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A novel electrochemical sensor based on ion imprinted polymer and gold nanomaterials for nitrite ion analysis in exhaled breath condensate
Journal Pre-proof A novel electrochemical sensor based on ion imprinted polymer and gold nanomaterials for nitrite ion analysis in exhaled breath condensate Alassane Diouf, Nezha El Bari, Benachir Bouchikhi PII:
S0039-9140(19)31210-X
DOI:
https://doi.org/10.1016/j.talanta.2019.120577
Reference:
TAL 120577
To appear in:
Talanta
Received Date: 18 July 2019 Revised Date:
18 November 2019
Accepted Date: 19 November 2019
Please cite this article as: A. Diouf, N. El Bari, B. Bouchikhi, A novel electrochemical sensor based on ion imprinted polymer and gold nanomaterials for nitrite ion analysis in exhaled breath condensate, Talanta (2019), doi: https://doi.org/10.1016/j.talanta.2019.120577. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
GRAPHICAL ABSTRACT :
1
A novel electrochemical sensor based on ion imprinted polymer and gold nanomaterials
2
for nitrite ion analysis in exhaled breath condensate Alassane Dioufa,b, Nezha El Barib, Benachir Bouchikhia,*
3 4
a
5
Moulay Ismaïl University of Meknes, B.P. 11201, Zitoune, Meknes, Morocco.
6
b
7
Faculty of Sciences, Moulay Ismaïl University of Meknes, B.P. 11201, Zitoune, 50003
8
Meknes, Morocco.
Sensor Electronic & Instrumentation Group, Department of Physics, Faculty of Sciences,
Biotechnology Agroalimentary and Biomedical Analysis Group, Department of Biology,
9
*
Corresponding author: Postal address: B.P. 11201, Zitoune, Meknes, Morocco Tel: +212 535 53 88 70; Fax: +212 535 53 68 08 Email: [email protected] 1
10
ABSTRACT
11
Human Exhaled Breath Condensate (EBC) contains markers of several inflammatory
12
diseases. Its analysis is of interest to a number of researchers. Nitrite ions (NO2-), which are
13
widely used in our daily lives, are nevertheless among these indicators. In this study, a simple,
14
fast, portable, non-invasive and cheap electrochemical sensor is developed for the analysis of
15
the nitrite profile in EBC. In this regard, sodium nitrite (NaNO2) was first immobilized on
16
self-assembled 2-aminothiophenol (2-ATP) on a screen-printed gold electrode (Au-SPE).
17
Then, a polymer matrix composed of polyvinyl alcohol (PVA) crosslinked with
18
glutaraldehyde (GA) was combined with gold nanoparticles (Au-NPs) to cover the modified
19
Au-SPE and complete the fabrication of the Ion Imprinted Polymer (IIP) sensor. The
20
electrochemical behaviour of the sensor was monitored using Cyclic Voltammetry (CV),
21
Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV)
22
methods, while the morphology and chemical composition of its layers were observed by
23
infrared Fourier transform (FTIR), Atomic Force Microscopy (AFM) and Scanning Electron
24
Microscopy coupled with energy dispersion X-Ray spectroscopy (SEM-EDS) techniques. In
25
addition, after a successful control test using a Non-Imprinted Ion Polymer (NIIP) sensor, the
26
obtained results demonstrated satisfactory sensitivity and selectivity to nitrite compared to co-
27
existing interfering substances in EBC, such as nitrate, acetate and ammonium nitrate. Under
28
improved experimental conditions, the nitrite IIP sensor exhibits responses proportional to
29
nitrite concentrations (R2 = 0.96) over a concentration range of 0.5 to 50 µg mL-1 with a
30
detection limit (LOD) of 4 µmol L-1 (signal-to-noise ratio S/N = 3). The proposed approach
31
was well applied for the nitrite determination in EBC samples with a relative standard
32
deviation (RSD = 4%) and could open clinical applications in respiratory medicine.
33
Keywords: Electrochemistry; Exhaled breath condensate; Ion imprinted polymer; Nitrite;
34
Screen-printed gold electrode.
35
2
36
1. Introduction
37
Diseases occur as a result of alterations in normal physiological processes due to metabolic
38
disorders or exposure to toxins (or microbial pathogens). The presence of typical chemicals
39
(biomarkers) in the body reveals the appearance of a disease. These markers go through the
40
circulatory system and are eventually expelled by the lungs. For this purpose, breath analysis
41
could provide very useful clues to the evolution of diseases in the human body. This leads
42
many researchers to focus on the analysis of human exhaled breath, which is non-invasive,
43
simple and useful compared to conventional methods of diagnosis and clinical treatment,
44
which are often invasive, costly, time-consuming, and require trained personnel. To trap and
45
concentrate volatiles from exhaled breath, it could be condensed by cooling to quantify
46
markers including nitrite and nitrate, hydrogen peroxide, eicosanoids, proteins, and so on [1].
47
Nitrite and nitrate have been shown to be important in understanding inflammatory diseases
48
[2,3]. Both are stable end products of the oxidative metabolism of nitric oxide that occurs
49
from L-arginine via nitric oxide synthase [4]. By consuming contaminated water and food,
50
excess of nitrite accumulates in the gastrointestinal tract of living organisms. The passage of
51
nitrite through the bloodstream can have two adverse effects on human health; it can either
52
combine with blood pigments producing methemoglobin in which oxygen is no longer
53
available for tissues [5,6], or interact in the stomach with amines and amides forming highly
54
carcinogenic N-nitrosamine compounds [7].
55
Due to the potential toxicity of nitrite in the human body, it is worthwhile for public health
56
to develop simple, rapid and accurate detection methods. Many analytical techniques have
57
been proposed including spectrophotometry (LOD 0.29 – 14.50 µmol L-1) [8], capillary
58
electrophoresis (LOD 0.72 – 4.35 µmol L-1) [9], electrochemistry [10] and ion
59
chromatography (LOD 0.07 − 0.14 µmol L-1) [11]. Because of the disadvantages, such as
60
operating costs and complexity, some of these methods are gradually being abandoned in
61
favour of electrochemical techniques that are simpler, more economical, faster and more
62
sensitive for nitrite detection. It is also important to note that with electrochemical techniques,
63
pre-treatments are minimized, instruments can be miniaturized, and measurements can be
64
performed on site. This solves many of the constraints encountered with conventional
65
methods [12-18]. Generally, the electrochemical detection of nitrite can be performed in two
66
ways; in most cases, oxidation of nitrite is preferred to its reduction, which is subject to
67
interference from other easily reducible species, such as nitrate ions and molecular oxygen
68
[19-21]. Nitrite is electroactive on certain metallic substrates, such as gold, copper, glassy
69
carbon. Therefore, its application on these substrates is likely to poison their surfaces 3
70
generating a high overvoltage [22,23]. This would reduce the sensitivity and accuracy of
71
nitrite detection [24]. Numerous studies have been carried out to optimize existing methods
72
for the determination of nitrite [14,25,26]. Therefore, the challenge is to overcome the
73
problems of low sensitivity, selectivity, and high potential caused by the direct application of
74
nitrite to the surfaces of metal electrodes. To achieve this goal, these surfaces must be
75
modified using appropriate electrocatalysts [23,27].
76
First, conductive polymers are undeniable factors because of their outstanding properties.
77
Polyaniline, poly-pyrrole and poly-thiophene are widely used. Among these polymers, amino-
78
thiophenol (ATP) has attracted particular attention because of its good electrical conductivity
79
[28,29]. Secondly, in order to increase the adsorption ability for electrochemical studies, these
80
polymers can even be combined with metallic nanoparticles. The nanoparticles advantages lie
81
in their ability to provide a large effective surface area, electrical conductivity, efficient
82
catalysis, and fast mass transport [30-33]. Among nanomaterials, the gold nanoparticles (Au-
83
NPs) are highly advantageous in several electrochemical implementations [34,35]. They are
84
also expected to increase the binding of NaNO2 to amine groups and allow the oxidation [36].
85
Since they can bind to functional groups, such as –CN, –NH2, or –SH by covalent bonding,
86
Au-NPs combined with conductive polymers should reduce overvoltage and allow faster
87
electron transfer kinetics for nitrite oxidation [37]. Third, compared to conventional
88
electrodes, screen-printed electrodes (SPEs) are becoming increasingly attractive thanks to
89
their low mass production cost, versatility, reproducibility, reliability for various practical
90
applications, and normally disposable after one or more uses [38-40]. Fourth, in molecular
91
imprinting technique (MIT), the interactions between the polymer and the target element are
92
hydrophobic, ionic, covalent or non-covalent, or a hydrogen bond [41]. The imprinting of
93
sites for the recognition of molecular or ionic elements in organic or inorganic polymer
94
matrices has been the subject of extensive studies [42-44]. The IIP technology initiated by
95
Nishide et al. [45] is the case in which the target is an ion. An IIP sensor is generally
96
constructed by binding the ionic element followed by copolymerization of a functional
97
monomer with a cross-linking agent to create recognition cavities in the polymer matrix.
