Simultaneous electrochemical sensing of ascorbic acid and uric acid under biofouling conditions using nanoporous gold electrodes

Journal of Electroanalytical Chemistry 846 (2019) 113160

Contents lists available at ScienceDirect

Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem

Simultaneous electrochemical sensing of ascorbic acid and uric acid under biofouling conditions using nanoporous gold electrodes

T

Tiago Almeida Silvaa,b, Md Rezaul Karim Khanc, Orlando Fatibello-Filhoa, ⁎ Maryanne M. Collinsonc, a

Department of Chemistry, Federal University of São Carlos, São Carlos, P.O. Box 676, 13560-970, SP, Brazil Department of Metallurgy and Chemistry, Federal Center for Technological Education of Minas Gerais, Timóteo, 35180-008, MG, Brazil c Department of Chemistry, Virginia Commonwealth University, Richmond, VA 23284-2006, United States b

ARTICLE INFO

ABSTRACT

Keywords: Biosensing Serum Blood Complex matrix Biosieving Voltammetry

Direct electrochemical sensing of biomolecules in samples with complex matrices such as human serum and blood is challenged by sensor surface biofouling resulting from the adsorption of concomitant macromolecules, particularly proteins. In this research, nanoporous gold (NPG) electrodes prepared from dealloying of a low-cost silver‑gold alloy are applied as antibiofouling electrodes for the simultaneous determination of ascorbic acid (AA) and uric acid (UA). From cyclic voltammetric assays, the antibiofouling features of the fabricated NPG were interrogated in the presence of biofouling agents, fibrinogen (FN) and bovine serum albumin (BSA) proteins using ascorbic acid and uric acid as the redox probes. It was shown that the porous surface of NPG ensured a stable signal for the redox probe even in the presence of proteins found in biological samples. The antibiofouling capability of NPG is credited to the biosieving effect of the morphology of the NPG, restricting the diffusion of large proteins into gold pores while permitting access of small electroactive molecules to efficiently exchange electrons. By applying differential pulse voltammetry (DPV) under optimum conditions, the AA and UA biomolecules were simultaneously determined with limits of detection of 63.0 and 9.0 μM, respectively. The DPV signals of AA and UA were stable on NPG in a biofouling environment and, the proposed NPG sensor was successfully applied for the simultaneous determination of AA and UA in the mimic human serum sample of fetal bovine serum (FBS).

1. Introduction Small molecules are involved in different biological processes of human metabolism and, therefore, the maintenance of normal concentrations of small molecules is required to preserve the physiological functions of the human body [1]. Among them, ascorbic acid (AA) and uric acid (UA) are examples of small biomolecules that play an important role in an individual's health. AA, more popularly known as vitamin C, is a water-soluble vitamin acting as a cofactor in different biosynthetic routes, including the pathways of collagen and carnitine synthesis as well as the conversion of neurotransmitters [2–5]. The reducing agent features of AA makes this vitamin an effective antioxidant in human blood plasma, protecting the human body against diseases and degenerative processes caused by oxidative stress [6,7]. The lack of AA causes scurvy, connective tissue damage, and even death in cases of acute AA deficiency [2]. Nevertheless, some authors have raised questions on the potential side effects of the intake of large

⁎

dosages of AA [8,9]. UA is the final product of complex purine metabolism, which can vary by different factors such as diet and intake of pharmaceutical drugs [10]. UA is a weak acid (pKa = 5.8), and at physiological pH, it exists mainly as a urate salt [11]. As the urate concentration increases in blood from abnormal overproduction or under-excretion, its crystal formation increases resulting in the pathologic conditions of hyperuricemia and gout [10,11]. The increase of UA content in blood has also served as a marker of cardiovascular disease risk [12,13]. Therefore, AA and UA concentrations in blood can serve as indicators of serious health problem conditions; the control of the content of these biomolecules in blood is important in clinical analysis. Electroanalytical methods appear as potential alternatives for the simultaneous determination of AA and UA. In literature, it is possible to find much work dedicated to the simultaneous voltammetric sensing of these biomolecules. Most work has focused on the proposition of new nanostructured modified surfaces with an enhanced response towards the oxidation of these biomolecules. From the use of those modified

Corresponding author. E-mail address: [email protected] (M.M. Collinson).

https://doi.org/10.1016/j.jelechem.2019.05.042 Received 24 January 2019; Received in revised form 15 May 2019; Accepted 16 May 2019 Available online 23 May 2019 1572-6657/ © 2019 Elsevier B.V. All rights reserved.

Journal of Electroanalytical Chemistry 846 (2019) 113160

T.A. Silva, et al.

sensors, wide linear ranges and limits of detection at micromolar levels could be achieved. Despite promising analytical figures-of-merit, it was noticed that the practical challenge associated with the electrode surface biofouling was not addressed. In some applications, the complex matrix can interfere with the analyte response or block the electrode surface due to adsorption. The adsorption of organic compounds from complex biological samples on electrode surfaces is called biofouling, and it can occur by hydrophobic or electrostatic interactions [14,15]. A major example of biofouling occurs precisely in the electrochemical sensing of biomolecules at in vitro or in vivo conditions, where the determinations are affected by the adsorption of platelet, fibrin and cells. The biofouling effect is very problematic because it can cause a decrease in the electron transfer rate as well as the analytical signal. In a more serious case, the signal is completely faded. Therefore, the accuracy and precision of the method are affected, and strategies are needed to eliminate this problem and ensure method reliability. In the literature, many studies have focused on the construction of antifouling electrodic surfaces. At the moment, the most relevant experimental approaches involve the physical/chemical modification of the surface with antifouling layers and manipulating the electrode surface topography [14]. With regard to electrode surface modification, the use of semipermeable membranes has been employed to prevent the biofouling by the size exclusion principle, where only the target analyte molecules are permeable and the fouling molecules are blocked in the membrane [16–19]. Despite satisfactory results, these strategies still present some drawbacks such as low coating life-time, poor reproducibility of (bio)sensor preparation and practical and financial limitations. In more recent years, the ability to manipulate the topography of electrode materials has opened new horizons regarding the construction of biofouling resistant electrodes. Collinson and collaborators [20–22] showed the concept that the electrode biofouling from protein adsorption can be minimized or eliminated by the adequate electrode topography manipulating of nanoporous gold surfaces. It is believed that the small nanopores in the 3D open framework of the porous gold restrict the transport of large biomolecules, thus minimizing passivation of the inner surfaces while permitting access to small redox probes to efficiently exchange electrons. From that, nanoporous gold has been successfully explored to conduct potentiometric measurements in a biofouling environment [23,24] and to the design of antibiofouling DNA-sensors [25–27]. In this research, we extended the range of electroanalytical possibilities of nanoporous gold-based sensors toward the simultaneous voltammetric sensing of biomolecules such as AA and UA containing proteins known to adsorb and biofoul metallic surfaces. We evaluated the voltammetric response of these biomolecules on the nanoporous gold surface and on a conventional flat gold surface, from which it was possible to diagnose the changes associated with electrochemical responsiveness. Moreover, we have carried out simultaneous studies toward AA and UA voltammetric response under biofouling conditions.

