electrochemical treatment exposes carbon-black conductive sites

Journal Pre-proof Improved electrochemical detection of metals in biological samples using 3Dprinted electrode: Chemical/electrochemical treatment exposes carbon-black conductive sites

Diego P. Rocha, André L. Squissato, Sarah M. da Silva, Eduardo M. Richter, Rodrigo A.A. Munoz PII:

S0013-4686(20)30079-7

DOI:

https://doi.org/10.1016/j.electacta.2020.135688

Reference:

EA 135688

To appear in:

Electrochimica Acta

Received Date:

04 October 2019

Accepted Date:

10 January 2020

Please cite this article as: Diego P. Rocha, André L. Squissato, Sarah M. da Silva, Eduardo M. Richter, Rodrigo A.A. Munoz, Improved electrochemical detection of metals in biological samples using 3D-printed electrode: Chemical/electrochemical treatment exposes carbon-black conductive sites, Electrochimica Acta (2020), https://doi.org/10.1016/j.electacta.2020.135688

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Journal Pre-proof

Improved electrochemical detection of metals in biological samples using 3D-printed electrode: Chemical/electrochemical treatment exposes carbon-black conductive sites

Diego P. Rocha, André L. Squissato, Sarah M. da Silva, Eduardo M. Richter, Rodrigo A. A. Munoz * 1

Institute of Chemistry, Federal University of Uberlandia, 38400-902, Uberlandia, Minas Gerais, Brazil 1

ISE Member

Corresponding author: E-mail: [email protected] (Rodrigo A. A. Munoz) Tel.: 55-34-3239-4385

1

Journal Pre-proof Abstract This work shows that the electrochemical activity of a 3D-printed electrode fabricated using a conductive composite of polylactic acid (PLA) containing carbon black (CB) can be substantially improved through a simple and fast chemical/electrochemical pretreatment in 0.5 mol L-1 NaOH. Scanning electron microscopy and infrared spectroscopy data showed that the pretreatment process promotes the removal of the nonconductive PLA material, providing greater exposure of the conductive particles. Cyclic voltammetry of the redox probe ferricyanide/ferrocyanide indicated faster electron transfer on the treated 3D-printed surface and increase in electroactive area. Moreover, electrochemical impedance spectroscopic results also confirmed faster electron transfer after surface pretreatment. As a proof-of-concept, a low-cost and sensitive method for the determination of cadmium and lead in real urine and saliva samples by square-wave anodic stripping voltammetry was developed. The chemical/electrochemical treatment provided an impressive 30-fold current increase in the detection of both metals. Acceptable limits of detection (2.9 μg L-1 for Cd2+ and 2.6 μg L-1 for Pb2+), wide linear ranges for both metals (30 μg L-1 to 270 μg L-1; R = 0.997), high stability (RSD lower than 4.5 %; n = 10), and adequate recovery values (between 93% and 112%) for the analysis of spiked samples were achieved. Moreover, interday (n = 3), intra-day (n = 3), inter-electrode (n = 2) and inter-treatment (n = 2) experiments revealed RSD values lower than 6.5%, which indicates high reproducibility of the proposed treated 3D-printed electrode. The strategy here proposed opens up new applications for 3D-printed electrode in analytical electrochemistry with improved electrochemical sensing properties in comparison to screen-printed electrodes.

Keywords: Cadmium; Lead; Anodic stripping voltammetry; 3D-printing; Biological fluids. 2

Journal Pre-proof 1. Introduction Additive manufacturing or three-dimensional (3D) printing technology has emerged as a powerful technique that enables the construction of a wide range of objects or devices that can be applied in different areas, including medicine, civil construction, mechanical engineering, electronics, biology, chemistry and so on [1,2]. The 3D-printing technology enables fast prototyping of customized objects under a rational consumption of the material used to print (polymer, metal, etc) and using an easy-to-use interface with accessible graphical software [3]. The most accessible 3D-printing technology is fused deposition modeling (FDM) due to the low-cost of printers and polymeric filaments [4– 6]. The 3D-printing popularity has been demonstrated by the recent increase in publications regarding many novel applications in chemistry, including microanalytical systems [7–12], chemical reactors [13], storage energy devices and electrochemical sensors [6,14–19]. Selective laser melting (SLM) 3D-printing has also been proposed to fabricate electrochemical sensors [16,19,20]; however, the extremely high-cost of the 3Dprinter and consumable materials for this 3D-printing process may be not attractive for the fabrication of electrochemical sensors. 3D printed materials have appeared as an innovative interface for electrochemical devices due to the feasibility of additive manufacturing using conductive filaments [1]. Polymeric filaments, such as poly-lactic acid (PLA) and acrylonitrile butadiene styrene (ABS), can be mixed with carbon nanostructures (nanographite or graphene) leading to conductive polymeric filaments that can be used for the construction of electrochemical sensors [1]. Some examples of ready-to-use FDM 3D-printed sensors have been reported in the literature demonstrating the electrochemical sensing of different analytes in standard solutions [5,18,21–26]. On the other hand, the obtained 3D-printed material generally contains excess of polymeric matrix that may result in inaccessible conductive 3

Journal Pre-proof sites at its interface. Some research groups have proposed the application of different procedures of surface treatment to overcome this drawback, and consequently to improve the electrochemical properties of the 3D-printed interface. Such procedures include solvent treatment (immersion in dimethylformamide for 10 min) [14,27], mechanical polishing (for 30 s) [25], solvent treatment followed by mechanical polishing (total time of 12 min) [25], enzymatic degradation (around 24 h) [28] and electrochemical treatment [22,29,30]. Different electrochemical treatments were reported, such as the application of voltammetric cycles within a wide potential range followed by a constant application of a negative potential to obtain reduced graphene oxide (total time of 15 min) [30], water electrolysis though the use of a battery of 9 V in 1 mol L-1 KCl electrolyte for 24 h [29], and using a chronoamperometry applying −2.5 V for 150 s in phosphate buffer solution (pH 7.2) [14]. In this context, we propose a novel electrochemical treatment of 3D-printed electrodes produced using PLA filaments containing carbon-black nanoparticles to improve their electrochemical sensing properties. Electrochemical treatment performed in 0.5 mol L-1 NaOH activates the 3D-printed surface by electro-degradation of PLA resulting in the exposure of carbon-black sites. Improved electrochemical sensing of metals using anodic stripping voltammetry is presented. In addition, to demonstrate the robustness and viability of the proposed 3D-printed sensor, the detection of metals in complex samples (human urine and saliva) was performed.

2. Experimental 2.1. Reagents and stock solutions All solutions were prepared using high purity deionized water (R ≥ 18 MΩ cm) obtained from a MilliQ water purification system (Millipore, Bedford, MA, USA).

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Journal Pre-proof Analytical grade reagents were used in the present work. Hydrochloric acid (37% w/v) was purchased from Synth (São Paulo, Brazil), hydrogen peroxide (30% v/v) from Modern Chemistry (Barueri, Brazil), sodium hydroxide (98% w/w) from Sigma-Aldrich Steinheim Germany) and acetic acid (99.7% w/v) from Vetec (Rio de Janeiro, Brazil). The electrochemical behavior of the 3D-printed electrode was evaluated using potassium ferrocyanide (K4[Fe(CN)6]) and potassium ferricyanide (K3[Fe(CN)6]) purchased from CAAL (São Paulo, Brazil) and Proquimios (Rio de Janeiro, Brazil), respectively. Aqueous standard solutions of cadmium (Cd2+), lead (Pb2+), manganese (Mn2+), iron (Fe3+), chromium (Cr3+), mercury (Hg2+), zinc (Zn2+), nickel (Ni2+) and copper (Cu2+) (1000 mg L−1) were obtained from Quimlab (Jacareí, Brazil). Standard working solutions were prepared daily and immediately prior to use by appropriate dilution of the stock solution in the supporting electrolyte. A solution of 0.1 mol L−1 acetate buffer (pH 4.7) was prepared using acetic acid solutions and pH adjustment was carried out using a 2.0 mol L−1 sodium hydroxide solution.

