12 (2022) 100275
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Biosensors and Bioelectronics: X
journal homepage: www.journals.elsevier.com/biosensors-and-bioelectronics-x
Electrochemical sensing of the maple syrup urine disease biomarker valine,
using saffron-silver nanoparticles
Sophia Karastogianni *, Ioanna Paraschi, Stella Girousi **
Aristotle University of Thessaloniki, Analytical Chemistry Laboratory, Chemistry Department, 54124, Thessaloniki, Greece
A R T I C L E I N F O
A B S T R A C T
Keywords:
Electrochemical sensing
Saffron
Silver nanoparticles
Urine samples
Maple syrup disease
Valine
Metabolic disorders are inherited disorders in which genetic defects prevent a metabolic pathway and cause
enzymes to malfunction. Maple syrup urine disease (MSUD) is a rare metabolic disease marked by high levels of
branched-chain amino acids (b-AAs), leucine, isoleucine, and valine. Elevated concentrations of b-AAs pose is­
sues including liver failure, neurocognitive impairment, and mortality. Given the unavoidable repercussions for
newborns, it is critical to establish quick and adaptable diagnoses in the early stages of life. Thus, this study
highlights the development of a novel electrochemical sensor for valine detection (MSDU biomarker) based on a
modified carbon paste electrode (CPE) with electropolymerized silver nanoparticles capped with saffron
(AgNPs@Sa) synthesized using a green method. Thus, a modified carbon paste electrode (CPE) with a conductive
polymer posing silver nanoparticles was employed to sensitively determine Val in this paper. Saffron capped with
silver nanoparticles (AgNPs@Sa) were synthesized, using a green method and studied using cyclic and square
wave voltammetry. Cyclic voltammetry (CV) was used in the electropolymerization of AgNPs@Sa on CPE (polyAgNPs@Sa-CPE) and utilized in the detection of Val. The modified electrode under the selected conditions
produced square wave signals with valine mass concentrations ranging from 0.258 to 11.94 ng L− 1 in the case of
buffer solutions and it was successfully applied in urine samples. The assay proved to be simple, rapid, and costeffective, sensitive with a low detection limit of 0.085 and 0.097 ng L− 1 for buffer and urine samples,
respectively.
1. Introduction
Metabolic disorders differ widely in terms of symptoms, prognosis,
and specific breakdown in catabolic pathways. These disorders are not
strictly classified, and the most popular classification takes into account
the principal compound that is affected (carbohydrates, fatty acids,
amino acids, and organic acids) [Jumbo-Lucioni et al., 2012; Burrage
et al., 2014].
Maple syrup urine disease (MSUD) is a rare chronic and progressive
disease caused by a defect in the branched-chain keto acid dehydroge­
nase complex (bkAD), which is involved in the catabolic pathway of
branched-chain amino acids (b-AAs), leucine (Leu) isoleucine (ileu), and
valine (Val) [Blackburn et al., 2017]. MSUD has five distinct phenotypes
with no obvious genotype-phenotype relationship. MSUD is classified
based on the age of onset, the severity of symptoms, the responsiveness
to thiamine supplementation, and biochemical findings [Blackburn
et al., 2017]. MSUD caused by E3 deficiency is most common in infants,
but intermediate, intermittent, and thiamine-responsive MSUD can
occur at any age [Blackburn et al., 2017]. To avoid these concerns, it is
critical to obtain a prompt and accurate diagnosis during the prime
periods of life [Blackburn et al., 2017, Burrage et al., 2014, García-­
Carmona et al. 2019, Scott 2006].
MSUD is characterized by neurological and developmental de­
ficiencies, encephalopathy, eating difficulties, and a maple syrup smell
in the urine. Elevated plasma b-AAs biochemically describe MSUD and
all b-AAs and allo-isoleucine are routinely examined, and inmates
generally have satisfactory clinical outcomes in addition to timely
therapy [Burrage et al., 2014; García-Carmona et al., 2019a].
