Supporting Information
The Hydroxyl Radical is a Critical Intermediate in the Voltammetric Detection of Hydrogen Peroxide
James G. Roberts, Maxim A. Voinov, Andreas C. Schmidt, Tatyana I. Smirnova, Leslie A. Sombers*
Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
E-mail: leslie_sombers@ncsu.edu
1.
Experimental Section
1.1.
Chemicals.
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used as
received, unless otherwise noted. A physiologically buffered solution (Tris Buffer: 15 mM Trisma HCl,
3.25 mM KCl, 1.2 mM CaCl2, 1.2 MgCl2, 2.0 mM Na2SO4, 1.25 mM NaH2PO4, and 145 mM NaCl) at pH 7.4
was used in all flow injection analysis. Tris buffer and phosphate buffered saline (0.1 M PBS) were used
for electron paramagnetic resonance (EPR) spectroscopy experiments. All aqueous solutions were made
using doubly deionized water (Millipore, Billerica, MA).
1.2.
Electrode Fabrication. Carbon-fiber disc microelectrodes with a polished angle of 30° were
fabricated using 10 µm P-55s pitch-based carbon fibers (Cytec Industries, Woodland Park, NJ) as
previously described.1 In brief, a single carbon fiber was inserted into a glass capillary tube (A-M
Systems, Sequim, WA), pulled with a micropipette puller (Narishige, Tokyo, Japan), exposed carbon was
trimmed, dipped in epoxy (Epoxy Technologies, Billerica, MA), oven dried, then polished (Sutter
Instrument Co., Novato, CA).
Platinum disc microelectrodes with a polished angle of 30° were
constructed using 25 µm hard platinum wire (Goodfellow, Huntingdon, England), following the same
procedure.
Carbon-fiber cylindrical microelectrodes were fabricated by aspirating a single 7 µm
diameter T-650 carbon fiber (Cytec Industries, West Patterson, NJ) into a glass capillary.2 A micropipette
puller was used to seal the microelectrodes and the exposed length of carbon fiber was cut to 400 µm.
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Electrical connections to the working electrode were made by backfilling the capillary with an ionic
solution (4 M potassium acetate and 150 mM potassium chloride) and inserting a lead. A Ag/AgCl pellet
reference electrode (World Precision Instruments, Sarasota, FL) was employed to complete the two
electrode electrochemical cell.
1.3.
Data Acquisition. All electrochemical data was collected in a custom flow injection system or
custom microliter electrochemical cell. In most experiments, a triangular cyclic waveform ranging from
+0.2 to +1.3 V with a holding potential of +0.2 V versus Ag/AgCl was applied at a scan rate of 400 V∙s-1
and at a frequency of 10 Hz using a custom built instrument for potential application to the
electrochemical cell and for current transduction (University of North Carolina at Chapel Hill,
Department of Chemistry, Electronics Facility). TH-1 software (ESA, Chelmsford, MA) was used for
waveform output with DAC/ADC cards and for synchronization of the electrochemical experiment with
the flow injection system (National Instruments, Austin, TX). Signal processing including background
subtraction, signal averaging, and digital filtering was software-controlled (low-pass filtered, 2 kHz).
Electrochemical experiments using a lower scan rate were performed with an eDAQ potentiostat
(ED410, EA161) (Denistone East, New South Wales, Australia).
1.4.
Flow Injection System. A syringe pump (New Era Pump Systems, Wantagh, NY) supplied a
continuous buffer flow of 1 mL∙min-1 across the working and reference electrodes.
The working
electrode was positioned within a custom electrochemical cell using a micromanipulator (World
Precision Instruments, Sarasota, FL). Two second bolus injections of analyte to the microelectrode
surface were accomplished with a six-port HPLC valve mounted on a two-position air actuator controlled
by a digital pneumatic solenoid valve (Valco Instruments, Houston TX). The entire apparatus was
housed within a grounded Faraday cage. All experiments were performed at room temperature.
1.5.
Spin-trapping Experiments.
