Analytical

Methods
PAPER

A novel all-3D-printed thread-based microfluidic

Cite this: Anal. Methods, 2021, 13, 1349
device with an embedded electrochemical
detector: first application in environmental analysis
of nitrite†
Rayan Marcel Carvalho, Valdir Souza Ferreira and Bruno Gabriel Lucca *

A microfluidic thread electroanalytical device (mTED) containing an embedded electrochemical detector is

Received 11th January 2021
Accepted 17th February 2021
DOI: 10.1039/d1ay00070e
rsc.li/methods
presented for the first time in this work. This novel device was entirely produced in an automated way using
the fused deposition modeling (FDM) 3D printing technique. The main platform was fabricated with
acrylonitrile butadiene styrene (ABS) filament, while the integrated electrochemical detector was
produced using a commercial conductive filament composed of carbon black and polylactic acid (CB/
PLA). The microfluidic channels consisted of cotton threads, which act as passive pumps, and the mTED
was used for microflow injection analysis (mFIA). As a proof of concept, this mFIA system was utilized for
the amperometric sensing of nitrite in natural waters. This is the first report on the use of both mTEDs
and 3D-printed CB/PLA electrodes to determine this species. This fully 3D-printed mTED was
characterized and all experimental and instrumental parameters related to the method were studied and
optimized. Using the best conditions, the proposed approach showed a linear response in the
concentration range from 8 to 200 mmol L
1 and a limit of detection (LOD) of 2.39 mmol L
1. The LOD
obtained here was ca. ten-fold lower than the maximum contaminant level for nitrite in drinking water
established by the Brazilian and US legislation. Moreover, the platform presented good repeatability and
reproducibility (relative standard deviations (RSDs) were 2.1% and 2.5%, respectively). Lastly, the 3D-
printed mTED was applied for the quantification of nitrite in well water samples and the results obtained
showed good precision (RSD < 3%) and excellent concordance (relative error was ca. 3%) with those
achieved by ion chromatography, used for validation.

substrate in microuidics has been largely explored in several

1. Introduction
Microuidics is nowadays a well-established area that has
brought benets to many elds. Some of the advantages on the
use of microuidic platforms are the reduced size, biocompat-
ibility, portability, high speed analysis, integration of several
analytical steps, extremely low consumption of samples and
reagents, and minimal waste generation.1,2 Microuidic
systems have been utilized for applications in many elds, such
as biomedical research,3 food analysis,4 forensic science,5 clin-
ical diagnosis6 and environmental monitoring.7
Among the various substrates used for the fabrication of
microuidic devices, cellulose-based materials such as paper
and, more recently, cotton threads, emerge as very interesting
options due to the low cost and large availability of the raw
material. Since the rst report in 2007,8 the use of paper as
Chemistry Institute, Federal University of Mato Grosso do Sul, Campo Grande, MS,
79074-460, Brazil. E-mail: bruno.lucca@ufms.br; Tel: +55 (67) 3345-3936
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d1ay00070e
applications.9,10 However, some limitations related to the use of
paper-based microuidic devices include low mechanical
resistance and the requirement of hydrophobic barriers to drive
and avoid leakage of solutions.11,12 On the other hand, cotton
threads are also an attractive option for applications in micro-
uidics. Some interesting features of cotton threads are high
mechanical resistance, the fact that no hydrophobic barrier is
necessary, and the high capillarity, which enable their use as
passive pumps without the need of external devices to transport
uids.13,14 These attributes make cotton threads a very inter-
esting option for use in low-cost microuidic devices.
Although several techniques can be utilized for detection in
microuidic devices, electrochemical methods are among the
most employed. Electrochemical detection is especially inter-
esting for sensing in microuidics due to advantages such as
ease of operation, low power consumption, low environmental
impact, good sensitivity, compatibility with miniaturization and
portability of instrumentation.15–17 Among the materials utilized
for electrochemical sensing, carbon-based electrodes are the
most used in electroanalytical applications due to features such