Tandem mass spectrometry (MS/MS), nuclear magnetic resonance
(NMR), enzyme activity assays, high-pressure liquid chromatography
(HPLC), capillary electrophoresis (CE), and genetic testing are the most
prevalent methods for determining Val [Azuma et al., 2016, Castellanos
et al., 2016, Delgado-Povedano et al., 2016, Fernández-Del-Campo-­
García et al., 2019, Le et al., 2019, Song et al., 2013, Synaridou et al.,
* Corresponding author.
** Corresponding author.
E-mail addresses: skarastogianni@hotmail.com, karastos@thea.auth.gr (S. Karastogianni), girousi@chem.auth.g (S. Girousi).
https://doi.org/10.1016/j.biosx.2022.100275
Received 4 August 2022; Received in revised form 24 October 2022; Accepted 25 October 2022
Available online 4 November 2022
2590-1370/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
S. Karastogianni et al.
Biosensors and Bioelectronics: X 12 (2022) 100275
2021, Tuma and Gojda, 2015, Ulusoy et al., 2016, Wei et al., 2017,
Wolak-Dinsmore et al., 2018], using various separation and detection
methods. Regardless, all these methods are time-consuming and, in
certain situations, require a large sample volume.
Another factor that complicates the diagnosis of MSUD is the fact
that newborn surveillance criteria vary by country and even within the
same country, making the diagnosis of MSUD more difficult. Further­
more, there is no cure, although dietary limitations are recommended,
and frequent monitoring of the target molecule should be done
throughout the patient’s life to minimize negative effects. In concluding,
Song et al. [Song et al., 2018] extensively reviewed current advance­
ments in standard amino acid analysis methods in biological samples,
providing thorough information on their analytical features.
Problems in MSUD diagnosis and monitoring, as well as the deter­
mination of b-AAs, highlight the need for new diagnostic procedures
that are simple to use and require small sample volumes, which is
especially relevant in neonates [Lv et al., 2019; Sandlers 2017]. Because
of their ease of use, simplicity, selectivity, sensitivity, and low cost,
electrochemical sensors represent a reliable means of determining
metabolic biomarkers to facilitate their use in the diagnosis and moni­
toring of rare diseases [Dincer et al., 2017; Zhang et al., 2016].
Interestingly, electrochemical sensors have hardly been put to
practical use for the determination of MSUD biomarker Val. This is
evidenced by the fact that Val is relatively electrochemically inactive on
bare electrodes [Xinying 2014]. This constraint can be overcome by
utilizing electrode modifiers like metal nanoparticles, polymer nano­
particles, and metals.
Consequently, Val had been detected using iron oxides [Hasanzadeh
et al., 2013], multiwalled carbon nanotubes (MWCNT) [Saghatforoush
et al., 2011], cobalt nanoparticles (CoNPs) [Hasanzadeh et al., 2009],
vertically aligned nickel nanowires [García-Carmona et al., 2018],
nickel oxide nanoparticles (NiONPs) [Tooley et al., 2018], modified
screen-printed electrodes (SPE) with iron [Naqvi et al., 2020], polymer
nanocrystals [Bi et al., 2016], enzyme-based microfluidic chip coupled
with graphene electrodes [García-Carmona et al., 2019b] with sufficient
analytical features. In summary, Karastogianni et al. 2020 extensively
reviewed current advancements in electrochemical sensing of MSUD
biomarkers like Val, providing thorough information on their analytical
features.
Conducting polymers (CP) are increasingly being used as sensitive
electrode surface modifiers on electrochemical sensors and biosensors
[Cosnier and Lepellec 2003]. They are differentiated by their strong
electrical conductivity and electrochemical reversibility, permitting
them to be used in sensor transducer signaling. Moreover, CPs can
generate functional groups that could act as "tags" to recognize biolog­
ical or chemical entities [Garnier 1989]. Yet, detecting small (bioactive)
analytes such as Val remains a challenge because their interaction with
detectable groups on the CP substrate is inadequate to create the
required electrochemical changes for detection.