98
Several advances have been made in IIP technology; however, a major challenge must be met
99
in the development of sensitive and selective devices for ion recognition in various
100
applications [46-48].
101
Taking all these considerations into account, the idea of this work is to functionalize Au-
102
SPE with 2-ATP and then synthesize and characterize a polymer combined with Au-NPs to
103
verify the ability of the obtained IIP sensor to recognize nitrite ions in EBC. The development 4
104
and retention capability of the nitrite IIP sensor is studied using CV, DPV, EIS, SEM-EDS,
105
AFM and FTIR techniques.
106 107
2. Experimental 2.1. Reagents and apparatus
108
Sodium nitrite (NaNO2), silver nitrate (AgNO3), acetate, ammonium nitrate, phosphate
109
buffered saline (PBS), gold chloride trihydrate (HAuCl43H2O) (99.99%), polyvinyl alcohol
110
(PVA) (98%), glutaraldehyde (GA) (25%), methanol, hydrochloric acid (HCl), and sulphuric
111
acid (H2SO4) were purchased from Sigma Aldrich. Dehydrated trisodium citrate was
112
purchased from Handel's Riedel, while potassium ferrocyanide trihydrate and potassium
113
ferricyanide trihydrate were from Fluka. Distilled water (DW) was used throughout the
114
experiments. All other chemicals were of analytical reactive quality and were used without
115
further purification.
116
Electrochemical measurements by CV, DPV and EIS were performed using a conditioning
117
circuit (Potentiostat, Palmsens3) interfaced to a computer controlled by PS-Trace software.
118
The Au-SPE consists of a gold working electrode (0.19 cm2), a saturated Ag/AgCl reference
119
electrode, and a gold-plate counter electrode (0.54 cm2).
120
For the morphology study, the chemical composition was studied using the FTIR method
121
via a HATR, ABB, MB3000 device with a resolution of 4 cm-1 at a spectral interval ranging
122
from 400 to 4000 cm-1 for 64 scans. In addition, atomic force microscopy (AFM) technique
123
was operated using a NANOVEA apparatus (from USA) with a maximum resolution of 110
124
µm in a scan area of (76 µm / 76 µm) scanned at 1 line/2s. Furthermore, the electrode
125
topology was observed using a scanning electron microscope (SEM) coupled with an energy
126
dispersive X-ray spectroscope (EDS, FEI QUANTA 250).
127
For the practical application of the IIP sensor, EBC samples were collected in the morning
128
from volunteers in our laboratory. After rinsing their mouths, the volunteers were asked to
129
blow into a mouthpiece. The blown air was trapped in a pipe immersed in ice. Finally, the
130
obtained liquid sample was recovered in a beaker. In order not to influence the measurement
131
of nitrite in EBC, the volunteers were asked to keep their mouths dry during sampling and
132
swallow the saliva from time to time. To avoid the chemical transformations in the EBC
133
samples, they were immediately analyzed by using the developed IIP sensor. All experiments
134
were performed at a room temperature of 25°C.
135
2.2. Preparation of the gold nanoparticles solution
5
136
The Au-NPs solution was prepared using the citrate reduction procedure. It was prepared
137
by mixing 5 mg of gold salt (HAuCl4) in 50 mL of distilled water and bringing to a boil.
138
Then, the solution was stirred by dropping a sodium citrate solution (10 mg in 1 mL of
139
distilled water) used as a stabilizing agent. The change in colour from light yellow to
140
burgundy red indicated the formation of nanoparticles [50]. The solution was stored in a
141
refrigerator until use.
142
2.3. Preparation of IIP and NIIP modified electrodes
143
The fabrication procedures of the IIP sensor are shown in Fig. 1.
144
Indeed, after the Au-SPE pre-treatment with ethanol and distilled water, the first step was
145
the self-assembly of 2-ATP on the gold electrode by Au-S bonding. The 2-ATP reagent was
146
chosen thanks to its good electrical conductivity [28]. For this purpose, an S1 solution
147
containing 10 mmol L-1 of 2-ATP was prepared by mixing 2-ATP (1.09 mg) in ethanol (1
148
mL) [51]. Then, 30 µL of S1 was deposited on the bare Au-SPE for a 12-hour incubation at a
149
room temperature of 25°C. << Here Figure 1>>
150 151
Then, a volume of 30 µL of a sodium nitrite solution (100 mg NaNO2 dissolved in 1 mL
152
distilled water) [52] was deposited on the electrode surface that was already modified by 2-
153
ATP. PVA was subsequently selected as a functional monomer because of its high
154
effectiveness in differentiating nitrite and nitrate species [53]. A solution of S2, containing
155
PVA (10 mg) dissolved in distilled water (100 mL), was first prepared by heating to boiling.
156
The reaction of PVA with dialdehydes is expected to form a cross-linked acetal structure.
157
Therefore, GA was chosen as the best cross-linking agent for PVA, owing to the absence of
158
heat treatment to trigger the reaction as well as its ability to covalently form cross-linked
159
networks with polymers containing hydroxyl groups. For this reason, 80 µL of (25% GA) was
160
secondarily added to 110 µL of S2 [54]. A volume of 200 µL of a sulphuric acid solution, used
161
as an acid solvent [55], was thirdly added to S2 to facilitate acetalization because PVA
162
polymerises in an acidic medium. In addition, 1 mL of Au-NPs and 1 mg of sodium sulphate
163
(Na2S04) were last added to S2 to prevent PVA from dissolving in water during acetalization.
164
Finally, 30 µL of the obtained S2 was deposited on the Au-SPE/2-ATP/NaNO2 electrode.
165
As a control test, a NIIP sensor was prepared using the same procedure without adding the
166
template. In order to remove the nitrite template, the IIP sensor was washed with a solution of
167
methanol/hydrochloric acid (4:1) [56]. The electrode was then rinsed with distilled water and
168
dried for ultimate use. 6
169
2.4. Characterization methods
170
In these studies, electrochemical characterizations were performed using potassium
171
ferri/ferrocyanide (5 mmol L-1) in 0.1 mol L-1 PBS (pH 7.4) to probe changes in the electrode
172
surfaces. In fact, CV and DPV characterizations were performed over a potential range of -0.4
173
to 0.6 V and -0.1 to 0.2 V at scan rates of 30 and 10 mV.s-1, respectively. EIS technique was
174
operated over a frequency range between 0.1 Hz and 50 kHz with 10 mV as AC voltage in an
175
open circuit.
176 177
The morphology of the electrodes was studied using characterization techniques, such as FTIR, AFM and SEM-EDS.
178
For this purpose, the FTIR spectra of the electrode surfaces were obtained based on the
179
sensitivity of the chemical functions at specific wavelength between 4000 and 400 cm-1. Thus,
180
by detecting the vibrations characteristic of chemical bonds, the apparatus was able to
181
generate the spectral characteristics of the electrode surfaces.
182
The AFM study was performed in contact mode where a tip is pressed against the electrode
183
surfaces. Thus, the tip sweeps and rubs the electrode surface following its relief. The
184
deformation of the lever, measured by a photo detector and recorded on a computer, allows a
185
3D image of the electrode surface to be reconstructed.
186
In the SEM-EDS analysis, an electron gun and an electron column emit an electron probe
187
on the working electrode, a microscope stage to move it in three directions and detectors to
188
capture and analyze the emitted radiation. By scanning the beam on the electrode, a map of
189
the scanned area is obtained (SEM image). Accordingly, the released X-rays as "signatures"
190
are exploited during the EDS analysis to identify the chemical elements that exist on the
191
electrode surface.
192
2.5. Optimisation of experimental and operational parameters
193
To improve the sensitivity and accuracy of the proposed method, some important
194
parameters that can affect the performance of the electrochemical sensor, such as the control
195
of the 2-ATP layer and polymer, the extraction time, and the pH of the buffer solution, have
196
been previously optimized.
197
The self-assembly of 2-ATP on the gold electrode was tested by using electrodeposition
198
and physical deposition methods. It was finally performed by physical deposition method for
199
12 hours because it admitted a better sensitivity than the electrodeposition method.
7
200
In addition, since the polymer consists of crosslinked PVA with GA reinforced with Au-
201
NPs, a better volume ratio between functional monomer (PVA) and crosslinking agent (GA)
202
was studied. Therefore, the GA/PVA ratio of 0.72 gave the best signal.
203 204
The duration of the extraction step is also a crucial factor in the adsorption ability of the sensor. Sufficient time has been set at 30 minutes.
205
A pH study was also required since the nitrite in exhaled breath is in an acidic medium
206
[24]. A comparative study of the best pH of the electrolyte was carried out using pH 6, 7.4
207
and 7. In this study, the physiological pH 7.4 gives better results [49].
208
3. Results and discussion
209
3.1. Characterization of the modified electrodes
210
The morphology and chemical composition of the electrode surfaces at the different stages
211
of the sensor development (bare Au-SPE, after polymer deposition and after the extraction
212
phase) were studied.
