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Size Control of Carbon Spherical Shells for Sensitive Detection of Paracetamol in Sweat, Saliva and Urine Anderson Massahiro de Campos, Paulo Augusto Raymundo-Pereira, Camila D. Mendonça, Marcelo L. Calegaro, Sergio A. S. Machado, and Osvaldo Novais Oliveira ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00139 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 13, 2018

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A simple method for size control (400–500 nm) of carbon spherical shells using centrifugation was proposed to sensitive detection of paracetamol in sweat, saliva and urine samples at a low detection limit with a performance similar to standard method (HPLC). The sensitivity in the paracetamol detection increased in agreement with the decreases of the size of the carbon spherical shells. 254x158mm (96 x 96 DPI)

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Size Control of Carbon Spherical Shells for Sensitive Detection of Paracetamol in Sweat, Saliva and Urine

Anderson M. Camposa, Paulo A. Raymundo-Pereirab,*, Camila D. Mendonçaa, Marcelo L. Calegaroa, Sergio A. S. Machadoa, Osvaldo N. Oliveira Jr.b,* a

São Carlos Institute of Chemistry, University of São Paulo (USP), CP 780, CEP 13566-590, São Carlos, São Paulo, Brazil b

São Carlos Institute of Physics, University of São Paulo(USP), CP 369, CEP 13560–970, São Carlos, São Paulo, Brazil

*Corresponding authors: São Carlos Institute of Physics, University of São Paulo(USP), CP 369, CEP 13560– 970, São Carlos, São Paulo, Brazil E-mail addresses: [email protected] (Paulo A. RaymundoPereira) and [email protected] (Osvaldo N. Oliveira Jr.) Tel.: +55 16 33518098; fax: +55 16 33518350.

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ABSTRACT We report on a facile strategy for separating carbon spherical shells (CSS) using centrifugation, with which shells were produced with diameter varying from 400 to 500 nm according to scanning and transmission electron microscopy. The shells were made of 79% carbon and 21% oxygen, and their surface was functionalized with carbonyl and hydroxyl groups. The CSS could form a homogeneous film on a glassy carbon (GC) electrode surface and be used in a sensing platform. In electroanalytical experiments, the sensitivity of the GC/CSS electrode for paracetamol increased with decreasing size of CSS. For 400 nm CSS, the sensitivity was 0.02 µA µmol–1 L and the limit of detection and quantification in sweat, saliva and urine samples were 120 and 400, 286 and 470, and 584 and 530 nmol L–1, respectively, which represents the highest performance among carbon-based sensors found in the literature. The GC/CSS electrodes were stable, robust against typical interferents and allowed detection of paracetamol in sweat, saliva and urine samples with a performance indistinguishable from conventional high-performance liquid chromatography. Keywords: Carbon nanoshells; size control; sweat; saliva; urine; paracetamol; sensors; non-invasive samples.

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1. INTRODUCTION The use of nanostructures to enhance the performance of electrochemical sensing, electrochemical energy conversion and storage devices is well documented, with useful features arising from large active surface areas and ultrathin thickness1-2. Of special interest for this work, carbon nanostructures exhibit useful physicochemical properties3-5, which can be

exploited in

signal-amplification

strategies

for

electrochemical devices6, as is the case of carbon-based electrode nanomaterials2. Carbon nanotubes

7

and graphenes8 are perhaps the most used of such nanostructures,

but others deriving from carbon black 9, Printex Carbon 8, biochar spherical shells (CSSs)

6

10

and carbon

have also been proven of value. CSSs, in particular, may be

advantageous owing to the inexpensive synthesis via hydrothermal routes that lead to homogeneous, porous particles 6, whose surface may be readily functionalized. CSSs are already applied in energy storage and conversion devices 13-14

, sensing

13, 15-16

and biosensing

6, 17-18

11

, solar cells 12, batteries

. In electrochemical sensing, they are

important due to their low cost, high-scale production, nontoxicity, large specific surface area, flexibility and chemical stability

15, 19

. Furthermore, CSSs resulting from

polymerization of carbohydrate may display increased interaction with analytes since they contain functional groups (carboxylic, formyl and hydroxyl)15, being therefore suitable electrode materials for ultrasensitive sensors and biosensors. In this study, we employ CSSs in electrochemical sensing, for which size control is important because their dimensions vary from nanometers to submicrometers, and the size and shape are crucial for electrochemical activity 15. We chose a challenging target as a proof-of-principle strategy, namely the detection of paracetamol (acetaminophen or N-Acetyl-4-aminophenol) in sweat, urine and saliva. The interest in paracetamol stems 3

