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1 DOI: 10.1002/elan.201800472
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Carbon Nanofiber and Meldola Blue Based
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Electrochemical Sensor for NADH: Application to the
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Detection of Benzaldehyde
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10 Ana Maria Titoiu,[b] Maxime Lapauw,[a] Georgiana Necula-Petrareanu,[c] Cristina Purcarea,[c] Pablo Fanjul-
11 Bolado,[d] Jean-Louis Marty,[e] and Alina Vasilescu(s)*[b]
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14 Abstract: Nanomaterials used in tandem with electro- graphene oxide prior to dropcasting. The “MB-erGO/
15 chemical mediators on screen-printed electrodes enable CNF” sensors obtained were characterized by a detection
16 sensitive, low cost detection of NADH with minimal limit of 0.5 mM, a linear range of 1–300 mM and a sensitivity
17 interferences in real-world samples. In this work we of 80.0  2.5 mA cm2 mmol1 L, 10 times higher than that of
18 investigated the combination between the mediator Meldo- commercial sensors. While the use of graphene oxide lead
19 la Blue and several types of commercial screen-printed to enhanced sensitivity and wider linear range, it didn’t
20 carbon electrodes, i. e. modified with mesoporous carbon, improve the operational stability as the mediator gradually
21 single wall carbon nanotubes, graphene or carbon nano- desorbed from the electrodes. Furthermore, the sensors
22 fibers (CNF) as NADH detectors. The sensors were were coupled with a new NAD + -dependent aldehyde
23 compared with bare carbon electrodes and with commer- dehydrogenase from a psychrophilic bacterium for the
24 cially available Meldola Blue-modified electrodes. The best analysis of benzaldehyde and proven to be advantageous
25 sensitivity for NADH detection by amperometry was over commercial electrodes with Meldola Blue in circum-
26 observed for Meldola Blue/CNF electrodes, and further stances where the detection was limited by NADH
27 improvement was obtained by mixing the mediator with detection, i. e. at pH 9.5.
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Keywords: Meldola Blue · carbon nanomaterial · NAD + -dependent dehydrogenase · amperometry
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1 Introduction
32 etc.). These were adsorbed [24], included in the carbon
33 Electrochemical detection of NADH with fast and low paste or ink during electrode production (either “as is”, as
34 cost sensors enjoyed high interest in the past 30 years due a salt with lower solubility or adsorbed on various
35 to the number of potential applications with NAD + materials) [25, 29] covalently immobilized [15, 25], en-
36 -dependent dehydrogenase enzymes. The direct electro- trapped by the sol-gel method[5] or electropolymerised on
37 chemical oxidation of NADH occurs however at high electrode surface [30–32].
38 positive potentials on conventional electrodes, where The phenoxazine Meldola Blue (8-dimethylamino-2,3-
39 enzymatically inactive products may form, adsorb on the benzophenoxazine) is a well-known and efficient electro-
40 electrode and lead to passivation. Successful solutions to chemical mediator for NADH, that was used with differ-
41 minimize the risk of interferences and electrode passiva-
42 tion rely on electrode modification with nanomaterials or
43 electrochemical mediators showing catalytic properties [a] M. Lapauw
44 towards NADH oxidation. Institut Universitaire de Technologie, University of Perpignan
45 Among the different types of nanomaterials, carbon- via Domitia, 77 Chemin de la Passio Vella, Perpignan France
[b] A. M. Titoiu, A. Vasilescu(s)
46 based ones offer several advantages in electrochemical
International Centre of Biodynamics, 1B Intrarea Portocalelor,
47 biosensors [1, 2]. Such nanomaterials including single 060101, Bucharest, Romania
48 walled (SWCNTs) or multi walled carbon nanotubes E-mail: avasilescu@biodyn.ro
49 (MWCNTs), graphene or carbon nanofibers (CNFs) have [c] G. Necula-Petrareanu, C. Purcarea
50 shown catalytic effects towards different analytes [3, 4], Institute of Biology, 296 Splaiul Independentei, 060031 Bu-
51 including NADH [5–13]. charest, Romania
52 Mediated detection of NADH is an alternative [d] P. Fanjul-Bolado
Metrohm Dropsens, S.L.,Ed.CEEI, Parque Tecnolgico de
53 approach to decrease the overpotential required for
Asturias, 33428-Llanera, Asturias, Spain
54 NADH oxidation [14], that is enabled by a variety of [e] J.-L. Marty
55 compounds including quinones [15], flavins [16], oxometa- BAE, University of Perpignan via Domitia, France, 52 av Paul
56 lates [17], phenazines [18], phenoxazines [16], phenothia- Alduy, Perpignan, France*
57 zines [19], nitrofluorenone derivatives [20], various tran- Supporting information for this article is available on the
58 sition metal complexes [21], phenolic compounds [22, 23] WWW under https://doi.org/10.1002/elan.201800472

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1 ent types of electrodes and NAD + -dependent enzymes in
2 electrochemical biosensors, e. g. for glutamate analysis in
3 foods [33], of acetaldehyde and D-lactate in wine [34],
4 lactate in blood [35], ethanol [36] etc .
5 Combining mediators with nanomaterials appears as
6 an effective strategy to exploit the advantages of both
7 components in a synergetic way for the sensitive and
8 selective detection of NADH [9, 13]. Indeed, electrodes
9 modified with MWCNTs for example promoted the
10 adsorption of aromatic molecules such as Meldola Blue by
11 p-p stacking and hydrophobic interactions, enabling stable
12 immobilization of high amounts of mediator [9]. Due to
13 their high electroactive area, the nanomaterial modified
14 electrodes offered enhanced surface for the reaction with
15 NADH moreover, the fast electron transfer at sensor
16 interface was facilitated by electron transport along the
17 nanotubes and the presence of catalytic sites on their
18 surface [5, 9]. Several studies reported higher sensitivity
19 for NADH detection, faster electron transfer rates or also
20 higher response when combined to enzymes, as compared
21 to the sensors without nanomaterials [5, 9]. For example,
22 ultrasensitive detection of NADH (detection limit of
23 1 pmol L1) was achieved with single wall carbon nano-
24 tube field effect transistors (SWCNT-FETs), where
25 SWCNTs in the FET were functionalized with a phenazine
26 compound [18]. Zhu et al. [9] adsorbed Meldola Blue on a
27 carbon nanotubes modified glassy carbon electrode (CNT/
28 GCE) and found that the heterogeneous electron transfer
29 rate constant (k(s)) of MB/CNT was around 3 times higher
30 than at a similar electrode made with graphite powder
31 instead of CNTs [9]. Arvinte et al. [2005] have shown that
32 a Meldola Blue-SWCNT sol-gel nanocomposite material
33 leads to enhanced sensitivity for NADH detection (from
34 0.195 for bare electrode to 0.465 mA mmol L1) and
35 provides moreover a good support for immobilizing a
36 NAD + -dependent D-lactate dehydrogenase. High sensi-
37 tivity was observed with the SWCNT-including sensors
38 compared to those lacking the nanomaterial and this was
39 attributed by the authors to the strong adsorption of the
40 mediator on the SWCNT [5].
