View Article Online

View Journal

Journal of
Materials Chemistry B
Materials for biology and medicine
Accepted Manuscript

This article can be cited before page numbers have been issued, to do this please use: N. M. Galdino, G.
S. Brehm, R. Bussamara, W. D. G. Gonçalves, G. A. Abarca Anjari and J. D. Scholten, J. Mater. Chem. B,
2017, DOI: 10.1039/C7TB02582C.

Volume 4 Number 1 7 January 2016 Pages 1–178 This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
Journal of accepted for publication.
Materials Chemistry B Accepted Manuscripts are published online shortly after
Materials for biology and medicine
www.rsc.org/MaterialsB

acceptance, before technical editing, formatting and proof reading.

Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.

You can find more information about Accepted Manuscripts in the

author guidelines.

Please note that technical editing may introduce minor changes

to the text and/or graphics, which may alter content. The journal’s
ISSN 2050-750X standard Terms & Conditions and the ethical guidelines, outlined
PAPER
in our author and reviewer resource centre, still apply. In no
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.

rsc.li/materials-b
 Page 1 of 6 Journal of Materials Chemistry B

Table of Contents View Article Online

DOI: 10.1039/C7TB02582C

Journal of Materials Chemistry B Accepted Manuscript

Published on 20 November 2017. Downloaded by University of Newcastle on 20/11/2017 23:55:34.

The sputtering deposition of Au nanoparticles onto ionic liquid-graphene oxide combined with
cholesterol oxidase affords an efficient biosensor for cholesterol detection.
 Please
Journal ofdo not adjustChemistry
Materials margins B Page 2 of 6
View Article Online
DOI: 10.1039/C7TB02582C

Journal Name

Journal of Materials Chemistry B Accepted Manuscript

COMMUNICATION
Published on 20 November 2017. Downloaded by University of Newcastle on 20/11/2017 23:55:34.

Sputtering deposition of gold nanoparticles onto graphene oxide

functionalized with ionic liquids: biosensor materials for
Received 00th January 20xx,
Accepted 00th January 20xx
cholesterol detection
DOI: 10.1039/x0xx00000x Nathália M. Galdino, Gabriele S. Brehm, Roberta Bussamara,* Wellington D. G. Gonçalves, Gabriel
Abarca and Jackson D. Scholten*
www.rsc.org/

