Gold Nanoparticle Modified Transparent Carbon Ultramicroelectrode

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Gold Nanoparticle Modified Transparent Carbon Ultramicroelectrode Arrays for the Selective and Sensitive Electroanalytical Detection of Nitric Oxide Janine Elliott, Jonathon Duay, Olja Simoska, Jason B. Shear, and Keith J. Stevenson Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03987 • Publication Date (Web): 19 Dec 2016 Downloaded from http://pubs.acs.org on December 21, 2016

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Analytical Chemistry

Gold Nanoparticle Modified Transparent Carbon Ultramicroelectrode Arrays for the Selective and Sensitive Electroanalytical Detection of Nitric Oxide Janine Elliott1†, Jonathon Duay1, Olja Simoska1, Jason B. Shear1, and Keith J. Stevenson2* 1

Department of Chemistry, Center for Nano- and Molecular Science and Technology The University of Texas at Austin, Austin, TX 78712, USA 2

Center for Electrochemical Energy Storage,

Skolkovo Institute of Science and Technology, Moscow, 143026, Russia ABSTRACT: Transparent carbon ultramicroelectrode arrays (T-CUAs) were made using a previously reported facile fabrication method (Duay et al. Anal. Chem. 2015, 87, 10109). Two modifications introduced to the T-CUAs were examined for their analytical response to nitric oxide (NO•). The first modification was the application of a cellulose acetate (CA) gas permeable membrane. Its selectivity to NO• was extensively characterized via chronoamperometry, electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). The thickness of the CA membrane was determined to be 100 nm and 88±15 nm using AFM and EIS, respectively. Furthermore, the partition and diffusion coefficients of NO• within the CA membrane were determined to be 0.0500 and 2.44x10-13 m2/s using EIS measurements. The second modification to the 1.54T-CUA was the introduction of chitosan and gold nanoparticles (CS/GNPs) to enhance its catalytic activity, sensitivity, and limit of detection (LOD) to NO•. Square wave voltammetry was used to quantify NO• concentration at the CA membrane covered 1.54T-CUA with and without CS/GNPs; the LODs were determined to be 0.2 ± 0.1 µM and 0.44 ± 0.02 µM (S/N=3), with sensitivities of 9 ± 9 and 1.2 ± 0.4 nA/µM, respectively. Our results indicate that this modification to the arrays result in a significant catalytic enhancement to the electrochemical oxidation of NO•. Hence, these electrodes allow for the in situ mechanistic and kinetic characterization of electrochemical reactions with high electroanalytical sensitivity.

Introduction. Nitric oxide (NO•) is a major biogenic reactive oxygen species (ROS). Thus, there is a lot of interest around the detection and monitoring of NO• within biological samples.1, 2 NO• plays many roles in advanced organisms. It is a messenger molecule for neuronal communication, the endothelium derived relaxing factor (EDRF), which is pivotal in vasodilation as it is released by the endothelium to relax smooth muscle, and it functions as an immunological defense mechanism by host organisms to fight pathogenic infections.1,3-7 NO• is generated by one of the four different classes of nitric oxide synthases (NOS), neuronal nitric oxide synthase (nNOS), immunological or inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), and mitochondrial nitric oxide synthase (mtNOS).8 The general mechanism for NOS consists of the conversion of Larginine and O2 to L-citrulline and NO•. Electroanalytical methods are proven as one of the most sensitive, robust, and direct detection methods for cellularly derived NO• among other ROS.1,9-11 Resembling other ROS molecules, NO• is an extremely diffuse molecule that is present in biological systems at low concentrations (≤ µM) with a short half-life (~6 s in physiological conditions).12 Therefore, the specifications of the detection method requires that the sensor has fast kinetics, high sensitivity, selectivity, as well as a low limit of detection