This journal is © The Royal Society of Chemistry 2021 Anal. Methods, 2021, 13, 1349–1357 | 1349
Analytical Methods
as a wide potential range, low cost, good chemical stability and
large availability.18 Within this topic, 3D printing technology, or
additive manufacturing, is a current hot topic that has been
widely explored as a very useful tool for the production of
analytical devices. The fabrication of electrochemical devices by
3D printing offers several advantages that include lower
production costs, quickness in prototyping and possibility of
large-scale implementation.19,20 This technology can be used
both in the fabrication of electrochemical sensors as well as for
production of auxiliary platforms, such as stationary or ow
cells.21 However, as stated by a recent review, a current chal-
lenge in using 3D printing for electrochemical applications is
the difficulty in fabricating devices with embedded electrodes.22
Fused deposition modeling (FDM) is the most popular 3D
printing technique and uses naturally non-conductive thermo-
plastic materials, such as acrylonitrile butadiene styrene (ABS)
and polylactic acid (PLA), to form three-dimensional objects.
These thermoplastics can also contain carbon-based materials,
such as graphene and carbon black, that provide conductive
properties and allow their use for the printing of electrodes.
Such applications have been oen reported in recent literature
and demonstrate the great potential of the use of additive
manufacturing in Analytical Chemistry.23–25
In this context, we present here, for the rst time, a micro-
uidic thread-based electroanalytical device (mTED) entirely
fabricated by 3D printing. The microuidic channels of this
device consisted of hydrophilic cotton threads. The concept of
mTED was rst presented in 2016 by Agustini et al., who
demonstrated its potential for use in analytical applications.26
The platform developed here presents advantages such as the
fact that it has an integrated detector and was fully fabricated in
an automated way by FDM 3D printing combining conductive
and non-conductive thermoplastics. The main base of the mTED
was printed with ABS while the embedded electrochemical
detector was fabricated utilizing a commercial carbon black/
PLA conductive lament. To the best of our knowledge, this is
the rst report of a fully 3D-printed mTED with an integrated
detector. This additively manufactured mTED was employed as
an efficient and inexpensive microow injection analysis (mFIA)
platform for amperometric sensing of nitrite. This ion is present
in soil, food, drinking water and physiological systems.27 The
toxic effects of nitrite on human health are well known: once in
the blood ow, nitrite has the ability to convert hemoglobin into
methemoglobin, which suppresses its capability to carry
oxygen; in addition, nitrite can be a carcinogenic species to the
stomach. So, the monitoring and control of nitrite levels is an
important parameter established in the legislation regarding
the quality of drinking water.28
As far as we know, this is the rst application of a mTED for
the sensing of nitrite. Furthermore, we also present here, for the
rst time, the electrochemical determination of nitrite using
3D-printed CB/PLA electrodes. The fully 3D-printed mTED
developed here presented excellent analytical performance and
the limit of detection achieved was ca. ten-fold lower than the
maximum contaminant level established by the legislation of
Brazil and US. The mFIA passive platform was successfully
employed to quantify nitrite in well water samples and the
1350 | Anal. Methods, 2021, 13, 1349–1357
Paper
results showed great concordance with the ion chromatography
technique, employed for validation of the method.
Therefore, according to the data presented here, we consider
this approach an interesting alternative for electroanalytical
procedures. This fully 3D-printed mFIA platform with an
embedded electrochemical detector demonstrates a novel and
promising application of 3D printing in Analytical Chemistry,
besides presenting advantages over conventional electro-
chemical ow systems that include portability, full integration
in an all-in-one platform, simple and automated fabrication,
easy replication, low cost, no need of external pumps, minimal
consumption of samples and reagents and reduced waste
generation, which is in accordance with the concept of green
chemistry. This way, the 3D-printed mFIA system demonstrated
here can be seen as an interesting new tool for analytical
applications and overcomes a limitation regarding the fabrica-
tion of electrochemical devices containing integrated electrodes
by 3D printing.
2. Experimental
2.1. Chemicals, materials, and solutions
All the chemicals used were of analytical grade and utilized as
received: sodium nitrite was purchased from Neon (Suzano, SP,
Brazil). Acetic acid was provided by Vetec (Duque de Caxias, RJ,
Brazil). Boric acid was obtained from Dinâmica (Diadema, SP,
Brazil). Sodium hydroxide and orthophosphoric acid were
supplied by Acros Organics (Geel, Belgium). Sodium carbonate
and sodium bicarbonate were purchased from J. T. Baker
(Phillipsburg, NJ, USA). The ABS lament (1.75 mm diameter)
was acquired from 3DFila (Belo Horizonte, MG, Brazil). The
conductive CB/PLA lament (1.75 mm diameter) was provided
by Protopasta (Vancouver, WA, USA). Hydrophilic cotton
bandage gauze and double-sided tape were purchased at local
stores. All the solutions utilized during the experiments were
prepared with puried water (resistivity $18.2 MU cm) supplied
by a Permution RO0310 reverse osmosis water purier system
(Permution, Curitiba, PR, Brazil). The stock solutions of the
buffers and analytes were prepared weekly. The solutions used
in the experiments were prepared daily by diluting the stock
solutions.
2.2. CAD design and fabrication of the 3D-printed mTED
The 3D modeling of the mTED platform with an embedded
three-electrode detector was performed using Fusion 360 free
3D CAD/CAM design soware (Autodesk, San Rafael, CA, USA).
The obtained CAD le was then converted to STL format and
processed with Ultimaker Cura 3.6 soware (Geldermalsen, The
Netherlands) to obtain the GCODE le containing the printing
commands. The 3D printing of the mTED was carried out in two
steps: rst, the base of the mTED was printed with a non-
conductive ABS lament; so, at the specic point, the printing
was paused, the ABS lament was changed to the CB/PLA
conductive lament and the process was resumed to print the
electrodes. A Creality Ender 3 FDM 3D printer (Creality,
Shenzhen, China) equipped with a 0.4 mm diameter brass
This journal is © The Royal Society of Chemistry 2021
Paper
Fig. 1 (A) CAD design of the base of the mTED with its respective
dimensions and (B) the fully 3D-printed mTED with the embedded
detector, cotton thread microchannels and electrical connections
such as used during the experiments. The 3D-printed CB/PLA elec-
trodes were 45 mm length  3 mm width  1.60 mm height. The
outlet reservoir is movable and fixed using two M3 screws.
extruder nozzle was utilized here. The layer resolution and
printing speed for both mTED and CB/PLA electrodes were