213
3.1.1. Characterization by FTIR
214
Figure 2 shows the obtained results after the FTIR analysis in transmittance mode. Peaks
215
at high frequency (around 4000 cm-1) are obtained corresponding to free −OH bonds because
216
the deposited layer on the electrode was prepared in an aqueous medium. By focusing on the
217
spectrum of the bare gold electrode (Fig. 2A), the main peaks are associated with CO2 at
218
about 2375 cm-1, reflecting the ambient experimental environment in which the analysis was
219
performed. After the immobilization of the polymer complex (Fig. 2B), peaks appear between
220
2820-2941 cm-1 related to PVA-GA complex. Similarly, a peak of the benzene group between
221
690 and 710 cm-1 is observed, which probably represents the 2-ATP used during the
222
development of the sensor. An acute peak and a lower peak are obtained at 1540 cm-1 and
223
1380 cm-1, respectively corresponding to the NO group. However, after the extraction step
224
(Fig. 2C), these peaks are no longer found, which indicates the absence of nitrite. This proves
225
the successful development of the nitrite IIP sensor.
226 227
<< Here Figure 2>> 3.1.2. Characterization by AFM
228
Figure 3 displays an overview of the surface characteristics of the sensor using the AFM
229
technique. As shown in Fig. 3A, the bare gold electrode has a more or less homogeneous
230
structure similar to a flat layer with an RMS value of 0.724 µm. After the immobilization of
231
the polymer complex, the relief changes and becomes much more homogeneous (Fig. 3B).
232
This can be interpreted by the presence of entangled polymer chains with an RMS of 1.35 µm.
233
This increase in roughness indicates modifications on the Au-SPE surface due to polymer 8
234
deposition. After the extraction stage, an interface with a less homogeneous morphology is
235
found (Fig. 3C). By comparing the RMS values of the polymerization and extraction steps, a
236
decrease in surface roughness from 1.35 µm to 0.822 µm is observed due to elution of the
237
template from the polymer. To sum up, the AFM analysis confirms that changes in the
238
electrode surface occurred after each step of the IIP sensor development.
239 240
<< Here Figure 3>> 3.1.3. Characterization by SEM-EDS
241
Figure 4 shows the results of the SEM-EDS analyses at a low magnification (x5000) and
242
at an accelerating voltage of 15 kV of the electrode surfaces (bare Au-SPE, after polymer
243
deposition and after the extraction phase). In the last two cases, the electrode surfaces are
244
significantly modified while compared to the first. They reveal two rough surfaces with
245
aggregated particles in the form of microspheres. Indeed, the SEM image of the polymer (Fig.
246
4B) shows a uniformly meshed structure with the appearance of gold nanoparticles on the
247
surface relative to the bare Au-SPE image (Fig. 4A), which shows a plate structure.
248
Concerning the SEM image of the elution stage (Fig. 4C), a more porous structure than that of
249
the polymer is observed. SEM studies clearly show that the surface of the electrode without
250
template has a relatively more porous structure with smaller particles that could promote
251
effective interaction with target ions.
252
<< Here Figure 4>>
253
Simultaneously, during the same analysis, the chemical compositions of the modified
254
electrodes were identified by the EDS technique (Spectra of Fig. 4A-C). The latter was used
255
to obtain indicators on the elementary chemical composition of a point or area of interest of
256
the electrode. Therefore, Table 1 shows the chemical composition of the Au-SPE surface with
257
90.74% by weight of gold (Au), 7.34% by weight of carbon (C) and 1.92% by weight of
258
aluminium (Al). This strong presence of Au proves that the bare Au-SPE has not been
259
modified. In Table 2, it can be noticed that after being modified with the polymer, the
260
electrode surface contains gold, carbon (C), oxygen, and nitrogen (N) in percentages 86.58%,
261
5.7%, 6.36%, and 1.36%, respectively. On one hand, the percentage of Au has decreased,
262
which demonstrates the electrode coverage. On the other hand, the appearance of N and O in
263
the chemical composition proves that the polymer containing NO2- was effectively
264
immobilized on the electrode surface. Table 3 shows that after the elution step, the electrode
265
surface contains 82.31% by weight of Au, 9.4% by weight of C, 4.76% by weight of O,
266
2.47% by weight of N and 1.06% by weight of Al. This presence of Au, O and N in a lower
267
percentage than in the polymerisation step reveals a successful elution of nitrite from the 9
268
electrode surface. Overall, the SEM images and EDS spectra show a complete distribution of
269
the elements on the electrode surface, demonstrating the successful modification of the
270
electrode surface.
271
<< Here Table 1>>
272
<< Here Table 2>>
273
<< Here Table 3>> 3.2. Electrochemical characterization of sensor in the fabrication process
274 275
Ferri/ferrocyanide ([Fe(CN)6]3-/4-), as redox probe, is used to monitor all stages of the IIP
276
sensor development. It provides information on the transfer of electrons between the
277
deposited layer and the electrode surface.
278
Figure 5 shows the results of these characterization steps using CV and EIS techniques.
279
Analysis of the cyclic voltammograms in Fig. 5A clearly show that the [Fe(CN)6]3-/4- signal
280
corresponding to the bare gold electrode displays two redox peaks. The potential peaks of the
281
anode (Epa) and cathode (Epc) are located respectively at 0.2 V and 0.08 V, with an oxidation
282
current peak of 46 µA. After the self-assembly of 2-ATP on the bare gold electrode, the
283
current peak of [Fe(CN)6]3-/4- significantly decreases (Ia = 32 µA) because of repulsive
284
interactions between the probe and the negatively charged amine groups (−NH2) of 2-ATP.
285
This explains the decrease in the electron transfer rate. These results demonstrate that the 2-
286
ATP layer was successfully self-assembled on the electrode. Complexing sodium nitrite
287
(NaNO2) with 2-ATP induces a slight decrease in the current peak (Ia = 31.7 µA) showing that
288
NaNO2 inhibits the diffusion of [Fe(CN)6]3-/4- on the electrode surface by electrostatic
289
interaction at an anodic potential of 0.2 V. When the polymer complex (PVA/GA+Au-NPs)
290
was immobilized on the previous modified electrode, a significant decrease in the redox
291
current peak is observed (Ia = 22 µA). On one hand, this may be due to the presence of
292
negatively charged amine groups that lead to an electrostatic repulsion preventing [Fe(CN)6]3-
293
/4-
294
that has been attached to the electrode. This indicates that the polymer was well immobilized
295
on the electrode.
296
access to the electrode surface. On the other hand, it can be explained by the compact mass
<< Here Figure 5>>
297
In addition, Fig. 5B shows the complex impedance curves corresponding to the
298
development stages of the sensor. The EIS characterization of the bare gold electrode displays
299
a low charge transfer resistance (Rct) value. The negative charges of the deposited elements
300
reject the anionic redox probe [Fe(CN)6]3-/4- and hinder electron transfer, which result in an 10
301
increase of electron transfer resistances (Rct) of 4439.1 Ω for 2-ATP, 5761.7 Ω for NaNO2 to
302
10485 Ω for the polymer. Thus, there is an agreement between the results obtained by the CV
303
and EIS techniques. The extraction stage followed and the IIP sensor was kept in the dark
304
until it was use.
305
3.3. IIP and NIIP sensor responses
306
The retention capability of the IIP sensor was evaluated through the use of the DPV
307
method under the probe [Fe(CN)6]3-/4- acting as an electro-active species at a scan rate of 100
308
mV/s between potentials ranging from -0.2 to 0.2 V.
309
In this regard, different nitrite concentrations over the range 0.5 to 50 µg mL-1 have been
310
prepared while taking into consideration that the nitrite in EBC is in micromolar
311
concentrations [57,58]. Then, 60 µL of each NaNO2 concentration was deposited on the
312
surface of the prepared IIP sensor for DPV measurements.
313
Figure 6 shows not only the obtained signals by the nitrite oxidation reaction on the sensor,
314
but also the corresponding calibration curves, where each point is the average of six different
315
measurements.
316
Figure 6A shows the dependence between the oxidation current and the NO2-
317
concentration. As noticed in DPV technique, the signals decrease when a higher concentration
318
of nitrite is applied to the sensor (Fig. 6A). This can be explained by the formed negative
319
membrane when the electrode was modified with nitrite anions. Ferrocyanide was hindered to
320
access the electrode surface due to the electrostatic repulsion of the negative charges. As a
321
result, the electrochemical response decreases on the electrode. This reduction is proportional
322
to the level of nitrite in solution.