(LOD).1 Conventionally, NO• is detected using expensive, time consuming methods notably fluorescence, chemiluminescence, and spectrophotometry.13,14 Due to their fast response times, easy manufacturing, low costs, and simple modification procedures for greater selectivity and sensitivity, electrochemical methods are an excellent alternative for the real time, continuous monitoring of NO•.13,15-17 These beneficial characteristics of electroanalytical sensors deem them an exceptional alternative to the conventional detection methods.1,12 Previously, we have reported a method for detecting the biogenic ROS molecule hydrogen peroxide (H2O2) using unmodified transparent carbon ultramicroelectrode arrays (TCUAs).18 T-CUAs offer advantages over bulk electrodes. The small sizes of microelectrodes decrease ohmic drops allowing for the use of less supporting electrolyte, achieving fast steady state responses, and enhanced mass transport to the electrode. The main issue with individual microelectrodes is that the current output runs in the picoamp range and is noise limited. T-CUAs possess the benefits of microelectrodes and have the additional advantage of amplified currents due to the individual electrodes in the array functioning in parallel.18 The active electrode material, a pyrolyzed photoresist, is similar to glassy carbon. It is hard, inert, has a high hydrogen overpotential, is highly conductive, chemically stable,

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biocompatible, and can be easily modified. The T-CUAs were made using a facile and versatile fabrication method,19 and characterized for their analytical response to the oxidation of H2O2, using cyclic voltammetry. The LOD for H2O2 obtained ~35 nM, which was lower than previously reported levels for array based ultramicroelectrodes (UMEs), the latter of which are extensively modified.1,13,14 Furthermore, the transparent arrays were analyzed for their spectroelectrochemical response showing a very good correlation between the optical and electrochemical response for ferrocenemethanol at UV wavelengths of 254 nm.18 Thus, these electrodes allow for the in situ mechanistic and kinetic characterization of heterogeneous electrochemical and intermediate homogeneous chemical reactions with high electroanalytical sensitivity. Here, we discuss the use of T-CUAs for the detection of NO• with the intention of integrating the device with biological systems for the in vitro detection of cellularly derived NO•. In this approach, the T-CUA is modified by a cellulose acetate (CA) gas permeable membrane that is selective to NO•.1,20 The small pore size of the membrane (6 Å) enhances the performance of the sensor by filtering out large non-gaseous molecules typically secreted by cells, such as proteins. Therefore, electrode fouling caused by the adsorption of large molecules on the working electrode surface is avoided.21 Extensive characterization of the membrane via chronoamperometry, electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) is performed. AFM and EIS are employed to determine the average membrane thickness. Chronoamperometry and EIS are used to verify the selectivity of the membrane to NO•, and to determine the partition and diffusion coefficients for both NO• and nitrite (NO2-).22 Being the oxidative product of NO•, NO2- is a major interferent.23 Comparing its diffusional and partitioning behavior across the membrane is paramount to confirming the membrane’s performance. This thorough investigation of the CA membrane’s permeability to NO• and NO2- is unprecedented. Thus our quantitative evaluation of the membrane gives kinetic parameters ascertaining why the membrane has a greater selectivity to NO•. A secondary modification to the electrode is introduced to enhance the sensitivity, increase catalytic activity, and lower the LOD of the electrode to NO•. This is achieved by electrochemically polymerizing chitosan in the presence of gold nanoparticles (GNP) onto the microelectrode.24 Numerous nanomaterials are currently used to enhance electrocatalytic oxidation of NO• for biosensory applications.25-28 Due to their unique attributes at small sizes, GNPs possess an electrocatalytic affinity to NO• by stabilizing intermediate redox species.29,30 They are also biocompatible,31 and thus are integrated in numerous NO• bioelectrochemical sensing schemes.32-35 Presently, we are using a simple, controllable method to electropolymerize chitosan (CS), a biocompatible polymer possessing anti-interference properties, around GNPs. 24 It can act as a metal-complexing agent and encapsulates the GNPs, forming a composite CS/GNP film on the electrodes.24 The films are characterized using scanning electron microscopy (SEM), and the catalytic enhancement is observed via cyclic voltammetry (CV) and square wave voltammetry (SWV). The one-step electrodeposition allows for the rapid modification to our arrays forming a film with significant catalytic enhancement to the electrochemical oxidation of NO•.24 Lastly, square wave voltammetry is used to enhance the signal to noise ratio of the current response to NO• oxidation at