0.20 mm and 45 mm s
1, respectively. The CB/PLA electrodes
were printed with 100% inll. Due to the practical characteris-
tics of this printing, the embedded CB/PLA detector must be
printed in horizontal orientation. As presented in Fig. 1A, the
base of the mTED has the dimensions of 95 mm length  60 mm
width  122 mm height and contains two reservoirs for the inlet
and outlet, with 22 mm diameter and a maximum volume of ca.
4 mL each. The outlet reservoir was designed to be movable in
order to facilitate the height difference adjustments. This part is
xed to the main platform by two M3 screws. The CB/PLA
electrodes have the dimensions of 45 mm length  3 mm
width  1.60 mm height.
2.3. Activation of the CB/PLA electrodes and assembling of
the mTED
Before use, the surface of the CB/PLA electrodes was submitted
to an activation procedure that consisted of polishing with 500
and 1500 grit sandpaper, respectively, in the presence of puri-
ed water. The electrodes were polished for ca. 1 min with each
sandpaper, aiming to obtain a smooth and homogeneous
surface and improve the sensitivity.
Aer the printing and activation steps, the assembly of the
mTED was completed by placing the hydrophilic cotton threads
that were obtained from cotton bandage gauze and served as
microow channels. No treatment or cleaning procedure was
performed before using the cotton threads, since during
manufacturing the hydrophilic gauze is already submitted to
a process that removes wax and other non-cellulosic
compounds that naturally cover the threads. It exposes the
hydroxyl groups on the cellulose structure, which are respon-
sible for the hydrophilicity.29–31 The desired number of cotton
treads was positioned perpendicular to the electrodes and the
ends were xed into the inlet and outlet reservoirs using double-
This journal is © The Royal Society of Chemistry 2021
Analytical Methods
sided tape. Fig. 1B shows an image of the fully 3D-printed mTED
with the cotton thread microchannel as utilized in this work.
2.4. Electrochemical setup
The electrochemical studies were carried out at room temper-
ature using an Autolab PGSTAT128N potentiostat/galvanostat
(Ecochemie, Utrecht, The Netherlands) connected to
a computer and managed by Metrohm Autolab Nova 2.1 so-
ware. All the measurements performed with the mTED were
recorded using the electrochemical detector composed of the
3D-printed CB/PLA sensors, which were employed as working,
counter and pseudo-reference electrodes. The electrical
connections were affixed directly to the CB/PLA electrodes, as
shown in Fig. 1B. The injections of the standards/samples were
performed using a micropipette.
2.5. Scanning electron microscopy
The structure and morphology of the CB/PLA electrodes were
investigated by scanning electron microscopy (SEM). Images at
different magnications were obtained using a JSM-6380LV
instrument (Jeol, Tokyo, Japan).
2.6. Application in environmental samples
Two samples of natural waters were collected from semi-
artesian wells located in the municipality of Nova Andradina,
MS, Brazil. The samples were ltered through a 0.22 mm
membrane syringe lter and submitted to electrochemical and
chromatographic analyses, before and aer spiking with known
concentrations of nitrite standard aqueous solution. For the
electrochemical determinations, the ltered samples were
directly diluted in the running buffer.
2.7. Chromatographic experiments
To compare and validate the results obtained with the 3D-
printed mTED, the well water samples were also analyzed by
ion chromatography. The chromatographic assays were per-
formed using an ICS 1100 instrument (Dionex, Sunnyvale, CA,
USA) equipped with an IonPac AS22 column (2 mm I.D. 
250 mm length) and suppressed conductivity detection (Dionex
AERS 500 2 mm). The mobile phase consisted of 4.5 mmol L
1
sodium carbonate/1.4 mmol L
1 sodium bicarbonate solution.
The ow rate was 0.25 mL min
1, the sample volume injected
was 125 mL and the temperature of the conductivity cell was
30  C.32
3. Results & discussion
3.1. Electrochemical behavior of nitrite, activation of the
electrodes and surface analysis
In this work, we present for the rst time a microuidic elec-
trochemical device based on cotton threads containing an
integrated electrochemical detector fully fabricated by 3D
printing. To demonstrate the performance and evaluate the
analytical applicability of this 3D-printed mTED, we have
selected the nitrite ion as a model analyte, since its
Anal. Methods, 2021, 13, 1349–1357 | 1351
Analytical Methods
Fig. 2 Cyclic voltammograms recorded with the embedded 3D-
printed CB/PLA electrochemical detector in the presence (solid line)
and absence (dashed line) of 1.5 mmol L
1 nitrite standard. Medium
was 0.1 mol L
1 Britton–Robinson (BR) buffer at pH ¼ 4.0 and the scan
rate was 100 mV s
1. The surface of the electrodes was polished
before the measurements.
quantication is an important topic regarding the quality of
natural waters.33,34 Initial experiments were performed in order
to investigate the performance of the embedded detector
towards the electrochemical sensing of nitrite. Fig. 2 exhibits
a cyclic voltammogram recorded with the CB/PLA three-
electrode system. As can be seen, nitrite presented electro-
chemical activity over the surface of the CB/PLA working elec-
trode. One single irreversible oxidation peak at Ep ¼ +1.08 V was
observed. This electrochemical behavior is quite similar to that
described in previous reports.25,35,36 So, it enables the use of this
platform for the analytical sensing of nitrite.
The poor electrochemical response of the CB/PLA electrodes is
an issue already known and reported in the literature.37 This way,
activation procedures are essential to improve the analytical
performance of these sensors. Here we evaluated mechanical and
electrochemical treatments to activate the electrode surface. The
mechanical treatment consisted of a polishing procedure using
sandpaper, as detailed in Section 2.3, while the electrochemical
treatment was based in a protocol described by Richter et al.,
which suggests the application of a sequence of potentials during
specic times (+1.4 V and
1.0 V, for 200 s each) in a 0.5 mol L
1
NaOH medium.24 The CB/PLA electrodes were activated in two
ways: only by mechanical polishing and by mechanical polishing
followed by electrochemical treatment. The efficiency of these
procedures was investigated through the sensing of nitrite using
the fully 3D-printed mTED platform and amperometric detection,
which was the technique employed in the analytical studies. The
activated electrodes were compared with non-activated electrodes
that were used as control. As can be seen in Fig. 3, the electro-
chemical response of the activated electrodes was signicantly
improved when compared to the non-activated electrodes.
However, no considerable differences in the current response
(687 vs. 680 nA) were observed between the electrodes activated
only by polishing and the electrodes activated by polishing and
electrochemical treatment. This result, which was different from
that described by Richter et al.24, may be attributed to the fact that