The objective in this respect was to construct particularly specific CP
recognition sites, enhancing the selectivity and sensitivity of the iden­
tification method. Silver nanoparticles can be employed in the fabrica­
tion of polymers generated by electrochemical techniques, and their
advantages comprise biocompatibility, excellent conductivity, and a
large surface-to-volume ratio [Rezaei et al., 2008]. As an outcome, they
are especially inspiring tools in electrochemical sensing and biosensing
[Barnes et al., 2003; Karastogianni and Girousi 2017; Lai et al., 2013; Li
et al., 2013; Papaioannou et al., 2022; Yang et al., 2010], since they can
be extensively used in modifying the surface of electrodes, establishing
approaches for detecting species biologically relevant, or fabricating
diagnostic elements for a variety of pathological states.
The novelty of this study lies in the fact that it is the first attempt to
determine valine using square wave voltammetry (SWV). Additionally,
the proposed electrochemical sensor was found to have high sensitivity,
as it presents a lower detection limit than existing methodologies re­
ported in the literature. Furthermore, the proposed procedure was
successfully applied in urine samples, indicating the potential applica­
bility of the sensor to real sample analysis. Moreover, the novelty of this
paper is the use of silver nanoparticles, fabricated with a simple, costeffective as well as green technology and successfully used in the con­
struction of an electrochemical sensor that selectively and sensitively
detects valine both in buffer solution and urine samples. Therefore,
silver nanoparticles capped with saffron seem to be a promising tool in
sensing since it was successfully applied by our team both in the
detection of Valine (present study) and mephedrone [Papaioannou
et al., 2022]. Finally, this work led to the improvement of the properties
of carbon paste electrodes as well as the accurate application of elec­
trochemical techniques in real sample analysis.
2. Material and methods
Unless otherwise noted, all reagents were analytical grade and were
used exactly as they were received. Merck provided the tris hydrox­
ymethyl amino-methane (Tris 99.8%, ACS), ethyl-diamino-tetra-acetic
acid (EDTA, ACS reagent, 99.4–100.06%), potassium dihydrogen
phosphate, and dipotassium hydrogen phosphate (Darmstadt, Ger­
many). Sigma-Aldrich (Saint Louis, MO, USA) provided mineral oil (IR
spectroscopy), sodium hydroxide, hydrochloric acid, nitric acid.
Graphite powder was purchased from Fluka (USA) (50870, p.a. purity
99.9% and particle size <0.1 mm, synthetic). Saffron was purchased
from a local store. As previously reported (Karastogianni and Girousi
2017; Papaioannou et al., 2022], silver nanoparticles were synthesized.
Briefly, saffron and silver nitrate (1:1 mass ratio) was dispersed in 25 mL
of NaOH aqueous solution (pH 10.0) in a 100 mL beaker with stirring,
for 3 min, and then exposed to the sun for 30 min in static condition. The
color of the mixture turned from wine red to brownish-yellow, showing
that silver nanoparticles were formatted. Afterward, the AgNPs@Sa was
preserved at ambient temperature, washed with water and ethanol, and
dried in a vacuum desiccator before being utilized. Deionized water was
used to make all of the aqueous solutions.
A Palm Sens potentiostat/galvanostat model 1 was used for vol­
tammetric experiments (Echo Chemie, The Netherlands). The electrodes
in a traditional three-electrode cell were a platinum wire counter elec­
trode, a 3 mol L− 1 KCl saturated Ag/AgCl reference electrode, a carbon
paste electrode (CPE) with a 3 mm inner and 9 mm outer diameter of the
PTFE sleeve, as well as the proposed modified electrode working elec­
trodes. All of the tests were carried out at room temperature. A Consort
C830 pH meter was used to determine the pH of all solutions (Consort
bvba, Turnhout, Belgium). The electrochemical cells were washed and
rinsed with deionized water after being cleaned with diluted nitric acid.
Prior to each experiment, ultrapure nitrogen was utilized to de-aerate
the liquids by purging the dissolved oxygen for 15 min.
Hand blending the right amount of graphite and paraffin oil in a mass
ratio of 75/25 resulted in the carbon paste electrode (CPE) [Svancara
et al., 2012]. The PTFE sleeve was filled with a portion of the resulting
mixture. Before use, the surface was polished smooth by hand on a piece
of weight paper. Stainless steel screws were used to make electrical
contact.
