Amperometric microsensor based on nanoporous gold for ascorbic acid detection in highly acidic biological extracts

Journal Pre-proof Amperometric microsensor based on nanoporous gold for ascorbic acid detection in highly acidic biological extracts Abhishek Kumar, Vinicius L. Furtado, Josué M. Gonçalves, Renata BannitzFernandes, Luis Eduardo S. Netto, Koiti Araki, Mauro Bertotti PII:

S0003-2670(19)31232-2

DOI:

https://doi.org/10.1016/j.aca.2019.10.022

Reference:

ACA 237156

To appear in:

Analytica Chimica Acta

Received Date: 30 August 2019 Revised Date:

24 September 2019

Accepted Date: 14 October 2019

Please cite this article as: A. Kumar, V.L. Furtado, J.M. Gonçalves, R. Bannitz-Fernandes, L.E.S. Netto, K. Araki, M. Bertotti, Amperometric microsensor based on nanoporous gold for ascorbic acid detection in highly acidic biological extracts, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.10.022. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier B.V. All rights reserved.

Amperometric microsensor based on nanoporous gold for ascorbic acid detection in highly acidic biological extracts Abhishek Kumar1*, Vinicius L. Furtado1, Josué M. Gonçalves1, Renata Bannitz-Fernandes2, Luis Eduardo S. Netto2, Koiti Araki1, Mauro Bertotti1* 1

Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, Av. Professor Lineu Prestes, 748, 05513-970, São Paulo - SP, Brazil. 2 Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo, Rua do Matão, 321, 05508-090, São Paulo - SP, Brazil.

Abstract: Tuning the electrocatalytic properties of high surface area porous metallic frameworks like Nanoporous Gold (NPG) by tailoring the structure is a convenient strategy to design electrochemical sensors. Accordingly, an NPG-based sensitive, selective and robust electroanalytical platform was designed for the detection of ascorbic acid (AA) in acidic extracts of Aspergillus fumigatus fungus and Arabidopsis thaliana leaves. NPG films were electrodeposited on a gold microelectrode by potentiostatic electrodeposition and characterized by electron microscopy techniques, which confirmed the morphology and highly porous structure resembling nanowires-type pure gold fractals. The electrodeposition parameters, particularly deposition potential and time, were optimized to achieve large and selective amperometric detection of AA on the NPG modified electrodes. Faster electron transfer kinetics was manifested on the 0.3 V shift in overpotential and remarkable enhancement of the oxidation peak current as compared with bare gold electrode. Amperometric measurements were performed at 0.3 V vs. Ag/AgCl(sat.

KCl)

in the highly acidic electrolyte solution employed to

extract ascorbate from biological samples and minimize its autoxidation. The sensitivity of conventional Au-microelectrodes was increased about one thousand-fold upon modification with NPG film, reaching 2 nA µmol-1 L-1. The detection limit for AA based on a linear currentconcentration calibration plot was found to be 2 µmol L-1. The NPG-based microsensor was 1

demonstrated to be selective, reproducible and stable, and was employed for determinations of AA concentration in highly acidic biological extracts. Keywords: Nanoporous gold, amperometric sensors, electrochemistry, nanomaterials, ascorbic acid, microelectrodes ∗Corresponding authors. Tel: + 5511-30912693 E-mail address: [email protected] (Abhishek Kumar), [email protected] (Mauro Bertotti)

1. Introduction Ascorbic acid (AA) is a vitamin that plays central roles in cellular redox metabolism. Initially, because of its involvement in scurvy, ascorbate was investigated as a cofactor of 2oxoglutarate-dependent dioxygenases, which are enzymes responsible for the hydroxylation of lysine and proline, a key step for collagen synthesis [1]. Later on, AA was described as an antioxidant [2] and as the reducing substrate of 1-Cys Peroxiredoxin (Prx) [3], an abundant and highly reactive Cystein (Cys)-based enzyme playing central role in the cellular metabolism of hydroperoxides, thereby been involved in a multitude of redox processes [4]. 1-Cys Prx enzymes do not form disulfides, since a single Cys residue participates in the catalytic process. Instead, 1-Cys Prx enzymes produce sulfenic acids (Cys-SOH) as a reaction intermediate [5]. The activity of 1-Cys Prx enzymes as biological reductants are still controversial and some of us proposed AA as a possible candidate [3]. The rate constant for reduction of sulfenic acid by AA is about 103 M-1 s-1 (unpublished data), such that high AA concentrations are required for this reaction to outcompete other relevant biological systems. Thus, a robust, highly sensitive and selective sensor is required to assess the relevance of AA in distinct biological systems. 2