1352 | Anal. Methods, 2021, 13, 1349–1357
Paper
Fig. 3 Amperometric responses for injections of 250 mmol L
1 nitrite
standard in the 3D-printed mFIA platform using non-activated and
activated CB/PLA electrodes. Detection potential: +1.15 V; time
interval: 0.1 s; supporting electrolyte: 0.1 mol L
1 BR buffer (pH ¼ 4.0);
injected volume: 4.0 mL.
in this work the analyte (nitrite instead dopamine), the electro-
chemical technique (amperometry instead cyclic and square-
wave voltammetry) and the measurement system (mFIA instead
stationary cell) were different. In addition, it is in agreement with
that presented in other recent studies describing the analytical
use of the same CB/PLA 3D-printed electrochemical sensors
without any electrochemical activation.38,39 Moreover, the intra-
electrode repeatability and inter-electrode reproducibility were
quite similar for all the activation protocols evaluated (relative
standard deviation (RSD) was <3% in all the cases). Thus, to make
the activation protocol simpler and faster, the CB/PLA electro-
chemical sensors used here were activated only by mechanical
polishing.
The supercial structure of the CB/PLA electrodes was
investigated by SEM. Fig. 4 presents images of the electrodes
before and aer the activation protocol utilized. As observed,
the surface of the electrodes is signicantly changed by the
polishing procedure. Before polishing (Fig. 4A), the PLA poly-
meric matrix is predominant on the surface. As an insulating
material, PLA decreases the conductivity and is responsible for
the low electrochemical response. Aer polishing (Fig. 4B), the
surface becomes more porous, rough and irregular, similar to
carbon-based electrodic surfaces.34 It is due to the partial
removal of the supercial PLA by polishing, which exposes the
CB particles, increasing the conductivity and improving the
sensitivity of the electrode.
3.2. Optimization of the mFIA platform for nitrite sensing
As a proof of concept, the fully additively manufactured mTED
proposed here was employed for the quantication of nitrite
This journal is © The Royal Society of Chemistry 2021
Paper
Fig. 4 SEM images obtained from the surface of the CB/PLA elec-
trodes before (A) and after (B) the activation procedure carried out by
polishing with sandpaper. The accelerating voltage was 10 kV and the
magnification was 5000.
ions by mFIA and amperometric detection. Since these analyses
are strongly dependent on parameters such as detection
potential, pH and sample injection, among others, the experi-
mental and instrumental conditions were studied. We selected
the most important parameters to be optimized in order to
achieve the best analytical performance during the analytical
determinations. While one condition was varied and studied,
all the others were kept constant.
First, the pH was optimized. This parameter has direct
inuence on the electrochemical reaction and also on the signal
intensity. Here, the dependence of the current signal on pH was
investigated in the range from 2.0 to 6.0. As shown in Fig. S1
(ESI†), it can be seen that as pH increases, the analytical signal
of nitrite also increases until it reaches its maximum at pH ¼
4.0. At pH values above 4.0, the current signal starts to decrease.
Thus, the pH ¼ 4.0 was chosen as the optimum for the analyt-
ical measurements.
Aer optimizing the pH, the detection potential was studied.
Based on the electrochemical prole of nitrite observed in the
cyclic voltammograms, the oxidation potential was varied in the
range from +0.90 V up to +1.30 V (versus CB/PLA pseudo-
This journal is © The Royal Society of Chemistry 2021
Analytical Methods
reference electrode). For each potential evaluated, injections
of 10 mmol L
1 nitrite standard were carried out. The results are
presented in Fig. S2A (ESI†). The oxidation of nitrite is favored
with the increase in the potential of detection and the highest
analytical signal was observed when +1.15 V was applied. So,
this value was adopted as the best detection potential, since the
analytical signal decreased at higher potential values. The
stability of the CB/PLA pseudo-reference electrode was also
investigated, since the constancy of detection potential directly
depends on this electrode. For this, consecutive cyclic voltam-
mograms (n ¼ 150 cycles) of nitrite were recorded. As shown in
Fig. S2B (ESI†), it can be seen that the peak potentials have not
been shied. The RSD regarding the peak potentials was ca.
2.0%, suggesting satisfactory stability for the pseudo-reference
electrode.
As the mFIA platform is based on passive pumping, the
height difference between the inlet and outlet reservoirs of the
mTED has great inuence on the ow rate, once the capillarity of
the solutions in the thread microchannels is affected by the
gravity action.40 The novel design of the platform developed
here allows the easy adjustment of this height difference in the
range from 20 to 100 mm, just by unscrewing two screws that
hold the outlet reservoir. So, the same 3D-printed platform can
be used under various conditions regarding this parameter.
According to the plot displayed in Fig. S3A (ESI†), the current
signal was notably inuenced by the height difference, showing
an expressive increase until 60 mm. For height differences
greater than this, the current signal signicantly decreased.
Hence, the height difference of 60 mm was utilized in further
studies.
The next variable optimized was the number of threads used
to create the microchannel of the mFIA platform. Measurements
using channels formed by 3 until 6 cotton threads were per-
formed, and the results are depicted in Fig. S3B (ESI†). As the
number of threads increases, the current signal becomes more
intense due to the higher ow rate. However, there is also
a greater dilution of the injected sample into the larger spaces of
the channel.29,41 Considering this, a good relationship between
the intensity of the analytical signal and the size of the micro-
channel must be found. According to the results obtained, the
maximum current response was achieved when the microuidic
channel was composed of 4 threads. When the number of
threads was greater than this, the current signal decreased,
probably due to the dilution of the sample. Thus, the optimal
microuidic channel consisted of 4 cotton threads.
The mFIA measurements also have a relevant dependence on
the way the threads are disposed, since the wicking of the
solutions directly depends on the spaces available between the
cotton bers.42 As such, the presence of twists in the thread
microuidic channel was considered. According to the results
shown in Fig. S3C (ESI†), it is evident that the current signal is
substantially reduced as the number of twists per meter
increases from 0 (threads in parallel) to 200 (twisted threads).
This behavior can be explained by the fact that, as the number
of twists increases, the space between the bers diminishes. It
results in a higher ow resistance, which decreases the amount
of sample that reaches the detector and may also require longer
Anal. Methods, 2021, 13, 1349–1357 | 1353
Analytical Methods Paper