Using cyclic voltammetry (CV), modified CPE with AgNPs@Sa was
created by depositing the electropolymerized silver nanoparticles on the
surface of the CPE (poly-AgNPs@Sa-CPE). In the AgNPs@Sa solution
(0.100 mol L− 1 acetate buffer pH 5.6 containing 0.010 mol L− 1 NaNO3),
cyclic voltammetric electrodeposition from − 0.300 to + 1.300 V for one
cycle with a scan rate of 0.200 V s− 1 and a step potential of 0.005 V was
performed to immobilize the polymeric form of silver nanoparticles
capped with saffron on the surface of the CPE (poly-AgNPs@Sa-CPE).
The electrode was then dried for 5 min at room temperature.
Following the electrodeposition of poly-AgNPs@Sa on CPE, an in­
cubation step was performed and the dissolving solution of Val (0.100
mol L− 1 Tris-HCl buffer pH 8.0) was stirred for 180 s without applying a
potential to allow Val to be transferred to the proposed modified elec­
trode (Val-poly-AgNPs@Sa-CPE).
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The prepared Val-poly-AgNPs@Sa-CPE was then transferred to the
measurement solution (0.100 mol L− 1 buffer solution pH 4.0), where
signal transduction was accomplished using square wave voltammetry
and anodic scanning of the working electrode potential between − 0.300
and +1.300 V with a step potential of 0.005 V, a pulse potential of 0.035
V, and a frequency of 5 Hz. The voltammograms that accompanied each
measurement correspond to the oxidation peak of saffron-capped silver
nanoparticles and the oxidation occurred at about +0.330 V.
The raw data were processed using the PalmSens software’s Savitzky
and Golay filter (level 2), followed by a moving average baseline
correction with a peak width of 0.03. After the CPE surface was regen­
erated by cutting and polishing the electrode, replicated measurements
were taken.
A member of our laboratory team volunteered to provide urine
samples. The urine sample was first filtered. The mixture was then
diluted at 1:100 with 0.100 mol L− 1 acetate buffer pH 5.6 containing
0.010 mol L− 1 NaNO3, and sonicated for 10 min. Following that,
accompanied with a 45 mm Millipore membrane was conducted, fol­
lowed by a 1:25 dilution and then another 10 min in the ultrasonic bath.
The background concentration in the blank matrices was reduced by
diluting the urine samples before spiking with standards [Thakare et al.,
2016]. A known volume of a standard Val solution of 1.000 μg L− 1 was
added to each of seven volumetric flasks (50 mL) holding urine samples.
To the first one, no standard solution was added. The oxidation signal of
the above-mentioned solutions was used to construct the calibration
curve.
of the two irreversible oxidation peaks at about +0.369 and + 0.800 V
decrease progressively up to the fifteenth scan cycle. The current
response of the reduction peak approximately at +0.100 V also de­
creases progressively with increasing the number of scan cycles.
Furthermore, above the fifth scan cycle, the current reduction peak
disappears. These oxidation and reduction peaks could be attributed to
the oxidation and reduction of crocin, respectively [Armellini et al.,
2017; Dar et al., 2017].
This behavior of poly-AgNPs@Sa cannot necessarily be related to the
destruction of their formed polymeric film in CPE (peroxidation) at the
relatively high potentials of the experiment, nor to the fact that the
formed polymeric film on the CPE can remain electro-inactive and either
prevent the oxidation of an additional monomer from the solution or is
exposed to a small number of reactive monomers (depleted reservoir),
which is adjacent to the working electrode diffusion layer [Kannan et L.
2011, Velusamy et al., 2011; Wang et al., 2014]. This behavior may be
due to the process of structural reorganization carried out in the growing
polymer layer adjacent to the working electrode diffusion layer
[Velusamy et al., 2011]. . Meanwhile, the potential shifts to more
negative prices. This means that different species are formed at the
electrode, which is possibly due to the process of structural reorgani­
zation which is carried out in the growing polymer film [Martins et al.,
2005].
According to R. Armellini et al., 2017, crocin oxidation occurs via
two distinct one-electron oxidation pathways in ethanol:acetonitrile
(1:1) solvent system with LiClO4 as supporting electrolyte solution.