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the CA membrane covered electrodes (T-CUA and transparent macroelectrode (T-Macro)) with and without chitosan-gold nanoparticle (CA/CS/GNP and CA) modification, in order to determine the respective LOD of each for NO•. Experimental section. Reagents and Materials. All chemicals were used as received. Photoresist AZ 1518 was purchased from Microchemicals. Polystyrene spheres (Polybead®) with diameters of 1.54 and 90 µm were purchased from Polysciences, Inc. Potassium chloride, acetic acid, cyclohexanone, isopropanol, glucose (G), uric acid (UA), ascorbic acid (AA), hydrogen peroxide (H2O2), dopamine (D), and nitric acid were acquired from Sigma-Aldrich Co. Monosodium phosphate, disodium phosphate, cellulose acetate (CA), fetal bovine serum (FBS), and sodium nitrite were purchased from Thermo Fisher Scientific Inc. Fabrication of T-Macro and T-CUAs. Preparation of the T-CUAs and T-Macro follows a previously described procedure.18,19 Here, T-CUAs are labeled “1.54T-CUA” and “90T-CUA”, where the numeric value indicates the micronsized diameter of the polystyrene spheres (PSS) used during their fabrication, 1.54 and 90 µm, respectively. Additionally, “T-Macro” is the label for a planar carbon electrode where no PSSs were used during fabrication. Preparation of CS/GNPs. The CS/GNP solution was prepared in accordance with a modified procedure described in the literature.24 A 10.00 mg/mL solution of CS in 0.05 M acetic acid and a 3.76×10-3 M HAuCl4 were prepared and mixed in a 20:1 (v/v) ratio, respectively. The mixture was well shaken and placed in a heated sonic bath for 1 hr until the solution color went from cloudy white to pink, indicating GNP formation. The solution pH is adjusted to 2.0 using nitric acid (HNO3) to dissolve the CS. Preparation of the CS/GNP Modified 1.54T-CUA and TMacro. The CS/GNP film was electrodeposited on the 1.54TCUA and T-Macro via chronoamperometry by holding a potential at -1.00 V for 60 seconds in the aforementioned CS/GNP solution. The selected potential and time for the deposition was determined by Wang and collaborators. 24 The prepared 1.54T-CUA and T-Macro were rinsed with deionized water and gently dried with N2 gas. Preparation of the CA Membrane. The CA membrane precursor solution was prepared according to a previous procedure.20 Briefly, 7.5 µL of a precursor solution containing cyclohexanone, isopropanol, and cellulose acetate was cast on the surface of distilled water. It was then allowed to cure 5-10 minutes. The electrode was submersed in the water and then was slowly lifted out drawing the membrane on top of it (Scheme 1). N2 gas was lightly blown on top of the membrane/electrode for 5 minutes. The pressure of the gas created a fitted, taut, and dry physical contact between the membrane and the surface of the electrode.


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Analytical Chemistry Scheme 1.

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Preparation of a Stock Standard NO• Solution. The stock NO• solution was prepared as previously reported, with modifications.1 6 M sulfuric acid (H2SO4) was slowly added drop wise into a saturated sodium nitrite (NaNO2) solution, which generated NO• gas. The gas was passed through a 30% sodium hydroxide (NaOH) solution, the remaining NO• gas was bubbled through 10 mL of 0.1 M SPB, 7.0 pH for 40 min. The resulting NO• concentration is 2 mM at 22°C.18, 36 The entire procedure must be performed very carefully because the NO• gas is toxic. The saturated NaNO2, 30% NaOH, and SPB solutions were purged with N2 for 3 hours to remove any oxygen. The NO• stock solution was used immediately upon preparation to make the standard NO• solutions. Preparation of Standard NO• Solutions. Standard NO• solutions, 20 mL each, were prepared having the following concentrations: 5 µM, 25 µM, 50 µM, 75 µM and 100 µM NO• in SPB. The solutions were prepared by diluting from the 2 mM NO• stock solution, respectively. NO• gas easily autoxidizes in the presence of O2, therefore standard solutions were purged with N2 gas for 2 hours before adding any NO• stock solution. Electrochemical Measurements. The electrochemical measurements were performed using a three-electrode cell system. Electrochemical experiments including EIS, SWV, CV, and chronoamperometry were performed using an Autolab PGSTAT30 potentiostat. The CA/90T-CUA, CA/1.54T-CUA, CA/CS/GNP 1.54T-CUA, CA/CS/GNP TMacro CA/T-Macro, or glassy carbon rotating disc electrode (GCRDE) was used as the working electrode, platinum mesh was used as the counter electrode and a saturated calomel electrode (SCE) was the reference. The T-CUAs and the TMacro were used as the working electrodes with a total exposed geometric area of 0.495 cm2. The geometric area is defined by the electrode area exposed to solution in our homemade Teflon electrochemical well. Therefore, the electroactive area for the T-Macro is 0.495 cm2. For the TCUAs, the geometric area is not to be confused with the electroactive area, which is determined by the total exposed carbon area. The total exposed carbon area for the 1.54 and