2.2. Instrumentation, electrochemical cell and electrodes Cyclic voltammetric (CV) and square-wave anodic stripping voltammetric (SWASV) experiments were performed using μ-Autolab Type III potentiostat (Metrohm Autolab Utrecht, The Netherlands). Electrochemical impedance spectroscopic (EIS) measurements were performed using the FRA2 module coupled to the PGSTAT 128N potentiostat. Equipament control and data acquisition were performed using NOVA 1.11.0 software. All measurements were carried out in the presence of dissolved O2 and at room temperature. All electrochemical measurements were performed using a single chamber electrochemical cell (ca. 5 mL volume) 3D-printed with a commercial non-conductive

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Journal Pre-proof ABS filament [5]. The working electrode was 3D-printed using a conductive filament composed by a blend of carbon black and polylactic acid (CB/PLA) well-known as protopasta, purchased from ProtoPlant INC (Vancouver, Canada). The electrochemical cell and working electrodes were manufactured using an open-source Graber i3 RepRap 3Dprinter with a direct drive extruder (210 °C) [25]. The structures of the 3D-printed parts were made using Simplify 3D software or the Tinkercad online platform. The reference and auxiliary electrodes were a miniaturized Ag/AgCl (saturated KCl) and a platinum wire, respectively. A scheme of the complete electrochemical sensing device is illustrated in Figure 1, highlighting the 3D-printed components of the cell (top cover, screw nuts and body) before and after aseembling of the cell. The counter electrode (CE), reference electrode (RE), and the 3D-printed working electrode (WE) are identified in the cell as well as a rubber O-ring used to limit the WE area and a stainless-steel plate to establish the electric contact with WE. Figure 1 also shows the representation of electrode connections, potentiostat, computer screen and a micropipette used for injections of standard solutions or samples into the cell.

Insert Figure 1

Fourrier transform infrared (FT-IR) spectroscopic data in attenuated total reflectance (ATR) mode were obtained utilizing a PerkinElmer Frontier MIR/FIR equipment, using a Pike Technologies ATR accessory. Measurements were taken in the range of 4000 – 220 cm−1 with 4 cm−1 resolution. Scanning electron microscope (SEM) data were acquired using a JEOL JSM-5600LV model.

2.3. Fabrication of 3D-printed electrode

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Journal Pre-proof The working electrode was 3D-printed using the CB/PLA filament in the shape of a three-dimensional piece measuring 3.0 cm (length) x 3.0 cm (width) x 1.5 cm (height) and a thickness of 2.0 mm. In this condition, both cleaning (polishing on sandpaper) and electrochemical pretreatment steps of the WE can be performed many times (used over and over again). Before the electrochemical measurements, the 3D-printed working electrode (three-dimensional piece) was polished on a sandpaper (grit size: 600 followed by grit size: 1200) moistened with ultrapure water until obtaining a homogeneous surface (smooth and without grooves). Then, the 3D-printed WE was coupled to the 3D-printed electrochemistry cell. The geometric area of the 3D-printed working electrode is defined by the inner diameter (0.54 cm) of a rubber O-ring (area = 0.229 cm2) [31] (Figure 1). After the coupling of the WE to the cell along with the other electrodes, a 0.5 mol L-1 NaOH solution was placed in the cell, and an electrochemical treatment procedure was performed before subsequent experiments. This optimized electrochemical treatment consists of applying the following potential sequence: +1.4 V for 200 s followed by 1.0 V for 200 s. After this, the sodium hydroxide solution was removed and the cell was rinsed with excess of deionized water and next filled with background electrolyte to perform the measurements for the simultaneous determination of Cd2+ and Pb2+. Figure 2 shows a scheme of mechanical polishing of the 3D-printed WE just as obtained from the 3D-printer (3.0 cm (length) x 3.0 cm (width) x 1.5 cm (height)), which was next cut into a single retangular piece (3.0 cm x 1.5 cm), assembled in the 3D-printed cell, and electrochemically treated in a 0.5 mol L-1 NaOH solution.

Insert Figure 2

7

Journal Pre-proof 2.4. Electrochemical measurements First, the 3D-printed working electrode was electrochemically conditioned to obtain clear and reproducible signals in the SWV measurements. For this, cyclic voltammograms (20 cycles) were performed for baseline stabilization at a potential range between +1.2 V to -1.2 V, at 50 mV s-1 in 0.1 mol L-1 acetate buffer (pH 4.7). Electrochemical impedance spectroscopic measurements were performed in the presence of 1:1 mmol L−1 of potassium ferrocyanide and potassium ferricyanide in a 0.1 mol L−1 KCl solution using the 3D-printed sensor as the WE, before and after electrochemical treatment, in a frequency range between 0.1 and 50 KHz, with a signal amplitude of 10 mV and 10 data points per frequency decade. The diameter of the semicircle of the Nyquist graph is proportional to the resistance to charge transfer (Rct value) obtained by the electrochemical adjustment option of Nova 1.11 software using the equivalent circuit composed of Rct, solution resistance (Rs) and impedance of Warburg (W). The supporting electrolyte composed of 0.1 mol L-1 acetate buffer (pH 4.7) was selected in this work due to the sensitive and reproducible electrochemical responses of Cd2+ and Pb2+ on carbon surfaces in this medium as described in the literature [32–35]. The SWASV parameters were optimized based on univariate experiments in the presence of 30 μg L−1 Pb2+ and Cd2+. The variables were: frequency (10 – 100 Hz), step potential (1 – 10 mV) and amplitude (10 – 100 mV). The potential and deposition time were optimized between −0.8 to −1.3 V and 60 to 300 s, respectively. The stirring rate was evaluated in the range between 500 and 2500 rpm. The conditioning step (cleaning) was selected using +0.6 V for 30 s in order to avoid contamination of the electrodes by the previous measurement due to memory effects. All electrochemical measurements were performed without removal of oxygen.

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Journal Pre-proof 2.5. Sample preparation All samples (saliva and urine) were collected from healthy volunteers and coauthors of the present work. The collection of saliva samples was performed with a Salivette device (Sarstedt AG & Co) according to a procedure described in the literature [36]. This device consists of a collection tube and an appropriate sponge that was inserted into the mouth of volunteers for one minute to absorb the saliva. The sponge was then placed in the Salivette collector tube for centrifugation at 2000 rpm for 2 min to decant the saliva absorbed in the sponge. Saliva volumes can range from 0.5 mL to 1.5 mL. Real urine samples were collected on polyethylene vessels (Falcon tubes) and stored at −10 °C until the analysis as described in previous works [36] [37]. Saliva and urine samples were acid-digested with the aid of an ultrasonic bath based on a previous report [38]. Briefly, in 10 mL tubes, 1 mL of urine or saliva sample was treated with 50 µL of concentrated HCl and 50 µL of H2O2 (30% v/v) and kept under sonication for 15 min in the ultrasonic bath. Initially the urine samples showed a dark yellow color and at the end of the treatment, they became yellowish (almost colorless) due to the action of the oxidant mixture. At the end of the treatment, the samples were neutralized with a 0.5 mol L-1 NaOH solution until reaching a pH value close to 4.7, which was the pH value of the acetate buffer electrolyte. Subsequently, each sample was analyzed after a 5-fold dilution in the supporting electrolyte (1 mL of the digested and neutralized solution sample was diluted in 4 mL of supporting electrolyte). The standard addition method was used for the simultaneous quantification of Cd2+ and Pb2+. 3. Results and discussion 3.1. Performance of the 3D-printed CB/PLA working electrode for Cd and Pb detection.

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Journal Pre-proof After the 3D-printed electrode was manufactured, the cleaning procedure was performed by polishing the surface of the material with the aid of sandpaper and water, as mentioned in the experimental section and shown in Figure 2. Then, the performance of the 3D-printed electrode was evaluated by square-wave anodic stripping voltammetry (SWASV) for the detection of 100 µg L−1 of Cd2+ and Pb2+ using 0.1 mol L-1 acetate buffer (pH 4.7) as the supporting electrolyte (Figure 13). It can be observed that the stripping peaks obtained for both metals (black lines) were small and poorly defined (low detectability). Thus, studies were conducted with the objective of improving the performance of the 3D-printed material as a WE. As already discussed in the literature, surface characteristics and degree of aggregation of carbon black nanoparticles can be changed by chemical treatment with NaOH [39]. Furthermore, the PLA material (aliphatic polyester) is susceptible to saponification if treated with NaOH solution [29]. In this work [29], Wirth and coauthors proposed a saponification mechanism of the nonconductive PLA in alkaline media (NaOH solution). They concluded that the hydroxide ions act as a nucleophile and attack the electrophilic carbonyl present in the ester. The process is repeated several times to break the PLA into smaller polymer chains and ultimately into lactate. This saponification reation provides a higher exposure of CB particles improving the electrochemical performance of the sensor [40]. It can be seen from Figure 13 (red line), a considerable improvement in both signals of Cd2+ and Pb2+ (about 30-fold higher) was obtained after chemical/electrochemical treatment of the 3Dprinted electrode in 0.5 mol L−1 NaOH (+1.4 V/200 s and -1.0 V/200 s). Thus, it can be assumed that a combined chemical/electrochemical treatment occurs as the NaOH solution consumes PLA and the electrochemical treatment may activate carbon black nanoparticles and contribute to PLA consumption.