Furthermore, the creation of the free radical species, crocin., might be
attributed to the first irreversible oxidation peak at around +0.360 V
(Fig. 1). The peak current was then abruptly increased to approximately
+0.800 V, which corresponds to the crocin radical that was further
oxidized to crocin2+. Only a minor cathodic wave occurred at +0.100 V,
approximately +260 mV less negative than the previous anodic peak,
consistent with a pseudo-reversible mechanism [Jorgensen et al., 1997].
In order to clarify this issue, it is necessary to study the polymer film
in a solution that does not contain the monomer, so that any response of
current arises solely due to the connection of Sa@AgNPs to the surface of
CPEThe electrochemical response of the poly-Sa@AgNPs, formed on the
CPE surface was studied in the absence of monomer in 0.1 mol L− 1 ac­
etate buffer pH 4.0 using cyclic voltammetry. The polymer film gave two
oxidation peaks at about +0.290 and + 0.890 V and two reduction peaks
at about +0.100 and + 0.200 V, see Fig. S1. These peaks were similar to
those given by the polymerization solution with the difference that an
additional reduction peak also appeared around +0.896 V, see Fig. S1.
The oxidation and reduction peaks are attributed to the oxidation and
reduction of crocin, respectively, located in the polymer film [Armellini
et al., 2017; Dar et al., 2017]. In addition, it can be seen from the cyclic
voltammograms in Fig. S1 that the current response of the oxidation and
the reduction peaks remain almost constant with increasing the number
of scan cycles, and the potential of the oxidation and reduction peaks
slightly shifts to more positive values with increasing the number of scan
cycles. It is noted that the response of the second oxidation peak in­
creases until the ninth scan cycle of the potential (turquoise curve,
Fig. S1) and then decreases and finally disappears. This reduction is
probably due to the total consumption of crocin free radicals and
availability for further oxidation to [crocin]2+ [Armellini et al., 2017]
and the partial breakdown of the polymeric film [Mansouri Majd et al.,
2013].
Saffron capped with silver nanoparticles was cycled through twenty
scans consecutively to evaluate the existence of passivation events in its
oxidation process. The change in peak current between the first and last
oxidation peaks was found to be less than 3%, indicating a minor
passivation impact. Furthermore, when cycling the potential with a scan
rate ranging from 15 to 100 mV s− 1, the resulting peak current changed
linearly with the square root of the scan rate, consistent with a diffusioncontrolled oxidation process [Bard and Faulkner, 2001, Wopschall and
Shain 1967].
3. Results and discussion
3.1. Electropolymerization of silver nanoparticles
Poly-AgNPs@Sa was potentiodynamially polymerized on CPE with a
scan rate of 20 mV s− 1 and a potential range of − 0.300 V to +1.300 V vs.
Ag/AgCl. It’s worth noting that it wasn’t applied a pretreatment step
was not used on CPE [Kang et al., 2002; Sheberla et al., 2015]. Fig. 1
shows the CVs of poly-AgNPs@Sa on CPE, which demonstrate progres­
sive decreases in the peak current over the polymerization process.
Furthermore, it can be seen in the figure that the peak current responses
Fig. 1. Electrochemical deposition of AgNPs@Sa in acetate buffer pH 5.6
containing 0.010 mol L− 1 NaNO3 at 0.200 V s− 1 on CPE. (a): number of scan 1,
(b) number of scan 3, (c): number of scan 5, (d): number of scan 7, (e): number
of scan 10, and (g): number of scan 15. Other experimental conditions as
mentioned in the materials and methods section.
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The peak current intensity of Val on the modified electrode was
substantially pH-dependent, peaking at pH = 5.6. Peaks were absent at
pH levels greater than 7.6. Furthermore, the peak potential as a function
of pH revealed values similar to those predicted for a proton and twoelectron (ΔE/ΔpH) = (24,32 ± 0,668) mV/pH at 25 ◦ C).
The stability of poly-AgNPs@Sa was studied, Fig. S1. It was found
that the formatted film was stable since it did not lose its reactivity even
after two weeks in the air or after multiple cycles (up to 15 scans) with
scan rates ranging from 5 to 50 mV s− 1. Furthermore, organic solvents
such as ethanol, acetone, chloroform, acetonitrile, and dimethyl sulf­
oxide had no effect on it (data are not shown). These findings support a
stiff structure and, most likely, a large molar mass. Instead, when kept in
the refrigerator, the film deteriorated.