90T-CUAs are 0.0071 and 0.00038 cm2, respectively.18 CV was performed at a scan rate of 100 mV s-1, and SWV was performed using a current of 5 A, 3 mV step potential, and a frequency of 15 Hz. The potential ranged from 0 – 1.2 V vs. SCE. The background solution, SPB, pH 7.0, was purged with N2 gas for 3 hours. After a background CV or SWV curve was collected, subsequent NO• measurements were obtained. All experiments were carried out in the presence of N2. SWV calibration curve experiments had N2 gas blowing over the surface of an enclosed standard solution, and EIS experiments were carried out within a N2 filled glove bag. AFM Characterization of CA Membrane and SEM Characterization. Experimental details are discussed in the supporting information. Results. Geometry of CUAs Characterization of the Cellulose Acetate Membrane. The selectivity of the cellulose acetate membrane was tested using chronoamperometry and SWV. Figure 2A shows the potential of a CA/GCRDE held at 0.75 V vs. SCE. Conjointly to the addition of 100 μM NO•, at or above physiological and in vitro concentrations of common interferents to NO• were added to CA modified and unmodified GCRDEs every 100 s: 1 mM uric acid (UA), 1 mM ascorbic acid (AA), 5.5 mM glucose (G), 1 mM dopamine (D), 100 µM NO2-, 100 µM H2O2, and 1% fetal bovine serum (FBS). The initial current decay is likely due from the reduction of active oxidation sites on the GCRDE. We do not see this affecting the results presented here as they are only semi-quantitative in nature. Other than NO•, the only noticeable current response is from H2O2, indicating that the membrane has excellent resistance to biofouling. These electrodes are capable of detecting H2O2 with its onset oxidation potential at 0.9 V.18 The oxidation potential of NO• occurs before the onset potential of H2O2 (0.71 vs. 0.9 V) therefore the current response of NO• can be selectively observed using any potentiodynamic method such as SWV, which is used here (figure 2B). By employing SWV at the CA/T-Macro it is observed that H2O2 has an unappreciable influence on the signal for NO• at 0.71 V, because the current changes by only 7 ± 3% (figure 2B). To quantify the selectivity of the cellulose acetate membrane for NO•, a series of EIS experiments were performed. Multiple CA membranes were individually tested on a GCRDE with no additional support, which allowed for measurements such as EIS. Two types of EIS experiments were executed to characterize the faradaic and non-faradaic electrochemical elements of the membrane system.22 Supporting electrolyte solution without NO• or NO2- was initially used to define the non-



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Figure 1. AFM measurements of the cellulose acetate membrane A., top view, B., view of an edge of the membrane, C., profile of red line found in B.

(1)

Where Cm is the membrane Capacitance, A is the geometric area of the membrane, εef is the effective membrane dielectric constant, ε0 is vacuum permittivity, and δef is the effective membrane thickness.

Figure 3. Bode plot and corresponding Randles Circuit for a typical cellulose acetate membrane at four different concentrations of SPB.

Figure 2. A. Chronoamperograms of 1.54T-CUA electrode modified with and without (inset) a CA membrane. The current response was monitored where common biological interferents were added every 100 s: 1 mM UA, 1 mM AA, 5.5 mM G, 1 mM ∙ D, 100 μM NO , 100 μM NO2-, 100 μM of H2O2, and 1% FBS. The potential was held at +0.75 V vs. SCE. Hydrogen peroxide is the only interferent to permeate through the membrane. B. SWV ∙ voltammograms of 100 μM NO and 100 μM H2O2 at a CA/TMacro.