323
<< Here Figure 6>>
324
With the EIS method, the information is derived from the value of the Nyquist diagram
325
intersection with the abscissa axis. It can be seen in Fig. 6B that these values increase with
326
rising analyte concentration. For the DPV method, good linearity (R2 > 0.96) is observed over
327
the study range with analytical parameters, such as sensitivity and detection limit of -0.11 (µg
328
mL-1)-1 and 4 µmol L-1 (Fig. 6C). The observed increasing variation of Rct, therefore, confirms
329
the obtained results by the DPV method with a sensitivity of 0.57 (µg mL-1)-1 and an LOD of
330
49.27 µmol L-1 (Fig. 6D), which is calculated by considering three times the standard
331
deviation of the blank signal divided by the sensor sensitivity. This shows that this platform
332
could be used satisfactorily in new generations of electroanalytical sensors.
11
333
However, the NIIP sensor test did not show significant sensitivity to nitrite (Fig. 7). This
334
proves that the obtained responses by the IIP sensor were only due to the presence of memory
335
sites on the electrode that could interact with the nitrite solution. << Here Figure 7>>
336 337
In addition, with the use of species co-existing with nitrite in EBC, sensitivities of -0.09
338
(µg mL-1)-1 for nitrate and -0.05 (µg mL-1)-1 for acetate and ammonium nitrate were obtained.
339
These values are insignificant compared with those obtained after nitrite application (Fig. 8).
340
It is therefore possible to affirm that the developed sensor is very selective. << Here Figure 8>>
341 342
We could barely find any literature on nitrite electro-oxidation that use the technology
343
described in this work with good sensitivity, low overpotential and a simple process.
344
Moreover, the operating range and LOD of the relevant sensor is compared with those in the
345
literature (Table 4). We conclude that satisfactory results are achieved through simple
346
experimentation, easy implementation without specialized personnel, and thus at a lower cost.
347
<< Here Table 4>>
348
3.4. Reproducibility, repeatability and stability study
349
For better performance in a given application, a sensor must meet some important criteria
350
during its development. Some of these parameters are reproducibility, repeatability and
351
stability.
352
Reproducibility is the ability to fabricate a sensor several times and it maintain its
353
responses relatively identical. For this purpose, we reproduced the sensor four times and the
354
generated responses have a relative standard deviation RSD = 2%. This proves that the
355
developed sensor is reproducible.
356
Besides, the repeatability of a sensor is its capability to generate more or less similar
357
responses for the same measurement. Indeed, using the same concentration in our study, the
358
sensor responses were almost the same with an RSD of 4%. These results also confirm that
359
the measurements made with the sensor are repeatable.
360
Stability is the ability of a sensor to give close responses for the same measurement at
361
more or less long-time intervals. In this respect, after 2 months, the sensor gave 97% of the
362
initial response, which implies its stability.
363 364 365
These results are acceptable indicating that the sensor is well suited for practical application in EBC samples. 3.5. Application of the nitrite IIP sensor in exhaled breath samples 12
366
After the IIP sensor development and the optimization of all its analytical parameters, it
367
was tested for the measurement of the nitrite content in condensed exhaled breath samples.
368
After the morning collection of samples from consenting persons, three aliquots were made
369
for each sample. Thus, for each sample, three measurements were made by placing 30 µL of
370
its corresponding aliquot on the sensor. Using the DPV method, peaks in electrochemical
371
responses were recorded to calculate the nitrite content in the various condensed breath
372
samples. We find that the sensor admits different amplitude responses depending on the
373
deposited breath sample. An example of the IIP sensor response is shown in Fig. 9. << Here Figure 9>>
374 375
Relatively low concentrations of nitrite with RSD = 4% are found during the sensor
376
practical application in EBC (Table 5). Indeed, all nitrite values calculated in the EBC
377
samples are in micromolar order, validating the nitrite values in the literature [59-61]. All in
378
all, the sensor can be considered as a viable tool for the determination of the nitrite profile in
379
EBC. << Here Table 5>>
380 381
4. Conclusion
382
The particularity of the developed IIP sensor for the determination of nitrite in condensed
383
exhaled breath is its simplicity. The developed ion imprinted sensor for nitrite determination
384
is simple and very sensitive. The calculated detection limit is 4 µmol L-1. In addition, since
385
nitrate, acetate and ammonium nitrate are interfering substances that coexist in breath, a
386
selectivity test is carried out with satisfactory results. Using the ion imprinted sensor, its
387
practical application in condensate human exhaled breath samples was well performed with
388
results in micromolar order. This could be a good vista for medical research on inflammation
389
and respiratory diseases.
390
Acknowledgements
391
Authors gratefully acknowledge Moulay Ismaïl University of Meknes for financial support
392
of the project “Research support”. This work has been funded in part by TROPSENSE under
393
the H2020-MSCA-RISE-2014 project, grant agreement number: 645758. The authors thank
394
and wish to express their gratitude to Institutului de Metale Neferoase si Rare (IMNR) team of
395
ROMANIA namely Dr. Ioan Albert Tudor, Dr. Laura Madalina Popescu, Dr. Ghita Mihai,
396
Lupu Andreea Nicoleta, and professor Driss Bouyahya of school of arts and humanities,
397
moulay ismail university for the excellent technical assistance of the characterization process
398
of the MIP-based sensors, and English paper correction. 13
399
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20
Table 1. Chemical composition by EDS for Au-SPE.
Element
Weight %
Atomic %
Net Int.
CK
7.34
53.47
111.915
Al K
1.92
6.22
132.51
Au M
90.74
40.31
478.11
Table 2. Chemical composition by EDS for the electrode after polymer deposit.
Element
Weight %
Atomic %
Net Int.
CK
5.7
33.68
35.29
NK
1.36
6.88
5.99
OK
6.36
28.23
46.72
Au M
86.58
31.21
596.6
Table 3. Chemical composition by EDS for the electrode after extraction stage.
Element
Weight %
Atomic %
Net Int.
CK
9.4
45.65
103.37
NK
2.47
10.29
18.16
OK
4.76
17.37
58.98
Al k
1.06
2.3
35.7
Au M
82.31
24.38
993.47
Table 4. Comparison of performance between this IIP sensor and reported nitrite sensor. Linear range
LOD
(µg mL-1)
(µmol L-1)
Graphene nanoplatelet
3.4 – 172
10
[62]
Reduced graphene oxide
6.9 – 69
0.83
[60]
Glassy Carbon Electrode
0.345 – 465
1.65
[63]
2.22 – 13.04
5.61
[49]
0.069 – 0.69
5
[64]
8.97 – 3036
45
[65]
0.5 – 50
4
This work
Electrodes
Edge plane pyrolytic graphite electrode
Gold electrode
Pre-treated glassy carbon electrode Gold electrode
Reference
Table 5. EBC nitrite levels by means of DPV method. Nitrite
IBlank
Isample
(µA)
(µA)
P77
22.62
20.04
-0.110
0.062
P83
17.60
16.36
-0.071
0.027
P88
21.71
17.73
-0.183
0.289
P118
18.93
16.90
-0.107
0.059
P128
24.26
21.49
-0.114
0.068
P89
5.313
4.994
-0.060
0.021
P99
7.304
5.995
-0.179
0.266
P128 bis
9.644
8.515
-0.117
0.072
Sample
(Isample - IBlank)/ IBlank
Concentration (µmol L-1)
Fig. 1. Procedures of the IIP sensor preparation.
Fig. 2. FTIR spectra obtained for: (A) Bare gold, (B) After polymer deposit, (C) After
extraction.
Fig. 3. AFM images obtained for: (A) Bare gold, (B) After polymer deposit, (C) After
extraction.
Fig. 4. SEM images and corresponding EDS spectra obtained for: (A) Bare gold, (B) After (A) Bare gold
polymer deposit, (C) After extraction.
(B) Polymer
(C) Extraction
Fig. 5. (A) Cyclic voltammograms of 5 mM [Fe(CN)6]3-/4- solution at: Bare gold, After 2-ATP deposit, After NaNO2 deposit, and after polymer deposit; (B) Nyquist plots of 5 mM [Fe(CN)6]3-/4- solution at: Bare gold, After 2-ATP deposit, After NaNO2 deposit, and after
polymer deposit.
Fig. 6. Electrochemical responses of the IIP sensor as a function of increasing nitrite concentration using: (A) DPV, (B) EIS in the presence of 5 mM of [Fe (CN) 6]3-/4- as redox probe. Calibration curves between the sensor responses and Logarithm of nitrite concentrations by: (C) DPV, (D) EIS in PBS buffer at pH 7.4.
Fig. 7. (A) By the presence of 5 mM of [Fe (CN) 6]3-/4- as redox probe, electrochemical responses of the NIIP sensor by using DPV, (B) Calibration curve between the sensor responses and Logarithm of nitrite concentrations in PBS buffer at pH 7.4.
Fig. 8. Calibration curves of (A) nitrite, and interfering molecules: (B) Silver nitrate, (C)
Acetate, (D) ammonium nitrate.
Fig. 9. Example of the IIP sensor response after an EBC sample exposure for nitrite determination.