The electrochemical characteristics of the modified electrode were
also investigated using cyclic voltammetry in the presence of 1.000 ×
10− 3 M K3[Fe(CN)6] containing 0.200 mol L− 1 KCl, Fig. S2. The CPE
exhibit two distinct peaks, with an anodic peak potential of +0.309 V
and a cathodic peak potential of +0.174 V (black curve, Fig. 2), while
the AgNPs@Sa-CPE gave an oxidation peak at +0.339 V and a reduction
peak at +0.154 V (pink curve, Fig. S2). Peak current rose for the polyAgNPs@Sa-CPE electrode (pink curve, Fig. S2), whereas the peak po­
tential difference between the anodic and cathodic peaks increased from
135 mV to 185 mV.
The result illustrates that poly-AgNPs@Sa covered the surface of CPE
and favoured the oxidation and reduction of the electroactive indicator.
The enhanced peak current was caused by a pinhole effect, which occurs
when larger pores are present and permits [Fe(CN)6]4–/3– to diffuse
through the poly-AgNPs@Sa film and to the electrode surface [Bonné
et al., 2009]. The increased peak current intensity, also, suggested that
the produced electrode had higher electrochemical activity due to its
bigger electroactive surface [Poriel et al., 2004]. The electroactive sur­
face area (A) of the poly-AgNPs@Sa-CPE electrode (Randles-Sevcik
equation) was calculated to be 0.0220 cm2 and 0.0480 cm2 for CPE
[Papaioannou et al., 2022] and poly-AgNPs@Sa-CPE, respectively. Once
those two parameters were contrasted, it is clear that adding
poly-AgNPs@Sa to CPE increased its electroactive surface area.
3.2. Morphology of Sa@AgNPs and poly-AgNPs@Sa-CPE
The morphological characteristics of CPE (Fig. 2a), AgNPs@Sa-CPE
(Fig. 2b, c, 2d, 2e) were studied by scanning electron microscopy
(SEM). As shown in Fig. 2b, c, 2d, and 2e, spherical-shaped Ag nano­
particles were synthesized. Fig. 2b and c shows the layer particles found
in the form of organized aggregates. In addition, Fig. 2a and b, 2c show
morphological differences between CPE and poly-AgNPs@Sa-CPE,
confirming the deposition of AgNPs@Sa on the surface of CPE. The
Fig. 2. SEM images of (a) CPE, (b), (c), (d), (e) AgSa@NPs-CPE at different magnifications and (f) EDX diagram of AgSa@NPs-CPE
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CPE electrode (Fig. 2a) is characterized by irregularly shaped graphite
particles with significant cohesion and small pore size. The polymer film
is homogeneously distributed in the mineral oil on the poly-AgNPs@SaCPE electrode with a fairly compact structure (Fig. 2b and c), as is also
the case with CPE (Fig. 2a), but with larger pores. The size of the par­
ticles varied as shown in Fig. 2d and e. Smaller-sized particles were seen,
but their dimension could not be precisely calculated with the existing
organology.
The compositional analysis of AgNPs@Sa-CPE was performed by
energy dispersive X-ray (EDX) analysis (Fig. 2f). EDX data (Fig. 2f)
showed the presence of silver in the synthesized material, confirming the
inclusion of the metal in the saffron structure. The silver content re­
ported in the EDX analysis was determined on a small portion of the
sample, assuming its uniform composition.
AgNPs@Sa starts with the electrooxidation where the first oxidation
peak of polyAgNPs@Sa may be attributed to the formation of the free
radical species, probably [AgNPs-crocin -Sa]. Afterward, coupling oc­
curs between [AgNPs-crocin.-Sa] and the [AgNPs-crocin2+-Sa], formed
from the subsequent oxidation of [AgNPs-crocin.], [Papaioannou et al.,
2022; Armellini et al., 2017; Jorgensen et al., 1997]. Then, termination
of the polymerization happens and polyAgNPs-[crocin]n-Sa is formed.