2
Ionic liquid (IL)-functionalized graphene oxide (GO) containing for home or in situ detection. The development of new
gold nanoparticles (Au NPs) (5.4-5.9 nm) deposited by sputtering materials that allow efficient enzyme immobilization to
combined with cholesterol oxidase appears to be a suitable and maintain high activity and stability during the process is of
efficient biosensor for total cholesterol detection. These fundamental importance in order to build a high performance
biosensors showed good linear range (25-470 μmol/L) for total biosensor for portable measurements with the same accuracy
cholesterol determination by colorimetric tests. In addition, as clinical analysis. For this purpose, several kinds of hybrid
control experiments in the presence of interferents demonstrated biosensors have been prepared and tested, such as
that the hybrid materials provided a selective detection of total Au/cysteamine/Au NPs/ChOx electrode (ChOx = cholesterol
6
cholesterol. The biosensing activities obtained for the hybrid oxidase), Pt/LDHs-chitosan/ChOx electrode (LDHs = layered
7
materials indicate that these compounds are potential biosensors double hydroxides), L-cysteine-rGO/GCE/Au NPs/ChOx (rGO =
8
for clinical applications. reduced graphene oxide, GCE = glassy carbon electrode),
5
Au@Ag core-shell NPs, ChOx/NiFe2O4/CuO/FeO-chitosan/ITO
Nowadays, non-transmissible chronic diseases are a worldwide 9
(ITO: indium-tin-oxide glass substrate), ChOx-
problem and a threat to health and human development. In ChEt/GPTMS/ITO (GPTMS: (3-
particular, one of the risk factors for the development of 10
glycidoxypropyl)trimethoxysilane), ChOx-ChEt/NiFe2O4 NPs-
cardiovascular disease, diabetes, atherosclerosis and chitosan/ITO,11 ChOx-ChEt/Cu2O NPs-chitosan/ITO12 and ChOx-
hypertension is a high level of cholesterol.1, 2 On the other ChEt/Tm2O3 nanorods/ITO.13 More examples of biosensors for
hand, low level of cholesterol in blood may be associated with cholesterol determination can be found in a previous review.2
depression, cancer and cerebral hemorrhage.3 Cholesterol is a Ionic liquids (ILs) have been used as alternative materials in
long-chain polycyclic alcohol, which is transported in the blood biosensing applications.14, 15 Moreover, these ionic compounds
plasma of animals. Also, it is an essential component for the have also been employed as modifiers in hybrid supports16-19
cell membrane and a precursor for the synthesis of other applied as cholesterol biosensors. However, to the best of our
biological materials.4 Rapid and accurate determination of knowledge, there are no examples of the use of a sputtering
cholesterol level is fundamental to prevent diseases as technique to generate small and clean Au NPs on IL-modified
mentioned above. The colorimetric method has been used for supports and their application in the detection of cholesterol.
cholesterol sensing because it is easy to apply, more In fact, the most common procedure to prepare Au NPs is from
economical when compared to electrochemical tests, uses the reduction of gold metal precursors, which generates
simple instrumentation and makes naked eye detection undesired by-products and remaining ligands. These undesired
possible without requiring additional measurements.5 In this compounds can coordinate to the metal surface and decrease
context, biosensors present many advantages compared to the particle’s activity. Motivated by the advantages of ILs20 and
classical methods: low cost, rapid detection, high selectivity GO,21 in this work we combined these compounds and
and sensitivity, and the possibility to produce portable devices sputtered Au NPs to produce a simple and efficient material to
be used as a biosensor for colorimetric determination of total
cholesterol. In fact, the synergistic effect of the IL fragments
covalently attached to the GO (rGO) together with the
presence of small and clean Au NPs provides a suitable hybrid
E-mail: [email protected] material.
E-mail: [email protected]; Tel: +55 51 3308 9633.

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1

Please do not adjust margins

 Page 3 of 6 Please
Journal ofdo not adjustChemistry
Materials margins B
View Article Online
DOI: 10.1039/C7TB02582C
COMMUNICATION Journal Name

H 2N H 2N NH 2 2.0 nm on IL 1-GO and 5.9 ± 2.1 nm on IL 2-GO (Fig. 3). Moreover, it

N + N H N + N
was observed well-dispersed particles with a spherical-like shape
Br - Br - deposited on the IL-GO sheets, although few large particles could
IL 1 IL 2 also be detected. The controlled size distribution for both samples

Journal of Materials Chemistry B Accepted Manuscript

is a significant advantage of the sputtering method, because in this
Fig. 1 Amine-functionalized ILs used as modifiers in the GO supports. case it was possible to control the experimental parameters such as
current, voltage and deposition time in order to obtain particles
The preparation of the amine-functionalized ILs (Fig. 1) and the GO with a similar average size. It is expected that the IL groups
modified with ILs was performed according to the methods attached onto the GO provide additional stabilization for the
Published on 20 November 2017. Downloaded by University of Newcastle on 20/11/2017 23:55:34.

described in Supporting Information (SI). The functionalized GO supported Au NPs, similar to that observed in other metal NPs
27, 28
supports were characterized by FTIR, Raman, X-ray diffraction (XRD) immobilized in IL-functionalized supports.
and X-ray photoelectron spectroscopy (XPS) (see SI). By Raman
analyses of the IL-GO supports (Fig. S2), it was possible to evaluate
2
the disorder and defects using the signals D and G – sp carbon
-1 -1 22, 23
vibrations – localized at 1360 cm and 1560 cm , respectively.
-1
The harmonic signal 2D (2680 cm ) is related to the number of
graphene sheets24 while the signal D+D’ is due to defects in the
23
carbon structure. Comparing the oxide precursors (GO) before
functionalization, only luminescence could be observed, which is
characteristics of GO.25 Concerning to evaluate the defects, we
considered the relation between the intensity of signals D and G
(ID/IG); a higher value was obtained for IL 1-GO, indicating the
presence of more structural defects, decreasing the sp2 sites in the
matrix and consequently the graphitic sites. In both cases, IL 1-GO
and IL 2-GO, the signals D and D+D’ were significant, which
indicates the presence of defects and low sp2 conjugation of
graphene sites. The size of the graphitic sites was estimated by
23
using the Tuinstra and Koenig relation, achieving 7.43 and 8.60 for
IL 1-GO and IL 2-GO, respectively (for more details, see SI). The
minor graphitic size observed for the sample IL 1-GO suggests that
functionalization with IL 1 provides a minor interaction among the
carbon sheets. In other words, IL 1-GO adopts a less organized Fig. 3 TEM micrographs of the Au NPs supported on (a) IL 1-GO and (b) IL 2-GO
with their respective size distribution histograms.
structure of graphene sheets while for IL 2-GO the IL may act as a
spacer among them, providing a graphite-like structure. The Au NPs
XRD measurements confirmed the presence of Au NPs on the
were deposited onto the IL-functionalized GO support by a
supports showing crystalline particles with fcc structure, where Au
sputtering technique (Fig. 2). This deposition method was chosen o
NPs presented their two characteristic peaks at 38.2 (111) and
because it is a facile and clean procedure to generate well- o
distributed nanoscale particles.26 40.5 (200), respectively (Fig. S3). After the oxidation of graphite by
the Hummers method, the XRD pattern of GO showed a sharp
o
diffraction peak at 2Ɵ = 9.5 (d-spacing = 0.93 nm) due to the
intercalation of functional groups containing oxygen between the
layers of graphite. Subsequently, the functionalization of GO (IL 1-
o
GO and IL 2-GO) showed a shift at 2Ɵ = 7.5 (d-spacing = 1.18 nm)
o
and 2Ɵ = 7.3 (d-spacing = 1.21 nm), respectively, which indicates
the intercalation of the ILs among the graphene layers. The peak at
o
2Ɵ = 28.3 (d-spacing = 0.315 nm) provides similar spacing to a
graphite structure, which indicates that the graphene oxide sample
functionalized with IL 2 adopted a partially crystalline system, which
can be related to simultaneous interaction of the amine groups of
the imidazolium cation with the carbon sheets. It is reasonable to
suppose that the IL cation acts as a bridge crosslinking the two
Fig. 2 Sputtering deposition of Au NPs onto the IL-functionalized GO supports. carbon sheets, modifying the interplanar spacing of the GO. It is
noteworthy that this 28.3° signal was not observed for IL 1-GO,
The supported Au NPs were analyzed by TEM, XRD and XPS analysis.
which suggests a preferred graphene-like arrangement in this case.
TEM measurements evidenced the formation of Au NPs with 5.4 ±

2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

 Please
Journal ofdo not adjustChemistry
Materials margins B Page 4 of 6
View Article Online
DOI: 10.1039/C7TB02582C
Journal Name COMMUNICATION

In order to evaluate and compare the surface chemical composition and tested for total cholesterol detection by colorimetric
of the GO, IL-GO and Au NPs/IL-GO samples, XPS analyses were assay. Biosensor responses were measured using standard
performed. XPS peaks for O 1s, N 1s, C 1s and Au 4f core-level cholesterol solutions at body temperature (see details in SI).
regions were observed. In Fig. 4 is shown the typical XPS spectra of Fig. 5 shows the plot of enzyme response vs. cholesterol

Journal of Materials Chemistry B Accepted Manuscript

the C 1s region of graphite and GO. High-resolution C 1s spectra concentration. Both free enzyme (free-ChOx), ChOx-Au NPs/IL
were decomposed into five peaks, corresponding to C-C, C=C, C-O- 1-GO and ChOx-Au NPs/IL 2-GO biosensors showed linear
29, 30
C, C=O and O=C-O, which were in agreement with the literature cholesterol detection at concentrations between 10 and 95
and confirm significant structural changes during the oxidation mg/L with correlation coefficients of 0.9991, 0.9951 and
process from graphite to GO. Indeed, the C/O atomic ratio 0.9890, respectively. The sensitivity values were determined as
Published on 20 November 2017. Downloaded by University of Newcastle on 20/11/2017 23:55:34.