electrolyte solution (0.1 M phosphate buffer, pH 7.4) and the 10.0 mM UA stock solution was prepared in 0.1 M NaOH solution; at this concentration level the UA compound is not completely solubilized at pH 7.4. 2.2. Instrumentation Scanning electron microscopy (SEM) analysis was performed on a Hitachi SU 70 field emission scanning electron microscope. For the Xray diffraction (XRD) measurements was used a PANalytical MPD X'Pert Pro equipment. Electrochemical assays were carried out using a Metrohm Autolab Potentiostat/Galvanostat (Model PGSTAT 128 N) driven by the NOVA software. A 10 mL three-electrode electrochemical cell was adopted. Planar Au or NPG were used as the working electrode while a Pt wire and a Ag/AgCl (1.0 M KCl) were applied as counter and reference electrodes, respectively. The geometric area of planar Au and NPG electrodes was defined by a 1/8-in. circle hand punched into a piece of tape, which was carefully placed on the electrode surface. 2.3. Preparation of NPG electrode Planar Au slides (EMF, Inc.) were used as a substrate to capture the NPG leaf after the dealloying process. The planar Au slides were first cut into rectangular pieces and successfully sonicated for 10 min in acetone, ethanol and ultrapure water followed by drying with a stream of nitrogen gas and exposure to an oxygen plasma for 5 min (Southbay, 18 W) in order to remove any residual impurities [28]. A commercial AgeAu alloy leaf (1 cm × 1 cm) was dealloyed in concentrated HNO3 for 14 min followed by multiple exposures to DI water in order to completely remove Ag+ and NO3− residual ions [20,23]. Finally, the NPG leaf was captured on the planar gold slide and cleaned under UV radiation for at least 6 h. All the described steps should be gently performed as the commercial gold leaves are very fragile. 2.4. Electrochemical assays Cyclic voltammetry (CV) was used to explore the electrochemical features of planar Au and NPG electrodes first towards the inorganic redox probe, potassium hexacyanoferrate (III) (K3Fe(CN)6). To evaluate the antibiofouling properties of planar Au and NPG, the CV profile of both was compared before and after exposure to known biofouling agents in supporting electrolyte. Following this initial characterization step, CV was then used to investigate the voltammetric response of AA and UA biomolecules on planar Au and NPG electrodes again in the absence and presence of biofouling agents. Posteriorly, the analytical features of NPG electrode towards the simultaneous determination of AA and UA were evaluated by applying the pulsed voltammetric technique of differential pulse voltammetry (DPV). Therefore, the main technical parameters affecting the responsivity of this voltammetric tool were subjected to optimization, these being scan rate (v), amplitude (a) and modulation time (tm). Under optimized experimental conditions, the respective AA and UA analytical curves were constructed from successive injections of aliquots of AA and UA stock solutions in the electrochemical cell previously filled with 6.0 mL of supporting electrolyte solution. From the obtained analytical curves, the analytical parameters for AA and UA simultaneous sensing were recorded (linear concentration range, sensitivity and limit of detection, LOD). The LOD was estimated from the Eq. (3 σ)/m. In this relation, σ is the standard deviation obtained for the current signal recorded through ten successive DPV measurements performed for the blank solution (only supporting electrolyte) and m is sensitivity (slope of the analytical curve).

2. Materials and methods 2.1. Reagents and solutions All chemical reagents employed in this research were of analytical grade and used as received from the respective suppliers (Fisher Scientific or Sigma-Aldrich). Bovine serum albumin (BSA) was purchased as a lyophilized powder and bovine fibrinogen (FN, 90% clottable) was from MP Biomedicals. Manetti 12 Karat gold leaf (50:50 Ag:Au alloy) was acquired from Fine Art Store. Fetal bovine serum (FBS) (Quality Biological Inc.) was kindly provided by Dr. Farrell (Department of Chemistry, VCU). Aqueous solutions were prepared using ultrapure water (resistivity not less than 18.2 MΩ cm) obtained from a Millipore Milli-Q® water purification system. Stock solutions of AA and UA at concentrations of 20.0 mM and 10.0 mM were prepared daily. The 20.0 mM AA stock solution was prepared in supporting 2

Journal of Electroanalytical Chemistry 846 (2019) 113160

T.A. Silva, et al.

Fig. 1. (a, b) SEM micrographs recorded for NPG at different magnifications. (c) XRD patterns of planar Au, NPG and the Ag:Au alloy. (d) Cyclic voltammograms recorded in 0.5 M H2SO4 using NPG (solid line) and planar Au electrodes (dashed line). Scan rate = 50 mV s−1.

