Electrochemical detection of peroxynitrite using hemin-PEDOT functionalized boron-doped diamond microelectrode

Abstract

Peroxynitrite is a potent nitroxidation agent and highly reactive metabolite, clinically correlated with a rich pathophysiology. Its sensitive and selective detection is challenging due to its high reactivity and short sub-second lifetime. Boron-doped diamond (BDD) microelectrodes have attracted interest because of their outstanding electroanalytical properties that include a wide working potential window and enhanced signal-to-noise ratio. Herein, we report on the modification of a BDD microelectrode with an electro-polymerized film of hemin and polyethylenedioxythiophene (PEDOT) for the purpose of selectively quantifying peroxynitrite. The nanostructured modified polymer layer was characterized by Raman spectroscopy and scanning electron microscopy (SEM). The electrochemical response to peroxynitrite was studied by voltammetry and time-based amperometry. The measured detection limit was 10 ± 0.5 nM (S/N = 3), the sensitivity was 4.5 ± 0.5 nA nM(-1) and the response time was 3.5 ± 1 s. The hemin-PEDOT BDD sensors exhibited a response variability of 5% or less (RSD). The stability of the sensors after a 20-day storage in 0.1 M PB (pH 7.4) at 4 °C was excellent as at least 93% of the initial response to 50 nM PON was maintained. The presence of PEDOT was correlated with a sensitivity increase.

Figure 1

Schematic of the BDD microelectrode architecture after insulation with a polypropylene micropipette tip and the electrochemical formation of the hemin-PEDOT layer on the exposed microelectrode.

Figure 2

The oxygen-dependent mechanism of peroxynitrite generation from 3-morpholino-sydnonimine (SIN-1). Reproduced from reference [27] with permission of The Royal Society of Chemistry.

Figure 3

The hemin-PEDOT film (A) is formed by the electropolymerization of EDOT with hemin. (B) Cyclic voltammograms recorded during the formation of the hemin-PEDOT conducting polymer film. The electropolymerization was performed over 20 cycles at 50 mV s⁻¹ between −1.5 and +1.5 V vs. Ag/AgCl in a hemin-EDOT monomer solution. The measurements were made with the solution under a N₂ blanket. The curves show increasing anodic and cathodic currents with increasing cycle number indicative of polymer growth.

Figure 4

(Left) SEM image of the hemin-PEDOT film on a BDD microelectrode at low magnification. The image reveals a nodular morphology of the conducting polymer that covers the entire microelectrode surface. (Right) The higher magnification SEM image shows the typical nanostructured ‘cauliflower’ morphology characteristic of a 3D branched-multiglobular polymer film with features within the 100–300 nm range.

Figure 5

Raman spectra of (A) a hemin-PEDOT BDD microelectrode, (B) a hemin-only BDD microelectrode, and (C) an unmodified BDD microelectrode. λ_ex = 532 nm. Integration time = 10 s. The hemin-only microelectrode was performed by the same potential cycling as the hemin-PEDOT microelectrode without any EDOT monomer.

Figure 6

Cyclic voltammetric i-E curves recorded for different concentrations of PON in 0.1 M PB (pH 7.4) from 50 to 400 nM at a hemin-PEDOT BDD microelectrode. Scan rate = 100 mV/s. The PON concentration was estimated from the known concentration of SIN-1 and assuming a 1/100 ratio of PON to SIN-1 under steady-state conditions [8,24,25,46,47].

Figure 7

(left) Cyclic voltammogram of 1.5 mM hemin in dichloromethane with 0.1 M tetrabutylammonium tetrafluoroborate at an unmodified BDD microelectrode (blue curve). The initial scan is shown. The background voltammogram in the absence of any added hemin is also shown (black curve). Scan rate = 100 mVs⁻¹. (right) Schematic showing the catalytic redox reaction mechanism for the hemin-mediated oxidation of PON.

Figure 8

(A) Continuous amperometric i-t curves recorded at a hemin-PEDOT BDD microelectrode for varying concentrations of PON generated by adding aliquots of a SIN-1 solution of known concentration to a mechanically-stirred 0.1 M PB (pH 7.4) solution. (B) Response curve shown for PON concentrations from 0-800 mM generated from the steady-state oxidation current at each concentration. (C) Response curve shown for PON over a more narrow concentration range with a linear regression fit. All currents were measured at +1.35 V vs. Ag/AgCl.

Figure 9

A comparison of the sensitivity of differently modified BDD microelectrodes for PON recorded in 0.1 M PB (pH 7.4). Response curves are shown for unmodified, hemin-only, PEDOT-only, hemin-PEDOT type A and hemin-PEDOT type BDD microelectrodes. The hemin-PEDOT type B film was formed using a 3× greater EDOT concentration in solution as compared to hemin-PEDOT type A. The PON concentration was estimated from the known concentration of SIN-1 in solution and assuming a 1/100 ratio of PON to SIN-1 under steady-state conditions [8,24,25,46,47]. All currents were measured at +1.35 V vs. Ag/AgCl.

Figure 10

Selectivity of the hemin-PEDOT-PEI BDD microelectrode during a continuous amperometric measurement of PON (50 and 500 nM) in 0.1 M PB (pH 7.4) in the absence and presence of three potential interfering species: norepinephrine, serotonin and uric acid. The concentration of each was 70 μM. The detection potential was +1.35 V vs. Ag/AgCl. Arrows indicate the time at which a solution of the interferent was added to the PON solution.