faradaic elements of the film such as the capacitance C and resistance R. Figure 3 shows the Bode plot and corresponding Randles Circuit for a typical membrane at four different concentrations of SPB.37 As is typical for a Bode plot, a slope of zero represents resistance, a slope of -1 represents capacitance, and a slope of -1/2 represents the Warburg or diffusional impedance, indicating a faradaic reaction. As the concentration of the buffer increases, the slope, representing the membrane capacitance (Cm), also increases to a value nearing -1. Here, one can see the advantage of EIS, as it becomes easy to separate out the double layer capacitance, Cdl, from the membrane capacitance, Cm and the membrane resistance, Rm, from the solution resistance, RS. From the membrane capacitance obtained from the Bode plot, the thickness of the membrane can be calculated using the following equation:

Using equation 1, the membrane thickness was calculated to be 88±15 nm, which is supported by the AFM measurements shown in Figure 1 where the thickness of the membrane was approximated to be ~100 nm. It should be noted that the thickness determined by EIS is the effective average thickness, which is a more relevant metric than the thickness obtained by AFM. In order to probe the intrinsic permeability of the membrane to NO• and NO2- the faradaic processes of these analytes were investigated at the RDE using EIS and chronoamperometry. The following equation is used: 22

(2)

Where P is the intrinsic diffusion permeability of the membrane to NO•, K is the partition coefficient of NO•, D is the diffusion coefficient of NO•, the R is the gas constant, T is the temperature, δ is the thickness of the membrane, R0 is the diffusion resistance taken from the steady state diffusion current obtained by chronoamperometry, F is Faraday’s constant, A is the geometric area of the electrode, and c is the concentration of NO•.


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Figure 4. Bode plots of solutions with 0 mM and 2 mM NO•, a, and the difference in admittance converted to impedances for the varying frequencies, b.

In order to separate out K and D, EIS and the following equations are used: 22

(3)

(4)

Where YO is the admittance of NO• by the membrane determined from EIS. This is obtained by taking the difference in admittance (reciprocal of the impedance, ZO) between the curves in Figure 4a. This difference was converted back to impedance and replotted, which can be observed in Figure 4b and displays a slope close to -1/2 indicating Warburg diffusion, and a faradaic event at the electrode. By dividing (4) by (2), K can be calculated and plugged back into (2) or (4) to find D. The same experiment and analyses were performed for NO2- (Figure S-2). The values for K and D were compared to that for NO2-, a major interferent, and presented in Table 1. As can be seen by the K value, the cellulose acetate membrane is nearly 10 times more permeable to NO• as compared to NO2-. The results indicate that the membrane is notably more impervious to the passage of NO2-. The direct oxidation of other oxidative products of NO, such as peroxynitrite (ONOO-), on glassy carbon occurs at potentials above 1.35 V vs. Ag/AgCl.38 Thus, possible interference with the signal at the electrode is avoided because its oxidation potential is outside the potential window for TCUAs. Table 1. The values for the Intrinsic diffusion permeability, P, the partition coefficient, K, and the diffusion coefficient, D, for NO• and NO2-.

Detection of NO• at CA coated CS/GNP Modified and Unmodified T-CUA and T-Macro Electrodes. The arrays are fabricated using microsphere lithography. The details of which are described previously.19 Briefly, the fabrication consists of drop casting PSSs, with varying diameters (1.54 and 90 µm) onto a conductive carbon pyrolyzed photoresist

film electrode. The diameters of the spheres dictate the size of the individual electrodes within the array as well as the distance between them.19 The smaller the diameter of the PSS, the denser the array and the smaller the individual electrode. The tunability and high-throughput fabrication of the T-Macro and T-CUA electrodes has allowed us to determine the best geometric dimensions for a given analyte and sensing scheme.18 Therefore, these electrodes can be easily adjusted in order to tune their figures of merit with the aim of attaining those required for the detection of NO•. Figure 5 shows the current-potential square wave voltammograms and calibration curves for the CA/1.54T-CUA, CA/90T-CUA and the TMacro. The limit of detection (LOD) for NO• with the CA/90T-CUA, CA/1.54T- CUA, and CA/T-Macro electrodes are 4, 0.4, and 0.8 µM, respectively. Figure 5 shows the square wave voltammeteric response for different concentrations of NO• and the corresponding calibration curve for NO•. Accordingly, as the concentration of NO• increased from 5100 µM the current response followed. The LOD was calculated as 3σ/slope of our calibration curve, utilizing standard approach 1 (SA1). 39 The LODs are comparable to or better than the LOD for the colormetric Greiss reagent (0.51 µM), 40-42 which is the current standard method for NO• detection. The Greiss reagent detects the oxidative product of NO•, NO2-. It is an in-direct detection method that measures the NO2- concentration as an index of NO• production. The reaction requires strong acid and a long period of time to proceed to completion. Therefore, real-time in vitro and in vivo analyses on NO• concentration are impossible to achieve with this current standard detection method. While physiologically present, such as in blood, NO• is known to have a half-life of 6-15 s.12, 43 Therefore the response time of sensor must be fast. Assessing the response time of the electrodes is a way to gauge their capability for detecting NO• in biological environments. We calculated the response times (Eq. S-2) from the RC time constants (Eq. S-1) for the CA/1.54T-CUA, CA/90T-CUA and the T-Macro to be approximately 105.5, 81.1, and 1695.6 µs, respectively (Table S-1). These response times are well below the NO• half-life, indicating that these electrodes would be suitable for the detection of NO• in biological samples. To further improve the LOD, a modification was introduced to the electrodes via one-step electrodeposition of CS and GNPs.24 CS can electropolymerize to form a stable, biocompatible hydrogel that physically encapsulates the GNPs and retains them within a film on the electrode surface.44 The literature procedure normally used to create the solution of CS/GNPs adjusts the pH to 2 with HCl to dissolve the CS. As HCl can cause the dissolution of the alumina insulating layer, the procedure was modified by using HNO3 instead. Additionally, HNO3 can solubilize CS better than HCl.45 GNPs are known for possessing selective catalytic properties for biogenic analytes and good biocompatibility.43 Gold is relatively inert in its bulk form.



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Figure 5. The graphs in the left column depict the square wave voltammetric response for five different concentrations of NO• at A., the CA/T-Macro, B., the CA/1.54T-CUA, and C., the CA/90T-CUA electrodes. The middle column illustrates the background corrected voltammograms at D., the CA/T-Macro, E., CA/1.54T-CUA, and F., CA/90T-CUA electrodes. The right column shows the corresponding calibration curves for NO• for each of the three electrodes, G., the CA/T-Macro, H., CA/1.54T-CUA, and I., CA/90T-CUA

When dispersed in the form of fine particles (≤ 10 nm) its catalytic activity increases resulting in a striking difference in its chemical structure.29 Table 2. A comparison of LODs to NO• between modified and unmodified T-Macro and T-CUAs to similarly modified optically transparent electrodes.

The majority of GNP’s catalytic activity is related to the high concentration of low coordinated active sites on the surface of small particles30 and to the fact that they can efficiently facilitate electron transfer.24 Figure 6 displays the SEM images of an unmodified 1.54TCUA, an individual electrode of CS/GNP 1.54T-CUA, and a CS/GNP 1.54T-CUA. The surface coverage of the nanoparticles on the electrodes is 232 ± 5 particles/µm2, with particle size ranging from 5-80 nm. Even though the sizes of the electrodeposited GNPs are varied, there is a significant enhancement in the electrooxidation of NO•. This effect is

observed in the cyclic voltammogram by the increase in the current value for the oxidation of NO• at the modified electrode (Figure 6(d.)). The T-CUAs could be completely covered with GNPs by using self-assembly of GNP monolayers to detect NO·.46 The procedure to modify the electrode using the aforementioned method is lengthy and the detection limit of it to NO· is higher (27 nM > 7.2 nM) than the reference that we chose to follow for modifying our electrodes. One hypothesis for the evident difference between the two LODs is that the CS encapsulates the GNPs, increasing the availability of active sites to NO·. Therefore, CS could have an enhancing effect on the catalytic capabilities of the individual GNP by homogeneously distributing them throughout the CS/GNP film. The GNPs that self-assemble into mono-/bi-layer films are organized in a packed fashion, where the GNPs are touching. This could limit the active sites available to NO· and decrease the efficiency of electron transfer through the GNPs. Therefore, we do not think that a fully GNP covered T-CUA would have better detection limits or sensitivity to the detection NO·. The enriching effect provided by the CS/GNP modification improves the LOD for the 1.54T-CUA by 50% (Table 2). Present GNP modified optically transparent electrodes33, 47, 48 for NO• detection have been characterized without the consideration of a membrane, therefore lacking the real concern for biological interferents and biofouling. When compared to modified and unmodified T-Macro and 1.54T-


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CUA electrodes, the latter have competitive LODs for the detection of NO• with the CA/CS/GNP 1.54T-CUA having the best LOD at 0.2 ± 0.1 µM with a sensitivity of 9 ± 9 nA/µM.

Figure 6. SEM images of a., unmodified 1.54T-CUA, b., individual electrode of CS/GNP 1.54T-CUA and c., CS/GNP 1.54T-CUA. The lower right graph d., displays cyclic voltammograms of NO• oxidation at a modified and at an unmodified electrode (scan rate 100 mV/s, 2 mM NO•).

The established physiological concentration range for NO• is 1-100 nM,49 and NO• concentrations of 1 µM have been determined in in vitro endothelial cell experiments.50 During an infection, pathophysiological pathways are activated within immune cells increasing the levels of NO• by several orders of magnitude (0.68-10 µM) via inducible nitric oxide synthase (iNOS).51, 52 The LODs of our electrodes are not within the non-stimulated (non-infectious or non-pathophysiological) physiological NO• concentration range. Though, they are within NO• concentrations under pathophysiological and in vitro endothelial cell environments. Therefore our immediate focus will be on applying our electrodes to investigate NO• under stimulated conditions.

techniques, such as chronoamperometry, and increasing the density of the T-CUAs may accomplish lower LODs. Conclusion. T-Macro and T-CUA electrodes were fabricated for the detection of NO•. The electrodes were modified by a CA gas permeable membrane selective to NO•. A comprehensive characterization of the membrane was performed via chronoamperometry, EIS, and AFM. AFM and EIS confirmed that the average membrane thickness is ~100 nm. The diffusional and partitioning behavior across the membrane of NO• and NO2- was determined. The CA membrane is 10 times more permeable to NO• than NO2-. This investigation of the CA membrane’s permeability to NO• and NO2- gives a quantitative understanding of the membrane’s kinetic parameters establishing why the membrane has a greater affinity to NO•. The sensitivity, catalytic activity, and LOD of the electrode to NO•, was enhanced by CS/GNP modification of the electrode. A simple, controllable one-step electrodeposition was used to form the CS/GNP film onto the surface of the T-Macro and 1.54T-CUA. The composite films were characterized using SEM showing that the GNP particle size ranges from 5-80 nm. The significant catalytic enhancement provided by the modification yields a 50% improvement in the LOD for the 1.54T-CUA.

AUTHOR INFORMATION Corresponding Author * [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT JD acknowledges support from “Understanding Charge Separation and Transfer at Interfaces in Energy Materials (EFRC:CST),” an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Award Number DE-SC0001091. JE acknowledges support from the Welch Foundation, grants F1331 and F-1529, respectively.

SUPPORTING INFORMATION

Figure 7. (a) Background corrected CA/CS/GNP T-Macro square wave voltammograms for various concentrations of NO•. (b) The calibration curve for the CA/CS/GNP 1.54T-CUA.

Thus, the T-CUAs have been engineered with the intent to observe and determine the in vitro cellular expression of NO•. The LOD of the CA/CS/GNP 1.54T-CUA is within the range of NO• concentrations generated during endothelial cell and pathophysiological processes alluding to the probable detection of NO• in cellular and under pathophysiological environments. Future work will consist of in vitro experiments that will employ the CA/CS/GNP 1.54T-CUA to investigate various cellular behaviors that hinge upon the secretion of NO•. We will continue to work on decreasing the LODs of our electrodes until they are within the NO• concentration range of un-stimulated physiological circumstances. The application of other electroanalytical

Figure S-1. Scanning electron microscopy (SEM) images of the CS/GNP 1.54T-CUAs. Figure S-2. Electrochemical impedance spectroscopy (EIS) spectra for nitrite. Figure S-3. Charging current decays for the various electrodes. RC Time Constant Calculation. Response Time Calculation. Table S-1. The RC time constants and response times for the different electrodes. Figure S-4. Stability measurements of 3 1.54T-CUAs covered with CA membranes. This material is available free of charge via the Internet at http://pubs.acs.org.

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