3. Results and discussion

rather than (200). This could be the reason for the intense (111) peak, particularly with respect to the (200) in the x-ray diffraction pattern of planar Au. The electrochemically active surface area (ECSA) of NPG was evaluated via CV carried out in 0.5 M H2SO4 solution. For comparison purposes, the same experimental approach was applied at a planar Au electrode. In Fig. 1(d), the CV curves recorded for NPG and planar Au electrodes in the potential range of 0.0 to +1.6 V at 50 mV s−1 are shown. A typical CV response of metallic gold in sulfuric acid was noted, which consists of gold oxide formation during anodic potential scanning and a sharp cathodic peak upon inversion of the potential scanning direction that is related to the reduction of gold oxides formed in the anodic scan. Using the charge required for the gold oxide reduction and a conversion factor of 386 μC cm−2 [32], the ECSA of planar Au and NPG electrodes were measured as being 0.097 cm2 and 0.83 cm2. From the obtained ECSA, the roughness factors (Rf = ECSA/ geometric area) of 1.2 and 10.5 were determined for planar Au and NPG, respectively. Therefore, the NPG provided a surface area at least 8.8 times larger than a conventional planar Au, as a result of the porous nanostructured 3D-network of NPG (Fig. 1 (a) and (b)).

3.1. Material characterization The main objective of this work is to evaluate the analytical performance of a NPG based electrochemical sensor towards the simultaneous voltammetric determination of AA and UA under biofouling conditions. For the preparation of NPG, a dealloying process, which involves keeping 12 K gold leaf (Ag:Au alloy) submerged in concentrated HNO3 for a specific interval time (14 min in our case), was utilized. During this time, the less noble metal silver is removed, and the remaining gold atoms reorganize to form a nanoporous structure with a sieve-like nanostructure. The mechanism for the evolution of pores involves the dissolution/atom reorganization phenomena established by Erlebacher et al. [29]. In Fig. 1 (a) and (b), SEM micrographs of the NPG surface under different magnifications are shown. From these images, it is evident that after the dealloying procedure, a network of inter-connected gold pores with a diameter ranging between 10 and 40 nm (average pore size = 19.9 nm) was formed. Crystallographic information about commercial Ag:Au alloy, NPG and planar Au materials was evaluated by XRD. Fig. 1 (c) shows the XRD patterns recorded for Ag:Au alloy, NPG and planar Au surfaces. The diffraction peaks verified for NPG are consistent with a single face-centered-cubic (f.c.c.) Au phase. The XRD patterns observed for Ag:Au alloy and NPG are similar, and this data is understandable as Ag and Au have equivalent crystallographic profiles. However, the intensity of the diffraction peak assigned to (111) plane in NPG is approximately half the intensity observed for the Ag:Au alloy. This result supports the idea that the majority of the silver atoms were dissolved from the 50:50 Ag:Au alloy leaf during the dealloying process. Still, on the (100) crystallographic plane, a slight positive shift in 0.013° for NPG compared to planar Au was observed, which has been related to surface strain resulting from the curvature of nanoporous Au structure [30,31]. The planar Au electrode, which is 100 nm thick Au coated microscope slide, was prepared by sputter coating on 5 nm thick Ti adhesive layer on a glass substrate. During sputter coating Au particles are mainly oriented to (111) facets

3.2. Antibiofouling features of NPG Biological fluids such as serum and blood are known by their complex chemical composition, including salts, metabolites and proteins. Proteins, in particular, adsorb on metallic surfaces, particularly those often used in electrochemical experiments (platinum and gold). As a result of adsorption, a total or partial loss of electron transfer is often observed. Therefore, the application of electrochemical sensors in the sensing of small molecules in biological fluids is frequently hindered. In the specific case of gold, it is known that adsorption of proteins, such as human serum albumin, can influence its electrochemical response [33,34]. Nevertheless, recent work has demonstrated that NPG electrodes are suitable candidates for the electrochemical sensing of small molecules in complex biological matrix samples [23–27]. The antibiofouling features of NPG electrodes prepared in this work 3

Journal of Electroanalytical Chemistry 846 (2019) 113160

µ

µ

T.A. Silva, et al.

Fig. 2. CVs obtained for 1.0 mM K3Fe(CN)6 in 0.1 M KCl in the absence and presence of (a, b) 1.0 mg mL−1 FN or (d, e) 1.0 mg mL−1 BSA using planar Au and NPG electrodes. Scan rate = 100 mV s−1. Plots of the evolution of normalized cathodic peak currents during 1 h of exposure to (c) 1.0 mg mL−1 FN and (f) 1.0 mg mL−1 BSA using NPG electrodes.

were first evaluated using the redox probe, potassium hexacyanoferrate (III) in the presence of the biofouling agents, FN and BSA. FN and BSA were selected because they are ideal representatives of the majority protein constitution of human blood [35]. The biofouling studies were simultaneously carried out on planar Au electrodes, for comparison purposes. Fig. 2(a) and (b) shows the CVs acquired at planar Au and NPG electrodes in potassium hexacyanoferrate (III) before and after the addition of FN at 1.0 mg mL−1 concentration in the supporting electrolyte. At planar Au, the voltammetric profile of the redox probe changed dramatically when FN was added to the supporting electrolyte. The previous reversible diffusion-controlled redox reactions observed on planar Au disappeared instantly after the addition of FN. In this case, the FN protein quickly adsorbed on the gold surface, creating a negatively charged layer that exerted repulsion to the negatively charged hexacyanoferrate(III) redox probe [20]. In contrast, at NPG electrodes, the voltammetric response before and after addition of FN was nearly identical. Fig. 2(c) depicts a plot of normalized peak current as a function of time following the addition of FN. The peak current stays nearly constant for NPG electrodes, whereas it drops quickly for planar gold electrodes consistent with previous results [20]. Similar results were also observed for potassium hexacyanoferrate(III) in the presence of 1.0 mg mL−1 BSA as shown in Fig. 2 (d–f). The antibiofouling properties of NPG surfaces result from a biosieving effect due to the unique morphology of this nanostructured electrode [20]. The biosieving effect is based on the simple concept that in a complex solution containing chemical species of different molecular sizes, only those molecules with a hydrated diameter equal or smaller than the NPG pore size will be able to enter into the interior of the pores, whereas large molecules like proteins will remain outside. It is worth mentioning that the average pore size of NPG (19.9 nm) was smaller than the length of the protein such as FN (48 ± 3 nm) [36] Therefore, small electroactive molecules may exchange electrons in the interior of the porous gold framework without apparent loss of electron transfer. In addition, the electron transfer kinetics for small molecules can also be improved due to the nanoconfinement effect; this aspect will be addressed in the next sections dedicated to electrochemistry of AA and UA. The results obtained using FN and BSA as biofouling agents demonstrate that the as-prepared NPG electrodes are able to make