Highlights Successful development of an electrochemical nitrite sensor-based ion imprinted polymer. CV, DPV, EIS, FTIR, AFM and SEM techniques for electrochemical and morphological characterizations. Utilization of the IIP sensor for nitrite determination in exhaled breath condensate. A lower LOD of 4 µmol L-1 in a working range from 0.5 to 50 µg mL-1 compared with related works.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0039-9140(19)31210-X
DOI:
https://doi.org/10.1016/j.talanta.2019.120577
Reference:
TAL 120577
To appear in:
Talanta
Received Date: 18 July 2019 Revised Date:
18 November 2019
Accepted Date: 19 November 2019
Please cite this article as: A. Diouf, N. El Bari, B. Bouchikhi, A novel electrochemical sensor based on ion imprinted polymer and gold nanomaterials for nitrite ion analysis in exhaled breath condensate, Talanta (2019), doi: https://doi.org/10.1016/j.talanta.2019.120577. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
GRAPHICAL ABSTRACT :
1
A novel electrochemical sensor based on ion imprinted polymer and gold nanomaterials
2
for nitrite ion analysis in exhaled breath condensate Alassane Dioufa,b, Nezha El Barib, Benachir Bouchikhia,*
3 4
a
5
Moulay Ismaïl University of Meknes, B.P. 11201, Zitoune, Meknes, Morocco.
6
b
7
Faculty of Sciences, Moulay Ismaïl University of Meknes, B.P. 11201, Zitoune, 50003
8
Meknes, Morocco.
Sensor Electronic & Instrumentation Group, Department of Physics, Faculty of Sciences,
Biotechnology Agroalimentary and Biomedical Analysis Group, Department of Biology,
9
*
Corresponding author: Postal address: B.P. 11201, Zitoune, Meknes, Morocco Tel: +212 535 53 88 70; Fax: +212 535 53 68 08 Email: [email protected] 1
10
ABSTRACT
11
Human Exhaled Breath Condensate (EBC) contains markers of several inflammatory
12
diseases. Its analysis is of interest to a number of researchers. Nitrite ions (NO2-), which are
13
widely used in our daily lives, are nevertheless among these indicators. In this study, a simple,
14
fast, portable, non-invasive and cheap electrochemical sensor is developed for the analysis of
15
the nitrite profile in EBC. In this regard, sodium nitrite (NaNO2) was first immobilized on
16
self-assembled 2-aminothiophenol (2-ATP) on a screen-printed gold electrode (Au-SPE).
17
Then, a polymer matrix composed of polyvinyl alcohol (PVA) crosslinked with
18
glutaraldehyde (GA) was combined with gold nanoparticles (Au-NPs) to cover the modified
19
Au-SPE and complete the fabrication of the Ion Imprinted Polymer (IIP) sensor. The
20
electrochemical behaviour of the sensor was monitored using Cyclic Voltammetry (CV),
21
Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV)
22
methods, while the morphology and chemical composition of its layers were observed by
23
infrared Fourier transform (FTIR), Atomic Force Microscopy (AFM) and Scanning Electron
24
Microscopy coupled with energy dispersion X-Ray spectroscopy (SEM-EDS) techniques. In
25
addition, after a successful control test using a Non-Imprinted Ion Polymer (NIIP) sensor, the
26
obtained results demonstrated satisfactory sensitivity and selectivity to nitrite compared to co-
27
existing interfering substances in EBC, such as nitrate, acetate and ammonium nitrate. Under
28
improved experimental conditions, the nitrite IIP sensor exhibits responses proportional to
29
nitrite concentrations (R2 = 0.96) over a concentration range of 0.5 to 50 µg mL-1 with a
30
detection limit (LOD) of 4 µmol L-1 (signal-to-noise ratio S/N = 3). The proposed approach
31
was well applied for the nitrite determination in EBC samples with a relative standard
32
deviation (RSD = 4%) and could open clinical applications in respiratory medicine.
33
Keywords: Electrochemistry; Exhaled breath condensate; Ion imprinted polymer; Nitrite;
34
Screen-printed gold electrode.
35
2
36
1. Introduction
37
Diseases occur as a result of alterations in normal physiological processes due to metabolic
38
disorders or exposure to toxins (or microbial pathogens). The presence of typical chemicals
39
(biomarkers) in the body reveals the appearance of a disease. These markers go through the
40
circulatory system and are eventually expelled by the lungs. For this purpose, breath analysis
41
could provide very useful clues to the evolution of diseases in the human body. This leads
42
many researchers to focus on the analysis of human exhaled breath, which is non-invasive,
43
simple and useful compared to conventional methods of diagnosis and clinical treatment,
44
which are often invasive, costly, time-consuming, and require trained personnel. To trap and
45
concentrate volatiles from exhaled breath, it could be condensed by cooling to quantify
46
markers including nitrite and nitrate, hydrogen peroxide, eicosanoids, proteins, and so on [1].
47
Nitrite and nitrate have been shown to be important in understanding inflammatory diseases
48
[2,3]. Both are stable end products of the oxidative metabolism of nitric oxide that occurs
49
from L-arginine via nitric oxide synthase [4]. By consuming contaminated water and food,
50
excess of nitrite accumulates in the gastrointestinal tract of living organisms. The passage of
51
nitrite through the bloodstream can have two adverse effects on human health; it can either
52
combine with blood pigments producing methemoglobin in which oxygen is no longer
53
available for tissues [5,6], or interact in the stomach with amines and amides forming highly
54
carcinogenic N-nitrosamine compounds [7].
55
Due to the potential toxicity of nitrite in the human body, it is worthwhile for public health
56
to develop simple, rapid and accurate detection methods. Many analytical techniques have
57
been proposed including spectrophotometry (LOD 0.29 – 14.50 µmol L-1) [8], capillary
58
electrophoresis (LOD 0.72 – 4.35 µmol L-1) [9], electrochemistry [10] and ion
59
chromatography (LOD 0.07 − 0.14 µmol L-1) [11]. Because of the disadvantages, such as
60
operating costs and complexity, some of these methods are gradually being abandoned in
61
favour of electrochemical techniques that are simpler, more economical, faster and more
62
sensitive for nitrite detection. It is also important to note that with electrochemical techniques,
63
pre-treatments are minimized, instruments can be miniaturized, and measurements can be
64
performed on site. This solves many of the constraints encountered with conventional
65
methods [12-18]. Generally, the electrochemical detection of nitrite can be performed in two
66
ways; in most cases, oxidation of nitrite is preferred to its reduction, which is subject to
67
interference from other easily reducible species, such as nitrate ions and molecular oxygen
68
[19-21]. Nitrite is electroactive on certain metallic substrates, such as gold, copper, glassy
69
carbon. Therefore, its application on these substrates is likely to poison their surfaces 3
70
generating a high overvoltage [22,23]. This would reduce the sensitivity and accuracy of
71
nitrite detection [24]. Numerous studies have been carried out to optimize existing methods
72
for the determination of nitrite [14,25,26]. Therefore, the challenge is to overcome the
73
problems of low sensitivity, selectivity, and high potential caused by the direct application of
74
nitrite to the surfaces of metal electrodes. To achieve this goal, these surfaces must be
75
modified using appropriate electrocatalysts [23,27].
76
First, conductive polymers are undeniable factors because of their outstanding properties.
77
Polyaniline, poly-pyrrole and poly-thiophene are widely used. Among these polymers, amino-
78
thiophenol (ATP) has attracted particular attention because of its good electrical conductivity
79
[28,29]. Secondly, in order to increase the adsorption ability for electrochemical studies, these
80
polymers can even be combined with metallic nanoparticles. The nanoparticles advantages lie
81
in their ability to provide a large effective surface area, electrical conductivity, efficient
82
catalysis, and fast mass transport [30-33]. Among nanomaterials, the gold nanoparticles (Au-
83
NPs) are highly advantageous in several electrochemical implementations [34,35]. They are
84
also expected to increase the binding of NaNO2 to amine groups and allow the oxidation [36].
85
Since they can bind to functional groups, such as –CN, –NH2, or –SH by covalent bonding,
86
Au-NPs combined with conductive polymers should reduce overvoltage and allow faster
87
electron transfer kinetics for nitrite oxidation [37]. Third, compared to conventional
88
electrodes, screen-printed electrodes (SPEs) are becoming increasingly attractive thanks to
89
their low mass production cost, versatility, reproducibility, reliability for various practical
90
applications, and normally disposable after one or more uses [38-40]. Fourth, in molecular
91
imprinting technique (MIT), the interactions between the polymer and the target element are
92
hydrophobic, ionic, covalent or non-covalent, or a hydrogen bond [41]. The imprinting of
93
sites for the recognition of molecular or ionic elements in organic or inorganic polymer
94
matrices has been the subject of extensive studies [42-44]. The IIP technology initiated by
95
Nishide et al. [45] is the case in which the target is an ion. An IIP sensor is generally
96
constructed by binding the ionic element followed by copolymerization of a functional
97
monomer with a cross-linking agent to create recognition cavities in the polymer matrix.
98
Several advances have been made in IIP technology; however, a major challenge must be met
99
in the development of sensitive and selective devices for ion recognition in various
100
applications [46-48].