signal was observed. This may be attributed to two factors: the

dispersion of the larger sample volumes and the saturation of
the microchannel capacity when receiving sample volumes
larger than 4.0 mL. This way, the injection volume of 4.0 mL was
selected as the optimum value. Ultimately, the distance between
the sample injection zone and the detection point was investi-
gated. This parameter was varied from 5 to 20 mm and the
results are presented in Fig. S4B (ESI†). As can be seen,
increasing this distance decreases the current signal, probably
due to the dispersion of the sample along the longer paths
before it reaches the detector. Since distances smaller than
5 mm caused noise and repeatability issues due to the proximity
of the embedded detector, the optimal distance was dened as 5
mm. All the parameters studied, as well as the optimized values,
are summarized in Table S1 (ESI†).

3.3. Analytical application

Fig. 5 Amperometric response obtained for nitrite standard solutions
in different concentrations: (a) 8 mmol L
1; (b) 10 mmol L
1; (c) 25 mmol
L
1; (d) 50 mmol L
1; (e) 100 mmol L
1; (f) 150 mmol L
1 and (g) 200
mmol L
1. The inset shows the respective analytical curve obtained
from the amperogram. Results for n ¼ 3 measurements. Other
conditions: see Table S1 (ESI†).
times for the analyses. So, in order to obtain the best analytical
performance and shorter analysis times, the threads used to
form the microuidic channel of the mTED were bundled
without twisting, in a parallel disposition.
The last two parameters evaluated were related to the sample
injection. First, the injection volume of the samples was studied
in the range from 2.0 to 7.0 mL. As exhibited in Fig. S4A (ESI†),
a linear increase in the current signal was observed for injection
volumes between 2.0 and 4.0 mL. It suggests that the central area
of the samples is less dispersed for these volumes. For injection
volumes larger than 4.0 mL, a slight decrease in the current
The analytical usefulness of the 3D-printed mFIA platform
containing the embedded CB/PLA electrochemical detector was
veried through the determination of nitrite in well water
samples. For this purpose, an analytical curve was constructed
in the concentration range from 8 to 200 mmol L
1. As shown in
Fig. 5, the analytical signal presented a good linear dependence
on concentration (R2 was 0.998). The linear regression of the
curve provided the equation Current signal/nA ¼
(82.92 
3.01) + (3.78  0.23)  [NO2
]/mmol L
1. The limits of detection
(LOD) and quantication (LOQ) were obtained experimentally
and also based on the equations 3  SDa/b and 10  SDa/b,
respectively, where SDa is the standard deviation of the intercept
and b is the slope of the calibration plot.43 The values achieved
for the LOD and LOQ were 2.39 and 7.61 mmol L
1, respectively.
To demonstrate the analytical efficiency of this novel fully
3D-printed mFIA platform, the results obtained here were
compared with other reports dealing with the electrochemical
determination of nitrite. As can be seen in Table 1, the LOD

Table 1 Comparison between the analytical performance of the fully 3D-printed mTED with the embedded electrochemical detector described
here and other electrochemical approaches applied in nitrite sensing

Linear range
Electrode Technique LOD (mmol L
1) (mmol L
1) Reference Year

Ag/H–C3N4/CCa Amperometry 0.216 5–1000 28 2020

3D-printed G/PLAb DPV 30 50–1300 25 2020
3D-printed G/PLAb BIA-MPA 0.03 0.5–250 25 2020
Au-SPE/IIP/AuNPsc DPV 4 10.9–1087 48 2020
CILE/BPE/hemind CV 330 1000–10 500 49 2019
SPCE/Ag/rGO/b-CDe LSV 0.24 1–2000 33 2019
CPE/Ag/HNT/MoS2f Amperometry 0.7 2–425 50 2018
SPCE/AgMCs-PAA/PVAg FI-Amperometry 4.5 2–800 51 2017
PEDOT-Gr/Tah DPV 7 10–2500 52 2017
3D-printed CB/PLAi Amperometry 2.39 8–200 This work —
a
Nanosilver combined with protonated carbon nitride supported on carbon cloth. b 3D-printed electrode composed of graphene/polylactic acid.
c
Gold screen-printed electrode modied with ion imprinted polymer/gold nanoparticles. d Carbon ionic liquid electrode modied with black
phosphorene/hemin. e Screen-printed carbon electrode modied with silver nanostructures/reduced graphene oxide/b-cyclodextrin. f Carbon
paste electrode modied with silver/halloysite nanotube/molybdenum disulde. g Screen-printed carbon electrode modied with silver
microcubics-polyacrylic acid/polyvinyl alcohol. h Poly(3,4-ethylenedioxythiophene)–graphene composite deposited over a Ta lm. i 3D-printed
electrode composed of carbon black/polylactic acid.