electrochemical measurements in biofouling environments consistent with that previously reported [20,26]. 3.3. Electrochemical response of AA and UA on NPG and planar Au Using CV, the electrochemical response of AA and UA in 0.1 M phosphate buffer solution at pH 7.4 was investigated at planar Au and NPG electrodes in the presence and absence of known biofouling proteins. At this pH, AA and UA exhibit irreversible 2e−/H+ oxidation reactions, with some occurrences of more complex oxidation mechanisms depending on gold crystallographic orientation [37,38]. Fig. 3(a) and (b) depict the CVs recorded for 2.0 mM AA and 1.0 mM UA at 50 mV s−1, respectively. These CVs are consistent with an irreversible oxidation reaction on both planar and NPG electrodes. It is clear by the peak current and peak position that AA and UA oxidation reaction kinetics are influenced by the electrode morphology. Comparatively, the irreversible AA and UA anodic peaks recorded at NPG exhibit a higher peak current and a less positive peak potential than observed at planar Au. The improved anodic response of AA and UA on NPG can be explained by the high surface area provided from the nanoporous morphology as well as nanoconfinement effects [39,40]. Compared to the relatively fast electron transfer kinetics of the hexacyanoferrate (III) redox probe, the slower electron transfer rates of AA and UA redox processes mean that more of the inner pore surface area is accessed, hence higher currents can be observed compared to that obtained on planar gold [40,41]. Likewise, confinement of the electroactive species in the nanoscale porous framework can lead to a larger contact time of electroactive species with the electrified gold surface and elevates the probability of electron transfer in the heterogeneous electrode/solution interface; consequently, the electron transfer kinetic becomes faster in the internal environment of gold nanopores [40]. This “nanoconfinement effect” results in improved electron transfer kinetics and explains the more negative peak potentials of AA and UA on NPG. The signal stability of NPG electrode towards the simultaneous voltammetric sensing of AA and UA was evaluated from successive DPV measurements carried out for a solution containing 1.4 mM AA and 0.46 mM UA. The DP voltammograms recorded during eighteen successive measurements are provided in Fig. 4 (a), and the monitoring of 4

Journal of Electroanalytical Chemistry 846 (2019) 113160

µ

µ

T.A. Silva, et al.

Fig. 3. CV obtained for (a) 2.0 mM AA and (b) 1.0 mM UA in 0.1 M phosphate buffer (pH = 7.4) using NPG and planar Au electrodes. Dashed lines indicate the CVs recorded for the blank solution (i.e. only supporting electrolyte). Scan rate: 50 mV s−1.

peak currents of AA and UA through the successive DPV measurements showed in Fig. 4 (b). Practically overlapped DP voltammograms were obtained, indicating excellent stability of the signal. Graphics of Fig. 4 (b) illustrate the stable behavior of AA and UA signals, with RSDs of only 2.4% and 2.1%, respectively.

constructed from the injection of aliquots from the respective stock solutions of AA and UA in supporting electrolyte. The DP voltammograms recorded as the concentration of both AA and UA increased is shown in Fig. 6(a) and the analytical calibration curves can be seen in Fig. 6(b) and (c). The voltammetric response for both AA and UA is linearly dependent on concentration in the range of 0.32 to 3.4 mM (AA) and 0.065 to 1.5 mM (UA). The regression equations are:

3.4. Simultaneous voltammetric sensing of AA and UA on NPG

AA: ip, µA =

The simultaneous determination of AA and UA at NPG was conducted by DPV as it is a highly sensitive electroanalytical tool. The relevant experimental parameters (e.g., scan rate, amplitude and modulation time) affecting the DPV response were first optimized. Initially, individual analytical curves for AA and UA were constructed by keeping the concentration of one of the two analytes constant and varying the other. Figs. 5(a) and (b) depict the DP voltammograms recorded for the individual determination of AA and UA when the concentration of the other analyte was fixed. In Fig. 5(a), the concentration of AA was changed from 0.31 to 2.9 mM while UA concentration was constant at 0.49 mM. From the DP voltammograms of Fig. 5(a), it is clear that AA peak current at ~0.05 V increases with concentration while the UA peak current at ~0.35 V was nearly constant, showing a RSD of only 2.4%. In Fig. 5(b) the UA concentration was varied from 0.060 to 1.2 mM, while the AA concentration was fixed at 1.4 mM. Similarly, two peaks are observed in the DPV. The UA peak at ~0.35 V increases with concentration while the AA peak near ~0.05 V stays approximately constant with a RSD = 9.1%. Next, the simultaneous analytical curves for AA and UA were

2.75 µA + 9.22 µA mM 1 [AA], mM, r = 0.999

UA: ip, µA = 4.62 µA + 19.4 µA mM 1 [UA], mM, r = 0.995

∆

µ

From the analytical sensitivities, the limits of detection for AA and UA were calculated as 63 μM and 9.0 μM, respectively. The obtained limits of detection are lower than the concentration ranges which these compounds are found in the human blood at normal or disturbed physiological conditions. The normal UA concentration in blood is found in the range of 0.16 to 0.37 mM in women and of 0.22 to 0.45 mM in men [42], while reference AA concentration values are in the range of 36.1 to 79.4 μM [43]. In Table 1, analytical parameters obtained at different electrochemical sensors from the literature are shown. Although the analytical parameters of most of the studies were better than those reported by us, there was no mention in any study on the anti-biofouling properties of the modified electrodes, which is of fundamental importance for practical purposes and represents the greatest novelty of the proposed voltammetric procedure. 5-HTP/GCE: glassy carbon electrode modified with 5-hydroxytryptophan; GNP/LC/GCE: gold nanoparticles-l-cysteine-modified glassy carbon electrode; PAN-ABSA/GCE: polyaniline nano-networks/