On the other hand, the oxidation of valine on CPE gives two low
intense oxidation peaks at about +0.120 and + 0.260 V. The first
oxidation peak could be ascribed to the oxidation of Val, accordingly to
equation 1 [Xinying, 2014] which is also in agreement with the results
from cyclic voltammetry.
In addition, the second peak could be ascribed to the oxidation of
[AgNPs-(crocin)n-Sa] [Papaioannou et al., 2022].
3.3. Electrochemical behavior of valine
3.4. Analytical performance of the proposed sensor
Square wave voltammetry (SWV) was used to evaluate the electro­
chemical characteristics of Val on CPE and poly-AgNPs@Sa-CPE in pH
4.0 acetate buffer. Anodic square wave voltammograms of CPE (Fig. 3a),
of Val on CPE (Fig. 3, b), of AgNPs@Sa-CPE, and Val on poly-AgNPs@SaCPE (Fig. 3, green curve) are given in Fig. 3. Val on CPE produced an
oxidation peak with a low peak current at about an oxidation peak with
a low peak current at approximately +0.294 V. (Fig. 3, black curve). It
should be noted that when Val was tested on poly-AgNPs@Sa-CPE, an
anodic peak was observed at +0.345 V, which had a bigger peak current
than Val on CPE (Fig. 3, green curve). These findings suggest that Val
interacted with AgNPs@Sa-CPE. To summarize, the presence of silver
nanoparticles improved the sensitivity of the detection of Val.
The electrochemical behavior of crocin expresses its capacity to
donate electrons accordingly to Armellini et al., 2017. Therefore, the
anodic process of crocin occurs through two one-electron oxidation and
one proton process processes [Papaioannou et al., 2022; Armellini et al.,
2017; Jorgensen et al., 1997]. Based on that, electropolymerization of
SWV was used in the developed electrochemical sensor to determine
Val in solution under the selected conditions. The sensor’s oxidation
currents rose as the concentration increased (Fig. 4). With a correlation
coefficient of 0.9997, the calibration graph revealed a linear relation­
ship between the SWV peak current and the Val mass concentration
ranging from 2.575 10− 10 g L− 1 to 1.194 10− 8 g L− 1. Equation y (A) =
17.041(±0.078)x (g L− 1) + 1.000 10− 7(±4.388 10− 10) was the linear
regression relationship derived from the SWV calibration curve. The
detection limit was calculated as 3sb/slope, where sb represents the
standard deviation of the blank and slope represents the slope of the
calibration curve. The detection limit was determined to be 8.498 10− 11
g L− 1.
The relative standard deviations of Val at 9.199 10− 9 g L− 1 and 1.194
− 8
10
ng L− 1 were 4.3% and 4.0%, respectively, showing sufficient
reproducibility of the assay (intraday precision). As demonstrated in
Table 1, the linear range for the Val of the proposed sensor is greater
than that obtained in various earlier reports. Furthermore, the detection
limit is much lower than the electroanalytical methods published in the
literature, as shown in Table 1.
The selectivity of the modified electrode was also investigated. The
results showed that the 100 times of K+, Na+, Ca2+, Fe3+, Cu2+, Cl− ,
NO−3 , sucrose, glucose, fructose, citric acid, ascorbic acid, leucine,
isoleucine, had no obvious influence on the results of the determination
of vanillin within the ±5% error (Table S1). Other amino acids such as
phenylalanine, and tryptophane due to their different oxidation poten­
tials, did not affect its determination.
3.5. Application in real samples
By using the standard addition method, the suggested assay was used
to determine Val in urine samples, demonstrating the sensor’s applica­
bility in real sample analysis. Equation I(A) = (32.60 ± 0.478)x (ng L− 1)
+ (8.450 10− 8±9.550 10− 10) with a linear correlation coefficient of
0.9997 was found to be the regression equation for the urine samples
(Fig. 5).
The LOD was calculated to be 0.097 ng L− 1. The standard deviation
between replicates of the same Val content in urine samples did
demonstrate some heterogeneity within the data.