41 Among the various applications where NADH detec-
42 tors are useful, detection of aldehydes is important in
43 several fields, from air quality monitoring to breath
44 analysis and up to the analysis of food and beverages.
45 While typically aldehydes are detected via chromato-
46 graphic methods [37, 38], different enzyme based ap-
47 proaches have been proposed relying on either optical or
48 electrochemical detection to simplify the analysis and
49 decrease costs. The enzyme used determines the selectiv-
50 ity of the assay. Formaldehyde dehydrogenase is highly
51 selective for formaldehyde, the aldehyde dehydrogenase
52 from baker’s yeast has a broad selectivity towards
53 aliphatic aldehydes while the detection of aromatic
54 aldehydes was reported with a microbial aldehyde oxidor-
55 eductase isolated from E. coli. [39] Novel enzyme
56 catalysts are required to meet not only the selectivity
57 requirements for practical applications but also those
58 related to the stability and operational conditions. In this
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context novel enzymes from extremophilic microorgan-
isms, able to withstand extreme conditions (temperature,
pH, salinity etc) and showing new selectivity are highly
desirable.
A few examples of aldehyde detection with enzyme-
based biosensors were reported in literature. For example,
aldehyde oxidoreductase PaoABC was electrically wired
to a screen printed carbon electrode via an osmium-
containing electrochemical mediator [39]. The reagentless
biosensor thus obtained was able to detect benzaldehyde
with a detection limit of 5 mmol L1 in the linear range
from 10 to 150 mmol L1 [39]. A NAD + -dependent
formaldehyde dehydrogenase from Pseudomonas putida
was immobilized on a screen-printed electrode modified
with 1-pyrene butyric acid and MWCNTs and allowed
reaching a detection limit of 6 ppb formaldehyde [40].
Mitsubayashi et al. developed amperometric biosensors
including aldehyde dehydrogenase [41] or formaldehyde
dehydrogenase [42] for measuring acetaldehyde in ex-
haled air after drinking [43] or for measuring gaseous
formaldehyde [44] For example, acetaldehyde was de-
tected in breath with biosensors in the range from 0.11 to
10 ppm [43]. Noguer et al. [45] reported reagentless bi-
enzymatic biosensors for analysis of alcoholic beverages
where aldehyde dehydrogenase was co-immobilized with
NADH oxidase and a high molecular weight NAD + on a
Pt electrode. The biosensor detected acetaldehyde in the
range from 0.5 mmol L1 to 330 mmol L1. Screen-printed
graphite electrodes modified with Meldola Blue or
Meldola Blue-Reinecke salt (included in the screen-
printing ink) were coupled with aldehyde dehydrogenase
from baker’s yeast to build disposable or reusable
biosensors for acetaldehyde. Detection limits of
6 mmol L1, respectively 1 mmol L1 acetaldehyde were
reached with these biosensors [46].
In this work we investigated the combination between
the electrochemical mediator Meldola Blue and several
types of screen-printed electrodes modified with carbon
nanomaterials for NADH detection, focusing on CNFs
modified electrodes. Mediator adsorption and the role of
graphene oxide in promoting stable and high mediator
loading were studied in relation to the physical and
electrochemical characteristics of the sensors and their
performance for the amperometric detection of NADH.
Furthermore, the sensors were used to develop an assay
for benzaldehyde based on a novel NAD (P) + -dependent
aldehyde dehydrogenase from a Flavobacterium sp. strain
isolated from the Antarctic sea water [47].
2 Experimental
2.1 Material
The reduced b-nicotinamide adenine dinucleotide
(NADH, purity > 98 %), b-nicotinamide adenine dinu-
cleotide(NAD +, purity > 98 %), benzaldehyde(purity
> 99 %), as well as b-mercaptoethanol (purity > 99.0 %),
potassium chloride, TRIS and phosphate salts (reagent
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1 grade) were obtained from Sigma (Merck), Germany. The
2 following buffer solutions were prepared: 0.1 M phosphate
3 buffer pH 7.4, 0.1 M TRIS pH 8.0, 8.5, 9.0 and 9.5 and
4 0.1 M phosphate buffer pH 10. All buffer solutions
5 contained 0.1 M KCl. A 1 mol L1 benzaldehyde solution
6 in ethanol was prepared that was further diluted with
7 buffer to the required concentrations. The recombinant
8 aldehyde dehydrogenase (E.C. 1.2.1.3) from the Flavobac-
9 terium sp PL002 strain isolated from Antarctic sea water
10 was obtained by cloning and expression of the aldh3 gene
11 from the psychrophilic bacterium in E. coli BL21(DE3)
12 (to be published elsewhere). At 25 8C, this enzyme has a
13 Michaelis-Menten constant Km = 0.269 mmol L1 when
14 using benzaldehyde and NAD + (1 mmol L1) as cofactor,
15 in 0.1 mol L1 glycine/KOH buffer pH 9.5. After purifica-
16 tion, enzyme stocks (2 mg mL1) were obtained in 50 mM
17 potassium phosphate buffer, pH 7, containing 20 % glycer-
18 ol and 10 mM b-mercaptoethanol, and stored at 20 8C.
19 Screen printed graphite electrodes modified with
20 graphene (GPH), single walled carbon nanotubes
21 (SWCNT), mesoporous carbon (MC), carbon nanofibers
22 (CNF), as well as bare graphite electrodes (DRP-110) and
23 graphite electrodes modified with Meldola Blue (DRP-
24 610) were purchased from Metrohm Dropsens, Oviedo,
25 Spain. All were screen-printed 3 electrode systems printed
26 on alumina substrate, had a round working electrode of
27 4 mm diameter, a carbon counter electrode and a pseudo-
28 reference electrode made from Ag ink. The potential of
29 the pseudo-reference electrode, further indicated in the
30 paper as “Ag/AgCl” was determined experimentally in
31 0.01 M PBS buffer pH 7.4 to be 0.05 V versus a classic
32 Ag/AgCl (3MKCl).
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2.2 Electrochemical Determinations
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36 All electrochemical experiments were performed with a
37 VSP potentiostat (Princeton Applied Research, USA),
38 equipped with the EC-Lab software. Cyclic voltammetry
39 experiments were performed with the screen-printed 3-
40 electrode setup mentioned in section 2.1, where the
41 working electrode was the graphite-based electrode inves-
42 tigated. Cyclic voltammetry tests at different pH were
43 performed in deoxygenated buffer in the range from
44 0.95 V to 0.25 V vs Ag/AgCl. Amperometry measure-
45 ments for NADH were conducted in a cell containing
46 6 mL of 0.01 M PBS buffer pH 7.4, under medium stirring
47 with the working electrode polarized at + 0.05 V versus
48 Ag/AgCl, unless otherwise specified in the text. For
49 chronoamperometry experiments with benzaldehyde, the
50 electrodes were held horizontally and a 50 mL volume of
51 NAD + -aldehyde dehydrogenase mixture in 0.1 M TRIS
52 buffer pH 9.5, containing 0.1 M KCl was deposited on top
53 of the electrodes. A potential of 0.05 V was applied and
54 after a minute, 10 mL of sample containing benzaldehyde
55 was added in the cell. The current intensity was recorded
56 for a total time of 10 minutes. The variation in current
57 intensity from t = 1 min (when the sample was introduced
58 in the cell) and t = 10 min was taken as the analytical
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parameter and correlated with the concentration of
benzaldehyde in the sample. The NAD + -aldehyde dehy-
drogenase mixture used in the test contained 3.2 mmol L1
NAD + and 0.7 mU enzyme.
2.3 Measurement of Enzymatic Activity
For measuring the enzymatic activity of aldehyde dehy-
drogenase, a TECAN plate reader equipped with a
340 nm adsorption filter was used and the plate temper-
ature was set at 25 8C. The activity was determined from
the rate of NADH formation measured at 340 nm, using a
value of e340b-NADH = 6.22 3 103 L mol1 cm1 for the absorp-
tivity coefficient of NADH. One unit of activity corre-
sponds to the transformation of 1 mmol benzaldehyde per
minute at 25 8C.
2.4 Electrode Characterization by Raman, TEM and SEM
Raman spectroscopy measurements were performed with
a SPELEC RAMAN instrument with a 785 nm laser
(Metrohm DropSens, Oviedo, Spain). Screen-printed
electrodes (reference DRP-110, DRP-110SWCNT, DRP-
110OMC, DRP-110GPH, DRP-110CNF) were placed in a
specific cell (DRP-RAMANCELL) coupled with a Ram-
an Probe (DRP-RAMANPROBE). The integration time
was 20 s.
A Jeol JEOC-200-EX-II transmission electron micro-
scope (TEM) was used to characterize carbon nano-
materials and a JEOL JSM-6100 scanning electron micro-
scope (SEM) was used to characterize the electrode
surface after gold sputtering.
3 Results
3.1 Characterization of Carbon Nanomaterial Modified
Electrodes
3.1.1 Direct Electrochemical Oxidation of NADH by
Cyclic Voltammetry
Electrode modification with carbon nanomaterials has
several advantages in biosensing related to good electrical
conductivity, enhanced surface area, electrocatalytic effect
towards direct oxidation/reduction of various analytes and
multiple functionalization possibilities. In this work,
several commercial carbon based screen printed electro-
des modified with various nanomaterials (graphene-GPH,
single walled carbon nanotubes-SWCNT, mesoporous
carbon-MC, carbon nanofibers-CNF) were screened by
cyclic voltammetry in the presence of NADH, searching
for the best electrocatalytic behavior and sensitivity.
Systems with the working electrode of 4 mm diameter, of
same base carbon material and including the same
reference and counter electrodes, from the same supplier
were chosen to avoid bias due to different type of carbon
paste, sensor design or fabrication process.
With a bare carbon electrode (DRP-110) the current
due to the direct electrochemical oxidation of NADH
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37 Fig. 1. Cyclic voltammograms recorded with various carbon nanomaterial-modified electrodes at 0.1 V/s in the range between 0.25
and 0.95 V vs. Ag/AgCl, in 0.01 mol L1 PBS pH 7.4 in the absence (black) and the presence (red) of 10 mmol L1 NADH. (A) Bare
38
carbon electrodes. (B) CNF-electrodes. (C) MC-electrodes. (D) GPH-electrodes and (E) SWCNT-electrodes. The dashed curve in (B)–
39 (E) represents the voltammogram recorded with a bare graphite electrode in the presence of NADH.
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41
42 reaches a maximum intensity at 0.603  0.032 V vs. Ag/ observations made with electrodes from different batches,
43 AgCl (Figure 1A). The capacitive current in 0.01 M PBS not shown).
44 buffer pH 7.4 is negligible for bare graphite electrodes, The largest cathodic shift (about 196 mV) is recorded
45 but is significant for all the nanomaterial-modified elec- with the CNF-modified electrode (Figure 1B). To explain
46 trodes and is particularly high for GPH electrodes. A the catalytic effect for NADH oxidation the electrodes
47 catalytic effect towards NADH oxidation is observed with were characterized by several techniques.
48 all the nanomaterial-modified electrodes, for which the
49 anodic peak potential of NADH is shifted towards more
3.1.2 Characterization by SEM, TEM, Raman
50 negative values, to 0.407  0.014 V, 0.463  0.034 V, 0.485 
Spectroscopy and Cyclic Voltammetry
51 0.041 V and 0.527  0.040 V (n = 3 electrodes) for CNF,
52 GPH, MC and SWCNT electrodes, respectively (Fig- All electrodes modified with carbon nanomaterials, in this
53 ure 1B–E). For CNF and GPH electrodes a second peak work, were characterized by Raman spectroscopy, while
54 at the potential corresponding to the oxidation of NADH more detailed studies by SEM, TEM and cyclic voltam-
55 on bare graphite is also observed (Figure 1B and D). The metry were focused on CNF electrodes exclusively.
56 relative height and separation between the 2 peaks are The Raman spectrum (Figure 2) shows a large D-band
57 related to the amount of nanomaterial modifier (based on at around 1300 cm1 for the CNF electrodes, and a higher
58 ID/IG ratio in comparison with the SWCNT and GPH

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21 Fig. 2. Raman spectroscopy analysis of the various graphite electrodes modified with carbon nanomaterials.
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24 modified electrodes. It is indicative of a highly functional- tion of NADH. This provides further evidence of the
25 ized surface and a high structural disorder near the edges electrocatalytic effect observed for the oxidation of
26 of the microcrystalline structure that decreases the NADH due to the edges of graphite structures located in
27 symmetry of the structure, in good agreement with the the external side of the nanofiber.
28 TEM observations. In contrast, in SWCNT and GPH Furthermore, the properties of CNF electrodes were
29 modified electrodes the D band is significantly smaller investigated by cyclic voltammetry in order to explain the
30 than the G band, so the defects density in the structure is good characteristics observed for NADH detection. Cyclic
31 much lower (Figure 2). Therefore, the shift in the anodic voltammetry experiments were performed at different
32 peak potential of NADH with CNF electrodes as scan rates in the range from 0.005 to 0.250 V/s in the
33 compared with bare carbon electrodes can be mainly presence of 5 mmol L1 potassium ferricyanide in order to
34 attributed to the structure of the highly functionalized determine the electroactive area of CNF and bare graph-
35 CNFs. ite electrodes, based on the Randles-Sevcik equation:
36 SEM picture of the CNF modified electrode surface
37 (Figure 3A) illustrates the homogeneous distribution of I ¼ 2:69  105 n3=2 Ac D1=2 v1=2 ,
38 CNF over the complete surface of the working electrode.
39 TEM analysis allows observing in more detail the carbon where I represents the current intensity (expressed in A),
40 nanofibers which have a bamboo-like structure (Fig- n = the number of electrons involved in the reaction (1),
41 ure 3B), with a high number of graphite edges oriented to A is the electrode area in cm2, C is the concentration of
42 external side of the nanofiber. On these edges are located analyte (ferricyanide) in mol cm3, D is the diffusion
43 the most of the oxygen containing functional groups coefficient of ferricyanide, taken here as 7.26 3 106 cm2 s1
44 (COOH, OH) with catalytic properties for the oxida- [48] and v is the scan rate in V s1. The electroactive area
45 of CNF-modified electrodes was 0.079  0.012 cm2 (n =
46 5 electrodes), around 41 % higher than the area of bare
47 graphite electrodes, which was calculated as 0.056 
48 0.006 cm2. Various values of the electrochemically active
49 area within 0.07–0.11 cm2 were reported in the literature
50 for the bare DRP-110 [8, 49–50]. The lower active area
51 compared to the geometric area of 0.126 cm2 is explained
52 by the electrochemically inactive binders included in the
53 screen-printing ink. Nonetheless, the enhanced electro-
54 chemically active area of carbon nanofiber-modified
55 electrodes (CNF) compared to the bare graphite electro-
56 des is correlated with a higher surface due to the use of
57 Fig. 3. (A) SEM analysis of the CNF electrodes. (B) TEM analysis the nanomaterial (CNF). Additionally, the increase in the
58 of the CNFs. electroactive area compared to bare electrodes is more

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25 Fig. 4. Cyclic voltammograms recorded at 0.1 V/s in the range between 0.4 and 0.2 V vs. Ag/AgCl, in 0.01 mol L1 PBS pH 7.4 in the
26 absence (black) and the presence (red) of 10 mmol L1 NADH. (A) Carbon nanofiber-electrodes with adsorbed Meldola Blue (“MB/
CNF” electrodes). (B) Graphene modified electrodes with adsorbed Meldola Blue electrodes. (C) Graphite electrodes with adsorbed
27
Meldola Blue (D) Carbon nanofiber-electrodes modified by adsorbing a mixture of Meldola Blue-graphene oxide, followed by
28 electrochemical reduction of graphene oxide (“MBerGO/CNF” electrodes).
29
30
31 substantial for CNF-modified electrodes than for other when analyzing real samples. Another drawback of the
32 carbon nanomaterial modified electrodes from the same direct electrochemical oxidation of NADH is that enzy-
33 supplier, for example around 4 % higher electroactive matically inactive products may be formed [51], adsorbing
34 area was reported for SWCNT and GPH electrodes by on the electrode and leading to passivation [52].
35 Grimaldi et al. [50] While the composition of the screen- A widely used strategy to enhance the selectivity and
36 printing paste used to obtain CNF electrodes is propri- sensitivity for NADH oxidation combines the use of
37 etary information, the ink was optimized by the electrode nanomaterials with an electrochemical mediator for
38 manufacturer to provide the highest electrocatalytic effect NADH [5, 9]. We have hereby chosen to investigate the
39 with a reasonable capacitive current, as increasing the effects of adsorbing a well-known and efficient electro-
40 amount of carbon nanofibers will also increase the chemical mediator for NADH, i. e. Meldola Blue on the
41 capacitive current. various screen-printed electrodes included in our initial
42 Taken together, the Raman spectroscopy, SEM, TEM screening. The screen-printed electrodes -CNF, MC, GPH
43 and cyclic voltammetry measurements emphasize the and SWCNT were modified by dropcasting 8 mL of a
44 characteristics of CNF modified electrodes as advanta- 5 mg mL1 aqueous solution of Meldola Blue, leaving it to
45 geous detectors for NADH over bare graphite electrodes dry at room temperature for 24 hours, followed by copious
46 of the same design, emphasizing a higher electroactive washing with water (to remove any weakly adsorbed
47 area and catalytic effect due to homogeneous surface mediator molecule) and drying.
48 coverage with bamboo-like nanofibers presenting a high The cyclic voltammograms of the nanomaterial modi-
49 number of catalytic sites (defects with functional groups) fied electrodes with adsorbed Meldola Blue (MB) in the
50 at the edges of graphite structures. absence and the presence of 10 mmol L1 NADH, respec-
51 tively were compared to those recorded with unmodified
52 graphite electrodes with the mediator adsorbed in the
3.1.3 Study of Various Electrodes with Adsorbed Meldola
53 same conditions. Moreover, commercial carbon electrodes
Blue
54 with the mediator included in the screen-printing ink,
55 Despite the catalytic effect observed with nanomaterial “DRP 610” were also evaluated. The recorded voltammo-
56 modified electrodes, the peak potential for the direct grams for CNF, GPH and graphite electrodes with
57 electrochemical oxidation of NADH remains high adsorbed mediator are shown in Figure 4 (for simplicity,
58 enough, in the region of significant risk of interferences the voltammograms recorded with the other electrodes-

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1 MC, SWCNT and DRP 610 are shown in Figure S1 in The mediator adsorbed on the electrode reacts with
2 Supplementary info). NADH and is reduced in the chemical reaction. Once the
3 All the electrodes with adsorbed Meldola Blue show applied potential exceeds the formal potential of MBox/red
4 clear oxidation and reduction waves, with a formal couple, the reduced mediator is electrochemically reoxi-
5 potential of the adsorbed mediator between 0.147  dized, becoming again available to participate in the
6 0.009 V (graphite electrodes) and 0.136  0.022 V(CNF reaction with NADH. As shown by cyclic voltammograms
7 electrodes). By comparison, the commercial graphite in Figure 4 this approach enables to determine NADH at
8 electrodes with the mediator included in the graphite ink much lower overpotentials compared to the direct electro-
9 (DRP 610) display barely visible oxidation and reduction chemical oxidation of NADH, minimizing thus the risk of
10 waves probably due to the lower quantity of Meldola Blue interferences and electrode passivation.
11 available (Figure S1C). Aiming to choose the best sensor for NADH among
12 The highest peak current intensities attributed to the all these electrodes, we set as selection criteria the ratio
13 adsorbed mediator were recorded with GPH-modified between the magnitude of the catalytic current in the
14 electrodes, however for these electrodes the difference in presence of 10 mmol L1 NADH (Icat) to the intensity of
15 the potential of the anodic and cathodic peaks is also the the anodic peak of Meldola Blue (Ipa) in the absence of
16 largest, 0.096 V compared to only 0.042 V for bare graph- NADH, at a potential of E = 0.0 V where electrochemical
17 ite or 0.047 V for CNF electrodes (Figure 4A–C). Larger interferences are minimal.