31
decreased from 24.13 for graphite to 3.84 for GO, in accordance 0.0127 L/mg and 0.0097 L/mg for ChOx-Au NPs/IL 1-GO and
with FTIR spectra (see Fig. S1). After reduction and functionalization ChOx-Au NPs/IL 2-GO, respectively. These results reveal that
with IL, the peak intensities of oxide moieties decreased, both materials developed in this work allowed efficient action
2
accompanied by an increase of the sp carbon peak, which indicates of the enzyme in the reaction medium, showing high potential
the presence of cationic IL fragments attached to the carbon for application as biosensors. Even though the percentage of
network. The N 1s peak region showed signals associated to the ChOx immobilized in both materials was similar (93% for ChOx-
32
imidazolium ring, a shoulder at 398.9 eV confirms the presence of Au NPs/IL 1-GO and 92% for ChOx-Au NPs/IL 2-GO), the
30, 33
NH and a peak at 405.2 eV confirms the presence of NH2. The biosensor ChOx-Au NPs/IL 1-GO showed a greater response to
IL-GO supports containing Au NPs deposited by sputtering were also cholesterol compared to the ChOx-Au NPs/IL 2-GO biosensor
analyzed (Fig. 4). The Au 4f7/2 photoelectron peak appeared at 83.87 (Fig. 5). In Fig. 5 (b and c) it is observable by naked eye the red
0
eV (SI) and peak doublet separation was consistent with the Au color gradient due to the increase of cholesterol concentration
34
state (3.7 eV). The relative Au content in the samples was in the samples.
estimated to be around 4% by XPS. Although only metallic Au was
0
found in the Au NPs/IL 1-GO sample, for Au NPs/IL 2-GO, Au and
δ+ δ+
Au components were observed. The presence of Au component
may have an effect on the biosensing activity of the Au NPs/IL 2-GO
material.

Fig. 5 (a) Detection of cholesterol at different concentrations: (○) free-ChOx, (▵)

ChOx-Au NPs/IL 1-GO, (□) ChOx-Au NPs/IL 2-GO; inset: (▿) ChOx-GO and (◊)
ChOx-Au NPs/GO. Typical colorimetric tests using (b) ChOx-Au NPs/IL 1-GO and
(c) ChOx-Au NPs/IL 2-GO.

Regarding to evaluate the biosensor response, the recovered

activity (activity percentage of the enzyme in the material relative
to the free enzyme) was calculated. Compared with free-ChOx, the
ChOx-Au NPs/IL 1-GO and ChOx-Au NPs/IL 2-GO biosensors retained
a maximum of 76% and 54% of ChOx activity, respectively. The
higher recovered activity for the ChOx-Au NPs/IL 1-GO biosensor is
probably due to the graphene-like structure of this material, which
Fig. 4 XPS measurements in the C 1s, N 1s and Au 4f regions of the IL-GO possibly provides more availability and better diffusion of
supports and the Au NPs/IL-GO materials. Black points represent the cholesterol to the enzyme active site. It is also possible that the
experimental data and the gray line represents the best fitting. In the C 1s
graphite-like structure of the ChOx-Au NPs/IL 2-GO biosensor
region: red, green, light blue, purple and yellow solid lines represent C=C, C-C C-
O-C, C=O and O=C-O chemical components, respectively. The C 1s region of the results in less exposure of the enzyme’s active sites, decreasing the
rGO represents typical behavior after functionalization of GO samples (IL- activity. Moreover, the presence of Auδ+ may facilitate the
modified GO). In the N 1s region: red, green and light blue solid lines represent coordination of additional electron-donating groups to the surface
NH (anchored to graphene), N (imidazolium ring) and NH2 chemical components,
respectively. In the Au 4f region: red and green solid lines represent Au0 and Auδ+
of the Au NP, which possibly interfere in the biosensing behavior of
chemical components, respectively. this material compared to Au NPs/IL 1-GO. As another important
aspect, the isoelectric point of the cholesterol oxidase is 5.4,
For biosensor construction, the ChOx enzyme was immobilized indicating that the enzyme has a negative charge in the working pH
on the hybrid materials Au NPs/IL 1-GO and Au NPs/IL 2-GO (7.0). This allows the enzyme to bind to a positively charged matrix

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

Please do not adjust margins

 Page 5 of 6 Please
Journal ofdo not adjustChemistry
Materials margins B
View Article Online
DOI: 10.1039/C7TB02582C
COMMUNICATION Journal Name
δ+
as the Au NP’s surface. Then, it is expected that the enzyme will to the presence of surface Au species observed by XPS.
interact more strongly with the Au NPs in the IL 2-GO material due

Table 1 Comparison of the analytical response in cholesterol detection of various biosensors

Journal of Materials Chemistry B Accepted Manuscript

Entry Material Determination method Linear range (μmol/L) Reference
1 ChOx-Au NPs/IL 1-GO Colorimetric 25-470 Present work
2 ChOx-Au NPs/IL 2-GO Colorimetric 25-470 Present work
5
3 ChOx/Au@Ag core-shell NPs Colorimetric 0.3-300
35
Published on 20 November 2017. Downloaded by University of Newcastle on 20/11/2017 23:55:34.

4 ChOx-IL-capped Au NPs/GCE Voltametric 0.1-50

5 ChOx/catalase/rGO-IL/GCE Chronoamperometric 0.25-5/5-215a 18

6 ChOx-ChEtb/Pt NPs/graphene-nafion/GCE Amperometric 5-35 36

a b
Two range for the same material; ChEt = cholesterol esterase.

In addition, the biosensor’s stability was tested. The response of Scheme 1 Proposed interaction of cholesterol with the ChOx-Au NPs/IL-GO
materials.
both materials to cholesterol was analyzed after 24 and 115 h. At
115 h, cholesterol detection decreased to 52% and 81% of the initial
Notably, the materials produced in this work showed a
value for ChOx-Au NPs/IL 1-GO and ChOx-Au NPs/IL 2-GO
cholesterol detection range from 25 to 470 µmol/L, which is a
biosensors, respectively, probably due to enzyme desorption of the
large range compared to similar state-of-the-art graphene-
material or a decrease of ChOx activity. Therefore, the hybrid
based materials found in the literature (Table 1). This large
materials showed good stability under the tested conditions,
linear range demonstrates that the present materials can be
indicating that these biosensors can be used in clinical analyses. The 3
used to detect low (<150 µmol/L), medium and high (>260
superior stability of the ChOx-Au NPs/IL 2-GO biosensor may be 1
µmol/L) levels of cholesterol, considering an analogous test
associated with a possible stronger interaction between the enzyme
performed in clinical analysis (with a plasma dilution factor of
and the Au NPs in this material. To improve the knowledge about
1:20).37 As control experiments, the hybrid materials were
the IL role in biosensor efficiency, the enzyme was immobilized only
tested in the presence of common interferents such as
on graphene oxide (ChOx-GO) and on graphene oxide containing
glucose, ascorbic acid and uric acid in order to verify a possible
gold nanoparticles (ChOx-Au NPs/GO). Besides the percentage of
interference effect in the biosensing activities. The results
ChOx immobilized on both materials (73% for ChOx-GO and ChOx-
indicate that the detection of the interferents is negligible and
Au NPs/GO) being slightly less than for ChOx-Au NPs/IL 1-GO and
the hybrid materials were selective towards total cholesterol
ChOx-Au NPs/IL 2-GO materials, the response of ChOx-GO and
(Fig. S7). Besides the large detection range, a considerable
ChOx-Au NPs/GO biosensors to cholesterol detection was not linear
advantage of these hybrid materials is the high enzyme
(Fig. 5). These results evidence that the presence of IL in biosensor
recovered activity, sensitivity and selectivity for total
materials has a positive synergistic effect, which is important to
cholesterol that allow the application of a typical clinical
distinguish the variation of cholesterol quantities. In addition, it is
method and further reuse the material. The robustness and
important to note that in the cases where non-functionalized GO
efficiency of these hybrid materials at high cholesterol
(without IL) was employed, the ChOx recovered activity was
concentration can be associated to the synergistic effect of IL-
superior to the free ChOx (see Table S1). These observations cannot
functionalized GO and the small/clean Au NPs obtained by the
be related to the enzyme activity on the cholesterol, but due to the
sputtering technique. Therefore, the combination of IL-GO and
oxidizing agents present at the GO surface that may react with the
Au NPs produced by the sputtering method generated
color reagent. Considering all the results obtained in this work,
biosensors with a large detection range that will be able to
Scheme 1 provides a schematic proposal for the interaction of the
extend the cholesterol quantification using other
cholesterol molecule with the functionalized hybrid materials.
methodologies.

Conclusions
In summary, the sputtering deposition of Au NPs onto GO
modified with amine-functionalized ILs generates promising
hybrid materials to be used as efficient biosensors for
cholesterol detection. This physical method employed to
generate NPs has the advantage of producing small, clean and
controlled size particles, which are difficult to obtain by using
the classical chemical reduction of metal precursors. The ChOx

4 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

 Please
Journal ofdo not adjustChemistry
Materials margins B Page 6 of 6
View Article Online
DOI: 10.1039/C7TB02582C
Journal Name COMMUNICATION

was efficiently immobilized on Au NPs/IL 1-GO and Au NPs/IL 18. M. B. Gholivand and M. Khodadadian, Biosens. Bioelectron., 2014, 53,
2-GO materials and showed a good recovered activity on both 472-478.
19. S. Y. Wu, Y. X. Wang, H. Mao, C. Wang, L. X. Xia, Y. Zhang, H. Ge and X.
supports. Preliminary experiments applying these hybrid M. Song, RSC Adv., 2016, 6, 59487-59496.
materials as cholesterol biosensors showed satisfactory results 20. J. Dupont, Acc. Chem. Res., 2011, 44, 1223-1231.

Journal of Materials Chemistry B Accepted Manuscript

to identify individuals with low to high cholesterol levels using 21. D. R. Dreyer, S. Park, C. W. Bielawski and R. S. Ruoff, Chem. Soc. Rev.,
2010, 39, 228-240.
a practical naked eye colorimetric test. Indeed, the target 22. A. Eckmann, A. Felten, A. Mishchenko, L. Britnell, R. Krupke, K. S.
biomolecule was detected with excellent efficiency and Novoselov and C. Casiraghi, Nano Lett., 2012, 12, 3925-3930.
linearity. Moreover, the presence of IL in the material 23. L. G. Cançado, A. Jorio, E. H. M. Ferreira, F. Stavale, C. A. Achete, R. B.
Capaz, M. V. O. Moutinho, A. Lombardo, T. S. Kulmala and A. C. Ferrari,
composition allows a synergistic effect with the enzyme that is
Published on 20 November 2017. Downloaded by University of Newcastle on 20/11/2017 23:55:34.

Nano Lett., 2011, 11, 3190-3196.

essential for the linear detection of cholesterol. Therefore, the 24. A. Gupta, G. Chen, P. Joshi, S. Tadigadapa and P. C. Eklund, Nano Lett.,
combination of Au NPs and IL-GO produces robust and 2006, 6, 2667-2673.
selective biosensors with large detection range for total 25. S. Hermanova, M. Zarevucka, D. Bousa, M. Pumera and Z. Sofer,
Nanoscale, 2015, 7, 5852-5858.
cholesterol, opening new possibilities for the design of active 26. K. I. Okazaki, T. Kiyama, K. Hirahara, N. Tanaka, S. Kuwabata and T.
materials to detect biochemical target compounds in clinical Torimoto, Chem. Commun., 2008, 691-693.
applications. 27. L. Luza, A. Gual, D. Eberhardt, S. R. Teixeira, S. S. X. Chiaro and J. Dupont,
ChemCatChem, 2013, 5, 2471-2478.
28. L. Foppa, L. Luza, A. Gual, D. E. Weibel, D. Eberhardt, S. R. Teixeira and J.
Dupont, Dalton Trans., 2015, 44, 2827-2834.
Conflicts of interest 29. C. K. Chua, Z. Sofer and M. Pumera, Angew. Chem. Int. Ed., 2016, 55,
10751-10754.
There are no conflicts of interest to declare. 30. H. F. Yang, C. S. Shan, F. H. Li, D. X. Han, Q. X. Zhang and L. Niu, Chem.
Commun., 2009, 3880-3882.
31. S. Shamaila, A. K. L. Sajjad and A. Iqbal, Chem. Eng. J., 2016, 294, 458-
Acknowledgements 477.
32. K. R. J. Lovelock, I. J. Villar-Garcia, F. Maier, H. P. Steinruck and P.
The authors thank the Brazilian agencies CNPq, CAPES and Licence, Chem. Rev., 2010, 110, 5158-5190.
FAPERGS for financial support and LNLS (Campinas-SP) for the 33. Z. J. Wang, Q. X. Zhang, D. Kuehner, X. Y. Xu, A. Ivaska and L. Niu, Carbon,
XPS analysis. 2008, 46, 1687-1692.
34. A. Y. Klyushin, M. T. Greiner, X. Huang, T. Lunkenbein, X. Li, O. Timpe, M.
Friedrich, M. Havecker, A. Knop-Gericke and R. Schlogl, ACS Catal.,
2016, 6, 3372-3380.
References 35. M. J. Dong, Z. H. Nan, P. P. Liu, Y. J. Zhang, Z. H. Xue, X. Q. Lu and X. H.
1. S. Mann, C. Beedie and A. Jimenez, Sports Med., 2014, 44, 211-221. Liu, Electrochim. Acta, 2014, 132, 465-471.
2. S. K. Arya, M. Datta and B. D. Malhotra, Biosens. Bioelectron., 2008, 23, 36. R. S. Dey and C. R. Raj, J. Phys. Chem. C, 2010, 114, 21427-21433.
1083-1100. 37. C. C. Allain, L. S. Poon, C. S. G. Chan, W. Richmond and P. C. Fu, Clin.
3. M. A. Crook, U. Velauthar, L. Moran and W. Griffiths, Ann. Clin. Biochem., Chem., 1974, 20, 470-475.
1999, 36, 613-616.
4. J. Hu, Z. H. Zhang, W. J. Shen and S. Azhar, Nutr. Metab., 2010, 7, 47.
5. X. H. Zhang, M. Wei, B. J. Lv, Y. J. Liu, X. Liu and W. Wei, RSC Adv., 2016, 6,
35001-35007.
6. N. Zhou, J. Wang, T. Chen, Z. G. Yu and G. X. Li, Anal. Chem., 2006, 78,
5227-5230.
7. S. N. Ding, D. Shan, T. Zhang and Y. Z. Dou, J. Eletroanal. Chem., 2011, 659,
1-5.
8. M. Zhang, R. Yuan, Y. Chai, S. Chen, H. Zhong, C. Wang and Y. Cheng,
Biosens. Bioelectron., 2012, 32, 288-292.
9. J. Singh, M. Srivastava, P. Kalita and B. D. Malhotra, Process Biochem.,
2012, 47, 2189-2198.
10. S. Kumar, J. Singh, V. V. Agrawal, M. Ahamad and B. D. Malhotra, Anal.
Methods, 2011, 3, 2237-2245.
11. J. Singh, A. Roychoudhury, M. Srivastava, V. Chaudhary, R. Prasanna, D.
W. Lee, S. H. Lee and B. D. Malhotra, J. Phys. Chem. C, 2013, 117, 8491-
8502.
12. J. Singh, M. Srivastava, A. Roychoudhury, D. W. Lee, S. H. Lee and B. D.
Malhotra, J. Phys. Chem. B, 2013, 117, 141-152.
13. J. Singh, A. Roychoudhury, M. Srivastava, P. R. Solanki, D. W. Lee, S. H.
Lee and B. D. Malhotra, Nanoscale, 2014, 6, 1195-1208.
14. M. Ratel, A. Provencher-Girard, S. S. Zhao, J. Breault-Turcot, J.
Labrecque-Carbonneau, M. Branca, J. N. Pelletier, A. R. Schmitzer and J.
F. Masson, Anal. Chem., 2013, 85, 5770-5777.
15. D. T. Wu, P. F. Cai, Z. H. Tao and Y. J. Pan, Anal. Chim. Acta, 2016, 902,
174-181.
16. A. I. Gopalan, K. P. Lee and D. Ragupathy, Biosens. Bioelectron., 2009, 24,
2211-2217.
17. A. Safavi and F. Farjami, Biosens. Bioelectron., 2011, 26, 2547-2552.

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 5

Please do not adjust margins