1354 | Anal. Methods, 2021, 13, 1349–1357 This journal is © The Royal Society of Chemistry 2021
Paper Analytical Methods

achieved here was similar or even lower than most of the reports 3.4. Repeatability, reproducibility, stability and ow rate
presented. Furthermore, the LOQ and the linear concentration studies
range obtained with the mTED reported here also allow the
The intra-electrode repeatability of the amperometric
application of this approach to quantify nitrite in drinking
measurements obtained with the mFIA system was investigated
water. The LOQ was around three-fold lower than the maximum
by carrying out successive injections (n ¼ 20) of a 50 mmol L
1
contaminant level (MCL) determined by the Brazilian Govern-
nitrite standard solution, as presented in Fig. S6 (ESI†). The
ment and the US Environmental Protection Agency (EPA), which
background current remained stable and the RSD for the
is 1.0 mg L
1 (equivalent to 21.7 mmol L
1).44 signals was 2.1%, which suggests excellent replicability for the
The integrated 3D-printed mFIA system proposed here was injections. Furthermore, it also indicates no fouling or passiv-
utilized for the determination of nitrite in well water samples.
ation of the working electrode, conrming the advantages of
The preliminary assays carried out in the samples using the
using a ow-based system, which promotes a constant cleaning
mTED did not show contamination by nitrite at detectable levels.
of the working electrode, increasing its lifetime and reducing
This way, the well water samples were spiked with known
the costs of analysis.29 This is especially important in the case of
concentrations of nitrite standard and the recovery rates were
the embedded CB/PLA sensors used here, which are solid
determined. To avoid any matrix effect and verify the precision
electrodes that can be used several times. The inter-electrode
of the results, the quantications were carried out in triplicate reproducibility of the platform was also evaluated. For this,
using the standard addition method. The samples were the amperometric response of 50 mmol L
1 nitrite standard
analyzed before and aer spiking, and the spiking levels were
solution was recorded using four different 3D-printed mFIA
based on the Brazilian and US legislation regarding contami-
systems. As can be seen in Fig. S7A (ESI†), an RSD of 2.5% was
nation of drinking waters with nitrite. Fig. S5 (ESI†) shows the
obtained. This result indicates great reproducibility in the
amperometric measurements and the standard addition curve
fabrication and assembly of the platform, and makes the
obtained in one of the determinations. The full results for the
approach proposed here suitable for large scale implementa-
analyses of the samples are detailed in Table 2. As observed, the
tion. The stability of the 3D-printed mTED was studied using the
recovery rates were ca. 99%, indicating excellent accuracy. same device for a period of 16 consecutive days. For this study,
Furthermore, the RSD values were ca. 3%, indicating great the polishing activation in the CB/PLA embedded detector was
precision. The water samples were also analyzed by ion chro-
carried out only on the rst day. As observed in Fig. S7B (ESI†),
matography, which was employed as the reference technique
no signicant variation was observed during injections of 50
for validation. The results obtained with the mFIA platform
mmol L
1 nitrite standard solution. The calculated RSD was
presented good agreement with the values found by ion chro-
4.0%. It indicates that there was no loss in the sensitivity of the
matography (relative error was ca. 3%).
electrodes nor decrease in the analytical efficiency during this
The effect of some possible interferent species that might be
time, suggesting a long lifetime for the platform. Moreover,
present in natural waters was evaluated to investigate the
when some signicant decrease in the electrochemical response
selectivity of the proposed mFIA approach. The response of
is observed over time, the embedded CB/PLA detector can be
nitrite was monitored in standard solutions containing 100-fold
reactivated again by the polishing procedure.
mass ratios of NO3
, SO42
, Cl
, PO43
, F
, K+, Na+ and Mg2+
Under the optimized conditions, the ow rate provided by
and 10-fold mass ratio of Mn2+, Fe3+, Pb2+, Cu2+, Co2+, Cd2+ and
the 3D-printed mTED was ca. 0.41 mL s
1. This value is higher
Zn2+. The response of nitrite in the presence of these interfer-
than those stated in previous reports that also describe ow
ents was compared to the nitrite response in their absence. The
devices based on passive pumps.29,40,45 Regarding the analytical
results showed that the relative response of nitrite was not
frequency, the value of 50 injections per hour was estimated for
signicantly affected by the presence of these species. As the mFIA platform, which is similar or even superior to other
detailed in Table S2 (ESI†), the values ranged between 95 and approaches.46,47 The cost for fabrication and assembling of each
104%, suggesting good selectivity for the method. It conrms
fully additively manufactured mFIA device (considering the
the feasibility of this novel 3D-printed mTED with the integrated
main platform, the embedded electrochemical detector, screws
electrochemical detector for the quantication of nitrite in
and ow channel) was calculated to be ca. $ 3. It is an extremely
drinking water.
low value for a ow device, especially considering that the

Table 2 Results of the determination of nitrite in well water samples using the 3D-printed mFIA platform and the reference technique employed
for validation

Method Founda (mmol L
1) Spiked (mmol L
1) Recovery  SDb (mmol L
1) RSD (%) Relative errorc (%)

3D-printed mTED <LOQ 15.0 14.8  0.4 2.7 +3.5

<LOQ 30.0 29.8  0.6 2.0
2.6
Ion chromatography — 15.0 14.3  0.2 1.4 —
— 30.0 30.6  0.2 0.6 —
a
Before spiking. b Aer spiking. Average of three determinations. c Calculated using the ion chromatography results as reference values.

This journal is © The Royal Society of Chemistry 2021 Anal. Methods, 2021, 13, 1349–1357 | 1355
Analytical Methods
hydrophilic channel and the 3D-printed parts can be used
several and several times. Thus, considering that the 3D-printed
mFIA platform presented here is inexpensive, fully integrated,
fabricated in an automated way, and consumes extremely low
volumes of samples and reagents, we can affirm that our
approach is a very interesting alternative for electrochemical
ow analysis and a promising novel application of 3D printing
in Analytical Chemistry. In addition, this mTED showed excellent
analytical performance and satisfactory sensitivity for the
determination of nitrite in drinking water, suggesting feasibility
for application in the determination of other electroactive
species.
4. Conclusions
We demonstrated in this work, for the rst time, a thread-based
microuidic device containing an embedded electrochemical
detector fully fabricated by FDM 3D printing. This platform was
combined with cotton threads and employed as a simple and
inexpensive mFIA analytical system. The novel additively man-
ufactured mTED described here showed some advantages that
include fabrication in an automated way, feasibility for large-
scale implementation, low consumption of samples and
reagents and low waste generation. Moreover, this mFIA plat-
form does not require external and expensive pumps to work.
As a proof of concept, the analytical usefulness of the mFIA
system proposed here was tested in the quantication of nitrite
in drinking water. To the best of our knowledge, this is the rst
analytical method regarding the use of a mTED for the deter-
mination of nitrite ions. All the experimental and instrumental
parameters were studied and optimized. Under the best
conditions, the mTED presented great precision (RSD of 2.1%),
excellent reproducibility (RSD of 2.5%) and good stability (RSD
of 4.0%). Furthermore, the LOQ achieved (7.61 mmol L
1) was
almost three-fold lower than the maximum contaminant level
established by the Brazilian and US legislation regarding the
presence of nitrite in drinking waters. The mFIA platform was
applied for the analysis of nitrite in well water samples and the
results showed great precision and accuracy. The validation of
the method was performed with ion chromatography. Thus,
according to the data reported here, this novel 3D-printed mFIA
platform proved to be an interesting and advantageous alter-
native for electrochemical ow studies since it is fully inte-
grated, easily fabricated, inexpensive, provides rapid analyses in
few steps and complies with the green chemistry concept. This
way, the device proposed here has promising potential to be
explored in other electrochemical/electroanalytical ow
applications.
Author contributions
Rayan Marcel Carvalho: investigation, methodology, conceptu-
alization, data curation, formal analysis, validation, soware,
Valdir Souza Ferreira: resources, project administration, fund-
ing acquisition, Bruno Gabriel Lucca: conceptualization,
methodology, investigation, resources, data curation, supervi-
sion, writing – original dra, writing – review & editing.
1356 | Anal. Methods, 2021, 13, 1349–1357
Paper
Conflicts of interest
There are no conicts to declare.
Acknowledgements
The authors would like to thank the nancial support given by
Conselho Nacional de Desenvolvimento Cientı́co e Tec-
nológico (CNPq) and Coordenação de Aperfeiçoamento de
Pessoal de Nı́vel Superior – Brasil (CAPES) – Finance code 001.
The authors also thank the Multiuser Laboratory for Analysis of
Materials (MULTILAM) of the Physics Institute (INFI) of the
Federal University of Mato Grosso do Sul (UFMS) by the SEM
images.
References
1 M. J. Beauchamp, A. V. Nielsen, H. Gong, G. P. Nordin and
A. T. Woolley, Anal. Chem., 2019, 91, 7418–7425.
2 N. M. Neris, R. D. Guevara, A. Gonzalez and F. A. Gomez,
Electrophoresis, 2019, 40, 296–303.
3 B. Nasseri, N. Soleimani, N. Rabiee, A. Kalbasi, M. Karimi
and M. R. Hamblin, Biosens. Bioelectron., 2018, 117, 112–128.
4 K. Kant, M.-A. Shahbazi, V. P. Dave, T. A. Ngo,
V. A. Chidambara, L. Q. Than, D. D. Bang and A. Wolff,
Biotechnol. Adv., 2018, 36, 1003–1024.
5 P. de T. Garcia, E. F. M. Gabriel, G. S. Pessôa, J. C. Santos
Júnior, P. C. Mollo Filho, R. B. F. Guidugli, N. F. Höehr,
M. A. Z. Arruda and W. K. T. Coltro, Anal. Chim. Acta, 2017,
974, 69–74.
6 C. Qian, S. Wu, H. Chen, X. Zhang, R. Jing, L. Shen, X. Wang,
S. Ju, C. Jia and H. Cong, Clin. Exp. Med., 2018, 18, 191–202.
7 M. Almeida, B. M. Jayawardane, S. D. Kolev and
I. D. McKelvie, Talanta, 2018, 177, 176–190.
8 A. W. Martinez, S. T. Phillips, M. J. Butte and
G. M. Whitesides, Angew. Chem., Int. Ed. Engl., 2007, 46,
1318–1320.
9 J. C. Jokerst, J. A. Adkins, B. Bisha, M. M. Mentele,