Fig. 4. (a) Successive DP voltammograms recorded in 0.1 M phosphate buffer (pH = 7.4) containing both 1.4 mM AA and 0.46 mM UA. DPV parameters: scan rate = 50 mV s−1, amplitude = 80 mV and modulation time = 50 ms. (b) Plot of Δip vs. measurement number. 5

Journal of Electroanalytical Chemistry 846 (2019) 113160

∆ µ

T.A. Silva, et al.

∆

µ

∆ µ

Fig. 5. (a) DP voltammograms recorded in 0.1 M phosphate buffer (pH = 7.4) containing 0.49 mM UA and different concentrations of AA (0.31 mM (line 1) to 2.9 mM (line 9)) using a NPG electrode. (b) DP voltammograms recorded in 0.1 M phosphate buffer (pH = 7.4) containing 1.4 mM AA and different concentrations of UA (0.060 mM (line 1) to 1.2 mM (line 9)) using a NPG electrode. DPV parameters: scan rate = 50 mV s−1, amplitude = 80 mV and modulation time = 50 ms.

Fig. 6. (a) DP voltammograms recorded in 0.1 M phosphate buffer (pH = 7.4) containing different concentrations of both AA (0.33 mM (line 1) to 3.4 mM (line 12)) and UA (0.065 mM (line 1) to 1.5 mM (line 12)) using a NPG electrode. DPV parameters: scan rate = 50 mV s−1, amplitude = 80 mV and modulation time = 50 ms. (b) Analytical curve obtained for AA (Δip vs. [AA]) and (c) Analytical curve obtained for UA (Δip vs. [UA]).

p-aminobenzene sulfonic acid modified glassy carbon electrode; pTSAGCE: glassy carbon electrode modified with electropolymerized film of p-toluene sulfonic acid; L-Cys/Au: L-cysteine self-assembled monolayers modified gold electrode; MB-SGGCE: sol-gel ceramic film incorporating methylene blue on glassy carbon electrode; Glu/GCE: covalently modified glassy carbon electrode with glutamic acid; EPPG: Edge Plane Pyrolytic Graphite Electrode; NEMGCE: norepinephrine modified glassy carbon electrode.

3.5. Simultaneous voltammetric sensing of AA and UA on NPG in the presence of FN The antibiofouling features of the NPG electrode towards the simultaneous voltammetric determination of AA and UA were first evaluated using FN. In this case, the DPV response was acquired before and after the electrode was exposed to 1.0 mg mL−1 FN solution for 1 h. The adsorption of proteins like FN on gold surfaces normally takes place quickly; however, the use of a long exposure time of 1 h is tied to the 6

Journal of Electroanalytical Chemistry 846 (2019) 113160

T.A. Silva, et al.

Table 1 Comparison of analytical parameters recorded by electrochemical sensors. Electrode

LOD (μM)

AA

UA

AA

UA

0.006 to 0.1 0.008 to 5.5 0.035 to 0.175 0.1 to 1.2 0.14 to 0.5 0.6 to 2.75 5.0 × 10−6 to 0.1 0.001 to 0.4 1.0 × 10−4 to 1.0 0.04 to 1.0 0.32 to 3.4

0.0007 to 0.011 0.0007 to 0.85 0.05 to 0.25 0.001 to 0.1 0.054 to 0.15 0.01 to 0.1 1.0 × 10−6 to 0.05 0.002 to 0.4 11.0 × 10−4 to 1.0 0.01 to 0.6 0.065 to 1.5

4.2 3.3 7.5 80 11 5.0 0.00045 0.92 0.12 – 63

0.28 0.2 12 0.5 2.0 10 0.00025 1.1 0.05 – 9.0

Biofouling tests

Reference

No No No No No No No No No No Yes

[44] [45] [46] [47] [48] [49] [50] [51] [52] [53] This work

∆ µ

5-HTP/GCE GNP/LC/GCE PAN-ABSA/GCE pTSA-GCE L-Cys/Au Sonogel-Carbon-L-Cysteine electrode MB-SGGCE Glu/GCE EPPG NEMGCE NPG

Linear range (mM)

Fig. 7. DP voltammograms recorded in 0.1 M phosphate buffer (pH = 7.4) containing 1.4 mM AA and 0.46 mM UA using a (a) NPG electrode and (b) planar Au electrode before (solid line) and after (dashed line) the electrode was soaked for 1 h in a buffered solution containing 1.0 mg mL−1 FN. DPV parameters: scan rate = 50 mV s−1, amplitude = 80 mV and modulation time = 50 ms.