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Journal Pre-proof Insert Figure 3

The effect of the chemical/electrochemical treatment on the surface of the 3Dprinted CB/PLA WE was also examined using SEM and FT-IR spectroscopy. According to SEM images, the carbon black nanoparticles are poorly available before the chemical/electrochemical treatment (Fig. 4A). On the other hand, after the treatment (Fig. 4B), the PLA material was partially removed (due to the saponification reaction) from the electrode surface providing a visible increase in porosity and consequent increase in the availability of carbon black nanoparticles (increase in electrode area).

Insert Figure 4

The data obtained from SEM analysis are in agreement with FT-IR results (Figure 5), which indicates the removal of the polymeric matrix by the chemical/electrochemical treatment.

Insert Figure 5

The FT-IR spectrum of the 3D-printed electrode before chemical/electrochemical treatment (black line) presented antisymmetric (2992 cm−1) and symmetrical (2915 cm−1) CH3 stretch vibrations related to the polymeric matrix of the PLA. The presence of oxygenated groups is revealed by the band obtained at 1743 cm−1 corresponding to the vibration of C=O. The C−O−C elongation is attributed to the intense bands at 1177, 1076 and 1036 cm−1 related to the structure of the polymeric matrix containing oxygenated functional groups, mainly the carboxylic group. These results are in agreement with a

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Journal Pre-proof previous study that investigated the 3D-printing using a conductive filament composed of graphene-doped polylactic acid (G-PLA) [5]. After the electrochemical treatment (red line), the intensity of the bands C=O and C−O−C, attributed to the oxygenated groups, decreased substantially. This can be explained by the removal of PLA (as the oxygenated groups belong to PLA) from the 3D-printed electrode surface and exposure of the conductive carbon material. The 3D-printed electrode, before and after chemical/electrochemical treatment, was also evaluated by EIS. Figure 6A and 6B show, respectively, Nyquist plots and cyclic voltammograms obtained in the presence of 1:1 mmol L−1 K3Fe(CN)6 /K4Fe(CN)6 in a 0.1 mol L−1 KCl solution for the 3D-printed electrode before (black line) and after (red line) chemical/eletrochemical treatment.

Insert Figure 6

It can be observed that the impedance graphs presented different profiles for the electrode surface before (black line) and after (red line) chemical/electrochemical treatment. The 3D-printed CB/PLA surface before treatment showed higher impedance and, therefore, higher resistance to charge transfer in relation to the sensor surface after treatment. The Rct value obtained for the untreated sensor was calculated as 1354 Ω, while the estimation of Rct value for the electrode after electrochemical treatment was not possible to be performed due to the absence of a semicircle on the Nyquist graph. These results indicate a strong evidence that the chemical/electrochemically-treated electrode surface with 0.5 mol L−1 NaOH provides a faster electron transfer compared to the untreated electrode. This fact is confirmed by Figure 6B which shows a significant increase in cathodic and anodic peak current and changes in the reversibility (ΔE: from

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Journal Pre-proof 0.911 V to 0.297 V) properties of the K3Fe(CN)6/K4Fe(CN)6 probe. Therefore, the considerable improvement in the detection of Pb2+ and Cd2+ on the CB/PLA 3D-printed electrode is probably due to the effects of chemical/electrochemical treatment of the electrode surface exposing the carbon black sites. Figure 7 shows the cyclic voltammetric recordings as function of scan rate (10 to 100 mV s-1) in the presence of 1 mmol L−1 K3Fe(CN)6/K4Fe(CN)6 in 0.1 mol L−1 KCl on the 3D-printed electrode before (A) and after chemical/electrochemical treatment with 0.5 mol L−1 NaOH (B). It can be observed that the plots of the anodic and cathodic peak current versus the square root of the scan rate showed linear behavior for both electrodes (before and after NaOH treatment), indicating that the electrochemical behavior of ferri/ferrocyanide on the surface of the 3D-printed CB/PLA electrode is diffusion-limited. Signal profiling was considerably improved after treatment, resulting in the reduction of the peak-to-peak separation (ΔEp) of the redox probe. The electroactive area of the 3Dprinted electrode (before and after treatment) was calculated using the Randles-Ševčík equation applied to cyclic voltammograms of the redox probe (data from Fig. 7). The geometric surface area defined by the O-ring (0.229 cm2) is much greater than the effective electrochemical area before (0.0293 cm2) and after electrochemical activation (0.118 cm2). This result agrees with the SEM images that showed that the untreated 3Dprinted CB/PLA surface is mainly composed of non-conductive PLA. Moreover, the significant increase in electroactive area (4-fold) after electrochemical treatment indicates that PLA was removed and carbon black nanoparticles are more exposed and contributted to the dramatic current increase. Insert Figure 7

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Journal Pre-proof The heterogeneous electron transfer rate (HET) constants (k0 obs) of each surface, before and after electrochemical treatment, were calculated by the Nicholson method [30,31,41]. The obtained values were 5.79 x 10−3 cm s−1 and 8.33 x 10−3 cm s−1 before and after treatment, respectively. These results are in agreement with the EIS measurements which showed that the electrode HET constant is higher after electrochemical treatment with NaOH. The slower electrode transfer kinetics of the electrode before treatment can be explained by the low exposure of carbon black nanoparticles on the surface and the increased amount of the polymeric matrix that blocked electron transfer [30].

3.2. Optimization and analytical features of the proposed method After the evaluation and conclusion of the superior performance observed for the 3D-printed electrode chemical/electrochemically pretreated in NaOH, the parameters that influence on the SWASV method were optimized for both metals. The optimized SWASV parameters in acetate buffer (pH 4.7) were as follows: frequency = 10 Hz, amplitude = 40 mV, step potential = 1 mV, conditioning potential = +0.6 V (for 30 s), equilibration time = 15 s, deposition potential = −1.1 V (for 180 s) and stirring rate = 1500 rpm. Table 1 summarizes the selected variables and the respective studied ranges.

Insert Table 1

A linear behavior with well-separate stripping peaks was obtained over the concentration range between 30 and 270 µg L−1 (R = 0.997) for both analytes. The limits of detection (LOD) were 2.9 μg L−1 and 2.6 μg L−1 and the limits of quantification (LOQ) were 8.9 μg L−1 and 7.9 μg L−1 for Cd2+ and Pb2+, respectively, under 180 s as deposition

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Journal Pre-proof time. The LOD and LOQ values were calculated as 3 × SD/S and 10 × SD/S, respectively, where SD is the standard deviation of blank samples (n = 10) and S the slope (sensitivity) of the analytical curve (S = 0.0206 μA/μg L−1 for Cd2+ and 0.0233 μA/μg L−1 for Pb2+). Considering the 5-fold dilution of all samples, the LOD and LOQ values of the method were 14.5 μg L−1 and 44.5 μg L−1 for Cd2+ and 13.0 μg L−1 and 39.0 μg L−1 for Pb2+ (LOD and LOQ values were multiplied by 5 due to the 5-fold dilution to obtain a more realistic estimation considering the analysis of real samples). A repeatability study was evaluated for two levels of concentration. For 50 μg L−1, the relative standard deviation (RSD) values were 4.3% and 3.5% and for 100 μg L−1 the RSD values were 4.4% and 4.5% for Cd2+ and Pb2+, respectively, for ten consecutive measures at each concentration level. The reproducibility of the proposed method as well as of the chemical/electrochemical activation of the electrode was evaluated by interday (n = 3), intra-day (n = 3), interelectrode (n = 2) and inter-activation (n = 2) experiments. Satisfactory results were obtained with RSD values lower than 6.5%. The good stability of the measurements shows that the electrode treatment process and/or the electrode exchange by a new sensor did not provide significant distortions between measurements, resulting in low RSD values. A summary of the analytical characteristics of the method using the treated 3Dprinted electrode for the simultaneous determination of Cd2+ and Pb2+ is shown in Table 2.

Insert Table 2

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Journal Pre-proof 3.3. Application of the treated 3D-printed electrode for the analysis of urine and saliva After obtaining the analytical parameters, the proposed method using the treated 3D-printed electrode was applied for the determination of Cd2+ and Pb2+ in real urine and saliva samples. Analysis by the SWASV technique showed that the concentrations of Cd2+ and Pb2+ in the samples were below the LOD values of the proposed method, even with the application of higher deposition times (240 s). Thus, the accuracy of the method was evaluated by recovery tests analyzing samples spiked with two known levels (50 and 100 μg L−1) of Cd2+ and Pb2+ concentrations. Table 3 reports the concentration of Cd2+ and Pb2+ in the 5-fold diluted samples in supporting electrolyte and their recovery values.