L. D. Goodridge and C. S. Henry, Anal. Chem., 2012, 84,
2900–2907.
10 C. K. Chiang, A. Kurniawan, C. Y. Kao and M. J. Wang,
Talanta, 2019, 194, 837–845.
11 L. G. Aguilar, J. M. Petroni, V. S. Ferreira and B. G. Lucca,
Talanta, 2020, 219, 121246.
12 D. Agustini, L. Fedalto, D. Agustini, L. G. de Matos dos
Santos, C. E. Banks, M. F. Bergamini and L. H. Marcolino-
Junior, Sens. Actuators, B, 2020, 304, 127177.
13 L. M. Ochiai, D. Agustini, L. C. S. Figueiredo-Filho,
C. E. Banks, L. H. Marcolino-Junior and M. F. Bergamini,
Sens. Actuators, B, 2017, 241, 978–984.
14 X. Li, J. Tian and W. Shen, ACS Appl. Mater. Interfaces, 2010,
2, 1–6.
15 N. Dossi, R. Toniolo, F. Terzi, N. Sdrigotti, F. Tubaro and
G. Bontempelli, Anal. Chim. Acta, 2018, 1040, 74–80.
16 R. S. Martin, A. J. Gawron, S. M. Lunte and C. S. Henry, Anal.
Chem., 2000, 72, 3196–3202.
17 J. Wang, Talanta, 2002, 56, 223–231.
This journal is © The Royal Society of Chemistry 2021
Paper
18 N. C. de Moraes, E. N. T. da Silva, J. M. Petroni, V. S. Ferreira
and B. G. Lucca, Electrophoresis, 2020, 41, 278–286.
19 H. H. Hamzah, S. A. Shaee, A. Abdalla and B. A. Patel,
Electrochem. Commun., 2018, 96, 27–31.
20 A. Ambrosi and M. Pumera, Chem. Soc. Rev., 2016, 45, 2740–
2755.
21 R. M. Cardoso, D. M. H. Mendonça, W. P. Silva,
M. N. T. Silva, E. Nossol, R. A. B. da Silva, E. M. Richter
and R. A. A. Muñoz, Anal. Chim. Acta, 2018, 1033, 49–57.
22 A. D. Ambaye, K. K. Kefeni, S. B. Mishra, E. N. Nxumalo and
B. Ntsendwana, Talanta, 2021, 225, 121951.
23 R. G. Rocha, R. M. Cardoso, P. J. Zambiazi, S. V. F. Castro,
T. V. B. Ferraz, G. O. Aparecido, J. A. Bonacin,
R. A. A. Munoz and E. M. Richter, Anal. Chim. Acta, 2020,
1132, 1–9.
24 E. M. Richter, D. P. Rocha, R. M. Cardoso, E. M. Keefe,
C. W. Foster, R. A. A. Munoz and C. E. Banks, Anal. Chem.,
2019, 91, 12844–12851.
25 R. M. Cardoso, P. R. L. Silva, A. P. Lima, D. P. Rocha,
T. C. Oliveira, T. M. do Prado, E. L. Fava, O. Fatibello-
Filho, E. M. Richter and R. A. A. Muñoz, Sens. Actuators, B,
2020, 307, 127621.
26 D. Agustini, M. F. Bergamini and L. H. Marcolino-Junior, Lab
Chip, 2016, 16, 345–352.
27 C. S. Bruning-Fann and J. B. Kaneene, Vet. Hum. Toxicol.,
1993, 35, 521–538.
28 Y. Shen, C. Ma, S. Zhang, P. Li, W. Zhu, X. Zhang, J. Gao,
H. Song, D. Chen, D. Pang and A. Li, Sci. Total Environ.,
2020, 742, 140622.
29 D. Agustini, M. F. Bergamini and L. H. Marcolino-Junior,
Anal. Chim. Acta, 2017, 951, 108–115.
30 R. Mitchell, C. M. Carr, M. Partt, J. C. Vickerman and
C. Jones, Cellulose, 2005, 12, 629–639.
31 G. R. Gamble, J. Agric. Food Chem., 2003, 51, 7995–7998.
32 J. M. Petroni, B. G. Lucca and V. S. Ferreira, Anal. Chim. Acta,
2017, 954, 88–96.
33 T. Zhe, X. Sun, Q. Wang, Y. Liu, R. Li, F. Li and L. Wang,
Mikrochim. Acta, 2019, 186, 319.
34 J. M. Petroni, B. G. Lucca and V. S. Ferreira, Electroanalysis,
2017, 29, 1762–1771.
This journal is © The Royal Society of Chemistry 2021
Analytical Methods
35 Y. Zhang, J. Nie, H. Wei, H. Xu, Q. Wang, Y. Cong, J. Tao,
Y. Zhang, L. Chu, Y. Zhou and X. Wu, Sens. Actuators, B,
2018, 258, 1107–1116.
36 X. Li, N. Zou, Z. Wang, Y. Sun, H. Li, C. Gao, T. Wang and
X. Wang, Chem. Zvesti, 2020, 74, 441–449.
37 F. M. de Oliveira, E. I. de Melo and R. A. B. da Silva, Sens.
Actuators, B, 2020, 321.
38 V. Katseli, A. Economou and C. Kokkinos, Electrochem.
Commun., 2019, 103, 100–103.
39 V. Katseli, A. Economou and C. Kokkinos, Talanta, 2020, 208,
120388.
40 J. Lankelma, Z. Nie, E. Carrilho and G. M. Whitesides, Anal.
Chem., 2012, 84, 4147–4152.
41 D. R. Ballerini, X. Li and W. Shen, Biomicrouidics, 2011, 5,
014105.
42 S. S. Banerjee, A. Roychowdhury, N. Taneja, R. Janrao,
J. Khandare and D. Paul, Sens. Actuators, B, 2013, 186, 439–
445.
43 J. Mocak, A. M. Bond, S. Mitchell and G. Scollary, Pure Appl.
Chem., 1997, 69, 297–328.
44 M. Shariati-Rad, M. Irandoust and S. Mohammadi,
Spectrochim. Acta, Part A, 2015, 149, 190–195.
45 C. Zhou, Y. Mu, M. Yang, Q. Song, Y. Zhang, Z. Wu, L. Xiang,
W. Jin and Q. Jin, Talanta, 2013, 112, 95–100.
46 A. Wisitsoraat, P. Sritongkham, C. Karuwan,
D. Phokharatkul, T. Maturos and A. Tuantranont, Biosens.
Bioelectron., 2010, 26, 1514–1520.
47 E. A. Carneiro, D. Agustini, L. C. S. Figueiredo-Filho,
C. E. Banks, L. H. Marcolino-Junior and M. F. Bergamini,
Electroanalysis, 2018, 30, 101–108.
48 A. Diouf, N. El Bari and B. Bouchikhi, Talanta, 2020, 209,
120577.
49 X. Li, G. Luo, H. Xie, Y. Niu, X. Li, R. Zou, Y. Xi, Y. Xiong,
W. Sun and G. Li, Mikrochim. Acta, 2019, 186, 304.
50 M. Ghanei-Motlagh and M. A. Taher, Biosens. Bioelectron.,
2018, 109, 279–285.
51 K. Promsuwan, P. Thavarungkul, P. Kanatharana and
W. Limbut, Electrochim. Acta, 2017, 232, 357–369.
52 F. Tian, H. Li, M. Li, C. Li, Y. Lei and B. Yang, Synth. Met.,
2017, 226, 148–156.
Anal. Methods, 2021, 13, 1349–1357 | 1357