reality of routine clinical analysis where the electrochemical sensor is exposed to biological matrices for a long time. For comparison, the same assays were performed using a planar Au electrode. The DP voltammetric profile of AA and UA on planar Au and NPG electrodes before and after exposure to FN is shown in Fig. 7(a) and (b). At NPG, a well-defined voltammetric profile for both AA and UA was achieved both before and after exposure to FN. Good peak-to-peak potential separation, constant baseline and, most importantly, practically the same peak current intensities can be noted. At planar Au, however, significant differences in the DP voltammograms before and after electrode exposure to FN can be noted (Fig. 7(b)). The peaks for both UA and AA are well defined before exposure while after exposure, the AA peak is clearly diminished. Interestingly, the loss of electron transfer because of the adsorption of the protein layer on the planar Au surface was observed only in the AA case. The anodic peak current for UA remained constant after the electrode was modified with FN. At present we do not fully understand why AA is greatly affected by the adsorbed protein layer while UA is not. One reason could be due to differences in the pKa values and thus differences in the proportion of the negatively charged ion to its protonated counterpart. At pH 7.4, both analytes will be predominately anions. However, the pKa of AA is 4.1 while for UA the pKa is 5.8. Thus, at pH 7.4, the ratio of the urate ion/uric acid concentrations is ~ 40 to 1 while the ratio of ascorbate ion/ascorbic acid is ~1700 to 1. Alternatively, the ability of urate/uric acid to bind to gold via the N group may play a factor. Additional investigations are needed to fully understand the reasons behind this observation. Even though UA could be apparently determined under biofouling conditions using a planar Au electrode, the real simultaneous

voltammetric sensing of AA and UA biomolecules under biofouling conditions on planar Au electrodes is impracticable. These observations confirm that the NPG electrode could completely minimize or eliminate any negative effect due to the adsorption of protein structures on its surface. As discussed previously, the surface of the NPG electrode is composed of pores with a diameter smaller than the average molecular size of FN. As such, only the small molecules (AA and UA) can penetrate into the nanopores and undergo electron transfer. This biosieving effect guarantees the electroactivity of both AA and UA under biofouling conditions; the potential of NPG for the direct and simultaneous determination of these small biomolecules in complex biological samples becomes possible. 3.6. Simultaneous determination of AA and UA in fetal bovine serum (FBS) The applicability of the NPG electrode for the simultaneous determination of AA and UA biomolecules in a complex sample was demonstrated using spiked FBS. In our experiments, the methodology applied by Seker and collaborators [25–27] involving the detection of DNA on NPG was adopted. A 10% (v/v) FBS sample was prepared in 0.1 M phosphate buffer solution (pH 7.4) and spiked with a known amount of AA and UA. In Fig. 8(a), the DP voltammograms obtained in the FBS sample before and after spiking with AA and UA are shown. Before addition of AA and UA, no peaks are observed. Posteriorly, after spiking the FBS sample with 2.9 mM AA and 1.3 mM UA, two anodic peaks corresponding to AA and UA are observed. The experiments were performed in triplicate and, by interpolation of the recorded peak currents in the respective analytical curves, the added AA and UA concentrations were recovered. Excellent average recoveries of 7

Journal of Electroanalytical Chemistry 846 (2019) 113160

T.A. Silva, et al.

Fig. 8. (a) DP voltammograms recorded at NPG in 10% (v/v) FBS (dashed line) and 10% (v/v) FBS spiked at 2.9 mM AA and 1.3 mM UA (solid line). Inset: DP voltammogram at a planar Au electrode in 10% (v/v) FBS spiked at 2.9 mM AA and 1.3 mM UA. DPV parameters: scan rate = 50 mV s−1, amplitude = 80 mV and modulation time = 50 ms. (b) Comparison of DP voltammograms recorded at NPG in spiked 10% (v/v) FBS (solid line) and 0.1 M phosphate buffer (pH = 7.4) containing 2.9 mM AA and 1.3 mM UA (dashed line).

95 ± 1% and 97 ± 3% were achieved for AA and UA, respectively. In Fig. 8(b), the DP voltammograms recorded for 2.9 mM AA and 1.3 mM UA in buffer solution and in the spiked FBS sample are compared. The similarity of the voltammetric profile and peak current intensities emphasizes the absence of any interference or biofouling effect, validating the excellent recovery percentages. This experiment was also performed at planar gold to confirm that the use of NPG is needed to simultaneously determine AA and UA in a complex matrix. The DP voltammogram at planar gold is shown in the inset of Fig. 8(a). Only UA yielded an anodic response; no oxidation peak was noted for AA. It is evident from these experiments that a NPG electrochemical sensor with antibiofouling features is required to properly perform the simultaneous quantification of AA and UA at complex biological samples.