Insert Table 3

As can be seen in Table 3, the proposed method using the 3D-printed electrode was accurate and provided acceptable values of recovery in the spiked saliva and urine samples. Recovery values were obtained in the range between 93 and 108% for Cd2+ and between 96 and 112% for Pb2+. Figure 8 shows the SWASV recordings and respective calibration curves for the simultaneous determination of Cd2+ and Pb2+ in urine (A, B and C) and saliva (D, E and F) samples that were 5-fold diluted in 0.1 mol L-1 acetate buffer (pH 4.7).

Insert Figure 8

The voltammetric signals showed good resolution and remained separated with increasing concentrations of both metals. Similar results were obtained in the detection

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Journal Pre-proof of cadmium and lead using a Hg2+/carbon-nanotube modified graphite electrode [42]. The stripping peaks for Cd2+ (−0.86V) and Pb2+ (−0.60V) are comparable to those obtained in this work, approximately at −0.77 V for Cd2+ and −0.58 V for Pb2+. The linear correlation coefficients were greater than 0.996. The standard addition method was applied for the simultaneous quantification of Cd2+ and Pb2+ in urine and saliva samples. In addition, undesirable signals from the matrix of complex samples were not observed. It is noteworthy that a single electrode was able to analyze all saliva and urine samples, requiring

only

a

polishing

with

sandpaper

and

water,

and

subsequent

chemical/electrochemical treatment every day before the beginning of the experiments. Monitoring of toxic metals, such as cadmium and lead, in biological samples provides evidence of possible sources of contamination and intoxication of the population, whether by food, atmospheric air or directly with harmful substances in the home or work environment. Thus, the method developed using treated 3D-printed electrode for the determination of Cd2+ and Pb2+ in real urine and saliva samples proved to be efficient for such applications.

Insert Table 4

A comparison of the analytical characteristics of the treated 3D-printed CB-PLA electrode with other reports from the literature that used electrochemical stripping analysis for the simultaneous or individual determination of Cd2+ and Pb2+ is presented in Table 4. The detection limits obtained in techniques that involves electrochemical stripping analysis are dependent on the deposition time. Thus, when we compare the

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Journal Pre-proof proposed work with others that used 3D-printed electrodes manufactured using FDM and nanografite within different polymeric filaments, this work provided 100-fold lower detection limits (2.9 µg L−1 and 2.6 µg L−1 for Cd2+ and Pb2+, respectively), which is a great evidence of the improvement of electrochemical properties obtained after the proposed chemical/electrochemical treatment [41,43]. The SLM 3D-printing technique fabricates sensors made of stainless-steel that are not the best material for electrochemical sensing, especially for electrochemical stripping analysis of metals [20]. For this reason, this surface needs to be modified by electrodeposition with Au or Bi to obtain filmmodified sensors capable of trace metal detection with proper analytical characteristics. Even tough, the obtained 3D-printed electrode after chemical/electrochemical treatment provided higher detectability using a low-cost filament and 3D-printing process (it is relevant to highlight that SLM 3D-printing is highly expensive and the obtained 3Dprinted material required further modification). One possible explanation for the great detectability of the proposed 3D-printed electrode for metal detection is the PLA matrix, which presents carboxylic groups that may be responsible for the facilitated metal preconcentration during the deposition step of stripping analysis. Although part of PLA is removed after the chemical/electrochemical treatment in NaOH as stated before by IR spectra, the 3D-printed electrode is predominantly composed of PLA, thus carboxylic groups are likely available. Regarding linear range, this work provides wider or similar linear ranges (between 30 µg L−1 and 270 µg L−1 for both metals) compared with the same previous published works. In addition, we must emphasize that some works employed organic solvents, such as xylene and chloroform, and laborious steps (reflux, sonication and solvent drying) in the manufacture of 3D-printed CB-PLA electrodes [41,43] while we propose a simpler protocol. It can also be observed from Table 4 that the analytical characteristics (LOD

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Journal Pre-proof and linear range) of the proposed 3D-printed electrode are in the same order of magnitude with those obtained using electrodes modified with bismuth or antimony film, which are commonly used for metal determination by electrochemical stripping analysis [44–46]. In other cases, the 3D-printed CB-PLA electrode presented superior performance in comparison with screen-printed electrodes [47,48], modified carbon-paste electrode [49] and diamond/graphite-based electrode [50]. It is also possible to compare the proposed 3D-printed electrode with conventional electrodes (such as glassy-carbon or graphite) modified with different complex materials (bismuth nanoparticles, Bi2O3/Fe2O3 at graphene oxide, metal-organic framework on a self-doped polyaniline) [51–55], which requires additional steps (and often time-consuming protocols). As shown in Table 4, even using such modified electrodes, the proposed method presented a wider linear range in most cases although the LOD value was not superior in comparison with these electrochemical sensors. However, the same chemical modifiers used to modify glassycarbon or graphite surfaces could be employed on the treated 3D-printed surface and probably improved LOD values would be obtained. For this reason, this work opens up new strategies to improve even more its dectability if lower LOD values are required depending on the analyzed sample. It is also important to mention that the LOD of the proposed method can be improved by extending the deposition time to 600 s, as it was explored to improve the LOD of a sensor cited in Table 4 [53]. Comparing with traditional methods, it can be noted that the LODs estimated in this work are higher than the ones obtained by graphite-furnace atomic absorption spectrometric methods; however, the LODs are in the same order of magnitude with some published works that employed optical emission spectrometry [56] or flame atomic absorption spectrometry [57]. In addition, the equipment used for the analysis in this work is not bulky, less expensive, and is portable compared to traditional methods which is

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Journal Pre-proof essential for in field analysis. Additionally, the 3D-printing technology can be used to fabricate affordable electrochemical sensors in large-scale and free of design with electrochemical sensing properties better than commercial screen-printed electrodes. Urine and saliva are biological fluids used to assess environmental exposure to pollutants, including toxic metals. Some metals, such as Pb, Cd, Hg, Fe, Zn, Cr, Mn, Ni, Cu among others, are indicative of environmental pollution, including the burning of fossil fuels, and may be present in biological fluids as a consequence of exposure to the work environment or contaminated locations. Even at low concentrations, the presence of toxic metals can lead to kidney and bone diseases, gastrointestinal disorders and even cancer [58,59]. In this work, the presence of Fe3+, Ni2+, Cr3+, Zn2+, Hg2+, Mn2+ and Cu2+ at two concentration levels (250 and 500 µg L−1) was evaluated as interfering species in the simultaneous determination of 50 µg L−1 Cd2+ and 50 µg L−1 Pb2+ as shown in Table 5. Insert Table 5

Fe3+, Ni2+, Zn2+ and the lowest level of Cr3+ concentration did not affect significantly the response of Cd2+ and Pb2+ (variation below 10%). No stripping signals of Fe3+, Ni2+, Zn2+ and Cr3+ or peak deformation of Cd2+ and Pb2+ were observed under the optimized experimental conditions. Thus, the treated 3D-printed electrode presents selectivity for the determination of Cd2+ and Pb2+ in the presence of the mentioned interferents (Fe3+, Ni2+, Zn2+ and 250 µg L−1 Cr3+). In the presence of Hg2+, Cu2+, at the highest level of Cr3+ concentration and the lowest level of Mn2+, the interference effects were more intense on the signals of Cd2+ and Pb2+ resulting in signal variations greater than 10%. However, no stripping signals for Cr3+ and Mn2+ or loss of resolution of the analytical signals of Cd2+ and Pb2+ were observed under the optimized conditions. On the

20

Journal Pre-proof other hand, other stripping signals were observed in the potential window studied for the higher concentration levels of Hg2+ and Cu2+. In addition, the shape of the Pb2+ stripping signal changed in the presence of Cu. Therefore, Cr3+, Mn2+, Hg2+ and Cu2+ interfere in the detection of Cd2+ and Pb2+ in the high concentration level (10:1). Nevertheless, it should be emphasized that the treated 3D-printed electrodes were applied for the analysis of real urine and saliva samples and no interferences were verified as the recovery values were in the range between 93 and 112 %. Thus, the presence of interferents originally found in the samples were evaluated in the analysis of real samples. Moreover, the standard addition method can solve possible interference effects caused by these metals in the determination of Cd2+ and Pb2+ in real samples.

4. Conclusions We have demonstrated that a simple chemical/electrochemical treatment with a 0.5 mol L-1 NaOH solution improved significantly the electrochemical response of 3Dprinted electrodes fabricated using conductive PLA filaments (proto pasta). Different techniques proved the change of the treated surfaces and exposure of nanographites from carbon black presented in the polymeric matrix. Cyclic voltammetry and EIS using the redox probe ferricyanide/ferrocyanide indicated faster electron transfer and increase in electroactive area after the treatment. The voltammetric responses of Cd2+ and Pb2+ dramatically increased (30-fold) after treatment which enabled the sensitive determination of both metals. Carboxylic groups naturally occurring on the PLA matrix may have contributed to the facilitated preconcentration of both metals during the electrochemical deposition step of stripping analysis. The treated 3D-printed electrodes were applied for the simultaneous determination of Cd2+ and Pb2+ in biological fluids to demonstrate the feasibility of the 3D-printed sensors. Other metallic species, such as Cu2+ 21

Journal Pre-proof and Hg2+, can be detected on this platform as revealed by the study of interferents. Moreover, the nature of PLA presenting free carboxylic groups may be investigated as a plataform to attach biomolecules for the construction of biosensors as recently demonstrated [60]. Hence, this work opens up a simple strategy to produce highperformance 3D-printed electrodes for analysis of a wide range of samples, here in demonstrated for biological samples, with sensing properties better than commercial SPEs. Moreover, the electrochemically-treated 3D-printed surface can serve as substrates to incorporate a wide range of chemical modifiers, including bismuth-based modifiers to improve even more the detectability of metals by the sensor if lower LOD values are required. It is also important to emphasize that such 3D-printed surfaces are inexpensive and thus serve as a source of disposable electrochemical sensors.

Acknowledgements The authors are grateful to CAPES (001 and Pró-Forense 25/2014 23038.007073/2014-12), CNPq (307271/2017-0 and 427731/2018-6), FAPEMIG (PPM 00640-16), and INCTBio (CNPq grant no. 465389/2014-7) for financial support.

5. References [1]

B. Gross, S.Y. Lockwood, D.M. Spence, Recent advances in analytical chemistry by

3D

printing,

Anal.

Chem.

89

(2017)

57–70.

doi:10.1021/acs.analchem.6b04344. [2]

A. Ambrosi, J.G.S. Moo, M. Pumera, Helical 3D-printed metal electrodes as custom-shaped 3D platform for electrochemical devices, Adv. Funct. Mater. 26 (2016) 698–703. doi:10.1002/adfm.201503902.

22

Journal Pre-proof [3]

A.A. Yazdi, A. Popma, W. Wong, T. Nguyen, Y. Pan, J. Xu, 3D printing: an emerging tool for novel microfluidics and lab-on-a-chip applications, Microfluid. Nanofluidics. 20 (2016) 1–18. doi:10.1007/s10404-016-1715-4.

[4]

Y. He, Y. Wu, J.Z. Fu, Q. Gao, J.J. Qiu, Developments of 3D Printing Microfluidics and Applications in Chemistry and Biology: a Review, Electroanalysis. 28 (2016) 1658–1678. doi:10.1002/elan.201600043.

[5]

R.M. Cardoso, D.M.H. Mendonça, W.P. Silva, M.N.T. Silva, E. Nossol, R.A.B. da Silva, E.M. Richter, R.A.A. Muñoz, 3D printing for electroanalysis: From multiuse electrochemical cells to sensors, Anal. Chim. Acta. 1033 (2018) 49–57. doi:10.1016/j.aca.2018.06.021.

[6]

C.L. Manzanares Palenzuela, F. Novotný, P. Krupička, Z. Sofer, M. Pumera, 3DPrinted Graphene/Polylactic Acid Electrodes Promise High Sensitivity in Electroanalysis,

Anal.

Chem.

90

(2018)

5753–5757.

doi:10.1021/acs.analchem.8b00083. [7]

A.S. Munshi, R.S. Martin, Microchip-based electrochemical detection using a 3-D printed

wall-jet

electrode

device,

Analyst.

141

(2016)

862–869.

doi:10.1039/C5AN01956G. [8]

A.J.L. Morgan, L.H. San Jose, W.D. Jamieson, J.M. Wymant, B. Song, P. Stephens, D.A. Barrow, O.K. Castell, Simple and versatile 3D printed microfluidics using fused filament fabrication, PLoS One. 11 (2016) e0152023. doi:10.1371/journal.pone.0152023.

[9]

A.A. Dias, T.M.G. Cardoso, R.M. Cardoso, L.C. Duarte, R.A.A. Muñoz, E.M. Richter, W.K.T. Coltro, Paper-based enzymatic reactors for batch injection analysis of glucose on 3D printed cell coupled with amperometric detection, Sensors Actuators, B Chem. 226 (2016) 196–203. doi:10.1016/j.snb.2015.11.040.

23

Journal Pre-proof [10]

S.Y. Lockwood, J.E. Meisel, F.J. Monsma, D.M. Spence, A Diffusion-Based and Dynamic 3D-Printed Device That Enables Parallel in Vitro Pharmacokinetic Profiling

of

Molecules,

Anal.

Chem.

88

(2016)

1864–1870.

doi:10.1021/acs.analchem.5b04270. [11]

F. Li, N.P. Macdonald, R.M. Guijt, M.C. Breadmore, Using Printing Orientation for Tuning Fluidic Behavior in Microfluidic Chips Made by Fused Deposition Modeling

3D

Printing,

Anal.

Chem.

89

(2017)

12805–12811.

doi:10.1021/acs.analchem.7b03228. [12]

J. Zhong, M. Yi, H.H. Bau, Magneto hydrodynamic (MHD) pump fabricated with ceramic tapes, Sensors Actuators, A Phys. 96 (2002) 59–66. doi:10.1016/S09244247(01)00764-6.

[13]

C. Parra-Cabrera, C. Achille, S. Kuhn, R. Ameloot, 3D printing in chemical engineering and catalytic technology: structured catalysts, mixers and reactors, Chem. Soc. Rev. 47 (2018) 209–230. doi:10.1039/C7CS00631D.

[14]

M.P. Browne, F. Novotný, Z. Sofer, M. Pumera, 3D Printed Graphene Electrodes’ Electrochemical Activation, ACS Appl. Mater. Interfaces. 10 (2018) 40294– 40301. doi:10.1021/acsami.8b14701.

[15]

B. Schmidt, D. King, J. Kariuki, Designing and Using 3D-Printed Components That Allow Students to Fabricate Low-Cost, Adaptable, Disposable, and Reliable Ag/AgCl Reference Electrodes, J. Chem. Educ. 95 (2018) 2076–2080. doi:10.1021/acs.jchemed.8b00512.

[16]

B.R. Liyarita, A. Ambrosi, M. Pumera, 3D-printed Electrodes for Sensing of Biologically Active Molecules, Electroanalysis. 30 (2018) 1319–1326. doi:10.1002/elan.201700828.

[17]

H.H. Bin Hamzah, O. Keattch, D. Covill, B.A. Patel, The effects of printing

24

Journal Pre-proof orientation on the electrochemical behaviour of 3D printed acrylonitrile butadiene styrene

(ABS)/carbon

black

electrodes,

Sci.

Rep.

8

(2018)

9135.

doi:10.1038/s41598-018-27188-5. [18]

T.S. Cheng, M.Z.M. Nasir, A. Ambrosi, M. Pumera, 3D-printed metal electrodes for electrochemical detection of phenols, Appl. Mater. Today. 9 (2017) 212–219. doi:10.1016/j.apmt.2017.07.005.

[19]

A. Ambrosi, M. Pumera, 3D-printing technologies for electrochemical applications, Chem. Soc. Rev. 45 (2016) 2740–2755. doi:10.1039/c5cs00714c.

[20]

K.Y. Lee, A. Ambrosi, M. Pumera, 3D-printed Metal Electrodes for Heavy Metals Detection by Anodic Stripping Voltammetry, Electroanalysis. 29 (2017) 2444– 2453. doi:10.1002/elan.201700388.

[21]

V. Katseli, A. Economou, C. Kokkinos, Single-step fabrication of an integrated 3D-printed device for electrochemical sensing applications, Electrochem. Commun. 103 (2019) 100–103. doi:10.1016/j.elecom.2019.05.008.

[22]

K.C. Honeychurch, Z. Rymansaib, P. Iravani, Anodic stripping voltammetric determination of zinc at a 3-D printed carbon nanofiber–graphite–polystyrene electrode using a carbon pseudo-reference electrode, Sensors Actuators, B Chem. 267 (2018) 476–482. doi:10.1016/j.snb.2018.04.054.

[23]

C.L. Manzanares Palenzuela, M. Pumera, (Bio)Analytical chemistry enabled by 3D printing: Sensors and biosensors, TrAC - Trends Anal. Chem. 103 (2018) 110– 118. doi:10.1016/j.trac.2018.03.016.

[24]

H.H. Hamzah, S.A. Shafiee, A. Abdalla, B.A. Patel, 3D printable conductive materials for the fabrication of electrochemical sensors: A mini review, Electrochem. Commun. 96 (2018) 27–31. doi:10.1016/j.elecom.2018.09.006.

[25]

R.M. Cardoso, S.V.F. Castro, M.N.T. Silva, A.P. Lima, M.H.P. Santana, E.

25

Journal Pre-proof Nossol, R.A.B. Silva, E.M. Richter, T.R.L.C. Paixão, R.A.A. Muñoz, 3D-printed flexible device combining sampling and detection of explosives, Sensors Actuators B Chem. 292 (2019) 308–313. doi:10.1016/j.snb.2019.04.126. [26]

E. Mattio, F. Robert-Peillard, C. Branger, K. Puzio, A. Margaillan, C. Brach-Papa, J. Knoery, J.L. Boudenne, B. Coulomb, 3D-printed flow system for determination of

lead

in

natural

waters,

Talanta.

168

(2017)

298–302.

doi:10.1016/j.talanta.2017.03.059. [27]

R. Gusmão, M.P. Browne, Z. Sofer, M. Pumera, The capacitance and electron transfer of 3D-printed graphene electrodes are dramatically influenced by the type of solvent used for pre-treatment, Electrochem. Commun. 102 (2019) 83–88. doi:10.1016/j.elecom.2019.04.004.

[28]

C.L. Manzanares-Palenzuela, S. Hermanova, Z. Sofer, M. Pumera, Proteinasesculptured 3D-printed graphene/polylactic acid electrodes as potential biosensing platforms: Towards enzymatic modeling of 3D-printed structures, Nanoscale. 11 (2019) 12124–12131. doi:10.1039/c9nr02754h.

[29]

D.M. Wirth, M.J. Sheaff, J. V. Waldman, M.P. Symcox, H.D. Whitehead, J.D. Sharp, J.R. Doerfler, A.A. Lamar, G. Leblanc, Electrolysis Activation of FusedFilament-Fabrication Spectroelectrochemical

3D-Printed Analysis,

Electrodes Anal.

for

Chem.

Electrochemical 91

(2019)

and

5553–5557.

doi:10.1021/acs.analchem.9b01331. [30]

P.L. dos Santos, V. Katic, H.C. Loureiro, M.F. dos Santos, D.P. dos Santos, A.L.B. Formiga, J.A. Bonacin, Enhanced performance of 3D printed graphene electrodes after electrochemical pre-treatment: Role of exposed graphene sheets, Sensors Actuators, B Chem. 281 (2019) 837–848. doi:10.1016/j.snb.2018.11.013.

[31]

D.P. Rocha, M.N.T. Silva, R.M. Cardoso, S.V.F. Castro, T.F. Tormin, E.M.

26

Journal Pre-proof Richter, E. Nossol, R.A.A. Munoz, Carbon nanotube/reduced graphene oxide thinfilm nanocomposite formed at liquid-liquid interface: Characterization and potential electroanalytical applications, Sensors Actuators, B Chem. 269 (2018) 293–303. doi:10.1016/j.snb.2018.04.147. [32]

L.A.J. Silva, W.P. da Silva, J.G. Giuliani, S.C. Canobre, C.D. Garcia, R.A.A. Munoz, E.M. Richter, Use of pyrolyzed paper as disposable substrates for voltammetric determination of trace metals, Talanta. 165 (2017) 33–38. doi:10.1016/j.talanta.2016.12.009.

[33]

S.A. Kitte, S. Li, A. Nsabimana, W. Gao, J. Lai, Z. Liu, G. Xu, Stainless steel electrode for simultaneous stripping analysis of Cd(II), Pb(II), Cu(II) and Hg(II), Talanta. 191 (2019) 485–490. doi:10.1016/j.talanta.2018.08.066.

[34]

N. Serrano, A. González-Calabuig, M. Del Valle, Crown ether-modified electrodes for the simultaneous stripping voltammetric determination of Cd(II), Pb(II) and Cu(II), Talanta. 138 (2015) 130–137. doi:10.1016/j.talanta.2015.01.044.

[35]

Y. Yao, H. Wu, J. Ping, Simultaneous determination of Cd(II) and Pb(II) ions in honey and milk samples using a single-walled carbon nanohorns modified screenprinted

electrochemical

sensor,

Food

Chem.

274

(2019)

8–15.

doi:10.1016/j.foodchem.2018.08.110. [36]

L.P. Caetano, A.P. Lima, T.F. Tormin, E.M. Richter, F.S. Espindola, F. V. Botelho, R.A.A. Munoz, Carbon-nanotube Modified Screen-printed Electrode for the Simultaneous Determination of Nitrite and Uric Acid in Biological Fluids Using Batch-injection Amperometric Detection, Electroanalysis. 30 (2018) 1870–1879. doi:10.1002/elan.201800189.

[37]

T. da Costa Oliveira, M.H.P. Santana, C.E. Banks, R.A.A. Munoz, E.M. Richter, Electrochemical Portable Method for on site Screening of Scopolamine in

27

Journal Pre-proof Beverage

and

Urine

Samples,

Electroanalysis.

31

(2019)

567–574.

doi:10.1002/elan.201800707. [38]

R.A.A. Munoz, F.S. Felix, M.A. Augelli, T. Pavesi, L. Angnes, Fast ultrasoundassisted treatment of urine samples for chronopotentiometric stripping determination of mercury at gold film electrodes, Anal. Chim. Acta. 571 (2006) 93–98. doi:10.1016/j.aca.2006.04.034.

[39]

S. Kim, S.J. Park, Effect of acid/base treatment to carbon blacks on preparation of carbon-supported platinum nanoclusters, Electrochim. Acta. 52 (2007) 3013– 3021. doi:10.1016/j.electacta.2006.09.060.

[40]

E.M. Richter, D.P. Rocha, R.M. Cardoso, E.M. Keefe, C.W. Foster, R.A.A. Munoz,

C.E.

Banks,

Complete

Additively

Manufactured

(3D-Printed)

Electrochemical Sensing Platform, Anal. Chem. 91 (2019) 12844−12851. doi:10.1021/acs.analchem.9b02573. [41]

C.W. Foster, H.M. Elbardisy, M.P. Down, E.M. Keefe, G.C. Smith, C.E. Banks, Additively manufactured graphitic electrochemical sensing platforms, Chem. Eng. J. 381 (2020) 122343. doi:10.1016/j.cej.2019.122343.

[42]

S.J.J.R. Prabakar, C. Sakthivel, S.S. Narayanan, Hg(II) immobilized MWCNT graphite electrode for the anodic stripping voltammetric determination of lead and cadmium, Talanta. 85 (2011) 290–297. doi:10.1016/j.talanta.2011.03.058.

[43]

Z. Rymansaib, P. Iravani, E. Emslie, M. Medvidović-Kosanović, M. Sak-Bosnar, R. Verdejo, F. Marken, All-Polystyrene 3D-Printed Electrochemical Device with Embedded Carbon Nanofiber-Graphite-Polystyrene Composite Conductor, Electroanalysis. 28 (2016) 1517–1523. doi:10.1002/elan.201600017.

[44]

G. Kefala, A. Economou, Polymer-coated bismuth film electrodes for the determination of trace metals by sequential-injection analysis/anodic stripping

28

Journal Pre-proof voltammetry,

Anal.

Chim.

Acta.

576

(2006)

283–289.

doi:10.1016/j.aca.2006.06.006. [45]

A. Economou, A. Voulgaropoulos, Stripping voltammetry of trace metals at bismuth-film electrodes by batch-injection analysis, Electroanalysis. 22 (2010) 1468–1475. doi:10.1002/elan.200970007.

[46]

M. Slavec, S.B. Hocevar, L. Baldrianova, E. Tesarova, I. Svancara, B. Ogorevc, K. Vytras, Antimony film microelectrode for anodic stripping measurement of Cadmium(II), Lead(II) and Copper(II), Electroanalysis. 22 (2010) 1617–1622. doi:10.1002/elan.200900583.

[47]

V. Sosa, C. Barceló, N. Serrano, C. Ariño, J.M. Díaz-Cruz, M. Esteban, Antimony film screen-printed carbon electrode for stripping analysis of Cd(II), Pb(II), and Cu(II)

in

natural

samples,

Anal.

Chim.

Acta.

855

(2015)

34–40.

doi:10.1016/j.aca.2014.12.011. [48]

M.R. Palomo-Marín, F. Rueda-Holgado, J. Marín-Expósito, E. Pinilla-Gil, Disposable sputtered-bismuth screen-printed sensors for voltammetric monitoring of cadmium and lead in atmospheric particulate matter samples, Talanta. 175 (2017) 313–317. doi:10.1016/j.talanta.2017.07.060.

[49]

Z. Koudelkova, T. Syrovy, P. Ambrozova, Z. Moravec, L. Kubac, D. Hynek, L. Richtera, V. Adam, Determination of zinc, cadmium, lead, copper and silver using a carbon paste electrode and a screen printed electrode modified with chromium(III)

oxide,

Sensors

(Switzerland).

17

(2017)

1832.

doi:10.3390/s17081832. [50]

Z. Zhai, N. Huang, H. Zhuang, L. Liu, B. Yang, C. Wang, Z. Gai, F. Guo, Z. Li, X. Jiang, A diamond/graphite nanoplatelets electrode for anodic stripping voltammetric trace determination of Zn(II), Cd(II), Pb(II) and Cu(II), Appl. Surf.

29

Journal Pre-proof Sci. 457 (2018) 1192–1201. doi:10.1016/j.apsusc.2018.06.266. [51]

U. Injang, P. Noyrod, W. Siangproh, W. Dungchai, S. Motomizu, O. Chailapakul, Determination of trace heavy metals in herbs by sequential injection analysisanodic stripping voltammetry using screen-printed carbon nanotubes electrodes, Anal. Chim. Acta. 668 (2010) 54–60. doi:10.1016/j.aca.2010.01.018.

[52]

Y. Wu, N.B. Li, H.Q. Luo, Simultaneous measurement of Pb, Cd and Zn using differential pulse anodic stripping voltammetry at a bismuth/poly(p-aminobenzene sulfonic acid) film electrode, Sensors Actuators, B Chem. 133 (2008) 677–681. doi:10.1016/j.snb.2008.04.001.

[53]

D. Yang, L. Wang, Z. Chen, M. Megharaj, R. Naidu, Anodic stripping voltammetric determination of traces of Pb(II) and Cd(II) using a glassy carbon electrode modified with bismuth nanoparticles, Microchim. Acta. 181 (2014) 1199–1206. doi:10.1007/s00604-014-1235-4.

[54]

X. Zhao, W. Bai, Y. Yan, Y. Wang, J. Zhang, Core-shell self-doped polyaniline coated

metal-organic-framework

(SPAN@UIO-66-NH2)

screen

printed

electrochemical sensor for Cd2+ ions, J. Electrochem. Soc. 166 (2019) B873– B880. doi:10.1149/2.0251912jes. [55]

T.R. Das, P.K. Sharma, Sensitive and selective electrochemical detection of Cd2+ by using bimetal oxide decorated Graphene oxide (Bi2O3/Fe2O3@GO) electrode, Microchem. J. 147 (2019) 1203–1214. doi:10.1016/j.microc.2019.04.001.

[56]

Z. Zhang, Z. Wang, Q. Li, H. Zou, Y. Shi, Determination of trace heavy metals in environmental and biological samples by solution cathode glow discharge-atomic emission spectrometry and addition of ionic surfactants for improved sensitivity, Talanta. 119 (2014) 613–619. doi:10.1016/j.talanta.2013.11.010.

[57]

V. Kazantzi, E. Drosaki, A. Skok, A.B. Vishnikin, A. Anthemidis, Evaluation of

30

Journal Pre-proof polypropylene and polyethylene as sorbent packing materials in on-line preconcentration columns for trace Pb(II)and Cd(II)determination by FAAS, Microchem. J. 148 (2019) 514–520. doi:10.1016/j.microc.2019.05.033. [58]

R.M. Cespón-Romero, M.C. Yebra-Biurrun, Determination of trace metals in urine with an on-line ultrasound-assisted digestion system combined with a flowinjection preconcentration manifold coupled to flame atomic absorption spectrometry,

Anal.

Chim.

Acta.

609

(2008)

184–191.

doi:10.1016/j.aca.2008.01.002. [59]

A. Habibiyan, M. Ezoddin, N. Lamei, K. Abdi, M. Amini, M. Ghazi-khansari, Ultrasonic assisted switchable solvent based on liquid phase microextraction combined with micro sample injection flame atomic absorption spectrometry for determination of some heavy metals in water, urine and tea infusion samples, J. Mol. Liq. 242 (2017) 492–496. doi:10.1016/j.molliq.2017.07.043.

[60] R.M. Cardoso, P.R.L. Silva, A.P. Lima, D.P.Rocha, T.C. Oliveira, T.M. do Prado, E.L. Fava, O. Fatibello-Filho, E.M.Richter, R.A.A. Muñoz, 3D-Printed graphene/polylactic acid electrode for bioanalysis: Biosensing of glucose and simultaneous determination of uric acid and nitrite in biological fluids, Sensors Actuators, B Chem. 307 (2020) 127621. doi: 10.1016/j.snb.2019.127621.

31

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Figure Captions

Figure 1. Schematic diagram of the 3D-printed cell, highlighting first the 3D-printed parts (top cover, body, bottom and screw nuts of the cell), platinum wire as counter electrode (CE), Ag/AgCl (3 mol L−1 KCl) as reference electrode (RE), 3D-printed working electrode (WE), O-ring, and contact plate to establish the electric contact of WE. At the center, the assembled cell with electrolyte and conections between electrodes and potentiostat. Images of micropipette used for injections of standards and samples and computer screen connected to the potentiostat are also illustrated.

Figure 2. Scheme of the surface treatment of the 3D-printed electrode: mechanical polishing using a hollow cube to enable easier to hold the 3D platform with dimensions of 3.0 cm (length) x 3.0 cm (width) x 1.5 cm (height); next a piece of 3.0 cm x 1.5 cm was cut and then assembled at the 3D-printed cell; 0.5 mol L-1 NaOH solution is placed in

the

cell

and

the

sequence

of

potentials

is

applied

to

perform

the

chemical//electrochemical treatment.

Figure 3. Background-corrected SWASV for Cd2+ and Pb2+ (100 µg L−1) before (black line) and after (red line) chemical/electrochemical treatment in 0.5 mol L−1 NaOH (+1.4V/200s and -1.0V/200s). SWASV conditions: deposition time: 180 s; deposition potential: -1.1 V; stirring rate: 1500 rpm; conditioning potential: +0.6 V; conditioning time: 30 s; step potential: 1 mV, modulation amplitude: 10 mV; frequency: 10 Hz. Supporting electrolyte: 0.1 mol L−1 acetate buffer (pH 4.7).

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Figure 4. SEM data of CB/PLA before (A) and after (B) electrochemical treatment.

Figure 5. Infrared spectrum (ATR mode) of CB/PLA surface before (▬) and after (▬) electrochemical treatment.

Figure 6. (A) EIS spectra acquired on 3D-printed sensor before (black line) and after (red line) chemical/electrochemical treatment using 0.5 mol L−1 NaOH (+1.4V/200s and 1.0V/200s). The inset represents an equivalent circuit used for EIS measurements. (B) Cyclic voltammetric recordings obtained on 3D-printed sensor before (black line) and after (red line) chemical/electrochemical treatment in the presence of 1:1 mmol L−1 K3Fe(CN)6/K4Fe(CN)6 in 0.1 mol L−1 KCl. Experimental conditions in (A): frequency range between 0.1 Hz and 50.000 Hz with signal amplitude of 10 mV with 10 data points per frequency decade; (B) scan rate: 50 mV s-1; step potential: 5 mV; dashed lines present in (B) correspond to the respective blanks.

Figure 7. Cyclic voltammetric measurements of 1:1 mmol L−1 K3Fe(CN)6/K4Fe(CN)6 in 0.1 mol L−1 KCl at different scan rates (10 to 100 mV s−1). Inset: Relation of anodic (■) and cathodic (●) peak currents versus square root of scan rate (ν0.5). Working electrode: (A) untreated and (B) treated 3D-printed CB/PLA.

Figure 8. SWASV recordings for urine (A) and saliva (D) samples, spiked with 50 µg L−1 and 5-fold diluted in electrolyte (1st scan) and after addition of standard solutions of Cd2+ and Pb2+ (2nd to 5th scans of both peaks). Standard addition curves for Cd2+ (B) and Pb2+ (C) in urine sample. Standard addition curves for Cd2+ (E) and Pb2+ (F) in saliva sample.

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Credit author statement Diego P. Rocha: Conceptualization, Methodology, Data curation, WritingOriginal draft preparation; Andre L. Squissato: Data curation, Writing- Original draft preparation; Sarah M. Da Silva: Methodology, Validation; Eduardo M. Richter: Investigation, Visualization, Supervision; Rodrigo A. A. Munoz: Investigation, Visualization, Supervision, Writing- Reviewing and Editing.

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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:

Journal Pre-proof Table 5. Variation of SWASV responses for 50 μg L−1 Cd2+ and Pb2+ in the presence of Fe3+, Ni2+, Zn2+, Cr3+, Hg2+, Mn2+ and Cu2+ at two concentration level, 250 (5:1) and 500 µg L−1 (10:1).

Interferents

Fe3+

Ni2+

Cr3+

Zn2+

Hg2+

Mn2+

Cu2+

Current signal

Current signal

variation

variation

for Cd2+ (%)

for Pb2+ (%)

5:1

− 2.5

− 1.9

10:1

− 3.7

− 4.2

5:1

− 0.3

− 3.4

10:1

− 5.8

− 5.2

5:1

− 8.9

− 7.8

10:1

−14.6

− 12.0

5:1

− 6.3

− 4.6

10:1

− 9.1

− 6.4

5:1

+ 28.0

− 18.6

10:1

+18.4

− 28.4

5:1

− 12.3

− 7.3

10:1

− 16.4

− 7.6

5:1

− 20.6

− 10.7

10:1

− 26.0

> 50

Interfering ratio

Table 4. Comparison of the analytical characteristics of the proposed 3D-printed CB/PLA electrode for the determination of Cd2+ and/or Pb2+ with other electroanalytical methods reported in the literature. Samples

Aqueous solutions

Technique

SWASV

Electrode

Selective lase melting 3D-printing of stainless-steel (SS) modified with Au or Bi film

Aqueous solutions

SWASV

Additive manufacturing NG/PLA filaments 3D-printed electrode

Aqueous solutions

DPASV

Polystyrene/CNF/graphite composite 3D‐printed electrode

Urine

SIA – ASV

GCE modified with polymer coated bismuth film

Fertiliser

SWASV

GCE modified with bismuth film

Analyte

Cd2+ Pb2+

Cd2+ Pb2+ Pb2+ Cd2+ Pb2+ Cd2+ Pb2+

Deposition time (s)

120

n.d.

60

120

60

Linear range

LOD

3D-SS: 200 – 500 (Cd) and 100 – 300 (Pb) µg L−1

3D-SS: 136 and 12 µg L−1

3D-Au and 3D-Bi: 50 – 500 (Cd) and 50 – 300 (Pb) µg L−1 0.79 – 4.5 mg L−1

3D-Au: 47 and 18 µg L−1 3D-Bi: 9.4 and 3.5 µg L−1 0.32 mg L−1

0.19 – 4.5 mg L−1

0.16 mg L−1

0.44 – 53.5 mg L−1

n.d.

2 – 60 µg L−1

2.0 µg L−1

2 – 60 µg L−1

2.0 µg L−1

10 – 70 µg L−1

10.0 µg L−1

10 – 70 µg L−1

10.0 µg L−1

Reference

[20]

[41]

[43]

[44]

[45]

Natural water

SWASV

SbFME

Groundwater

DPASV

in-situ SbSPCE

Atmospheric particulate matter Industrial wastewater Aqueous solutions

SWASV

BispSPE

SWASV

Cr-CPE

DPASV

Diamond/graphite nanoplatelets electrode

Cd2+ Pb2+ Cd2+ Pb2+ Cd2+ Pb2+ Cd2+ Pb2+ Cd2+ Pb2+

120

120

180

100

270

Cd2+ Herb samples

River water samples Real water samples

SIA-SWASV

SPCNTE

Pb2+

180

Cd2+ DPASV

Bi/poly(p-ABSA) film

Pb2+

NanoBiE

Pb2+

1.9 µg L−1

20 – 100 µg L−1

3.1 µg L−1

11.5 – 72.4 µg L−1

3.4 µg L−1

16.8 – 62.6µg L−1

5.0 µg L−1

20.0 – 80.0 µg L−1

11.82 µg L−1

20 – 150.0 µg L−1

6.07 µg L−1

10.0 – 800.0 µg L−1

3.0 µg L−1

10.0 – 800.0 µg L−1

3.0 µg L−1

10.0 – 250.0 µg L−1

2.45 µg L−1

25.0 – 250.0 µg L−1

3.05 µg L−1

2–100 µg L−1

0.8 µg L−1

2–100 µg L−1

0.2 µg L−1

1–110 µg L−1 240

Cd2+ SWASV

20 – 100 µg L−1

600

L−1

0.63 µg L−1 0.8 µg L−1

5–60 µg L−1

0.4 µg L−1

5–60 µg L−1

0.8 µg L−1

1–130 µg

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

Natural lake and urine samples

SWASV

SPAN@UIO-66-NH2/SPCE

Cd2+

120

0.5–100 µg L−1

0.17 µg L−1

[54]

Water, fruits, soil, and biological samples

SWASV

Bi2O3/Fe2O3@GO/PGE

Cd2+

90

6.2 – 1160.2 ng L−1

1.85 ng L−1

[55]

Biological samples

SWASV

3D-printed PLA material containing carbon-black nanoparticles

Cd2+

30 – 270 µg L−1

2.9 µg L−1

L−1

L−1

This Work

Pb2+

180

30 – 270 µg

2.6 µg

SIA-SWASV: Sequential injection analysis-square-wave anodic stripping voltammetry; SPCNTE: Screen-printed carbon nanotubes electrodes; DPASV: Differential-pulse anodic stripping voltammetry; Bi/poly(p-ABSA) film: Bismuth/poly(p-aminobenzene sulfonic acid); NanoBiE: Bi nanoparticles were mixed with Nafion and directly coated on GCE; SPAN@UIO-66-NH2/SPCE: Self-doped polyaniline (SPAN) modified Metalorganic Framework (SPAN@UIO-66-NH2); Bi2O3/Fe2O3@GO/PGE: Bimetal oxide decorated Graphene oxide modified pencil graphite electrode; NG/PLA: Nanographite (NG) containing polylactic acid (PLA) filament; CNF: Carbon Nanofiber; SIA: Sequential-injection analysis; GCE: Glassy-carbon electrode; SbFME: Antimony film microelectrode; SbSPCE: In-situ antimony film screen-printed carbon electrode; BispSPE: Bismuth sputtered screen-printed electrode; Cr-CPE: Oxide modified carbon paste electrode; SWASV: Square-wave anodic stripping voltammetry; DPV: Differential-pulse anodic stripping voltammetry.

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Table 1. Optimization of the SWASV parameters for the determination of Cd2+ and Pb2+ using chemical/electrochemically pretreated 3D-printed electrode. Parameters

Studied range

Selected value

Deposition potential (V)

−0.8 to −1.3

−1.1

Time of deposition (s)

60 to 300

180

Stirring rate (rpm)

250 to 2500

1500

Conditioning potential (V)

+0.4 to +0.7

+0.6

Conditioning time (s)

15 to 60

30

Equilibration time (s)

15 to 25

15

Step (mV)

1 to 10

1

Amplitude (mV)

10 to 100

40

Frequency (Hz)

10 to 100

10

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Table 2. Analytical characteristics of the method for simultaneous determination of Cd2+ and Pb2+ using the 3D-printed electrode. Analytical parameters

Cd2+

Pb2+

Sensitivity (µA µg-1 L)

0.0206

0.0233

Limit of detection (µg L-1)

2.9

2.6

Limit of quantification (µg L-1)

8.9

7.8

Linear range (µg L-1)

30 to 270

30 to 270

R

0.997

0.997

Repeatability (n = 10 for 50 µg L-1)

4.3 %

3.5 %

Repeatability (n = 10 for 100 µg L-1)

4.4 %

4.5 %

Inter-day (n = 3)

5.2 %

6.1 %

Intra-day (n = 3)

4.7 %

5.0 %

Inter-electrode (n = 2)

6.0 %

5.3 %

Inter-activation (n = 2)

6.4 %

5.1 %

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Table 3. Recovery values for the analysis of two urine samples and two saliva samples spiked with known concentrations of Cd2+ and Pb2+ using the 3D-printed electrode.

Samples

Found (µg

L−1)

Cd2+ fortified (µg L−1)

Found (µg

L−1)

Recovery (%)

Found (µg

L−1)

Pb2+ fortified (µg L−1)

Found (µg

L−1)

Recovery (%)

Urine A

< LOD

250

258 ± 9

103 ± 4

< LOD

250

240 ± 9

96 ± 4

Urine A

< LOD

500

543 ± 4

108 ± 1

< LOD

500

517 ± 8

103 ± 2

Urine B

< LOD

250

240 ± 1

96 ± 1

< LOD

250

278 ± 5

111 ± 2

Urine B

< LOD

500

484 ± 8

97 ± 2

< LOD

500

523 ± 4

105 ± 1

Saliva A

< LOD

250

256 ± 6

102 ± 2

< LOD

250

281 ± 11

112 ± 5

Saliva A

< LOD

500

499 ± 7

100 ± 2

< LOD

500

494 ± 9

99 ± 2

Saliva B

< LOD

250

234 ± 4

93 ± 1

< LOD

250

272 ± 14

111 ± 3

Saliva B

< LOD

500

491 ± 19

98 ± 4

< LOD

500

512 ± 5

102 ± 2