In fact, the determination of AA concentration in biological systems is a challenge, among other reasons because cellular extracts are made in very acidic conditions to minimize AA autoxidation. Attempts to measure AA by mass spectrometry failed in part due to its poor ionization in gas phase. Thus, considering the high electrochemical activity of AA, electroanalytical methods have demonstrated clear advantages over methods such as titration with an oxidant solution [6, 7], liquid chromatography [8], spectrophotometry [9] and chemiluminescence [10], demonstrating higher sensitivity, selectivity, simplicity and costeffectiveness. More importantly, electrochemical sensors can be miniaturized eventually enabling in-vivo and in situ quantification of a given analyte in the actual microenvironment. Traditionally, carbon or bare metal electrodes have been used to quantify AA in many samples, and the analytical performance of unmodified Glassy Carbon (GCE) and Carbon Paste electrodes (CPE) have been comprehensively evaluated by Wang et al. [11]. They concluded that higher sensitivity and faster responses are achieved with GCE, whereas CPE exhibits better stability and signal-to-background noise ratio. Conventional platinum [12] and gold [13] electrodes have also been used to study the AA electrooxidation at low pH, but the electrode process was found to be very slow in acidic electrolytic medium as confirmed by the large associated overpotential. Furthermore, dopamine (DA) and uric acid (UA) co-exist in biological samples and may act as severe interferants. Finally, the oxidation product of AA (dehydroascorbic acid; DHA) tends to adsorb on these electrode surfaces leading to significant suppression of the electrochemical response. Such limitations of bare electrodes have been overcome by using a single crystal gold (111) electrode, since the AA oxidation peak is shifted to less positive potentials leading to its discrimination from the voltammetric signals of those interferants [14]. However, such electrodes are expensive and the oxidation products of AA usually adsorb on the surface,

3

resulting in loss of sensitivity and reproducibility. Our group has also reported the fabrication of a ruthenium oxide hexacyanoferrate modified microelectrode for AA detection [15, 16], but such sensor showed no response in very acidic conditions such as those found in the solution used to extract AA from biological tissues (pH close to 1). To address the above-mentioned challenges, metal and carbon electrodes have been modified with electrocatalytically active materials and, particularly, metal-carbon composite nanomaterials to increase sensitivity and selectivity and minimize electrode fouling. Chu et al. [17] used a gold nanoparticles-polyaniline composite to detect AA in the presence of DA and reported high sensitivity with a detection limit of 9 µmol L-1. A voltammetric AA sensor based on platinum nanoparticles-decorated carbon nanotubes with selective response for AA and a detection limit of 20 µmol L-1 was reported by Dursun et al. [18]. A palladium nanoparticlessupported graphene oxide AA sensor was proposed by Wu et al. [19], and the device showed fast response, long linear range and detection limit of 14 µmol L-1. A recent work by Salahandish et al. [20] reported a biosensor based on 3-D graphene-silver nanoparticles-polyanniline composite for selective detection of AA in clinical samples, with a long linear range and detection limit of 8 µmol L-1. A carbon nanotube paste electrode for selective detection of AA in the presence of acetaminophen was proposed by Duarte et al. [21], and the sensor presented a linear range from 100 to 700 µmol L-1 and a detection limit of 7 µmol L-1. The use of such platforms based on nanomaterials allowed the detection of AA with high sensitivity, but no systematic study has been performed in acidic and biological medium. Moreover, metal nanoparticles used in these works require very long and complex chemical synthesis and processing (2-3 days). Additionally, drop casting of the nanomaterial ink was used in the majority of these works, which often leads to differential loading on the electrode surface resulting in poor

4

reproducibility. Finally, carbon nanotubes used in these works were solution-processed and the stability is adversely affected because of poor debundling of the carbon and low exfoliation efficiency, as reported previously [22]. Porous nanomaterials, particularly nanoporous gold (NPG), are another class of electrocatalytically active and high surface area materials which have drawn tremendous research interests in the development of electrochemical sensors [23, 24], including AA detection [25]. NPG materials are characterized by a bicontinuous network of interconnected nanometric gold grains and multiple sized pores, giving an appearance of a metallic sponge. These materials have been mainly synthesized by electrochemical methods such as dealloying of a gold containing alloy [26], electrodeposition from a gold precursor solution [27] and anodic corrosion of a gold surface [28]. The electrosynthesis method and experimental parameters determine the structure and morphology of such a porous material. NPG modified electrodes have been previously used by El-Said and Qiu et al. [29, 30] for selective detection of DA and AA, which gave separate voltammetric peaks in contrast with conventional gold disk electrodes. However, these works were more focused on DA detection and detailed AA sensing performance studies were not shown. Moreover, the experiments were performed in pH~7 buffer solution, a condition that favors the oxidation of AA [31], and no information was given about AA sensing in a harsh acidic biological assay conditions in the presence of several other ions and possible interferants. Accordingly, herein described is an NPG modified gold microelectrode-based electroanalytical platform for AA determination in real biological samples. NPG films were synthesized on a gold microelectrode by Dynamic Hydrogen Bubble Template (DHBT) method, [32] and the structure and morphology of the electrodeposited films were thoroughly characterized by electron microscopy and spectroscopy techniques. To obtain the most suitable 5

NPG material for AA amperommetric detection, a comprehensive optimization study was performed by preparing NPG films while adjusting the experimental electrodeposition parameters. The effect of pH and possible intereference of DA on the AA voltammetric response was also investigated. Finally, the performance of the NPG-based sensor was examined by determining relevant analytical paremeters and analyzing AA in biological samples, more especifically in Aspergilius fumigatus mycelia and Arabidopsis thaliana leaves extracts in highly acidic conditions.

Notably, the electrochemical data obtained by the new amperometric

microsensor based on nanoporous gold described herewere comparable with spectrophotometric measurements in those samples, thus validating this new approach.