18 peak separation indicates a slower electron transfer rate Among the various electrodes tested by cyclic voltam-
19 on GPH electrodes than on the other types of electrodes, metry (Figure 4A–C and Figure S1, Supplementary Infor-
20 with regards to the electrochemical oxidation/reduction of mation), CNF and GPH electrodes with adsorbed media-
21 Meldola Blue. More detailed investigations by cyclic tor were most promising for the detection of NADH
22 voltammetry in 0.01 mol L1 PBS buffer pH 7.4 empha- (Table S1, Supplementary Information). The largest cur-
23 sized a linear dependence between the anodic peak rent intensities in the presence of NADH were recorded
24 current and the scan rate in the range 0.010–0.2 V/s for with GPH electrodes with adsorbed Meldola Blue and
25 MB/GPH, MB/CNF and MB/graphite electrodes. Togeth- were attributed to the high loading of Meldola Blue on
26 er with peak separations of 0.042–0.096 V this indicates a graphene. Despite the better mediator coverage on GPH
27 typical electrochemical behavior for a quasi-reversible electrodes, when compared in the presence of NADH the
28 surface-adsorbed redox couple. In these conditions, the highest current density increase Icat/Ipa corresponds to MB/
29 surface coverage with the mediator G can be calculated CNF electrodes. Moreover, CNF-electrodes display lower
30 based on the magnitude of the peak current (Ip) recorded capacitive current than GPH as shown by cyclic voltam-
31 at different scan rates (v) and by applying the Laviron’s mograms recorded in PBS buffer pH 7.4 (Figure 1). Taken
32 equation [53]: together, MB/CNF electrodes seem more appropriate as
33 NADH detectors compared to the other devices inves-
34 Ip ¼ n2 F2 vAG=RT tigated.
35 To take advantage of the strong adsorption of Meldola
36 where n is the number of electrons transferred in the Blue observed on graphene, we proposed further to
37 electrochemical process, F is Faraday constant, A is the investigate whether modification of CNF electrodes with
38 surface area of the electrode, R is the universal gas both Meldola Blue and graphene could be more advanta-
39 constant and T is the temperature. geous in terms of stability and sensitivity towards NADH
40 Average loadings with Meldola Blue of 3.6 3 than modification with the mediator alone. For this
41 1010 mol cm2, 1.1 3 1010 mol cm2 and 1.0 3 1011 mol cm2 purpose, a mixture of mediator and graphene oxide
42 were calculated for the MB/GPH, MB/CNF and MB/ (2.5 mg mL1 Meldola Blue and 2 mg mL1 GO) was
43 graphite electrodes, respectively. While both types of dropcasted on the CNF electrodes. After drying and
44 nanomaterial-modified electrodes, i. e. CNF and GPH washing the excess mediator and graphene oxide the
45 enable significantly higher (11 to 36 times) coverage with electrode was submitted to cyclic voltammetry in the
46 Meldola Blue compared to graphite, the mediator loading range from + 0.2 V to 1.5 V for 10 cycles in 0.01 M PBS
47 on GPH is impressive and is explained by the strong pH 7.4 in order to reduce the graphene oxide and obtain
48 adsorption of the aromatic mediator on graphene by p-p MB-erGO/CNF electrodes. The MB-erGO/CNF electro-
49 stacking interactions. des, characterized by a formal potential of Meldola Blue
50 For all electrodes with adsorbed Meldola Blue, e. g. the of 0.177  0.020 V vs. Ag/AgCl show a high electro-
51 MB/CNF electrode shown in Figure 4A, in the presence of catalytic effect towards NADH oxidation (Figure 4D). In
52 NADH there is an increase in the anodic current starting the presence of 10 mmol L1 NADH, the anodic current
53 at a potential higher than the formal potential of Meldola increases greatly starting around the anodic peak potential
54 Blue, corresponding to the sequence of transformations: of the adsorbed Meldola Blue and becomes diffusion-
55 limited once the potential reaches around 0.05 V vs. Ag/
56 NADH þ MBþ ! NADþ þ MBH: ð1Þ AgCl. In the same time, the cathodic current decreases
57 E<E0 appreciably, similarly to the experiments with MB/CNF
58 MBH ƒƒ
ƒ! MBþ þ Hþ þ 2e : ð2Þ electrodes. Overall, the MB-erGO/CNF electrodes appear

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Fig. 5. (A) Detection of NADH with MB-erGO/CNF and MB/CNF electrodes by amperometry in stirred solutions at + 0.05 V in
14
0.01 M PBS pH 7.4. Aliquots of a 5 mmol L1 NADH solutions were added regularly in the electrochemical cell (final NADH
15 concentrations for the experiment shown were: 10, 20, 30, 69, 88, 108 and 127 mmol L1.) The current intensity recorded after addition of
16 NADH in the cell was correlated with NADH concentration. (B) Calibration plots for NADH recorded with DRP 610, MB-erGO/CNF
17 and MB/CNF electrodes.
18
19
20 to display similar catalytic performance towards NADH with MB/graphite (slope of the calibration curve: 9.2 
21 oxidation as the MB/CNF electrodes (Table S1, Supple- 0.4 mA cm2 mmol1 L) and DRP-610 electrodes (slope:
22 mentary information). 7.7  1.2 mA cm2 mmol1 L), respectively. The enhanced
23 Consequently, for the rest of this work we have current densities are attributed to the larger electroactive
24 continued with these two types of sensors, MB/CNF and area and higher mediator coverage of CNF-modified