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from the need to detect its possible accumulation in the human body, for it is a widely used – often indiscrimately - analgesic to combat fever and for pain relief20. The indiscriminate use of paracetamol yields severe consequences such as nephrotoxicity and hepatoxicity caused by intoxication of the liver with toxic metabolites20. Detection of paracetamol in sweat, urine and saliva is especially relevant owing to the noninvasive nature of the tests. Indeed, there has been a trend toward monitoring of biomarkers, such as electrolytes, metabolites and heavy-metals in saliva, urine, tears and sweat 9, 21-22. Sweat has been overlooked as a biospeciation in clinical analysis, probably because of the lack of complete and precise information about the presence of endogenous metabolites and the need for research to correlate pathological states with sweat metabolite composition 23. It is also worth mentioning that detection of paracetamol has been mostly done with high-performance liquid chromatography (HPLC), spectrophotometry, capillary electrophoresis and electrochemical devices9, 20, 24-25, but not in sweat. Electrochemical methods, such as the one employed here, are advantageous because of their easy operation, high sensitivity, low cost, potential for development of equipment for screening and in-field/on-site sensing. We shall show that the performance of CSSs depends on the particle size, with the optimized sensitivity being indistinguishable from the results of HPLC.

2. EXPERIMENTAL SECTION 2.1. Chemicals. Paracetamol, N,N-dimethylformamide (DMF), ethanol and glucose were acquired from Sigma–Aldrich (St. Louis, MO, USA) with analytical grade and 4

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high purity. Solutions were made with high-purity water (resistivity > 18 MΩ cm) from a Nanopure Ultrapurification System (Barnstead Inc., Waltham, MA, USA). The phosphate buffer solution (PBS) (pH 7.0) contained sodium phosphate dibasic and sodium phosphate monobasic monohydrate for 0.1 mol L−1 concentration (SigmaAldrich (St. Louis, MO, USA)) and then used as electrolytic solution.

2.2. Apparatus and procedures. The shape of CSS was investigated with a high-resolution transmission electron microscope (HR-TEM) model FEI TECNAI G2 F20 operating at 200 kV. The CSS was ultrasonicated for 2 h using DMF, and then the mixture was dispersed on carbon–coated copper grids. A Rigaku Rotaflex diffractometer model RU200B at 50 kV and 100 mA, with CuKα radiation, λ=1.542 Å was used in X-ray powder diffraction (XRD). All measurements were performed at room temperature (25ºC) with scanning between 10° and 80°. Scanning electron microscopy connected to an energy-dispersive X-ray spectroscopy (SEM-EDX) operating at 20 kV, model LEO-440 microscope (Zeiss-Leica), was used to analyze the homogeneity and assembly of CSS thin film. FTIR experiments were performed in a model IRAffinity 1 spectrophotometer (Shimadzu, Portland, USA), in the wavenumber range between 400 and 4000 cm−1, resolution 4.0 cm−1 and 36 scans. XPS analyses were obtained with a K-Alpha™+ X-ray photoelectron spectrometer system K- (Thermo Scientific, Waltham, Massachusetts, USA) equipped with a hemispherical electron analyzer. A potentiostat model PGSTAT 302 Autolab electrochemical (Eco Chemie, Utrecht, Netherlands) controlled with NOVA software was used in the electrochemical experiments with a conventional three–electrode cell. This cell comprised a GC 5

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electrode coated with a CSS ultrathin layer as working electrode, a reference electrode of Ag/AgCl (KCl 3.0 mol L−1), and a platinum wire as an auxiliary electrode. Differential pulse voltammetry (DPV) measurements were conducted between –0.2 and +0.7 V at a scan rate of 5 mV s−1, modulation time at 1 ms and pulse amplitude 50 mV for all electrochemical and electroanalytical studies. The chromatographic system used in HPLC measurements was a model Shimadzu with manual injector, two pumps (LC-10AD VP) and diode array detector (DAD), equipped with a reverse phase C18 column (250 × 4.6 mm ID, 5 μm particle size) C18 pre-column (2 cm × 4 mm ID, 5 μm particle), both from Ascentis® Supelco.

2.3. Synthesis of carbon spherical shells. The carbon spherical shells (CSS) were synthesized using the hydrothermal method

15, 26

with a few modifications (see

Figure 1). Specifically, 6.5 g of glucose were added in 72 mL of water under ultrasonication, followed by transfer and sealing in a Teflon-sealed autoclave (100 mL) maintained for 5 h at 180 °C before being cooled in air. The dense suspensions were dark brown in color. The CSS were separated by centrifuging at 4000, 8000 and 12000 rpm for 30 min. A cleaning procedure followed, which included three cycles of centrifugation/washing/redispersion in ultrapure water and in ethanol, before oven drying at 80 °C for 4 h.

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Figure 1 – Schematic diagram of preparation and separation of carbon spherical shells (CSS) with diameter of 400, 450 and 500 nm, and results from sensing paracetamol.

2.4. Electrode Preparation. The GC electrode was mechanically cleaned using silicon carbide (4000 mesh), washed thoroughly with ultrapure water and dried with N2 27

. Then, 2.0 mg of CSS were dispersed in DMF (2.0 mL) using ultrasonication during

120 min 8. A thin electroactive coating layer (6.0 µL) was dropped on the GC electrode surface and maintained at room temperature for complete solvent evaporation28.