[3] G. Shaw, A. Lee-Barthel, M.L. Ross, B. Wang, K. Baar, Vitamin C-enriched gelatin supplementation before intermittent activity augments collagen synthesis, Am. J. Clin. Nutr. 105 (1) (2017) 136–143. [4] C.J. Rebouche, Ascorbic acid and carnitine biosynthesis, Am. J. Clin. Nutr. 54 (6) (1991) 1147S–1152S. [5] J.M. May, Z.-c. Qu, M.E. Meredith, Mechanisms of ascorbic acid stimulation of norepinephrine synthesis in neuronal cells, Biochem. Biophys. Res. Commun. 426 (1) (2012) 148–152. [6] B. Frei, L. England, B.N. Ames, Ascorbate is an outstanding antioxidant in human blood plasma, Proc. Natl. Acad. Sci. 86 (16) (1989) 6377–6381. [7] A.M. Pisoschi, A. Pop, The role of antioxidants in the chemistry of oxidative stress: a review, Eur. J. Med. Chem. 97 (2015) 55–74. [8] G.-C. Yen, P.-D. Duh, H.-L. Tsai, Antioxidant and pro-oxidant properties of ascorbic acid and gallic acid, Food Chem. 79 (3) (2002) 307–313. [9] J. Knight, K. Madduma-Liyanage, J.A. Mobley, D.G. Assimos, R.P. Holmes, Ascorbic acid intake and oxalate synthesis, Urolithiasis 44 (4) (2016) 289–297. [10] M. Gliozzi, N. Malara, S. Muscoli, V. Mollace, The treatment of hyperuricemia, Int. J. Cardiol. 213 (2016) 23–27. [11] J. Maiuolo, F. Oppedisano, S. Gratteri, C. Muscoli, V. Mollace, Regulation of uric acid metabolism and excretion, Int. J. Cardiol. 213 (2016) 8–14. [12] A.H. Wu, J.D. Gladden, M. Ahmed, A. Ahmed, G. Filippatos, Relation of serum uric acid to cardiovascular disease, Int. J. Cardiol. 213 (2016) 4–7. [13] W. Doehner, E.A. Jankowska, J. Springer, M. Lainscak, S.D. Anker, Uric acid and xanthine oxidase in heart failure — emerging data and therapeutic implications, Int. J. Cardiol. 213 (2016) 15–19. [14] A. Barfidokht, J.J. Gooding, Approaches toward allowing electroanalytical devices to be used in biological fluids, Electroanalysis 26 (6) (2014) 1182–1196. [15] N. Wisniewski, F. Moussy, M.W. Reichert, Characterization of implantable biosensor membrane biofouling, Fresenius J. Anal. Chem. 366(6) 611–621. [16] J.A. Rather, S. Pilehvar, K. De Wael, A biosensor fabricated by incorporation of a redox mediator into a carbon nanotube/nafion composite for tyrosinase immobilization: detection of matairesinol, an endocrine disruptor, Analyst 138 (1) (2013) 204–210. [17] C. Sun, J. Miao, J. Yan, K. Yang, C. Mao, J. Ju, J. Shen, Applications of antibiofouling PEG-coating in electrochemical biosensors for determination of glucose in whole blood, Electrochim. Acta 89 (2013) 549–554. [18] V. Beni, A. Valsesia, P. Colpo, F. Bretagnol, F. Rossi, D.W.M. Arrigan, Electrochemical properties of polymeric nanopatterned electrodes, Electrochem. Commun. 9 (7) (2007) 1833–1839. [19] A.L. Gui, E. Luais, J.R. Peterson, J.J. Gooding, Zwitterionic phenyl layers: finally, stable, anti-biofouling coatings that do not passivate electrodes, ACS Appl. Mater. Interfaces 5 (11) (2013) 4827–4835. [20] J. Patel, L. Radhakrishnan, B. Zhao, B. Uppalapati, R.C. Daniels, K.R. Ward, M.M. Collinson, Electrochemical properties of nanostructured porous gold electrodes in biofouling solutions, Anal. Chem. 85 (23) (2013) 11610–11618. [21] R.K. Khan, S.P. Gadiraju, M. Kumar, G.A. Hatmaker, B.J. Fisher, R. Natarajan, J.E. Reiner, M.M. Collinson, Redox potential measurements in red blood cell packets using nanoporous gold electrodes, ACS Sensors 3 (8) (2018) 1601–1608. [22] A.A. Farghaly, R.K. Khan, M.M. Collinson, Biofouling-resistant platinum bimetallic alloys, ACS Appl. Mater. Interfaces 10 (25) (2018) 21103–21112. [23] A.A. Farghaly, M. Lam, C.J. Freeman, B. Uppalapati, M.M. Collinson, Potentiometric measurements in biofouling solutions: comparison of nanoporous gold to planar gold, J. Electrochem. Soc. 163 (4) (2016) H3083–H3087. [24] C.J. Freeman, A.A. Farghaly, H. Choudhary, A.E. Chavis, K.T. Brady, J.E. Reiner, M.M. Collinson, Microdroplet-based potentiometric redox measurements on gold nanoporous electrodes, Anal. Chem. 88 (7) (2016) 3768–3774. [25] P. Daggumati, Z. Matharu, L. Wang, E. Seker, Biofouling-resilient nanoporous gold electrodes for DNA sensing, Anal. Chem. 87 (17) (2015) 8618–8622.

4. Conclusions Nanoporous gold electrodes were prepared by a simple dealloying procedure from a commercial silver‑gold alloy. From different CV experiments, a high surface area along with antibiofouling features were verified for the proposed NPG by using the redox probe, potassium hexacyanoferrate (III) and the biofouling agents fibrinogen and albumin. Exploring via DPV, the small biomolecules AA and UA were determined in the linear ranges of 0.32 to 3.4 mM and 0.065 to 1.5 mM, with the limits of detection of 63 μM and 9.0 μM, respectively. The designed NPG electrode ensured excellent electrochemical activity towards the simultaneous voltammetric sensing of AA and UA under biofouling conditions, demonstrated especially from recovery experiments conducted for the complex biological sample FBS. Acknowledgments The authors are grateful to the financial support of the VCU Presidential Research Incentive Fund and Virginia Blood Foundation. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) Finance Code 001. Tiago A. Silva is especially grateful to CAPES foundation for a sandwich doctorate scholarship (Proc. N.: 88881.132463/2016-01). References [1] C. Feng, S. Dai, L. Wang, Optical aptasensors for quantitative detection of small biomolecules: a review, Biosens. Bioelectron. 59 (2014) 64–74. [2] Y. Li, H.E. Schellhorn, New developments and novel therapeutic perspectives for vitamin C, J. Nutr. 137 (10) (2007) 2171–2184.