25 MBerGO/CNF. electrodes.
26 Moreover, with MB-erGO/CNF electrodes, the detec-
27 tion limit was 0.3 mM (calculated as 3 times the signal to
28 3.2 Amperometric Detection of NADH with CNF and noise ratio) and the linear range extended from 1 to
29 Meldola Blue Modified Electrodes 300 mmol L1 (linear regression coefficient R2 = 0.9987). A
30 second linear range was observed at higher NADH
3.2.1. Calibration Plots at Neutral pH
31 concentrations, from 300 to 700 mmol L1 NADH (slope:
32 In order to obtain sensitive detectors for NADH that can 65.1  12.0 mA cm2 mmol1 L. R2 = 0.9966). By compari-
33 be used for detecting substrates of different NAD + - son, the MB/CNF electrodes present similar detection
34 dependent dehydrogenases, we have investigated the limit, 0.5 mmol L1 and two distinct linear ranges, 5–
35 performances of the sensors with CNF and adsorbed 100 mM (sensitivity: 42.2  8.1 mA cm2 mmol1 L, R2 =
36 Meldola Blue by amperometry at 0.05 V vs. Ag/AgCl, at 0.9991) and 100–700 mM (sensitivity: 25.1 
37 room temperature under constant stirring. Both types of 4.4 mA cm2 mmol1 L, R2 = 0.9952), respectively. The com-
38 sensors, MB/CNF and MB-erGO/CNF were obtained using mercial DRP 610 electrodes displayed a linear range of
39 solutions containing the same concentration of mediator, 10–500 mM but much smaller current densities as shown in
40 2.5 mg mL1 Meldola Blue. The potential value chosen for Figure 5B. These electrodes have Meldola Blue included
41 amperometry experiments provides enough sensitivity with in the screen-printing ink and therefore the reaction with
42 low interferences from real samples. Aliquots of NADH of NADH at the electrode surface is limited by the number
43 increasing concentrations were added in the cell containing of molecules of mediator available on the surface and the
44 6 mL of 0.01 M PBS buffer pH 7.4 and the current density diffusion of the mediator from the bulk of the screen-
45 recorded after each addition was plotted against the printed graphite-Meldola Blue layer to the electrode
46 concentration of NADH (Figure 5). surface. In contrast, electrodes with dropcasted Meldola
47 Control calibration experiments were performed using Blue have the mediator adsorbed on the electrode surface
48 the same conditions with carbon electrodes modified by readily available for the reaction with NADH. Addition-
49 adsorbed Meldola Blue (MB/graphite) and with commer- ally, CNF-electrodes provide a higher surface for mediator
50 cial electrodes where the mediator was included in the adsorption, enhancing the signal for NADH oxidation
51 screen-printing ink, DRP-610. even more. Furthermore, due to its aromatic structure,
52 The calibration plots for NADH in 0.01 M PBS buffer Meldola Blue is readily adsorbed on graphene oxide by p-
53 pH 7.4 have emphasized the superior sensitivity of the p stacking interactions.
54 MB-erGO/CNF electrodes (slope of the calibration curve: To rationalize the much larger (double) sensitivity
55 80.0  2.5 mA cm2 mmol1 L) compared to MB/CNF observed for the amperometric detection of NADH with
56 (slope: 42.7  8.7 mA cm2 mmol1 L). Both sensors allowed MB-erGO/CNF compared to MB/CNF electrodes we
57 to observe current densities that were 5–10 times higher have investigated the difference in mediator surface
58 than those obtained for the same NADH concentrations coverage and electrochemically active area between the

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1 Table 1. Comparison of the performances of MB-erGO/CNF electrodes with other screen-printed electrochemical sensors for NADH.
2
Mediator Operational conditions Detection limit Linear range Sensitivity Reference
3
(mmol L1) (mmol L1)
4
[30]
5 Poly(thionine) + 0.2 V, 0.1 M PBS pH 7 1.74 20–1000 0.34 mA mmol1 L
6 (10.83 mA mmol1 L cm2) [23]

7 Rosmarinic acid + 0.25 V, pH 7.25 5.62 18.72–220 10.94 mA mmol1 L

(87.1 mA mmol1 L cm2)
8 [54]
Poly(thionine) 0.1 V; PB[b] pH 6.9 3 5–100 1.14 mA mmol1 L [27]
9 Meldola Blue-Reinecke salt (in 0.1 V; 0.05 mol L1 PB 10 20–1000 2.03 mA mmol1 L
10 the screen-printing ink) pH 7.0; [34]
11 Meldola Blue 0 V, 0.1 M PB pH 7.0; 5 10–1000 1.98 mA mmol1 L
12 ( in the screen-printing ink) [5]
13 Sol-gel, Meldola Blue-SWCNT 0.05 V; 0.050 mol L1 6 50–1830 0.465 mA mmol1 L
14 PB, pH 7.5; [25]
MB (in the screen printing ink) 0.05 V; 0.050 mol L1 2.5 Up to 250 n/a[a]
15
PB pH 7
16 MB-erGO/CNF + 0.05 V; 0.1 M PB 0.3 1–300 300– 80.0  2.5 mA cm2 mmol1 L This work
17 pH 7.4 with 0.1 M KCl 700 mM 65.1  12.0 mA cm2 mmol1L
18
[a] n/a: not available. [b]: PB: phosphate buffer
19
20
21
22 two types of sensors. Surprisingly, the sensors have similar prior to drop-casting on the electrode has doubled
23 surface coverage with Meldola Blue: 3.2 3 1011 mol cm2 approximately the sensitivity for NADH detection but did
24 for MB-erGO/CNF and 3.5 3 1011 mol cm2 for MB/CNF not prevent the mediator leakage in a significant manner.
25 electrodes (Figure S2, Supplementary information). Addi- Thus, the gradual decrease in sensitivity has to be taken
26 tionally, there was no significant change of the electro- into account when the sensors are used for long studies,
27 chemically active area of a CNF electrode after modifica- i. e. by performing regular calibrations. Due to their higher
28 tion with GO alone by the same procedure as used to sensitivity, the MB-erGO/CNF electrodes were used for
29 obtain MB-erGO/CNF electrodes (i. e. dropcasting fol- the rest of this work.
30 lowed by electrochemical reduction). The surface area of The repeatability of the MB-erGO/CNF electrodes,
31 erGO/CNF electrodes was calculated as 0.081  0.016 cm2 estimated as the relative standard deviation (RSD) of the
32 (n = 5 electrodes) while that CNF electrodes was 0.079  slope of the calibration curve for NADH was quite good,
33 0.012 cm2. This result may be explained by procedure used RSD = 3.12 % (for n = 4 sensors from the same batch).
34 for the electrochemical reduction of graphene oxide that Compared to other screen-printed electrochemical sensors
35 has only lead to the partial reduction of graphene oxide. for NADH (Table 1), the performances of MB-erGO/
36 Taking into account all these experimental data, we CNF electrodes can be judged as similar, noting the
37 advance the hypothesis that the way the mediator is differences in the applied potential and experimental
38 immobilized at sensor surface-adsorbed flatly on graphene setup. The detection limit and sensitivity are better than
39 oxide by p-p stacking interactions interaction is favorable for most sensors without nanomaterials, highlighting the
40 for the fast kinetics of the reaction with NADH and the advantage of using CNF-modified as opposed to bare
41 subsequent electron transfer to the electrode. carbon electrodes. The linear range is wider than for the
42 Furthermore, we considered the issue of operational sensors modified with rosmarinic acid as mediator for
43 stability of the MB/CNF and MB-erGO/CNF sensors, as example [25], yet narrower than for sensors including
44 stable NADH detection is required for reusable biosen- Meldola Blue in the screen-printing ink [34] or in sol-gel
45 sors including NAD + -dependent dehydrogenases. Despite [5].