101
Taking all these considerations into account, the idea of this work is to functionalize Au-
102
SPE with 2-ATP and then synthesize and characterize a polymer combined with Au-NPs to
103
verify the ability of the obtained IIP sensor to recognize nitrite ions in EBC. The development 4
104
and retention capability of the nitrite IIP sensor is studied using CV, DPV, EIS, SEM-EDS,
105
AFM and FTIR techniques.
106 107
2. Experimental 2.1. Reagents and apparatus
108
Sodium nitrite (NaNO2), silver nitrate (AgNO3), acetate, ammonium nitrate, phosphate
109
buffered saline (PBS), gold chloride trihydrate (HAuCl43H2O) (99.99%), polyvinyl alcohol
110
(PVA) (98%), glutaraldehyde (GA) (25%), methanol, hydrochloric acid (HCl), and sulphuric
111
acid (H2SO4) were purchased from Sigma Aldrich. Dehydrated trisodium citrate was
112
purchased from Handel's Riedel, while potassium ferrocyanide trihydrate and potassium
113
ferricyanide trihydrate were from Fluka. Distilled water (DW) was used throughout the
114
experiments. All other chemicals were of analytical reactive quality and were used without
115
further purification.
116
Electrochemical measurements by CV, DPV and EIS were performed using a conditioning
117
circuit (Potentiostat, Palmsens3) interfaced to a computer controlled by PS-Trace software.
118
The Au-SPE consists of a gold working electrode (0.19 cm2), a saturated Ag/AgCl reference
119
electrode, and a gold-plate counter electrode (0.54 cm2).
120
For the morphology study, the chemical composition was studied using the FTIR method
121
via a HATR, ABB, MB3000 device with a resolution of 4 cm-1 at a spectral interval ranging
122
from 400 to 4000 cm-1 for 64 scans. In addition, atomic force microscopy (AFM) technique
123
was operated using a NANOVEA apparatus (from USA) with a maximum resolution of 110
124
µm in a scan area of (76 µm / 76 µm) scanned at 1 line/2s. Furthermore, the electrode
125
topology was observed using a scanning electron microscope (SEM) coupled with an energy
126
dispersive X-ray spectroscope (EDS, FEI QUANTA 250).
127
For the practical application of the IIP sensor, EBC samples were collected in the morning
128
from volunteers in our laboratory. After rinsing their mouths, the volunteers were asked to
129
blow into a mouthpiece. The blown air was trapped in a pipe immersed in ice. Finally, the
130
obtained liquid sample was recovered in a beaker. In order not to influence the measurement
131
of nitrite in EBC, the volunteers were asked to keep their mouths dry during sampling and
132
swallow the saliva from time to time. To avoid the chemical transformations in the EBC
133
samples, they were immediately analyzed by using the developed IIP sensor. All experiments
134
were performed at a room temperature of 25°C.
135
2.2. Preparation of the gold nanoparticles solution
5
136
The Au-NPs solution was prepared using the citrate reduction procedure. It was prepared
137
by mixing 5 mg of gold salt (HAuCl4) in 50 mL of distilled water and bringing to a boil.
138
Then, the solution was stirred by dropping a sodium citrate solution (10 mg in 1 mL of
139
distilled water) used as a stabilizing agent. The change in colour from light yellow to
140
burgundy red indicated the formation of nanoparticles [50]. The solution was stored in a
141
refrigerator until use.
142
2.3. Preparation of IIP and NIIP modified electrodes
143
The fabrication procedures of the IIP sensor are shown in Fig. 1.
144
Indeed, after the Au-SPE pre-treatment with ethanol and distilled water, the first step was
145
the self-assembly of 2-ATP on the gold electrode by Au-S bonding. The 2-ATP reagent was
146
chosen thanks to its good electrical conductivity [28]. For this purpose, an S1 solution
147
containing 10 mmol L-1 of 2-ATP was prepared by mixing 2-ATP (1.09 mg) in ethanol (1
148
mL) [51]. Then, 30 µL of S1 was deposited on the bare Au-SPE for a 12-hour incubation at a
149
room temperature of 25°C. << Here Figure 1>>
150 151
Then, a volume of 30 µL of a sodium nitrite solution (100 mg NaNO2 dissolved in 1 mL
152
distilled water) [52] was deposited on the electrode surface that was already modified by 2-
153
ATP. PVA was subsequently selected as a functional monomer because of its high
154
effectiveness in differentiating nitrite and nitrate species [53]. A solution of S2, containing
155
PVA (10 mg) dissolved in distilled water (100 mL), was first prepared by heating to boiling.
156
The reaction of PVA with dialdehydes is expected to form a cross-linked acetal structure.
157
Therefore, GA was chosen as the best cross-linking agent for PVA, owing to the absence of
158
heat treatment to trigger the reaction as well as its ability to covalently form cross-linked
159
networks with polymers containing hydroxyl groups. For this reason, 80 µL of (25% GA) was
160
secondarily added to 110 µL of S2 [54]. A volume of 200 µL of a sulphuric acid solution, used
161
as an acid solvent [55], was thirdly added to S2 to facilitate acetalization because PVA
162
polymerises in an acidic medium. In addition, 1 mL of Au-NPs and 1 mg of sodium sulphate
163
(Na2S04) were last added to S2 to prevent PVA from dissolving in water during acetalization.
164
Finally, 30 µL of the obtained S2 was deposited on the Au-SPE/2-ATP/NaNO2 electrode.
165
As a control test, a NIIP sensor was prepared using the same procedure without adding the
166
template. In order to remove the nitrite template, the IIP sensor was washed with a solution of
167
methanol/hydrochloric acid (4:1) [56]. The electrode was then rinsed with distilled water and
168
dried for ultimate use. 6
169
2.4. Characterization methods
170
In these studies, electrochemical characterizations were performed using potassium
171
ferri/ferrocyanide (5 mmol L-1) in 0.1 mol L-1 PBS (pH 7.4) to probe changes in the electrode
172
surfaces. In fact, CV and DPV characterizations were performed over a potential range of -0.4
173
to 0.6 V and -0.1 to 0.2 V at scan rates of 30 and 10 mV.s-1, respectively. EIS technique was
174
operated over a frequency range between 0.1 Hz and 50 kHz with 10 mV as AC voltage in an
175
open circuit.
176 177
The morphology of the electrodes was studied using characterization techniques, such as FTIR, AFM and SEM-EDS.
178
For this purpose, the FTIR spectra of the electrode surfaces were obtained based on the
179
sensitivity of the chemical functions at specific wavelength between 4000 and 400 cm-1. Thus,
180
by detecting the vibrations characteristic of chemical bonds, the apparatus was able to
181
generate the spectral characteristics of the electrode surfaces.
182
The AFM study was performed in contact mode where a tip is pressed against the electrode
183
surfaces. Thus, the tip sweeps and rubs the electrode surface following its relief. The
184
deformation of the lever, measured by a photo detector and recorded on a computer, allows a
185
3D image of the electrode surface to be reconstructed.
186
In the SEM-EDS analysis, an electron gun and an electron column emit an electron probe
187
on the working electrode, a microscope stage to move it in three directions and detectors to
188
capture and analyze the emitted radiation. By scanning the beam on the electrode, a map of
189
the scanned area is obtained (SEM image). Accordingly, the released X-rays as "signatures"
190
are exploited during the EDS analysis to identify the chemical elements that exist on the
191
electrode surface.
192
2.5. Optimisation of experimental and operational parameters
193
To improve the sensitivity and accuracy of the proposed method, some important
194
parameters that can affect the performance of the electrochemical sensor, such as the control
195
of the 2-ATP layer and polymer, the extraction time, and the pH of the buffer solution, have
196
been previously optimized.
197
The self-assembly of 2-ATP on the gold electrode was tested by using electrodeposition
198
and physical deposition methods. It was finally performed by physical deposition method for
199
12 hours because it admitted a better sensitivity than the electrodeposition method.
7
200
In addition, since the polymer consists of crosslinked PVA with GA reinforced with Au-
201
NPs, a better volume ratio between functional monomer (PVA) and crosslinking agent (GA)
202
was studied. Therefore, the GA/PVA ratio of 0.72 gave the best signal.
203 204
The duration of the extraction step is also a crucial factor in the adsorption ability of the sensor. Sufficient time has been set at 30 minutes.
205
A pH study was also required since the nitrite in exhaled breath is in an acidic medium
206
[24]. A comparative study of the best pH of the electrolyte was carried out using pH 6, 7.4
207
and 7. In this study, the physiological pH 7.4 gives better results [49].
208
3. Results and discussion
209
3.1. Characterization of the modified electrodes
210
The morphology and chemical composition of the electrode surfaces at the different stages
211
of the sensor development (bare Au-SPE, after polymer deposition and after the extraction
212
phase) were studied.