2. Experimental 2.1. Materials and reagents Analytical grade chemicals were used throughout this work without any further purification. All aqueous solutions were prepared using Milli-Q ultrapure water (resistivity ~18 MΩ cm). Acetic acid, metaphosphoric acid, DTPA, dopamine hydrochloride, L-ascorbic acid, gold (III) chloride trihydrate and Phosphate Buffer Saline (PBS) tablets were purchased from Sigma-Aldrich. One tablet of PBS was dissolved into 200 mL of water generating a pH~7.4 buffer solution which was used to prepare the AA and DA stock solutions (0.1 mol L-1). 2.2. Fabrication of gold microelectrodes A gold microelectrode was fabricated using a Pasteur glass pipette by placing a hard Aufiber inside and sealing the end with Araldite glue. In a typical procedure, the pipette was carefully washed in piranha solution and dried for 24 hours at 110 °C. Approximately 1 cm long 6

and 25 µm diameter gold fiber (purity, 99.99%; hard, Goodfellow, UK) was inserted at the sharp end of a pipette, which was then sealed with Araldite glue (purchased in a local supermarket) and kept upright for 24 hours. The eventually protruding gold fiber was cut-off and the pipette tip surface polished on P600 Blackstone Waterproof Sandpaper. Then, carbon powder was packed in and a nickel/chromium wire inserted to the other end of the pipette to establish the electric contact with the Au-fiber inside. The microelectrode was finally characterized using a 10 mmol L-1 K3[Fe(CN)6] + 0.1 mol L-1 KCl solution, at a scan rate of 20 mV s-1 (Fig. S1).

2.3. Ascorbate extracts preparation The fungus extract was prepared using an A. fumigatus wt strain. The fungus was grown in solid YAG complete medium (yeast extract 0.5% (w/v); glucose 2% (w/v); trace elements (micronutrients) 1 ml L-1, agar 2% (w/v)) at 37 °C for 72 h. The composition of trace elements solution was described previously [33]. The conidia were harvested after cultivation by stirring the culture with a sterile Milli-Q water, and the suspension gently filtered to separate them from hyphal fragments. Their concentration was determined using a Neubauer chamber, and 1.0 × 107 conidia were inoculated in 100 mL of liquid YAG complete medium (yeast extract 0.5%; glucose 2%; trace elements 1 ml L-1) and grown at 37 °C overnight, at 200 rpm in an orbital shaker. The mycelia were then collected by filtration using a vacuum pump, the cell mass was frozen in liquid nitrogen, and immediately macerated in the presence of liquid nitrogen. Finally, 0.5 mL of an extraction buffer (1 mmol L-1 DTPA; 3% metaphosphoric acid and 8% acetic acid) was added per gram of fresh fungus for AA extraction. Such extraction buffer was previously demonstrated to be the best medium for recovering the ascorbate present in biological

7

samples. Moreover, the acidic environment prevents the oxidation of AA and contributes for the precipitation of the proteins present in the sample [34]. The plant extract was prepared from A. thaliana leaves using a proportion of 20 mg of sample (leaves) per 500 µL of the extraction buffer. The leaves were cut using scissors, frozen in liquid nitrogen and the extraction buffer was added for further maceration with polypropylene pistil inside a microtube. Both fungus and plant solutions were centrifuged at 15,000 rpm for 20 min at 4 °C and filtered using 0.45 µm 13 mm Millex® filter (Merck).

2.4. Electrochemical experiments Electrochemical measurements were conducted with a PGSTAT-128N Autolab Metrohm workstation interfaced with Nova 11.1 software. A conventional three-electrodes cell comprising an NPG modified gold microelectrode, Ag/AgCl (KCl sat.) and platinum wire as working, reference and counter electrodes, respectively, was used in the electrochemical experiments. NPG

electrodes

were

fabricated

through

bottom-up

DHBT

potentiostatic

electrodeposition method, at fixed applied potentials, in a three-electrodes cell using a strongly stirred 5 mmol L-1 HAuCl4 in 0.5 mol L-1 H2SO4 solution, at room temperature, in the absence of external gas bubbling. Two types of NPG films were electrodeposited: (a) at applied potentials of -1.0 V, -2.0 V, -3.0 V and -4.0 V for a fixed time of 100 s and (b) at fixed applied potential of 4.0 V for electrodeposition time of 50 s, 100 s, 200 s, 400 s and 600 s. The respective modified gold electrodes were washed with water and dried at room temperature open to air. Care was taken in the NPG electrode preparation to keep the working electrode at exactly the same position relative to a rotating magnetic bead in order to ensure reproducible results.

8

NPG electrodes were characterized by cyclic voltammetry (CV) in 0.5 mol L-1 H2SO4 solution, in the 0.2 to 1.6 V potential range and scan rate of 50 mV s-1. CVs were also recorded to study the anodic oxidation of DA and AA at the NPG modified electrodes in 0.1 mol L-1 PBS, in the -0.2 to 0.6 V potential range, and scan rates from 5 to 200 mV s-1. Amperometric measurements were performed to get the current-concentration calibration plot for AA in the 10 µmol L-1 to 1.1 mmol L-1 range and the amounts present in the extraction solutions determined by the standard addition method.

2.5. Structural and morphological characterization High Resolution Transmission Electron Microscopy (HRTEM) imaging was performed in a JEOL JEM-2100 microscope equipped with a Field Emission Gun (FEG, ZrO/W(100) Schottky field emission electron source) operating at 200 kV to investigate the crystalline structure and morphology of the NPG materials deposited on the gold microelectrode surface. The point resolution of the instrument was 0.19 nm (point-to-point). Scanning transmission electron microscopy (STEM) images of a nanometer thick slice of NPG samples were taken at an FEITM Helios Nanolab 660 equipment in STEM mode, with applied voltage of 30 kV. The samples for TEM imaging were prepared as follows: The electrodeposited NPG layer was first removed in an ultrasonic bath and transferred into a PELCO 12 Eponate resin that was cured for 16 hours at 60 °C. The solid was cut into a few nm (30–40 nm) thick slices using an RMC PowerTome XL ultramicrotome equipped with a PELCO diamond knife, and transferred onto TEM copper grids (TedPella) for imaging.

9

The surface morphology of the NPG films was investigated using a JEOL JSM-FEG 7401F SEM instrument, while Energy Dispersive Spectroscopy (EDS) analyses were carried out in a FEI Helios Nanolab 660 equipment to assess the chemical purity of the materials. SEM images were recorded at accelerating voltage of 2 kV while performing horizontal and cross-sectional scans of the surface. 2.6. Spectrophotometric analysis of AA The concentrations of AA determined by the electrochemical sensor were compared with those obtained using a standard spectrophotometric method. Briefly, AA was firstly quantitatively oxidized to DHA by L-ascorbate oxidase, and the chromophore (methyl ketals) generated through the reaction of DHA with methanol in phosphate buffer was assayed spectrophotometrically [35]. In a typical procedure, 2 ml of AA aqueous aliquots were mixed with 50 µL of ascorbic acid oxidase solution (5% by weight in PBS, pH~7) followed by incubation at room temperature for 10 minutes. The reaction mixture was then added to 150 µL of 15.4 mmol L-1 desferrioxamine mesylate aqueous solution, 1 mL of methanol and 2 mL of PBS (pH~7) and the resulting solution was incubated for 30 minutes at 38° C. The UV-Vis spectra of the resulting solutions were recorded using a Hewlett Packard 8453A diode-array spectrophotometer with a 1 nm slit width and 0.1 nm accuracy in the range of 190-1100 nm using a 10 mm quartz cuvette.

3. Results and Discussions 3.1. Structure and morphology characterization of NPG

10

Considering that the generation of the NPG film was achieved from a gold chloride solution, chloride can be a potential contaminant in the electrodeposited film. To assess the chemical purity of the NPG films and evaluate whether any chloride can be present, EDS spectra were recorded. As shown in Fig. 1a, the EDS spectrum depicts a strong band at 2.12 keV, which is a characteristics optical absorption of gold nanocrystals involving the X-rays from gold Mshell [36]. The low intensity bands at 9.71 keV and 11.5 keV also correspond to gold in accordance with the EDS energy table associated with the characteristics X-rays from gold outer shells. Thus, any suspected contamination from chloride was ruled out and the electrodeposition process produced high purity NPG films.

Figure 1: EDS spectral profile and micrometric SEM image in the inset (a), magnified SEM image of a selected area (b), STEM image of a 40 nm thick cross-section (c) and SAED pattern (d) of NPG electrodeposited at -4.0 V for 400 s.

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The micrometric morphology of the NPG films shown in the inset of Fig. 1a represents a typical honeycomb-like feature exhibiting evenly distributed micropores, as expected for a NPG film prepared by the DHBT method [32]. The respective magnified SEM micrograph (Fig. 1b) shows large agglomerates of urchin-like structures constituted by few hundred nanometers long and densely branched nanowires, with extremely high surface area and nanoporosity. Such elongated and branched fractal structures can be a consequence of longer deposition time (400 s) and extremely high current density through a microelectrode surface, favoring a stable and progressive nucleation [37]. The smaller nanopores in the branched fractals were further revealed by the STEM image (Fig. 1c) of a 30 nm thick cross-section of an NPG film, which exhibited less than 20 nm diameter nanopores (highlighted in red circles), thus validating the effectiveness of the electrodeposition method in generating a sponge-like nanoporous structure. The degree of crystallinity of the NPG film microstructures was examined by Selected Area Electron Diffraction (SAED) implemented in HRTEM technique. The SAED pattern exhibits concentric rings, as shown in Fig. 1d, where each ring is associated with electrons diffracted by a unique gold crystal orientation and the interplanar distances (d-spacing) are proportional to the respective diameters. The four rings ((1), (2), (3) and (4)), as indicated in the image, correspond to the (111), (200), (220) and (311) planes of crystalline gold lattice.

3.2. Electrochemical characterization of the ascorbic acid oxidation process 3.2.1. Optimization of electrodeposition parameters

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Electrodeposition conditions (deposition potential (Ed) and time (td)) can strongly influence the morphology, structure and exposed catalytic active sites of the deposited NPG films, as discussed in detail previously [38]. Hence, one can modulate the electrochemical reaction of AA by careful optimization of such parameters, and a systematic study was performed to investigate the electrochemical behavior of the modified electrodes and the analytical parameters relevant for AA detection. Accordingly, NPG films were synthesized at different Ed (-1.0 V, -2.0 V, -3.0 V and -4.0 V) for a fixed td (100 s), and at different td (50 s, 100 s, 200 s, 400 s and 600 s) for a fixed Ed (-4.0 V). The formation of the NPG film after electrodeposition at different experimental conditions can be confirmed by examining the CVs recorded in 0.5 mol L-1 H2SO4 solution (Fig. 2a and 2c). The CVs depict anodic processes consisting of two peaks at 1.20 V and 1.45 V associated with oxidation of gold to gold oxide at low indexed crystalline facets ((200), (220) and (311)) and at (111) planes, respectively [39], followed by a cathodic wave peaked at 0.90 V attributed to regeneration of metallic gold in the reverse scan. On the other hand, the bare gold electrode exhibited only one oxidation peak at 1.45 V (CV given in the inset of Fig. 2a), demonstrating that the (111) facet is predominantly exposed at the solution interface. The higher cathodic currents of NPG electrodes in comparison to bare gold can be attributed to the nanostructuration and enhanced surface area. It can be also deduced from the charge calculated under the reduction wave that the NPG films area increases as a function of the negative Ed and longer td, and that the effect of td on the surface area is much more pronounced. Thus, NPG films with area and electrochemical properties suitable for a targeted application like AA sensing can be realized by adjusting the Ed and td values.

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Figure 2: CVs of NPG modified gold microelectrodes prepared at different Ed at fixed td (100 s) recorded in 0.5 mol L-1 H2SO4 electrolyte solution (a), and in 0.1 mol L-1 PBS solution containing 0.5 mmol L-1 AA (b). Inset of Fig. 2a shows the CV of bare gold microelectrode in 0.5 mol L-1 H2SO4 electrolyte solution and the inset in Fig. 2c shows the CV of bare gold microelectrode in 0.1 mol L-1 PBS solution containing 0.5 mmol L-1 AA. CVs of NPG modified gold microelectrodes prepared at different td at fixed Ed (E = -4.0 V) recorded in 0.5 mol L-1 H2SO4 electrolyte solution (c) and in 0.1 mol L-1 PBS solution containing 0.5 mmol L-1 AA (d). Inset of Fig. 2c and 2d shows the corresponding CVs at shorter times (50, 100 and 200 s). (Scan rate:20 mV s-1).

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The corresponding electrochemical behavior of AA at different NPG modified electrodes was investigated by recording CVs in 0.1 mol L-1 PBS solution in the -0.2 to 0.6 V potential range, at a scan rate of 20 mV s-1. Fig. 2b and 2d show the comparison of CVs of AA redox processes at NPG electrodes prepared at different Ed at a fixed td and vice-versa, respectively. A sharp anodic wave, typical of fast electron transfer kinetics associated with the electrooxidation of AA into DHA by a two electron process [40] at Eap~0 V, can be noticed in CVs recorded with the NPG modified electrodes. Such a process takes place at a potential that is approximately 0.3 V less positive as compared to the corresponding one obtained with the bare gold microelectrode (CV shown in the inset of Fig. 2b). Hence, this is a clear indication that AA anodic process is facilitated at NPG electrodes. The effect of the higher area of NPG electrodes is further manifested by the more than 1000-fold increase in AA electrooxidation current in comparison to the one obtained with the bare gold microelectrode. A more systematic analysis of the influence of the electrodeposition parameters on the AA electrochemical behavior was performed by plotting peak current (ip) and overpotential (Ep) as a function of Ed and td, as shown in Fig. S2. The influence of Ed on both ip and Ep is approximately linear, where an increase of ip and a shift of Ep to less positive values were observed as Ed was set more negative. This can be a consequence of the linear increase in the electrode area and the number of catalytic sites as Ed is more negative [41]. On the other hand, an exponential enhancement of ip and a shift of Ep with increasing td was noted, which can be attributed to progressive nucleation in the growth mechanism of NPG resulting in exponential increase of area and concentration of catalytic active sites as a function of td [42]. Thus, NPG films prepared at more negative potentials and longer deposition time td yielded the highest ip value and Ep shifted to ~0.0 V for the AA oxidation process. However, a thicker film is not 15

always desirable for sensor applications because of the increase in capacitive current and corresponding noise level of the measurements, which can be detrimental to achieve high sensitivity and low detection limits. Therefore, the best experimental parameters were found to be Ed = -4.0 V and td = 400 s, and sensors prepared at these conditions provided the best electrochemical response for AA.

3.2.2. Electrochemical detection of DA and AA DA is a neurotransmitter that plays several important roles in the brain and body, but is not found in fungus and plant samples. Nevertheless, the electrochemical determination of DA is difficult in some biological fluids because AA is present at high concentrations. Therefore, we found it interesting to examine whether our sensor could distinguish the response to both AA and DA. Thus, the selectivity of DA response in the presence of AA was assessed by recording CVs in a 0.1 mol L-1 PBS solution containing 0.5 mmol L-1 AA and 0.5 mmol L-1 DA for further comparison with the CV recorded in the absence of DA. A few main conclusions can be drawn from the analysis of the voltammograms shown in Fig. 3a and 3b: i) The currents are much larger in CVs shown in Fig. 3b as a result of the larger surface area provided by the nanoporous architecture; ii) The voltammograms corresponding to AA oxidation at 0.0 V change from a sigmoidal profile typical of microelectrodes (radial diffusion) (Fig. 3a) to a peak shaped profile (Fig. 3b) as consequence of the thin layer diffusion mechanism prevalent at thicker nanoporous film surface. On the other hand, the CVs corresponding to DA present two peaks at Ep ~0.23 V and Ep ~0.11 V associated with the quasi-reversible DA oxidation to dopamine-o-quinone and its reduction back to DA by a two-electrons process [43]. The presence of the cathodic component

16

would not be expected by recording CVs with a microelectrode and its appearance can be assigned to the adsorption of dopamine-o-quinone onto the Au surface; iii) The anodic process corresponding to AA oxidation is better resolved from the one associated to DA when electrodes modified with thicker NPG films are used (Fig. 3b). Moreover, the response loss observed in the CV recorded with the NPG film prepared at short td (Fig. 3a) can be attributed to the strong adsorption of DA on the film surface, leading to a partial blocking of the electrocatalytic sites employed for AA oxidation. Similar interference studies were performed with gold microelectrodes modified with NPG films deposited at different Ed and a fixed td (Fig. S3). Notwithstanding the loss of sensitivity, well-resolved AA and DA oxidation peaks were also observed, demonstrating the proposed platform is suitable for the detection of both compounds in biological matrices.

Figure 3: Comparison of CVs recorded with NPG modified gold microelectrodes prepared at Ed = -4.0 V and td= 50 s (a); and Ed = -4.0 V and td= 600 s (b) in 0.1 mol L-1 PBS (black curve), 0.1 mol L-1 PBS+0.5 mmol L-1 AA (red curve); and 0.1 mol L-1 PBS +0.5 mmol L-1 AA+0.5 mmol L-1 DA (blue curve), at scan rate of 20 mV s-1.

17

3.2.3. Effect of pH on AA voltammetric response AA is extracted from biological tissues in very acidic conditions to minimize autoxidation of this compound [2]. Therefore, we analyzed the influence of pH on AA electrochemical process. The voltammetric behavior of AA in 0.1 mol L-1 PBS solution was studied with an NPG modified Au-microelectrode at different pH values (1.2 to 7.4), as shown in Fig. 4a. A strong effect of pH is evident on the voltammograms, with changes in Ep and ip with varying pH. Such changes have been systematically shown in Fig. 4b, where a linear decrease in Ep (from 0.26 V to 0.0 V) and an initial decrease followed by an increase in ip can be noticed with increasing pH. Such trend in Ep is a clear evidence that protons participate in the electrode process, as previously reported by Compton et al. [44]. Considering the higher ip even in stronger acidic condition, NPG films are a suitable platform to fabricate sensors for AA detection in the targeted acidic samples.

Figure 4: Comparison of voltammograms recorded with an NPG modified electrode (Ed = -4.0 V for 400 s) in solutions of different pH values prepared by addition of H3PO4 to a 0.1 mol L-1 PBS+0.5 mmol L-1 AA solution at scan rate of 20 mV s-1 (a) and changes of peak current (ip) and anodic peak potential (Ep) as a function of pH (b).

18

The electrocatalysis of the AA oxidation process at NPG surfaces at different pH has been correlated with the change in the reaction mechanism. Based on the previous work of Compton et al. [44], a generalized reaction mechanism for the AA oxidation involves a combination of electrochemical and chemical reactions. To confirm the presence of a chemical reaction in this electron transfer process, the change in the AA electrooxidation peak potential (Ep) was plotted as a function of the scan rate in a 5 to 200 mV s-1 range, as shown in Fig. 5a. The presence of two linear regions with different slopes suggests a chemical reaction coupled with an electrochemical step, as reported previously for an irreversible electrochemical process [45]. Thus, based on these results, a generalized mechanism for the AA oxidation into DHA can be proposed, as shown in Fig. 5b, in which electron-transfer steps are represented in the horizontal direction, while chemical steps are represented in the vertical direction. Since the pK1 value of ascorbic acid is ~4.1, the prevalent species at pH~7 is the ascorbic acid monoanion, while in acidic medium (pH < 4.1), AA remains in the protonated form. Accordingly, the starting species in the reaction mechanism changes as indicated in the reaction scheme. At all pH values, the reactions sequence proceeds through Electrochemical-Chemical-Electrochemical (ECE) pathways, in which a chemical step involving deprotonation is the Rate Determining Step (RDS). To determine the RDS at different pH values, the charge transfer coefficient α was calculated through the linear correlation of log (I) vs potential (as shown in Fig. S4), also known as Tafel plot, for the current rising section of a voltammogram (kinetically controlled region) recorded at a scan rate of 5 mV s-1. The slope of the Tafel plot equals (1-α)nF/2.3RT, where n, F, R, and T represent the number of electrons involved in the rate-determining step, Faraday coefficient, universal gas constant and temperature, respectively. A large difference in α values was noted for neutral and acidic medium (αpH~7 = 0.31, αpH1.2 = 0.63). The α values close to zero or one indicate

19

that the structure of the transition state associated with the RDS is close to reactant or product, respectively. Hence, in neutral medium the RDS is quickly achieved resulting in decrease in activation energy of the reactions sequence and consequently AA is oxidized easily close to 0 V. On the other hand, at acidic conditions the α value is closer to one, indicating the structure of the transition state associated to the RDS resembles the one corresponding to the oxidized compound. As a consequence, the activation energy for the AA oxidation in acidic medium will be higher, making the oxidation more difficult, which is also demonstrated by the shift towards more positive values. This is also in agreement with the previously reported work of Karabinas et al. [12].

Figure 5: Changes of Ep for AA electrooxidation as a function of Log (scan rate) using the NPG electrode (Ed = -4.0 V for 400 s) at pH~7 (a). Reaction scheme of AA oxidation on the NPG electrode at different pH values (b). Adapted from [44]. 20

3.3. Amperometric sensing of AA

The determination of AA in different acidic extracts (pH~1.7) was achieved by amperometric measurements performed in a continuously stirred solution at a fixed potential of 0.3 V. Fig. 6 shows a typical amperometric response of the NPG sensor (Ed = -4.0 V; td= 400 s) corresponding to consecutive additions of a standard AA solution, in such way that the AA concentration in the electrochemical cell was increased from 10 µmol L-1 to 1.1 mmol L-1 during the course of the experiment. It is evident from inspection of Fig. 6 that with each addition of AA, an initial steep increase in current associated with oxidation of AA into DHA is followed by a current plateau consistent with the dispersion of the injected volume, confirming the short response time of the sensor.

21

Figure 6: Amperometric response of the NPG modified electrode (Ed = -4.0 V for 400 s) at pH~1.7 during additions of AA in a range from 10 to 1100 µmol L-1 (E = 0.3 V). The inset shows the corresponding calibration plot.

The calibration plot shown in the inset of Fig. 6 was characterized by a linear increase in sensor response as a function of AA concentration. Based on the linear fit parameters, the sensor response at any concentration within the linear range (10 to 1100 µmol L-1) can be predicted by the correlation equation iAA(nA) = 2.1*CAA (µmol L-1) + 29.6. The slope of the linear correlation defines the sensitivity of the sensor, which was estimated as 2.1 nA µmol-1 L-1. Such high sensitivity can be clearly noted by looking at the amperogram in Fig. 6, where changes in the current response can be observed for small changes in the ascorbic acid concentration (10 µmol L-1), such that a limit of detection (LOD) of 2 µmol L-1 was determined taking into account the signal to noise ratio of 3. Such value is much lower than the AA concentration usually found in the investigated extracts, hence the sensor can be applied for the desired goal as discussed below. The applicability of the sensor to measure AA in biological samples was further demonstrated by determining the AA concentration in acidic cellular extracts by the standard addition method. A typical amperometric experiment with current changes corresponding to the presence of AA in fungus and plant extracts, as well as the resulting calibration plot, are shown in Fig. S5. Based on the calibration plot, the concentration of AA was determined in each sample extract and the results compared with the ones obtained by spectrophotometry, as shown in Table 1. Details of the spectrophotometric analysis (spectra and the associated absorbanceconcentration calibration curve) are presented in Fig. S6. A close agreement in measured values

22

of AA in the samples by two independent methods can be noticed, which confirms the reliability of our proposed amperometric method in accurately determining AA in acidic biological medium. Measurements of the AA content in different acidic extracts of A. fumigatus fungus and A. thaliana leaves by HPLC with coulometric detection have been previously performed and the results were similar to those found in this work (unpublished data), reinforcing the usefulness of the developed electrochemical sensor towards the detection of AA in acidic biological medium. The AA concentration in plants varies between tissues and highest amounts of the compound can be found in leaves and flowers, which are more photosynthetically active tissues [2]. Typical values of AA concentration in A. thaliana leaves range from 2 to 10 µmol g-1 of fresh leave weight [2] and the obtained value for AA concentration by using the proposed electrochemical sensor (around 5 µmol g-1 of fresh leave weight) is within the mentioned concentration range. Considering the percentage of water in a leaf is close to 90%, the average AA concentration in the tissues is around 5 mmol L-1, which is consistent with previous descriptions [2]. To the best of our knowledge, there is no previous information regarding AA concentration in A. fumigatus. The obtained value here using the electrochemical sensor was 0.20 µmol g-1 of fresh fungus weight (around 0.2 mmol L-1 inside the fungus), or around 2 µmol g-1 of dry fungus weight. This concentration of AA is in the same order of magnitude as the one reported for the filamentous fungus Sclerotium rolfsii: 0.2-0.9 µmol g-1 of dry fungus weight [46].

23

Table 1: Determination of AA in real samples by amperometry (n = 3) and spectrophotometry (n= 3). Results are shown as the amount of ascorbate in fresh A. thaliana leaves and fresh A. fumigatus. AA concentration (µmol g-1)

Samples Amperometry

Spectrophotometry

Plant

4.9

4.8

Fungus

0.20

0.19

The reproducibility of the NPG sensor was assessed by comparing the current responses obtained at a fixed AA concentration using 5 different gold microelectrodes modified with an NPG film electrodeposited at similar experimental conditions (Ed = -4.0 V and td = 400 s). The bar plot in Fig. 7a shows the results, which are almost identical (relative standard deviation (RSD) < 2%). Such high reproducibility in the sensor response towards AA detection also highlights the effectiveness of the DHBT electrodeposition method in preparing NPG films with controlled and reproducible structure and morphology. The long-term stability of the sensor was evaluated by performing amperometric measurements in a 0.5 mmol L-1 AA solution once in a week up to 10 weeks, while the sensor was stored in open atmosphere at room temperature. As shown in Fig. 7b, the sensor lost approximately 10% of sensitivity in the first 5 weeks but a stable response was recorded in the subsequent weeks. Overall, a 12% loss in the sensor response was observed after 10 weeks and this can be attributed to the slow oxidation of NPG surface, which inhibited partially the electron transfer process [47]. The stability of the NPG electrode in acidic medium was further investigated by storing the electrode for 48 hours in a solution with pH~ 1.2 and comparing the electrochemical 24

response regarding AA detection before and after the storage. As depicted in Fig. S7, 92% of AA electrooxidation voltammetric response was retained and a 20 mV positive shift in Ep was noticed. The small variation in the electrochemical response was attributed to change in nanoporous morphology, which corresponds to a porous agglomeration of NPG struts (Fig. S8).

25

Figure 7: Comparison of AA electrooxidation current values for amperometric measurements with 5 different NPG electrodes prepared at the same experimental conditions (Ed = -4.0 V for 400 s) (a) and changes in the electrode response with storage time (b). Experiments performed in 0.1 mol L-1 PBS containing 0.5 mmol L-1 AA.

Conclusions NPG films synthesized onto the surface of Au microelectrodes through Dynamic Hydrogen Bubble Template (DHBT) method allowed us to fabricate a sensitive and selective amperometric sensor for detection of AA in acidic conditions. Optimization studies were performed by varying the electrodeposition parameters (potential and time). A strong correlation of Ed and td employed for NPG films preparation with the obtained electrooxidation responses for AA was noticed and an increased current response of 1000-fold for the NPG film prepared for longer td as compared to conventional Au-microelectrodes was found at optimized conditions. Moreover, a remarkable shift of the associated peak potential towards less positive

26

values was noticed, conferring high selectivity to the measurements. The NPG microsensor showed stable AA electrochemical responses at different pH, which was crucial for the implementation of such platform in detecting AA in acidic extracts. The optimized NPG microelectrode was tested for amperometric determination of AA in acidic conditions at a fixed potential of 0.3 V and the electrochemical sensing performance was evaluated by means of relevant analytical features. Finally, the NPG electrode applicability in accurately determining AA in acidic extracts of A. fumigatus fungus and A. thaliana leaves was demonstrated, and the obtained results were in close agreement with those found by spectrophotometric measurements. Hence, the results shown in this work have demonstrated the developed NPG-based microelectrode is a useful sensor for the determination of a large range of AA concentrations in acidic biological medium.

Notes: The authors declare no competing financial interest. All authors have contributed equally in the manuscript preparation.

Acknowledgments Authors are grateful to Sao Paulo State Research Foundation (FAPESP 2018/08782-1, 201818/21489-1, 2016-07461-1 and 2018/09027-2) and National Council for Scientific and Technological Development (CNPq 401581/2016-0 and 303137/2016-9) for providing generous funding throughout the research project. Authors are also thankful to Fabiano Montoro at LNNano, CNPEM, Campinas for performing electron microscopy characterizations. Josué M.

27

Gonçalves thanks CNPq for doctoral fellowship. Authors are grateful to Carlos Takeshi Hotta and Bruno Fernandes Matsukura for providing A. thaliana samples.

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Highlights 1. An NPG film microelectrode was prepared and used for ascorbic acid sensing 2. The peak potential was ~0.3 V shifted towards less positive values than bulk Au 3. Active catalytic sites formed in the NPG film are responsible for the potential shift 4. Real application was demonstrated using acidic extracts from fungus and leaves

Supplementary Information

Amperometric microsensor based on nanoporous gold for ascorbic acid detection in highly acidic biological extracts Abhishek Kumar1*, Vinicius L. Furtado1, Josué M. Gonçalves1, Renata Bannitz-Fernandes2, Luis Eduardo S. Netto2, Koiti Araki1, Mauro Bertotti1* 1

Department of Fundamental Chemistry, Institute of Chemistry, University of São Paulo, Av. Professor Lineu Prestes, 748, 05513-970, São Paulo - SP, Brazil. 2 Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo, Rua do Matão, 321, 05508-090, São Paulo - SP, Brazil.

Fig. S1: CV of gold microelectrode in 10 mmol L-1 K3[Fe(CN)6] + 0.1 mol L-1 KCl solution at a scan rate of 20 mV s-1.

Fig. S2: Variations of AA electrooxidation Eap and

ip

at different Ed for a fixed td of 100 s

(extracted from Fig. 2b) (a) and at different td for a fixed Ed of -4.0 V (extracted from Fig. 2d) (b).

Fig. S3: Comparison of CVs recorded with NPG modified gold microelectrodes prepared at different Ed (-4.0 V, -3.0 V, -2.0 V and -1.0 V) for a fixed td = 100 s in in 0.1 mol L-1 PBS +0.5 mmol L-1+0.5 mmol L-1 DA at scan rate 20 mV s-1.

Fig. S4: Tafel plots for electrooxidation of 0.5 mmol L-1 AA in 0.1 mol L-1 PBS at different pH values using an NPG modified gold microelectrode (Ed = -4.0 V for 400 s).

Fig. S5: Amperometric responses obtained at E = 0.3 V with an NPG modified electrode (Ed = 4.0 V for 400 s) at pH~1.7 for consecutive additions of 2 samples (plant extract and fungus extract) and 7 AA standard samples (a) (each addition of the AA standard solution caused an increase in the AA concentration of 10 µmol L-1). Plots of the standard addition method for both samples (b).

Fig. S6: UV-Vis spectra with increasing concentration of AA (from 44 to 270 µmol L-1) in the reaction mixture (a) and the associated calibration plot (b).

Fig. S7: Voltammograms recorded with an NPG film modified electrode (Ed = -4.0 V for 400 s) for 0.5 mmol L-1 AA at pH~1.2 before and after storage (48 h) in 0.1 mol L-1 PBS. Scan rate: 20 mV s-1.

Fig.8: SEM image of the NPG film after 48 hours storage in acidic solution (pH 1.2)

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Amperometric microsensor based on nanoporous gold for ascorbic acid detection in highly acidic biological extracts Abhishek Kumar, Vinicius L. Furtado, Josué M. Gonçalves, Renata Bannitz-Fernandes, Luis Eduardo S. Netto, Koiti Araki, Mauro Bertotti