46 the strong adsorption of Meldola Blue on both these Moreover, with these sensors there was no interfer-
47 sensors, a gradual decrease in the sensitivity towards ence observed by amperometry at 0.05 V when testing
48 NADH was observed upon more stringent testing, i. e. by wine samples diluted 10 times with 0.01 M PBS buffer
49 performing a series of 10 calibrations by amperometry in pH 7.4 (data not shown). This opens the way for
50 stirred solutions, in the same day and including 8 applications with NAD + -dependent dehydrogenase for
51 concentration levels per each calibration series. For MB/ wine analysis (e. g. for lactate, acetaldehyde, glycerol etc
52 CNF electrodes and MB-erGO/CNF electrodes, the slope detection as investigated previously by us and others).
53 of the corresponding calibration curves for NADH has
54 decreased to 69.5  5.6 % and 70.0  6.2 % of the initial
3.2.2 Influence of pH and the Applied Potential on the
55 value, respectively, after 10 calibrations (n = 3 electrodes,
Sensitivity of MB/CNF-SPCE Electrodes
56 Figure S3, Supplementary information). This is attributed
57 to the partial leakage of the mediator in solution. Based We have furthermore investigated how the pH and the
58 on these results, mixing the mediator with graphene oxide applied potential influence the sensitivity of MB-erGO/

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26 Fig. 6. (A) Cyclic voltammograms recorded with the MB-erGO/CNF electrodes at different pH values: from right to left:
7.4; 8.0; 8.5; 9.0; 9.5 and 10. (B) Dependence of the formal potential of redox couple I of MB on the pH. (C) Effect of the pH on
27
sensitivity of MB-erGO/CNF electrodes towards NADH detection, by amperometry at + 0.05 V vs. Ag/AgCl. (D) Influence of the
28 applied potential on the sensitivity of the MB-erGO/CNF electrodes. The results are reported as percentage of the sensitivity recorded
29 at the lowest potential investigated (0.05 V).
30
31
32 CNF electrodes towards NADH. Cyclic voltammetry rate of approximately 41 mV/pH (Figure 6B). This shift is
33 experiments performed with these electrodes in the pH consistent with the transformations in equations 1–2.
34 range from 7.4 to 10 and between 0.95 and + 0.25 V Notably, at pH 9.5 for example, the formal potential of
35 emphasize 3 redox waves. In the example illustrated in the redox couple I is Eo’ = 0.295  0.022 V. This value is
36 Figure 6A for a typical sensor, these redox waves corre- rather far from the potential corresponding to the direct
37 spond to formal potentials of approximately 0.213 V, electrochemical oxidation of NADH on CNF electrodes,
38 0.367 V and 0.499 V at pH 7.4. Redox couple I is 0.603 V vs. Ag/AgCl (at pH 7.4, Figure 1A). It is well
39 involved in catalyzing NADH oxidation [9], while the known that the kinetics of the reaction between NADH
40 origin of the other two redox waves is believed to be and phenoxazine mediators decreases with higher pH
41 related to the oxygen containing functional groups on [55]. Hence, a lower sensitivity towards NADH of the
42 graphite, onto which the mediator is adsorbed. This Meldola Blue-based sensors developed here is expected at
43 assumption is based on the observation that these redox alkaline pH as compared to the detection at neutral pH.
44 couples are also observed with graphite electrodes with Indeed, by comparing the slopes of the calibration curves
45 adsorbed Meldola Blue. (Figure S4, Supplementary info). obtained at different pH values in the range from 7.4 to
46 The redox couple II of Meldola Blue was reported in 10, a sharp decrease in sensitivity around pH 9 was
47 literature[9] while redox couple III reported here for the observed. (Figure 6C) At pH 9.5, the sensitivity for
48 first time is observed thanks to the wide negative potential NADH detection with MB-erGO/CNF electrodes repre-
49 window investigated in the cyclic voltammetry experi- sents only around 26 % of the value registered at neutral
50 ments. Redox couples II and III vary with 0.057 V/pH and pH. This is in agreement with results reported with other
51 0.060 V/pH, respectively, which indicates that an equal sensors containing the same electrochemical mediator
52 number of protons and electrons is involved in the [26]. The decreased performance of MB-erGO/CNF-
53 corresponding electrochemical reactions. The matter of electrodes in alkaline media should be taken into account
54 redox couples II and III deserves further investigation and when the intended use of NADH detector is with
55 will be reported in a further work. Here, bearing in mind dehydrogenases that perform optimally in such pH range.
56 NADH detection, we have focused exclusively on redox With regards to another critical operational parameter
57 couple I, for which a cathodic shift in the formal potential for the amperometric detection of NADH, i. e. the applied
58 was observed, with the increase in pH from 7.4 to 10 at a potential, tests performed in the range from 0.05 V to

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Fig. 7. (A) Chronoamperometric detection of benzaldehyde with MB-erGO/CNF electrodes in the presence of NAD + and F-ALDH.
25
The tests were performed at + 0.05 V, in 0.1 M TRIS-HCl pH 9.5 with 0.1 M KCl as described in the text. Traces shown correspond to
26
benzaldehyde concentrations of 0; 167 mmol L1, 333 mmol L1, 583 mmol L1, 833 mmol L1, 1167 mmol L1 and 1667 mmol L1; (B)
27 Influence of the amount of F-ALDH in the cell on the current density observed for 583 mmol L1 benzaldehyde with the MB-erGO/
28 CNF sensors. (C) Calibration plot for benzaldehyde. (D). Detection of 833 mM benzaldehyde by chronoamperometry with DRP 610
29 electrodes in the presence of NAD + and F-ALDH.
30
31
32 0.2 V vs. Ag/AgCl have shown that at + 0.05 V vs. Ag/ been examined by chronoamperometry in the following
33 AgCl the sensitivity of the MB-erGO/CNF sensors (i. e. setup: a volume of 50 mL of buffer 0.1 M TRIS-HCl
34 slope of the calibration curve) reaches a plateau (Fig- containing 0.1 M KCl, 4 mg mL1 (26 mU mL1) aldehyde
35 ure 6D). dehydrogenase and 3.2 mmol L1 enzymatic cofactor
36 These results justify the choice of + 0.05 V as the NAD + was placed on top of the screen-printed electrodes,
37 applied potential for the tests reported in this work, held horizontally. The working electrode, MB-erGO/CNF
38 allowing good sensitivity in a potential window where was polarized at 0.05 V vs Ag and after 60 s a volume of
39 minimal interferences from real samples are expected. 10 mL of benzaldehyde solution in the same TRIS-HCl
40 buffer pH 9.5 was added on top on the electrodes. The
41 experiment was stopped 10 minutes after applying the
3.4 Development of an Enzymatic Assay for
42 potential. The increase in current density from t1 = 1
Benzaldehyde
43 minute to t2 = 10 minutes was taken as the analytical
44 The potential of MB-erGO/CNF as NADH detectors was parameter (Figure 7A). The current density recorded for
45 evaluated by coupling the electrodes with a novel NAD + different concentrations of benzaldehyde added in the cell
46 -dependent aldehyde dehydrogenase isolated from a was plotted against the concentration of benzaldehyde.
47 psychrophilic bacterium. Benzaldehyde was used as a First, the amount of enzyme used in the tests was
48 substrate and its detection was based on measuring with optimized in order to have a good analytical response
49 MB-erGO/CNF electrodes the NADH formed in the with the lowest cost, i. e. lowest enzyme quantity. Tests
50 enzymatic reaction catalyzed by aldehyde dehydrogenase: performed with 10 mL of 3.5 mmol L1 benzaldehyde
51 added in the cell containing 50 mL of 3.2 mmol L1 NAD +
52 Benzaldehyde þ NADþ ! Benzoic acid þ NADH and different amount of enzyme, i. e. activities of 3–
53 87 mU mL1 aldehyde dehydrogenase have shown that
54 Chronoamperometry measurements in low volume of 11 mU mL1 in the reaction mixture, corresponding to
55 samples applied on horizontally held electrodes represent 0.7 mU enzyme per test enable the optimum response
56 an easy approach, preferred for disposable biosensors. (Figure 7B). Hence, this amount of enzyme was used for
57 The performances of screen-printed electrodes modified all further experiments.
58 with carbon nanofibers and adsorbed Meldola Blue have

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1 Calibration experiments illustrated in Figure 7C em-
2 phasize a linear range between 80 and 1000 mM benzalde-
3 hyde (linear regression coefficient of the calibration, R2 =
4 0.9985), a detection limit of 17 mM benzaldehyde and an
5 average sensitivity of 0.82  0.10 mA cm2 mmol1 L.
6 To check the advantage of MB-erGO/CNF in the
7 particular case of benzaldehyde detection with the
8 described biosensors, we have performed parallel tests
9 with the commercial DRP 610 electrodes instead of MB-
10 erGO/CNF electrodes. In this case, there was a much
11 smaller response, i. e. more than 10 times lower for a
12 concentration of 833 mM benzaldehyde in the same
13 conditions (Figure 7D). This indicates that the biosensor
14 response is limited by the detection of NADH and not by
15 the enzymatic reaction and proves that combining an
16 efficient mediator with a nanomaterial modified electrode
17 where the mediator is well adsorbed is a good strategy to
18 achieve sensitive detection of benzaldehyde, taken as an
19 example of substrate of NAD + -dependent dehydrogen-
20 ases. Thus, we attribute the different behavior of the two
21 types of electrodes to the higher mediator surface cover-
22 age, readily available for interaction with NADH from
23 solution and to the higher area of the MB-erGO/CNF
24 electrodes compared to the DRP 610 sensors.
25 Compared to other aldehyde biosensors described in
26 the literature [39–46], the analytical performances re-
27 ported here with MB-erGO/CNF electrodes and a novel
28 aldehyde dehydrogenase from psychrophilic bacteria may
29 not seem very impressive. However, a very low concen-
30 tration of enzyme is used in the test, only 0.7 mU as
31 compared for example to 130 mU for a similar disposable
32 biosensor for acetaldehyde previously developed by some
33 of us [46]. The assay is much simpler than the ones based
34 on two-enzymes systems [45]. Moreover, the pH at which
35 the MB-erGO/CNF sensors are operated is not optimal
36 for NADH electrochemical detection based on Meldola
37 Blue mediator, but for enzyme’s catalytic activity towards
38 benzaldehyde conversion to benzoic acid. Thus, better
39 performances with the MB-erGO/CNF electrodes in
40 biosensing might be obtained by sensors optimization and
41 by combination with dehydrogenases that have optimum
42 activity near neutral pH.
43
44
4 Conclusions
45
46 CNF-modified electrodes facilitated the direct electro-
47 chemical oxidation of NADH by lowering the oxidation
48 potential with 196 mV. Moreover, among several types of
49 screen-printed electrodes modified with MC, SWCNT,
50 GPH, CNF-modified electrodes with adsorbed Meldola
51 Blue enabled the detection of NADH at low overpoten-
52 tials and with reasonable capacitive currents. Mixing the
53 mediator with graphene oxide before dropcasting on the
54 CNF electrodes, followed by electrochemical reduction of
55 the graphene oxide, enabled to obtain sensitive ampero-
56 metric detectors for NADH operating at low potential
57 values of + 50 mV (detection limit 0.3 mmol L1 and
58 sensitivity 80.0  2.5 mA cm2 mmol1 L in the range 1–
www.electroanalysis.wiley-vch.de  2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
300 mmol L1). The performances are attributed to the
combination between the catalytic effect of Meldola Blue
and the high electroactive area and good electron transfer
properties of CNF electrodes, supported by cyclic voltam-
metry, Raman spectroscopy, SEM and TEM imaging. The
mediator is adsorbed in high quantity and in a strong
manner on both CNF and graphene oxide. At similar
surface coverage and electrochemically active area, MB-
erGO/CNF electrodes are advantageous in terms of
sensitivity over MB/CNF electrodes, presumably due to
most favorable exposure of mediator facilitating the
reaction with NADH and electron transfer. The sensitivity
of MBerGO/CNF electrodes is about 10 times higher than
that of commercial graphite electrodes containing the
same electrochemical mediator, DRP-610. Despite a
gradual decrease in performance upon prolonged testing
by amperometry in stirred solutions, the MB-erGO/CNF
sensors retain a good sensitivity of about 70 % of the
initial value even after 80 NADH injections. The potential
of the novel electrodes was illustrated by coupling with a
novel NAD(P) + -dependent aldehyde dehydrogenase from
a psychrophilic bacterium in an enzymatic assay for the
detection of benzaldehyde. Although employed at an
alkaline pH where their performance is far from optimal,
the MB-erGO/CNF sensors were still able to provide
sensitive NADH detection as opposed to commercial
electrodes containing the same mediator in a graphite ink,
for which a very small response was obtained in the same
conditions. Based on the results of this work, carbon
nanofibersmodified electrodes are indicated as a good
platform for the detection of NADH, when used in
synergy with the mediator Meldola Blue. Coupling with
NAD + -dependent dehydrogenases in bioassays and bio-
sensors is the next step to fully exploit the potential of
MB-erGO/CNF electrodes for various practical applica-
tions.
Acknowledgements
We thank Paris Lavin for providing the bacterial strain
Flavobacterium sp. PL002. A.M.T, G.PN, C.P and A.V
gratefully acknowledge the financial support by the
Romanian Executive Agency for Higher Education,
Research, Development and Innovation (UEFISCDI),
PN-IIIP2-2.1-PED-2016-0116 grant.
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