213
3.1.1. Characterization by FTIR
214
Figure 2 shows the obtained results after the FTIR analysis in transmittance mode. Peaks
215
at high frequency (around 4000 cm-1) are obtained corresponding to free −OH bonds because
216
the deposited layer on the electrode was prepared in an aqueous medium. By focusing on the
217
spectrum of the bare gold electrode (Fig. 2A), the main peaks are associated with CO2 at
218
about 2375 cm-1, reflecting the ambient experimental environment in which the analysis was
219
performed. After the immobilization of the polymer complex (Fig. 2B), peaks appear between
220
2820-2941 cm-1 related to PVA-GA complex. Similarly, a peak of the benzene group between
221
690 and 710 cm-1 is observed, which probably represents the 2-ATP used during the
222
development of the sensor. An acute peak and a lower peak are obtained at 1540 cm-1 and
223
1380 cm-1, respectively corresponding to the NO group. However, after the extraction step
224
(Fig. 2C), these peaks are no longer found, which indicates the absence of nitrite. This proves
225
the successful development of the nitrite IIP sensor.
226 227
<< Here Figure 2>> 3.1.2. Characterization by AFM
228
Figure 3 displays an overview of the surface characteristics of the sensor using the AFM
229
technique. As shown in Fig. 3A, the bare gold electrode has a more or less homogeneous
230
structure similar to a flat layer with an RMS value of 0.724 µm. After the immobilization of
231
the polymer complex, the relief changes and becomes much more homogeneous (Fig. 3B).
232
This can be interpreted by the presence of entangled polymer chains with an RMS of 1.35 µm.
233
This increase in roughness indicates modifications on the Au-SPE surface due to polymer 8
234
deposition. After the extraction stage, an interface with a less homogeneous morphology is
235
found (Fig. 3C). By comparing the RMS values of the polymerization and extraction steps, a
236
decrease in surface roughness from 1.35 µm to 0.822 µm is observed due to elution of the
237
template from the polymer. To sum up, the AFM analysis confirms that changes in the
238
electrode surface occurred after each step of the IIP sensor development.
239 240
<< Here Figure 3>> 3.1.3. Characterization by SEM-EDS
241
Figure 4 shows the results of the SEM-EDS analyses at a low magnification (x5000) and
242
at an accelerating voltage of 15 kV of the electrode surfaces (bare Au-SPE, after polymer
243
deposition and after the extraction phase). In the last two cases, the electrode surfaces are
244
significantly modified while compared to the first. They reveal two rough surfaces with
245
aggregated particles in the form of microspheres. Indeed, the SEM image of the polymer (Fig.
246
4B) shows a uniformly meshed structure with the appearance of gold nanoparticles on the
247
surface relative to the bare Au-SPE image (Fig. 4A), which shows a plate structure.
248
Concerning the SEM image of the elution stage (Fig. 4C), a more porous structure than that of
249
the polymer is observed. SEM studies clearly show that the surface of the electrode without
250
template has a relatively more porous structure with smaller particles that could promote
251
effective interaction with target ions.
252
<< Here Figure 4>>
253
Simultaneously, during the same analysis, the chemical compositions of the modified
254
electrodes were identified by the EDS technique (Spectra of Fig. 4A-C). The latter was used
255
to obtain indicators on the elementary chemical composition of a point or area of interest of
256
the electrode. Therefore, Table 1 shows the chemical composition of the Au-SPE surface with
257
90.74% by weight of gold (Au), 7.34% by weight of carbon (C) and 1.92% by weight of
258
aluminium (Al). This strong presence of Au proves that the bare Au-SPE has not been
259
modified. In Table 2, it can be noticed that after being modified with the polymer, the
260
electrode surface contains gold, carbon (C), oxygen, and nitrogen (N) in percentages 86.58%,
261
5.7%, 6.36%, and 1.36%, respectively. On one hand, the percentage of Au has decreased,
262
which demonstrates the electrode coverage. On the other hand, the appearance of N and O in
263
the chemical composition proves that the polymer containing NO2- was effectively
264
immobilized on the electrode surface. Table 3 shows that after the elution step, the electrode
265
surface contains 82.31% by weight of Au, 9.4% by weight of C, 4.76% by weight of O,
266
2.47% by weight of N and 1.06% by weight of Al. This presence of Au, O and N in a lower
267
percentage than in the polymerisation step reveals a successful elution of nitrite from the 9
268
electrode surface. Overall, the SEM images and EDS spectra show a complete distribution of
269
the elements on the electrode surface, demonstrating the successful modification of the
270
electrode surface.
271
<< Here Table 1>>
272
<< Here Table 2>>
273
<< Here Table 3>> 3.2. Electrochemical characterization of sensor in the fabrication process
274 275
Ferri/ferrocyanide ([Fe(CN)6]3-/4-), as redox probe, is used to monitor all stages of the IIP
276
sensor development. It provides information on the transfer of electrons between the
277
deposited layer and the electrode surface.
278
Figure 5 shows the results of these characterization steps using CV and EIS techniques.
279
Analysis of the cyclic voltammograms in Fig. 5A clearly show that the [Fe(CN)6]3-/4- signal
280
corresponding to the bare gold electrode displays two redox peaks. The potential peaks of the
281
anode (Epa) and cathode (Epc) are located respectively at 0.2 V and 0.08 V, with an oxidation
282
current peak of 46 µA. After the self-assembly of 2-ATP on the bare gold electrode, the
283
current peak of [Fe(CN)6]3-/4- significantly decreases (Ia = 32 µA) because of repulsive
284
interactions between the probe and the negatively charged amine groups (−NH2) of 2-ATP.
285
This explains the decrease in the electron transfer rate. These results demonstrate that the 2-
286
ATP layer was successfully self-assembled on the electrode. Complexing sodium nitrite
287
(NaNO2) with 2-ATP induces a slight decrease in the current peak (Ia = 31.7 µA) showing that
288
NaNO2 inhibits the diffusion of [Fe(CN)6]3-/4- on the electrode surface by electrostatic
289
interaction at an anodic potential of 0.2 V. When the polymer complex (PVA/GA+Au-NPs)
290
was immobilized on the previous modified electrode, a significant decrease in the redox
291
current peak is observed (Ia = 22 µA). On one hand, this may be due to the presence of
292
negatively charged amine groups that lead to an electrostatic repulsion preventing [Fe(CN)6]3-
293
/4-
294
that has been attached to the electrode. This indicates that the polymer was well immobilized
295
on the electrode.
296
access to the electrode surface. On the other hand, it can be explained by the compact mass
<< Here Figure 5>>
297
In addition, Fig. 5B shows the complex impedance curves corresponding to the
298
development stages of the sensor. The EIS characterization of the bare gold electrode displays
299
a low charge transfer resistance (Rct) value. The negative charges of the deposited elements
300
reject the anionic redox probe [Fe(CN)6]3-/4- and hinder electron transfer, which result in an 10
301
increase of electron transfer resistances (Rct) of 4439.1 Ω for 2-ATP, 5761.7 Ω for NaNO2 to
302
10485 Ω for the polymer. Thus, there is an agreement between the results obtained by the CV
303
and EIS techniques. The extraction stage followed and the IIP sensor was kept in the dark
304
until it was use.
305
3.3. IIP and NIIP sensor responses
306
The retention capability of the IIP sensor was evaluated through the use of the DPV
307
method under the probe [Fe(CN)6]3-/4- acting as an electro-active species at a scan rate of 100
308
mV/s between potentials ranging from -0.2 to 0.2 V.
309
In this regard, different nitrite concentrations over the range 0.5 to 50 µg mL-1 have been
310
prepared while taking into consideration that the nitrite in EBC is in micromolar
311
concentrations [57,58]. Then, 60 µL of each NaNO2 concentration was deposited on the
312
surface of the prepared IIP sensor for DPV measurements.
313
Figure 6 shows not only the obtained signals by the nitrite oxidation reaction on the sensor,
314
but also the corresponding calibration curves, where each point is the average of six different
315
measurements.
316
Figure 6A shows the dependence between the oxidation current and the NO2-
317
concentration. As noticed in DPV technique, the signals decrease when a higher concentration
318
of nitrite is applied to the sensor (Fig. 6A). This can be explained by the formed negative
319
membrane when the electrode was modified with nitrite anions. Ferrocyanide was hindered to
320
access the electrode surface due to the electrostatic repulsion of the negative charges. As a
321
result, the electrochemical response decreases on the electrode. This reduction is proportional
322
to the level of nitrite in solution.
323
<< Here Figure 6>>
324
With the EIS method, the information is derived from the value of the Nyquist diagram
325
intersection with the abscissa axis. It can be seen in Fig. 6B that these values increase with
326
rising analyte concentration. For the DPV method, good linearity (R2 > 0.96) is observed over
327
the study range with analytical parameters, such as sensitivity and detection limit of -0.11 (µg
328
mL-1)-1 and 4 µmol L-1 (Fig. 6C). The observed increasing variation of Rct, therefore, confirms
329
the obtained results by the DPV method with a sensitivity of 0.57 (µg mL-1)-1 and an LOD of
330
49.27 µmol L-1 (Fig. 6D), which is calculated by considering three times the standard
331
deviation of the blank signal divided by the sensor sensitivity. This shows that this platform
332
could be used satisfactorily in new generations of electroanalytical sensors.