2.5. Analytical procedure. GC electrodes were modified with carbon nanospheres with diameter of 400, 450 and 500 nm, and then used to detect paracetamol, with molecular structure depicted in Fig. 2, using DPV with optimized parameters. The limit of detection and quantification (or limit of determination) were calculated with the equations: LOD = a + 3 Sy/x and LOQ = a + 10 Sy/x with a and Sy/x 7

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being the intercept and the standard deviation of the linear regression, respectively 8, 22, 27, 29-30

. The repeatability studies were conducted to verify the precision of the proposed

sensor platform with intra-day (n = 10) and inter-day (n = 5) experiments. Analyses of paracetamol in synthetic sweat samples were prepared as reported by Mathew et al.

31

consisting of urea 1.0 g L–1, 1.0 g L–1 of KCl, 7.5 g L–1 of NaCl,

and lactic acid 1.0 mL L–1 solubilized in PBS. Artificial saliva samples consisting of 0.1 g urea, 0.081 g of CaCl2·2H2O, 0.043 g KCl, 0.078 g NaH2PO4·2H2O, 0.04 g NaCl, 1 mL of 0.05% of Na2S·9H2O in 100 mL of 0.1 mol L–1 PBS solution 22. Artificial urine samples were prepared based on the work of Laube et al. 32 containing 6.25 g urea, 0.35 g KH2PO4, 0.73 g NaCl, 0.56 g Na2SO4, 0.40 g KCl, 0.27 g CaCl2·2H2O and 25 g NH4Cl in a 250 mL of 0.1 mol L–1 PBS solution. The sweat, saliva and urine samples were used immediately after preparation. For comparison with the standard chromatographic method, paracetamol detection was verified with the sweat spiked with varied concentrations of this analyte. The parameters utilized for paracetamol analysis were: injection volume of 20 µL; mobile phase comprising methanol (A) and water (B) in isocratic mode (25% A), running of 7.5 min and retention time (tR) of 6.0 min at a flow rate of 1.0 mL min–1 and UV monitoring at λ = 248 nm

33

. The parameters used for DPV measurements were

recorded at a scan rate of 5 mV s–1, in the potential range from –0.2 to 0.7 V, pulse amplitude of 50 mV and modulation time of 50 ms. The concentration of paracetamol in these samples was obtained and compared with the value measured with HPLC, considered as a standard method33.

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3. RESULTS AND DISCUSSION 3.1. Separation and detection performance of carbon spherical shells. The carbon spherical shells (CSS) in the TEM images in Figure 2A had diameters of 400, 450 and 500 nm, being successfully separated by centrifugation at 4000, 8000 and 12000 rpm, respectively. The separation procedure was based on density gradient centrifugation where the most dense CSS (400 nm) shells were sedimented first, followed by less dense (450 and 500 nm) shells, indicating that particle density increased upon reducing their size 34. The carbonaceous nanospheres dispersed onto the electrode surface formed a homogeneous assembly as shown in Figure 2B. The size distribution histogram in Figure S1 (in the Supporting Information) demonstrates the suitability of this approach to separate 400-500 nm carbon spherical shells. Figure 2C depicts a considerably higher oxidation current for GC/CSS400nm in the presence of 4.5 µM paracetamol. Analysis of DPV´s for several paracetamol concentrations (Figure S2 in the Supporting Information) leads to linear plots for the current at the peak in Figure 2D, with high sensitivity (2.0×10–2 A mol L–1) for GC/CSS400nm. In contrast, the plots for GC/CSS450nm (1.5×10–2 A mol L–1) and GC/CNE500nm (1.1×10–2 A mol L–1) show lower sensitivity in the range analyzed due to the smaller area of these shells. Moreover, the bare GCE showed no linearity in the studied range (sigmoidal profile). The amount of electroactive species (Γ /mol cm–2) was estimated using Γ = Q/nFA

28, 35

, where Q (C) is the background-corrected electric charge, estimated by

integrating the anodic peaks of Figure 2C; n is the number of electrons; F is the Faraday constant (96,485.34 C mol–1); and A is the surface geometric area (0.071 cm2). Q was 1.72 × 10–6, 1.22 × 10–6 and 0.83 × 10–6 C, and the estimated surface concentration was 1.26 × 10–10, 0.89 × 10–10 and 0.61 × 10–10 mol cm–2 at the GC coated with CSS400nm, 9

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CSS450nm or CSS500nm, respectively. The decrease in diameter produces an increase in the number of electroactive CSS on the surface, thus increasing the sensitivity toward paracetamol as depicted in Figure 2E. The linear regression for the electroanalytical response using GC/CSS400nm, GC/CSS450nm and GC/CSS500nm gave: IP (A) = 1.0×10–8 + 2.0 × 10–2 Cparacetamol (mol L–1), r = 0.999, n = 20; IP (A) = 7.9×10–9 + 1.5×10–2 Cparacetamol (mol L–1), r = 0.999, n = 19; IP (A) = 9.8×10–9 + 1.1×10–2 Cparacetamol (mol L– 1

), r = 0.999, n = 17, respectively. The superior electroanalytical performance of

GC/CSS400nm in electrooxidation of paracetamol can be confirmed in the Inset of Figure 2D. The limit of detection and quantification were 2.13×10−7 and 7.13×10−7 mol L−1, respectively. The scheme in Figure 2F represents the electrooxidation mechanism of paracetamol involving transfer of two electrons and two protons

36

. Paracetamol is

oxidized at 450 mV resulting in N-acetyle-p-quinone imine 37-38.

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Figure 2 – A – HR–TEM images of the CSS with diameter of 400, 450 and 500 nm. B – SEM images of GC surface coated with a film containing CSS with 400 nm (a), 450 nm (b) and 500 nm (c) at a magnification of 25000×; C – DPV for 4.5×10−6 mol L−1 paracetamol with a GC coated with CSS400nm (a), CSS450nm (b) or CSS500nm (c). D – Calibration curves for paracetamol on CSS with different diameters. Conditions: 0.1 mol L-1 phosphate buffer solution, pH 7.0. E – Effect of CSS diameter on the number of electroactive species (Γ) and sensitivity. F – Scheme for electrooxidation of paracetamol on the sensing platform containing CSS with 400, 450 and 500 nm.

3.2. Chemical features of carbon spherical shells. The FTIR spectra in Figure 3A reveal the surface functionalization of the spherical shells with hydrophilic carbonyl and hydroxyl groups15, 39-40. The bands at 3412 and 2924 cm−1 correspond to O–H and C–H stretching vibrations, respectively. Bands at 1703 and 1620 cm−1 are assigned to C O and C C stretching vibrations, respectively15, which is consistent with the hypothesis of aromatization of the carbohydrate during the hydrothermal route39. The bands between 1000 and 1400 cm–1 are assigned to –C–OH stretching and –OH bending vibrations, which points to hydroxy groups at the surface39. The survey XPS spectrum in Figure 3B indicates 79.3% and 20.7% of carbon and oxygen, respectively, in the carbon nanospheres. The C 1s profile is basically constituted by three peaks at 284, 286 and 287 eV corresponding to C–C, C–OH and O–C–OH (Figure S3 in the Supporting Information), while the peaks at 532.4 and 533.8 eV can be indexed as C=O and C–OH bonds11, 15, respectively (Figure S4 in the Supporting Information). These results are consistent with the samples having hydroxyl and carboxylic functional

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groups, and with FTIR measurements. Finally, the XRD pattern in Figure 3C shows a broad peak at 20.4°, typical of amorphous carbon11, 15.

Figure 3 – A – FTIR spectra of carbon spherical shells (CSS). B – XPS survey scans of carbon spherical shells (CSS). C – Powder XRD pattern of carbon spherical shells (CSS).

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3.3. Determination and statistical comparison of sweat, saliva and urine samples using paracetamol as the standard. DPV´s for several paracetamol concentrations in sweat are shown in Fig. 4A, with the current at the peak increasing linearly with concentration as shown in Fig. 4B. Similar results for saliva and urine samples are shown in Figures S5 and S6. Regression diagnosis depicted in the inset indicated a suitable distribution of residuals in which 50% are above and 50% below zero, with the exception of y1 which might be an outlier. The sensitivity was the same for sweat, saliva and urine samples, viz. 0.02 A mol L–1, similar to the value for paracetamol in PBS (Fig. 2C) for GC/CSS400nm, which demonstrates the absence of the matrix effect. The limit of detection and quantification in sweat, saliva and urine samples were 120 and 400, 286 and 470, and 584 and 530 nmol L–1, respectively. Figure 4C shows the correlation between the results from the electroanalytical method proposed and the standard method (HPLC) with an intercept value of 3.1 × 10–8, a slope of 0.96 and product–moment correlation coefficient of 0.999, thus implying equivalence of the two techniques. The random errors lead to a deviation from the ideal case35. In the Student’s t-test, the value of 63.4 (higher than the t-critical value of 2.57 for 95% of significance for n = 10 (degrees of freedom = 8; α = 0.05)) showed no significant difference between the two techniques29,

35, 41-42

. Thus, the electroanalytical sensing

performance was similar to the standard HPLC for paracetamol in sweat samples. The selectivity of the GC/CSS sensor was assessed by measuring the DPV responses generated by five possible interfering substances at 2.4×10−6 mol L−1 concentration, and comparing with the oxidation peak of 2.4×10−6 mol L−1 paracetamol under identical measuring conditions. Other substances such as lactic acid and urea were not added because they are contained in the sweat sample. Firstly, DPV 14

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measurements were conducted with 2.4×10−6 mol L−1 of paracetamol electrooxidation. Subsequently, 2.4×10−6 mol L−1 of glucose, cholesterol, ascorbic acid, dopamine and uric acid were individually added, and the corresponding DPV responses were recorded as depicted in Figure 4D. In the potential window studied, no additional peaks were detected for the interfering compounds, suggesting that these substances do not interfere with paracetamol detection. The analytical signal recorded for paracetamol detection had negligible variation with addition of glucose (0.07%), cholesterol (0.97%), ascorbic acid (1.15%), dopamine (1.46%), and uric acid (2.1%) as seen in Figure 4D, suggesting a high selectivity for paracetamol which can be used in the quantification of real samples.

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Figure 4 – A – Differential pulse voltammograms obtained for paracetamol detection in sweat samples using GC coated with CSS400nm. B – Calibration curve for paracetamol detection in sweat sample; Inset: Residuals of regression diagnosis. C – Correlation between the results obtained of the sweat samples studied with the GC/CSS sensor and the standard (chromatography) method. D – Effect from adding 2.4 µmol L–1 of glucose, cholesterol, ascorbic acid, dopamine and uric acid on the voltammetric responses for 2.4 µmol L–1 of paracetamol.

Table 1 summarizes the analytical efficiency of carbon-based sensing layers relationship to the sensitivity (A L mol–1), detection limits (mol L–1) and linear range for paracetamol detection. The GC/CSS sensor shows the lowest detection limits and highest sensitivity. Furthermore, one should emphasize that the limit of detection can be compared with similar sensing layers for detecting paracetamol compared with most carbon materials used for this purpose.

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Table 1: Analytical performance of sensing layers made with carbon-based materials to detect paracetamol. Sensing Layer

Sensitivity

LOD

Linear range

(μA / μmol L−1)

(μmol L−1)

(μmol L−1)

DPV

-----

1.2

5.00 – 800.00

43

DLC:VACNT

CV

0.28

2.30

10.00 – 100.00

44

SWCNT/CCE

DPV

0.47

0.12

0.20 – 150.00

45

MWCNTs:graphite/GC

SWV

0.11

0.20

0.47 – 13.20

46

PEDOT/GO/GCE

CV

0.08

0.57

10.00 – 60.00

47

f-MWCNTs/GCE

DPV

-----

0.60

3.00 – 300.00

48

f-MWCNTs/GCE

DPV

0.06

0.52

1.00 – 90.00

49

CSS/GCE

DPV

0.02

0.12

0.37 – 7.52

This work

ERG/GCE

Technique

Ref.

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Note: GC: glassy carbon; ERG: electrochemically reduced graphene; GCE: glassy carbon electrode; DLC: diamond-like carbon; VACNT: vertically aligned multiwalled carbon nanotubes; SWCNT: single-walled carbon nanotube; CCE: carbon-ceramic electrode; MWCNTs: multiwalled carbon nanotubes; PEDOT: poly(3,4-ethylenedioxythiophene); GO: graphene oxide; f-MWCNTs: acid functionalized multi-wall carbon nanotubes; CSS: carbon spherical shells; DPV: differential pulse voltammetry; CV: cyclic voltammetry; SWV: square wave voltammetry.

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The relative standard deviation (RSD) obtained in the repeatability study of the sensor platform was 5.4% for peak current at 1.0×10−6 mol L−1 paracetamol for repeatability (n = 10). The stability was also conducted with the electrochemical response decreasing by only 3.01% (RSD) after 50 DPV profiles. The RSD for inter-day repeatability was 3.7% with five co–fabricated sensors utilized for detecting 1.0×10−6 mol L−1 paracetamol, which suggests high reproducibility of the fabrication procedure for GC/CSS sensors.

4. Conclusion We have proposed a simple method for separation based on density of carbon nanospheres by centrifugation with 400, 450 and 500 nm diameter that were deposited on glassy carbon (GC) electrodes. The GC/CSS sensor was then tested for detecting paracetamol, with sensitivity and detection limit of 0.02 μA/mol L−1 and 0.12 µmol L−1, respectively,

which

represents

the

best

performance

among

carbon-based

electrochemical sensors we could find in the literature (0.12 – 2.3 µmol L−1). In addition, the electroanalytical performance of CSS was sufficient to estimate paracetamol concentration in sweat samples. The GC/CSS sensory platform was also stable, reproducible, and robust with regard to relevant interferents for paracetamol sensing in artificial sweat, saliva and urine samples. The performance of CSS for electrodes is particularly important for developing miniaturized electroanalytical devices with simple fabrication procedures and low cost.

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Supporting Information Control experiments for electrochemical oxidation of paracetamol (DPV profiles); size distribution histogram of the CSS of 400, 450 and 500 nm; C 1s and O 1s XPS spectrum of carbon nanospheres. Differential pulse voltammograms obtained for paracetamol detection in saliva and urine samples using GC coated with CSS400nm. Calibration curves for paracetamol detection in saliva and urine samples.

Notes The authors declare no competing financial interest.

Acknowledgments The authors are thankful to FAPESP (2012/17689-9, 2016/01919-6, 2014/05197-0, 2013/14262-7 and 2016/12759-0), CNPq and CAPES for the financial support. The authors are grateful to Ms. Marcio de Paula and CAQI/IQSC/USP are specially acknowledged for SEM facilities. The authors thank the Brazilian Nanotechnology National Laboratory/Center for Research in Energy and Materials (LNNano/CNPEM) and Structural Characterization Laboratory/Department of Materials Engineering of the Federal University of São Carlos (LCE/DEMa/UFSCar) for technical support during XPS and TEM analyses, respectively.

References

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(1) Cakici, M.; Reddy, K. R.; Alonso-Marroquin, F. Advanced electrochemical energy storage supercapacitors based on the flexible carbon fiber fabric-coated with uniform coral-like MnO2 structured electrodes. Chem. Eng. J. 2017, 309, 151-158. (2) Khan, A. H.; Ghosh, S.; Pradhan, B.; Dalui, A.; Shrestha, L. K.; Acharya, S.; Ariga, K. Two-Dimensional (2D) Nanomaterials towards Electrochemical Nanoarchitectonics in Energy-Related Applications. Bull. Chem. Soc. Jpn. 2017, 90, 627-648. (3) Kumar, S.; Rani, R.; Dilbaghi, N.; Tankeshwar, K.; Kim, K. H. Carbon nanotubes: a novel material for multifaceted applications in human healthcare. Chem. Soc. Rev. 2017, 46, 158-196. (4) Komiyama, M.; Yoshimoto, K.; Sisido, M.; Ariga, K. Chemistry Can Make Strict and Fuzzy Controls for Bio-Systems: DNA Nanoarchitectonics and CellMacromolecular Nanoarchitectonics. Bull. Chem. Soc. Jpn. 2017, 90, 967-1004. (5) Bairi, P.; Minami, K.; Hill, J. P.; Nakanishi, W.; Shrestha, L. K.; Liu, C.; Harano, K.; Nakamura, E.; Ariga, K. Supramolecular Differentiation for Construction of Anisotropic Fullerene Nanostructures by Time-Programmed Control of Interfacial Growth. ACS Nano 2016, 10, 8796-8802. (6) Xu, Q. N.; Yan, F.; Lei, J. P.; Leng, C.; Ju, H. X. Disposable Electrochemical Immunosensor by Using Carbon Sphere/Gold Nanoparticle Composites as Labels for Signal Amplification. Chem-Eur. J. 2012, 18, 4994-4998. (7) Canevari, T. C.; Raymundo-Pereira, P. A.; Landers, R.; Benvenutti, E. V.; Machado, S. A. S. Sol-gel thin-film based mesoporous silica and carbon nanotubes for the determination of dopamine, uric acid and paracetamol in urine. Talanta 2013, 116, 726735. (8) Campos, A. M.; Raymundo-Pereira, P. A.; Cincotto, F. H.; Canevari, T. C.; Machado, S. A. S. Sensitive determination of the endocrine disruptor bisphenol A at ultrathin film based on nanostructured hybrid material SiO2/GO/AgNP. J. Solid State Electr. 2016, 20, 2503-2507. (9) Vicentini, F. C.; Raymundo-Pereira, P. A.; Janegitz, B. C.; Machado, S. A. S.; Fatibello, O. Nanostructured carbon black for simultaneous sensing in biological fluids. Sensor Actuat B-Chem 2016, 227, 610-618. (10) Kalinke, C.; Mangrich, A. S.; Marcolino-Junior, L. H.; Bergamini, M. F. Biochar prepared from castor oil cake at different temperatures: A voltammetric study applied for Pb2+, Cd2+ and Cu2+ ions preconcentration. J Hazard Mater 2016, 318, 526-532. (11) Xia, X. H.; Zhang, Y. Q.; Fan, Z. X.; Chao, D. L.; Xiong, Q. Q.; Tu, J. P.; Zhang, H.; Fan, H. J. Novel Metal@Carbon Spheres Core-Shell Arrays by Controlled SelfAssembly of Carbon Nanospheres: A Stable and Flexible Supercapacitor Electrode. Adv. Energy Mater. 2015, 5, 1-9. (12) Zhao, P. L.; Li, D.; Yao, S. T.; Zhang, Y. Q.; Liu, F. M.; Sun, P.; Chuai, X. H.; Gao, Y.; Lu, G. Y. Design of Ag@C@SnO2@TiO2 yolk-shell nanospheres with enhanced photoelectric properties for dye sensitized solar cells. J. Power Sources 2016, 318, 49-56. (13) Gan, T.; Lv, Z.; Deng, Y. P.; Sun, J. Y.; Shi, Z. X.; Liu, Y. M. Facile synthesis of monodisperse Ag@C@Ag core-double shell spheres for application in the simultaneous sensing of thymol and phenol. New J Chem 2015, 39, 6244-6252. (14) Ding, C.; Zeng, Y. W.; Cao, L. L.; Zhao, L. F.; Zhang, Y. Hierarchically porous Fe3O4/C nanocomposite microspheres via a CO2 bubble-templated hydrothermal approach as high-rate and high-capacity anode materials for lithium-ion batteries. J Mater Chem A 2016, 4, 5898-5908. 21

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(15) Gan, T.; Shi, Z. X.; Wang, K. L.; Chen, Y. Y.; Sun, J. Y.; Liu, Y. M. Sizecontrolled core-shell-structured Ag@carbon spheres for electrochemical sensing of bisphenol A. J. Solid State Electr. 2015, 19, 2299-2309. (16) Zhang, S.; Zheng, J. B. Synthesis of single-crystal alpha-MnO2 nanotubes-loaded Ag@C core-shell matrix and their application for electrochemical sensing of nonenzymatic hydrogen peroxide. Talanta 2016, 159, 231-237. (17) Mao, S. X.; Long, Y. M.; Li, W. F.; Tu, Y. F.; Deng, A. P. Core-shell structured Ag@C for direct electrochemistry and hydrogen peroxide biosensor applications. Biosens. Bioelectron. 2013, 48, 258-262. (18) Zhou, X.; Dai, X. X.; Li, J. G.; Long, Y. M.; Li, W. F.; Tu, Y. F. A sensitive glucose biosensor based on Ag@C core-shell matrix. Mat. Sci. Eng. C-Mate.r 2015, 49, 579-587. (19) Huang, H. P.; Cui, R. J.; Yu, L. J.; Mao, C. J. Preparation of Au-Decorated Ag@C Nanoparticle and Its Biosensing Application. Chem. Lett. 2014, 43, 781-783. (20) Amiri-Aref, M.; Raoof, J. B.; Ojani, R. Electrocatalytic oxidation and selective determination of an opioid analgesic methadone in the presence of acetaminophen at a glassy carbon electrode modified with functionalized multi-walled carbon nanotubes: Application for human urine, saliva and pharmaceutical samples analysis. Colloid Surface B 2013, 109, 287-293. (21) Kim, J.; Kumar, R.; Bandodkar, A. J.; Wang, J. Advanced Materials for Printed Wearable Electrochemical Devices: A Review. Advanced Electronic Materials 2017, 3, 1-15. (22) Raymundo-Pereira, P. A.; Shimizu, F. M.; Coelho, D.; Piazzeta, M. H. O.; Gobbi, A. L.; Machado, S. A. S.; Oliveira Jr., O. N. A Nanostructured Bifunctional platform for Sensing of Glucose Biomarker in Artificial Saliva: Synergy in hybrid Pt/Au surfaces. Biosens. Bioelectron. 2016, 86, 369-376. (23) Hooton, K.; Han, W.; Li, L. Comprehensive and Quantitative Profiling of the Human Sweat Submetabolome Using High-Performance Chemical Isotope Labeling LC-MS. Anal. Chem. 2016, 88, 7378-7386. (24) Cook, S. F.; King, A. D.; van den Anker, J. N.; Wilkins, D. G. Simultaneous quantification of acetaminophen and five acetaminophen metabolites in human plasma and urine by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry: Method validation and application to a neonatal pharmacokinetic study. J. Chromatogr. B 2015, 1007, 30-42. (25) Fujino, H.; Yoshida, H.; Nohta, H.; Yamaguchi, M. HPLC determination of acetaminophen in saliva based on precolumn fluorescence derivatization with 12-(3,5dichloro-2,4,6-triazinyl)benzo[d]benzo[1 ',2 '-6,5]isoindolo[1,2-b][1,3]thiazolidine. Anal. Sci. 2005, 21, 1121-1124. (26) Sun, X. M.; Li, Y. D. Ag@C core/shell structured nanoparticles: Controlled synthesis, characterization, and assembly. Langmuir 2005, 21, 6019-6024. (27) Raymundo-Pereira, P. A.; Campos, A. M.; Mendonça, C. D.; Calegaro, M. L.; Machado, S. A. S.; Oliveira Jr., O. N. Printex 6L Carbon Nanoballs used in Electrochemical Sensors for Simultaneous Detection of Emerging Pollutants Hydroquinone and Paracetamol. Sensor Actuat. B-Chem. 2017, 252, 165-174. (28) Raymundo-Pereira, P. A.; Campos, A. M.; Vicentini, F. C.; Janegitz, B. C.; Mendonça, C. D.; Furini, L. N.; Boas, N. V.; Calegaro, M. L.; Constantino, C. J. L.; Machado, S. A. S.; Oliveira Jr., O. N. Sensitive detection of estriol hormone in creek water using a sensor platform based on carbon black and silver nanoparticles Talanta 2017, 174, 652-659. 22

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Page 24 of 25

(29) Raymundo-Pereira, P. A.; Ceccato, D. A.; Junior, A. G. B.; Teixeira, M. F. S.; Lima, S. A. M.; Pires, A. M. Study on the structural and electrocatalytic properties of Ba2+- and Eu3+-doped silica xerogels as sensory platforms. RSC Adv. 2016, 6, 104529104536. (30) Raymundo-Pereira, P. A.; Lima, A. R. F.; Machado, S. A. S. A nanostructured label-free platform based on an ultrathin film for ultrasensitive detection of a secosteroid hormone. RSC Adv. 2016, 6, 34458-34467. (31) Mathew, M. T.; Ariza, E.; Rocha, L. A.; Fernandes, A. C.; Vaz, F. TiCxOy thin films for decorative applications: Tribocorrosion mechanisms and synergism. Tribol. Int. 2008, 41, 603-615. (32) Laube, N.; Mohr, B.; Hesse, A. Laser-probe-based investigation of the evolution of particle size distributions of calcium oxalate particles formed in artificial urines. J. Cryst. Growth 2001, 233, 367-374. (33) Ranjbari, E.; Golbabanezhad-Azizi, A. A.; Hadjmohammadi, M. R. Preconcentration of trace amounts of methadone in human urine, plasma, saliva and sweat samples using dispersive liquid-liquid microextraction followed by high performance liquid chromatography. Talanta 2012, 94, 116-122. (34) Majekodunmi, S. O. A Review on Centrifugation in the Pharmaceutical Industry. Am. J. Biom. Eng. 2015, 5, 11. (35) Raymundo-Pereira, P. A.; Mascarenhas, A. C. V.; Teixeira, M. F. S. Evaluation of the Oxo-bridged Dinuclear Ruthenium Ammine Complex as Redox Mediator in an Electrochemical Biosensor. Electroanal. 2016, 28, 562-569. (36) Eisele, A. P. P.; Clausen, D. N.; Tarley, C. R. T.; Dall'Antonia, L. H.; Sartori, E. R. Simultaneous Square-Wave Voltammetric Determination of Paracetamol, Caffeine and Orphenadrine in Pharmaceutical Formulations Using a Cathodically Pretreated BoronDoped Diamond Electrode. Electroanal. 2013, 25, 1734-1741. (37) Kalambate, P. K.; Sanghavi, B. J.; Karna, S. P.; Srivastava, A. K. Simultaneous voltammetric determination of paracetamol and domperidone based on a graphene/platinum nanoparticles/nafion composite modified glassy carbon electrode. Sensor Actuat. B-Chem. 2015, 213, 285-294. (38) Kalambate, P. K.; Srivastava, A. K. Simultaneous voltammetric determination of paracetamol, cetirizine and phenylephrine using a multiwalled carbon nanotubeplatinum nanoparticles nanocomposite modified carbon paste electrode. Sensor Actuat. B-Chem. 2016, 233, 237-248. (39) Sun, X. M.; Li, Y. D. Colloidal carbon spheres and their core/shell structures with noble-metal nanoparticles. Angew. Chem. Int. Edit. 2004, 43, 597-601. (40) Wei, Y.; Yang, R.; Guo, Z.; Gao, C.; Wang, L.; Liu, J. H.; Huang, X. J. A new "capturer" for electrochemical detection of organophosphate pesticides: The hydroxylation and carbonylation carbonaceous nanospheres. Anal. Methods 2012, 4, 353-356. (41) Miller, J. C.; Miller, J. N. Basic Statistical-Methods for Analytical-Chemistry .1. Statistics of Repeated Measurements - a Review. The Analyst 1988, 113, 1351-1356. (42) Miller, J. N. Basic Statistical-Methods for Analytical-Chemistry .2. Calibration and Regression Methods - a Review. The Analyst 1991, 116, 3-14. (43) Adhikari, B.-R.; Govindhan, M.; Chen, A. Sensitive Detection of Acetaminophen with Graphene-Based Electrochemical Sensor. Electrochim. Acta 2015, 162, 198-204. (44) Silva, T. A.; Zanin, H.; May, P. W.; Corat, E. J.; Fatibello-Filho, O. Electrochemical performance of porous diamond-like carbon electrodes for sensing 23

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hormones, neurotransmitters, and endocrine disruptors. ACS Appl. Mater. Interfaces 2014, 6, 21086-92. (45) Habibi, B.; Jahanbakhshi, M.; Pournaghi-Azar, M. H. Differential pulse voltammetric simultaneous determination of acetaminophen and ascorbic acid using single-walled carbon nanotube-modified carbon-ceramic electrode. Anal. Biochem. 2011, 411, 167-175. (46) Moghaddam, A. B.; Mohammadi, A.; Mohammadi, S.; Rayeji, D.; Dinarvand, R.; Baghi, M.; Walker, R. B. The determination of acetaminophen using a carbon nanotube:graphite-based electrode. Microchim. Acta 2010, 171, 377-384. (47) Si, W. M.; Lei, W.; Han, Z.; Zhang, Y. H.; Hao, Q. L.; Xia, M. Z. Electrochemical sensing of acetaminophen based on poly(3,4-ethylenedioxythiophene)/graphene oxide composites. Sensor Actuat. B-Chem. 2014, 193, 823-829. (48) Alothman, Z. A.; Bukhari, N.; Wabaidur, S. M.; Haider, S. Simultaneous electrochemical determination of dopamine and acetaminophen using multiwall carbon nanotubes modified glassy carbon electrode. Sensor Actua.t B-Chem. 2010, 146, 314320. (49) Baghayeri, M.; Namadchian, M. Fabrication of a nanostructured luteolin biosensor for simultaneous determination of levodopa in the presence of acetaminophen and tyramine: Application to the analysis of some real samples. Electrochim. Acta 2013, 108, 22-31.

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