Spin-trapping agents were obtained from Enzo Life Sciences
(Farmingdale, NY). A solution containing 5 mM 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) along with a
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mixed solution containing 5 mM hydrogen peroxide (H2O2) and 5 mM DMPO was prepared in pH 7.4
PBS. 200 µL of either solution was placed in a custom polypropylene microliter electrochemical cell,
which included a Ag/AgCl reference electrode. A platinum microdisc or carbon-fiber microelectrode was
lowered into the solution and a waveform from +0.2 to +1.3 V at 400 V∙s-1 was applied at 100 Hz. This
waveform was applied for 30 minutes, and the solution was immediately transferred to a
polytetrafluoroethylene (PTFE) 0.81 x 1.12 mm diameter tubing (NewAge Industries, Southampton, PA)
for EPR measurements. 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO) was also
employed as an additional spin-trapping agent.
1.6.
EPR Measurements. All measurements were taken at room temperature with a Varian (Palo
Alto, CA) Century Series E-109 spectrometer and Bruker (Billerica, MA) Biospin ELEXSYS E-500
spectrometer. In the experiments, the sample was drawn into PTFE tubing (NewAge Industries,
Southampton, PA), the tubing was folded twice and inserted into a standard 3 x 4 mm diameter quartz
EPR tube (Wilmad-LabGlass, Vineland, NJ). Data acquisition parameters for Varian instrument were set
as follows: 3388 G center of field; 9.523 GHz frequency; 0.8 G modulation amplitude; 2 mW microwave
power; 100 G scan width; 30 sec sweep time; 64 msec time constant. Data acquisition parameters for
Bruker instrument were set as follows: 3510 G center of field; 9.863231 GHz frequency; 1 G modulation
amplitude; 14.71 msec conversion time; 0.2 mW microwave power; 100 G scan width; 30.13 sec sweep
time; 10.24 msec time constant. All spectra represent 100 averaged scans.
1.7.
Voltammetric Simulations. Digital simulations of voltammograms were performed with DigiSim
V3 (Bioanalytical Systems, West Lafayette, IN). DigiSim cannot model irreversible reactions, so two
reactions were simulated:
the heterogeneous reaction of hydrogen peroxide oxidation and a
homogeneous reaction that rapidly depleted the oxidized species.
voltammograms was accomplished with Matlab (The MathWorks, Natick, MA).
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Filtering of simulated
1.8.
Voltammetric Simulation Parameters. Reactions: B + e- = A, B = C; Waveform: -0.4 to +1.4 to -
0.4 at 400 V∙s-1; Electrode: Planer 4E-5 cm2; Semi-infinite linear diffusion; Butler-Volmer kinetics; Room
temperature; Initial concentration of H2O2: 100 µM; Eo: 0.85 V; α/λ: 0.55 eV; ks: 1E-4 cm∙s-1; D (all
species): 1E-5 cm2∙s-1; Homogeneous reaction rates: Keq: 100, Kf: 1E+4, Kb: 100.
1.9.
Statistics. All values are given as the mean ± standard deviation, unless otherwise noted. Two-
tailed paired Student’s t tests were used, with significance designated as p < 0.05. Multiple linear
regression was used to extract concentration values from complex mixtures (Matlab, The MathWorks,
Natick, MA). Statistical and graphical analysis was accomplished using GraphPad Prism 5 (GraphPad
Software, Inc., La Jolla, CA).
2.
Supporting Figures and Tables
Figure S1. (A) Representative voltammograms (scan rate: 400 V∙s-1) for 100 µM H2O2 with filters enabled
(red line), the analog filter disabled (black dashed line), and both the analog and digital filters disabled
(grey dashed line). (B) Simulated voltammogram for H2O2 (grey dashed line) and lowpass filtering of the
simulated data (black line).
Figure S1A shows representative voltammogram for 100 µM H2O2 with both a 2 kHz analog
Sallen-Key low-pass filter and a 2 kHz digital 4th Order Bessel low-pass filter enabled (red line). Peak
current is evident on the return scan, at about +1.2 V. As the filters are individually removed, the
oxidative peak shifts toward the switching potential (black and grey dashed lines).
To further
demonstrate this, commercially available software (DigiSim) that utilizes a finite difference model3,4 was
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employed to simulate a voltammogram (grey dashed line, Figure 1B), using a scan rate of 400 V∙s-1 and
the parameters listed in the supporting information. Briefly, two reactions were simulated including a
heterogeneous reaction for H2O2 oxidation and a homogeneous reaction that rapidly depletes the
oxidized species to force an irreversible voltammogram. The simulated voltammogram is similar in
shape to the unfiltered fast scan experimental data. When a digital low-pass filter is applied to the
simulated voltammogram (black line), the peak shifts in a manner that closely approximates the
experimental data (Figure 1A, red line).
Figure S2. Representative voltammograms of (A) 200 µm H2O2 on carbon and (B) 20 µm H2O2 on
platinum microelectrodes, collected at various scan rates.
Peak current increases with increased scan rates on platinum electrodes, in agreement with
theory.5 Interestingly, the opposite trend is evident on carbon, again suggesting a critical dependence
on the state of the electrode surface.
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Figure S3. Representative EPR spectra of solutions containing 5 mM DMPO and 5 mM H2O2 (red line) or
5 mM DMPO, 5 mM H2O2 and 0.1 M DMSO (blue line), where the cyclic waveform was applied to a
platinum electrode for 30 minutes.
Figure S4.
pH dependence of H2O2 oxidation on carbon-fiber electrodes.
Representative
voltammograms at 400 V∙s-1 of 100 µM H2O2 showing an increase in current for higher pH values. (n = 6
electrodes)
Table S-1. A) Least-squares simulated data of EMPO•-OH adduct.
Species
1
Experimental
Hyperfine Coupling Constants
Literature6
Hyperfine Coupling Constants
A(N) = 14.11 ± 0.02 G
A(N) = 14.0 G
A(H) = 15.00 ± 0.04 G
A(H) = 15.1 G
A(H) = 0.82 ± 0.03 G
A(H) = 0.9 G
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A(N) = 14.02 ± 0.03 G
A(N) = 14.0 G
A(H) = 12.62 ± 0.02 G
A(H) = 12.7 G
2
(n = 2 spectra)
B) Least-squares simulated data of DMPO•-OH adduct.
Experimental
Hyperfine Coupling Constants
Literature7
Hyperfine Coupling Constants
A(N) = 14.97 ± 0.01 G
A(N) = 14.9 G
A(H) = 14.86 ± 0.01 G
A(H) = 14.9 G
(n = 3 spectra)
Table S-2. Predicted concentration of H2O2 within a solution containing only H2O2, and within a solution
containing H2O2 plus a trapping agent (DMPO or DMSO).
Solution
Predicted H2O2 Concentration (µM)
1 mM DMPO + 100 µM H2O2
92 ± 2
100 µM H2O2
99 ± 3
10 mM DMSO + 100 µM H2O2
96 ± 1
100 µM H2O2
100.0 ± 0.9
(n = 3 electrodes)
Table S-3. Predicted concentration of histamine within a solution containing only histamine, and within
a solution containing histamine plus DMPO.
Solution
Predicted Histamine Concentration (µM)
1 mM DMPO + 1 µM histamine
0.9 ± 0.2
1 µM histamine
0.9 ± 0.2
(n = 4 electrodes)
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3.
References
(1) Zachek, M. K.; Hermans, A.; Wightman, R. M.; McCarty, G. S. Journal of Electroanalytical Chemistry
2008, 614, 113-120.
(2) Roberts, J. G.; Lugo-Morales, L. Z.; Loziuk, P. L.; Sombers, L. A. Methods in molecular biology 2013,
964, 275-94.
(3) Maloy, J. T. In Laboratory Techniques in Electroanalytical Chemistry, Kissinger, P. T.; Heinemann, W.
R., Eds.; Marcel Dekker, Inc.: New York, 1996.
(4) Feldberg, S. W. In Electroanalytical Chemistry: a Series of Advances, Bard, A. J., Ed.; Marcel Dekker:
New York, 1969.
(5) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; John
Wiley: New York, 2001, p xxi, 833 p.
(6) Olive, G.; Mercier, A.; Le Moigne, F.; Rockenbauer, A.; Tordo, P. Free Radical Bio Med 2000, 28, 403408.
(7) Minotti, G.; Aust, S. D. J Biol Chem 1987, 262, 1098-104.
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