8

Journal of Electroanalytical Chemistry 846 (2019) 113160

T.A. Silva, et al. [26] Z. Matharu, P. Daggumati, L. Wang, T.S. Dorofeeva, Z. Li, E. Seker, Nanoporousgold-based electrode morphology libraries for investigating structure–property relationships in nucleic acid based electrochemical biosensors, ACS Appl. Mater. Interfaces 9 (15) (2017) 12959–12966. [27] P. Daggumati, S. Appelt, Z. Matharu, M.L. Marco, E. Seker, Sequence-specific electrical purification of nucleic acids with nanoporous gold electrodes, J. Am. Chem. Soc. 138 (24) (2016) 7711–7717. [28] R.K. Khan, A.A. Farghaly, T.A. Silva, D. Ye, M.M. Collinson, Gold-nanoparticledecorated titanium nitride electrodes prepared by glancing-angle deposition for sensing applications, ACS Appl. Nano Mater. 2 (3) (2019) 1562–1569. [29] J. Erlebacher, An atomistic description of dealloying: porosity evolution, the critical potential, and rate-limiting behavior, J. Electrochem. Soc. 151 (10) (2004) C614–C626. [30] X. Yan, X. Ge, S. Cui, Pt-decorated nanoporous gold for glucose electrooxidation in neutral and alkaline solutions, Nanoscale Res. Lett. 6 (1) (2011) 313. [31] E.J. Schofield, B. Ingham, A. Turnbull, M.F. Toney, M.P. Ryan, Strain development in nanoporous metallic foils formed by dealloying, Appl. Phys. Lett. 92 (4) (2008) 043118. [32] R. Szamocki, A. Velichko, C. Holzapfel, F. Mücklich, S. Ravaine, P. Garrigue, N. Sojic, R. Hempelmann, A. Kuhn, Macroporous ultramicroelectrodes for improved electroanalytical measurements, Anal. Chem. 79 (2) (2007) 533–539. [33] S.E. Moulton, J.N. Barisci, A. Bath, R. Stella, G.G. Wallace, Investigation of protein adsorption and electrochemical behavior at a gold electrode, J. Colloid Interface Sci. 261 (2) (2003) 312–319. [34] P. Ying, A.S. Viana, L.M. Abrantes, G. Jin, Adsorption of human serum albumin onto gold: a combined electrochemical and ellipsometric study, J. Colloid Interface Sci. 279 (1) (2004) 95–99. [35] J.T. Nix, F.C. Mann, J.L. Bollman, J.H. Grindlay, E.V. Flock, Alterations of protein constituents of lymph by specific injury to the liver, Am. J. Phys. 164 (1) (1950) 119–122. [36] C.E. Hall, H.S. Slayter, The fibrinogen molecule: its size, shape, and mode of polymerization, J. Biophys. Biochem. Cytol. 5 (1) (1959) 11–16. [37] B. Kumar Jena, C. Retna Raj, Morphology dependent electrocatalytic activity of au nanoparticles, Electrochem. Commun. 10 (6) (2008) 951–954. [38] V.C. Ferreira, J. Solla-Gullón, A. Aldaz, F. Silva, L.M. Abrantes, Progress in the understanding of surface structure and surfactant influence on the electrocatalytic activity of gold nanoparticles, Electrochim. Acta 56 (26) (2011) 9568–9574. [39] J.H. Bae, J.-H. Han, D. Han, T.D. Chung, Effects of adsorption and confinement on nanoporous electrochemistry, Faraday Discuss. 164(0) (2013) 361–376. [40] S. Park, H.C. Kim, T.D. Chung, Electrochemical analysis based on nanoporous structures, Analyst 137 (17) (2012) 3891–3903.

[41] M.M. Collinson, Nanoporous gold electrodes and their applications in analytical chemistry, ISRN Anal. Chem. (2013) (2013) 21. [42] G. Desideri, G. Castaldo, A. Lombardi, M. Mussap, A. Testa, R. Pontremoli, L. Punzi, C. Borghi, Is it time to revise the normal range of serum uric acid levels, Eur. Rev. Med. Pharmacol. Sci. 18 (9) (2014) 1295–1306. [43] J. Pincemail, #235, S. Vanbelle, F. Degrune, J.-P. Cheramy-Bien, C. Charlier, J.-P. Chapelle, D. Giet, G. Collette, A. Albert, J.-O. Defraigne, Lifestyle behaviours and plasma vitamin C and -carotene levels from the ELAN population, J. Nutr. Metab. 2011 (2011) 10. [44] X. Lin, Y. Li, Monolayer covalent modification of 5-hydroxytryptophan on glassy carbon electrodes for simultaneous determination of uric acid and ascorbic acid, Electrochim. Acta 51 (26) (2006) 5794–5801. [45] G. Hu, Y. Ma, Y. Guo, S. Shao, Electrocatalytic oxidation and simultaneous determination of uric acid and ascorbic acid on the gold nanoparticles-modified glassy carbon electrode, Electrochim. Acta 53 (22) (2008) 6610–6615. [46] L. Zhang, C. Zhang, J. Lian, Electrochemical synthesis of polyaniline nano-networks on p-aminobenzene sulfonic acid functionalized glassy carbon electrode: its use for the simultaneous determination of ascorbic acid and uric acid, Biosens. Bioelectron. 24 (4) (2008) 690–695. [47] L. Wang, P. Huang, J. Bai, H. Wang, X. Wu, Y. Zhao, Voltammetric sensing of uric acid and ascorbic acid with poly (p-toluene sulfonic acid) modified electrode, Int. J. Electrochem. Sci. 1 (2006) 334–342. [48] Y. Zhao, J. Bai, L. Wang, P. Huang, H. Wang, L. Zhang, Simultaneous electrochemical determination of uric acid and ascorbic acid using L-cysteine self-assembled gold electrode, Int. J. Electrochem. Sci. 1 (2006) 363–371. [49] M. Choukairi, D. Bouchta, L. Bounab, M. Ben atyah, R. Elkhamlichi, F. Chaouket, I. Raissouni, I.N. Rodriguez, Electrochemical detection of uric acid and ascorbic acid: application in serum, J. Electroanal. Chem. 758 (2015) 117–124. [50] S.B. Khoo, F. Chen, Studies of sol–gel ceramic film incorporating methylene blue on glassy carbon: an electrocatalytic system for the simultaneous determination of ascorbic and uric acids, Anal. Chem. 74 (22) (2002) 5734–5741. [51] L. Zhang, X. Lin, Covalent modification of glassy carbon electrode with glutamic acid for simultaneous determination of uric acid and ascorbic acid, Analyst 126 (3) (2001) 367–370. [52] R.T. Kachoosangi, C.E. Banks, R.G. Compton, Simultaneous determination of uric acid and ascorbic acid using edge plane pyrolytic graphite electrodes, Electroanalysis 18 (8) (2006) 741–747. [53] H.R. Zare, F. Memarzadeh, M.M. Ardakani, M. Namazian, S.M. Golabi, Norepinephrine-modified glassy carbon electrode for the simultaneous determination of ascorbic acid and uric acid, Electrochim. Acta 50 (16) (2005) 3495–3502.

9