11
333
However, the NIIP sensor test did not show significant sensitivity to nitrite (Fig. 7). This
334
proves that the obtained responses by the IIP sensor were only due to the presence of memory
335
sites on the electrode that could interact with the nitrite solution. << Here Figure 7>>
336 337
In addition, with the use of species co-existing with nitrite in EBC, sensitivities of -0.09
338
(µg mL-1)-1 for nitrate and -0.05 (µg mL-1)-1 for acetate and ammonium nitrate were obtained.
339
These values are insignificant compared with those obtained after nitrite application (Fig. 8).
340
It is therefore possible to affirm that the developed sensor is very selective. << Here Figure 8>>
341 342
We could barely find any literature on nitrite electro-oxidation that use the technology
343
described in this work with good sensitivity, low overpotential and a simple process.
344
Moreover, the operating range and LOD of the relevant sensor is compared with those in the
345
literature (Table 4). We conclude that satisfactory results are achieved through simple
346
experimentation, easy implementation without specialized personnel, and thus at a lower cost.
347
<< Here Table 4>>
348
3.4. Reproducibility, repeatability and stability study
349
For better performance in a given application, a sensor must meet some important criteria
350
during its development. Some of these parameters are reproducibility, repeatability and
351
stability.
352
Reproducibility is the ability to fabricate a sensor several times and it maintain its
353
responses relatively identical. For this purpose, we reproduced the sensor four times and the
354
generated responses have a relative standard deviation RSD = 2%. This proves that the
355
developed sensor is reproducible.
356
Besides, the repeatability of a sensor is its capability to generate more or less similar
357
responses for the same measurement. Indeed, using the same concentration in our study, the
358
sensor responses were almost the same with an RSD of 4%. These results also confirm that
359
the measurements made with the sensor are repeatable.
360
Stability is the ability of a sensor to give close responses for the same measurement at
361
more or less long-time intervals. In this respect, after 2 months, the sensor gave 97% of the
362
initial response, which implies its stability.
363 364 365
These results are acceptable indicating that the sensor is well suited for practical application in EBC samples. 3.5. Application of the nitrite IIP sensor in exhaled breath samples 12
366
After the IIP sensor development and the optimization of all its analytical parameters, it
367
was tested for the measurement of the nitrite content in condensed exhaled breath samples.
368
After the morning collection of samples from consenting persons, three aliquots were made
369
for each sample. Thus, for each sample, three measurements were made by placing 30 µL of
370
its corresponding aliquot on the sensor. Using the DPV method, peaks in electrochemical
371
responses were recorded to calculate the nitrite content in the various condensed breath
372
samples. We find that the sensor admits different amplitude responses depending on the
373
deposited breath sample. An example of the IIP sensor response is shown in Fig. 9. << Here Figure 9>>
374 375
Relatively low concentrations of nitrite with RSD = 4% are found during the sensor
376
practical application in EBC (Table 5). Indeed, all nitrite values calculated in the EBC
377
samples are in micromolar order, validating the nitrite values in the literature [59-61]. All in
378
all, the sensor can be considered as a viable tool for the determination of the nitrite profile in
379
EBC. << Here Table 5>>
380 381
4. Conclusion
382
The particularity of the developed IIP sensor for the determination of nitrite in condensed
383
exhaled breath is its simplicity. The developed ion imprinted sensor for nitrite determination
384
is simple and very sensitive. The calculated detection limit is 4 µmol L-1. In addition, since
385
nitrate, acetate and ammonium nitrate are interfering substances that coexist in breath, a
386
selectivity test is carried out with satisfactory results. Using the ion imprinted sensor, its
387
practical application in condensate human exhaled breath samples was well performed with
388
results in micromolar order. This could be a good vista for medical research on inflammation
389
and respiratory diseases.
390
Acknowledgements
391
Authors gratefully acknowledge Moulay Ismaïl University of Meknes for financial support
392
of the project “Research support”. This work has been funded in part by TROPSENSE under
393
the H2020-MSCA-RISE-2014 project, grant agreement number: 645758. The authors thank
394
and wish to express their gratitude to Institutului de Metale Neferoase si Rare (IMNR) team of
395
ROMANIA namely Dr. Ioan Albert Tudor, Dr. Laura Madalina Popescu, Dr. Ghita Mihai,
396
Lupu Andreea Nicoleta, and professor Driss Bouyahya of school of arts and humanities,
397
moulay ismail university for the excellent technical assistance of the characterization process
398
of the MIP-based sensors, and English paper correction. 13
399
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20
Table 1. Chemical composition by EDS for Au-SPE.
Element
Weight %
Atomic %
Net Int.
CK
7.34
53.47
111.915
Al K
1.92
6.22
132.51
Au M
90.74
40.31
478.11
Table 2. Chemical composition by EDS for the electrode after polymer deposit.
Element
Weight %
Atomic %
Net Int.
CK
5.7
33.68
35.29
NK
1.36
6.88
5.99
OK
6.36
28.23
46.72
Au M
86.58
31.21
596.6
Table 3. Chemical composition by EDS for the electrode after extraction stage.
Element
Weight %
Atomic %
Net Int.
CK
9.4
45.65
103.37
NK
2.47
10.29
18.16
OK
4.76
17.37
58.98
Al k
1.06
2.3
35.7
Au M
82.31
24.38
993.47
Table 4. Comparison of performance between this IIP sensor and reported nitrite sensor. Linear range
LOD
(µg mL-1)
(µmol L-1)
Graphene nanoplatelet
3.4 – 172
10
[62]
Reduced graphene oxide
6.9 – 69
0.83
[60]
Glassy Carbon Electrode
0.345 – 465
1.65
[63]
2.22 – 13.04
5.61
[49]
0.069 – 0.69
5
[64]
8.97 – 3036
45
[65]
0.5 – 50
4
This work
Electrodes
Edge plane pyrolytic graphite electrode
Gold electrode
Pre-treated glassy carbon electrode Gold electrode
Reference
Table 5. EBC nitrite levels by means of DPV method. Nitrite
IBlank
Isample
(µA)
(µA)
P77
22.62
20.04
-0.110
0.062
P83
17.60
16.36
-0.071
0.027
P88
21.71
17.73
-0.183
0.289
P118
18.93
16.90
-0.107
0.059
P128
24.26
21.49
-0.114
0.068
P89
5.313
4.994
-0.060
0.021
P99
7.304
5.995
-0.179
0.266
P128 bis
9.644
8.515
-0.117
0.072
Sample
(Isample - IBlank)/ IBlank
Concentration (µmol L-1)
Fig. 1. Procedures of the IIP sensor preparation.
Fig. 2. FTIR spectra obtained for: (A) Bare gold, (B) After polymer deposit, (C) After
extraction.
Fig. 3. AFM images obtained for: (A) Bare gold, (B) After polymer deposit, (C) After
extraction.
Fig. 4. SEM images and corresponding EDS spectra obtained for: (A) Bare gold, (B) After (A) Bare gold
polymer deposit, (C) After extraction.
(B) Polymer
(C) Extraction
Fig. 5. (A) Cyclic voltammograms of 5 mM [Fe(CN)6]3-/4- solution at: Bare gold, After 2-ATP deposit, After NaNO2 deposit, and after polymer deposit; (B) Nyquist plots of 5 mM [Fe(CN)6]3-/4- solution at: Bare gold, After 2-ATP deposit, After NaNO2 deposit, and after
polymer deposit.
Fig. 6. Electrochemical responses of the IIP sensor as a function of increasing nitrite concentration using: (A) DPV, (B) EIS in the presence of 5 mM of [Fe (CN) 6]3-/4- as redox probe. Calibration curves between the sensor responses and Logarithm of nitrite concentrations by: (C) DPV, (D) EIS in PBS buffer at pH 7.4.
Fig. 7. (A) By the presence of 5 mM of [Fe (CN) 6]3-/4- as redox probe, electrochemical responses of the NIIP sensor by using DPV, (B) Calibration curve between the sensor responses and Logarithm of nitrite concentrations in PBS buffer at pH 7.4.
Fig. 8. Calibration curves of (A) nitrite, and interfering molecules: (B) Silver nitrate, (C)
Acetate, (D) ammonium nitrate.
Fig. 9. Example of the IIP sensor response after an EBC sample exposure for nitrite determination.
Highlights Successful development of an electrochemical nitrite sensor-based ion imprinted polymer. CV, DPV, EIS, FTIR, AFM and SEM techniques for electrochemical and morphological characterizations. Utilization of the IIP sensor for nitrite determination in exhaled breath condensate. A lower LOD of 4 µmol L-1 in a working range from 0.5 to 50 µg mL-1 compared with related works.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: