but forming a thinner film and increasing carbon surface oxides may have inhibited the Hb electroactivity in electrolytes containing high 1-PrOH concentrations. The combined interpretation of the data suggests that the improvement of electrochemical Hb detection based on incorporation of an alcohol relies mainly on removal of inhibiting C=O and C-O groups from the carbon surface and modification of the Hb structure. The ideal situation is one where both these mechanisms work together to increase Hb electroactivity, as with 60% MeOH. However, high concentrations of EtOH and 1-PrOH can cause these mechanisms to counteract, and this should be avoided for Hb detection purposes.

Conclusions In this work, we provided a detailed analysis of the mechanisms by which water-miscible primary alcohols improve Hb electroactivity on carbon electrodes. Changes in carbon surface oxides were examined by XPS, while evidence of a film formation was examined using spectroscopic ellipsometry. Structural changes in Hb were examined using both UV-Vis absorption and fluorescence spectroscopy. Hb denaturation is important in increasing Hb electroactivity in electrolytes containing any one of MeOH, EtOH, or 1-PrOH. Although clear evidence was presented for many subtle changes in the Hb structure upon exposure to 1-PrOH, EtOH, and high concentrations of MeOH, 23



these alone were not sufficient to explain the changes in Hb-reduction. Changes to the carbon surface oxides on GC are also important in affecting Hb electroactivity. For example, the addition of (at least 40%) MeOH led to increased Hb electroactivity (decreased C-O and C=O surface groups), but 1-PrOH and high concentrations of EtOH led to decreased electroactivity (increased C-O and C=O surface groups). There was also clear evidence of an electroactive Hb film adsorbed onto GC after incubation in Hb with or without alcohol. The incubation solutions containing 1-PrOH created thicker and more electroactive films than either EtOH or MeOH. Nevertheless, electrolytes containing 60% MeOH resulted in the highest Hb electroactivity of all the alcohols and concentrations tested because of both Hb denaturation and decreased inhibiting carbon surface oxides favoring Hb electroactivity. This work clearly shows that extra caution is required when using alcohols in studies of Hb electroactivity since both Hb and the carbon surface are changed upon alcohol exposure, affecting Hb electroactivity. These alcohols are commonly present as binder solvents (e.g. in Nafion suspensions), and we show for the first time that enhanced Hb electroactivity is due to alcohol-induced Hb film formation, changes in the carbon electrode surface, and Hb structure. It is clear that knowledge of these mechanisms is important because of the common use of Nafion to immobilize Hb on electrode surfaces;13 if the impact of alcoholic solvent is not recognized, the higher Hb currents may be erroneously attributed to other film components. The alcoholic solvent present in binders, especially in Nafion, likely also affect the electroactivity of other redox-active proteins and perhaps their synthetic analogs. Researchers should be aware of any alcohol solvents used in preparing electrochemical sensors involving redox-active proteins.

24


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Associated Content Supporting Information -

Spectroscopic Ellipsometry Model Considerations (SI.1)

-

Protein Film Thickness from Spectroscopic Ellipsometry (SI.2)

-

UV-Vis Absorption and Fluorescence Spectra (SI.3)

-

C1s XPS Spectra of Alcohol-modified Glassy Carbon (SI.4)

(SI.DOCX File)

Acknowledgements This work was supported financially by Dalhousie University, the Natural Sciences and Engineering Research Council (NSERC), and the Nova Scotia Health Research Foundation (NSHRF).

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For TOC only

29



Denatured Hemoglobin

e-

Carbon Electrode

e-

Hemoglobin C=O

e- e-

Alcohol

C=O

O

e - e-

Carbon Electrode

C=O

C=O

Hemoglobin C=O

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 30 of 36

e-

O O

Carbon Electrode


Page 31 of 36

Volume Percent Alcohol 20%

10 0 -5 20 30

(a) 0

(b) 0 -0.60

50 -1

-0.8

-0.75

-0.4

-30

10 20 30 40 50 60 Methanol Content / v/v %

-0.2

0

0.2

50%

-1

-0.8

-0.6

60%

-0.60 -0.6 -0.65 -0.65 -0.70 -0.7 -0.75 -0.75

-35

0.4

E vs. Hg/Hg2SO4 (sat. K2SO4) / V

(c) 0

40 60 50

-25 0

-0.6

30

-15 -20

-0.70

60

-35

I / µA

Hb Peak Potential / V

40

-30

10 20

-10

-0.65

-0.4

0 10 20 30 40 50 60 0 10 20 30 40 50 60 Ethanol Content / v/v % Ethanol Content / v/v %

-0.2

0

0.2

0.4


(d) 400

0 10 20

-15

60

-20 -25

50

30

-30

40

-35 -1

-0.8

300

-0.60

-0.6

-0.6


-10

-Charge / µC

-5


I / µA

-15

-25

0

-5

-10

-20

40%

30%

Hb Peak Potential / V Hb Peak Potential / V

10%

0%

I / µA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


-0.65

-0.65

-0.70 -0.7

100

-0.75 -0.75 10 20 20 30 0 0 10 30 40 40 5050 6060 1-Propanol Content %% 1-Propanol Content/ v/v / v/v

-0.4

200

-0.2

0

0.2


0.4

0 0


10

20 30 40 50 Alcohol Content / v/v %

60

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Mechanisms of Enhanced Hemoglobin Electroactivity on Carbon

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