There was a lot of variation in measurements, making it difficult to
accurately determine Val concentrations. This procedure, though, could
be used to confirm the presence of Val in mass concentrations greater
than 0.258 ng L− 1 in aqueous samples and 0.293 ng mL− 1 in urine
samples.
The different slopes of the calibration curve in buffer solutions and
urinary spiked samples indicate the influence of the matrix effect.
Therefore, this difference was calculated and estimated to be 15.56 ng
L− 1 A− 1. This value is low and indicative that the matrix impact is
minimal [Paul et al., 2014].
Fig. 3. Anodic square wave voltammograms of CPE (a) 0.4 ng L-1 Val on CPE
(b) 0.4 ng L-1 Val on poly-AgNPs@Sa-CPE (c) in acetate buffer pH 4.0.
Experimental conditions as described in the material and methods section.
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Fig. 4. (A) Anodic square wave voltammograms of poly-AgNPs@Sa-CPE using the selected conditions for a mass concentration between 2.575 10-10 g L-1 to 1.194
10-8 g L-1, of Val: 2.575 10-10 g L-1 (a), 9.199 10-10 g L-1 (b), 3.475 10-9 g L-1 (c), 5.939 10-9 g L-1 (d), 7.187 10-9 g L-1 (e), 9.687 10-9 g L-1 (f) and 1.194 10-8 g L1 (g) mass concentration of Val. (B): Calibration curve. Experimental conditions as described in the material and methods section.
Table 1
Analytical features of selected Val detections.
Detection method
Electrode
Limit of
detection (LOD)
Linear range
Sample
Reference
HPLC-fluorescence
LC-MS/MS
-
0,002 μg/mL
11.5 10− 3 mg/L
(0.01–80) μg/mL
(38.3–200) μg/L
Chocolate
Water
Amperometry
Differential pulse
voltammetry
Differential pulse
voltammetry
Cyclic voltammetry
MCM-41-Fe2O3/GCEa
GCE/MWCNTsc
84 nM
1.67 μM
(200–510) nM
25–1000 μM
Human blood
-
Synaridou et al. (2021)
Fernández-Del-Campo-García
et al. (2019)
Hasanzadeh et al. (2013)
Saghatforoush et al. (2011)
Cobalt hydroxide on glassy carbon
electrode
Modified glassy carbon electrode and
platinum with nickel and iron oxide
nanoparticles and
Iron-modified screen-printed electrode on
glass slab
TOCNC/l-Cys/Aub
9.86 μM
(25–10000) μM
-
Hasanzadeh et al. (2009)
-
-
Phosphate
buffer saline
Tooley et al. (2018)
0.001 M
Buffer solution
pH 8.0
Blood
Naqvi et al. (2020)
-
(0.1–0.5) M and (10–100)
M
-
Bi et al. (2016)
Copper microwires electrodes
Silver nanoparticles capped with saffron
modified carbon paste electrode
0.006 mM
0.085 ng L− 1 (i.
e. 0.073 pM)
(0.02–3.3) mM
(0.258–11.94) ng L− 1 [(i.
e. 0.073–1019) pM]
Plasma
Urine
García-Carmona et al. 2019
This work
Cyclic voltammetry
Differential pulse
voltammetry
Chronoamperometry
Square wave
voltammetry
a
b
c
MCM-41-Fe2O3/GCE: Magnetic (Fe2O3) mobile crystalline material-41 (MCM-41) glassy carbon electrode (GCE).
2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized cellulose nanocrystals (TOCNCs) and l-cystine (l-Cys) modified Au electrode.
GCE/MWCNTs: glassy carbon/multiwall carbon nanotubes.
6
S. Karastogianni et al.
Biosensors and Bioelectronics: X 12 (2022) 100275
Fig. 5. Standard addition method curve of Val in urine samples. 0.00 ng L-1 (a), 0.220 ng L-1 (b), 0.400 ng L-1 (c), 1.000 ng L-1 (d), 1.398 ng L-1 (e), 2.984 ng L-1 (f),
and 3.984 ng L-1 (g) of Val in urine samples. Selected conditions as mentioned in the material and methods section. Other experimental conditions as mentioned in
the material and methods section.
4. Conclusions
Acknowledgments
In this paper, the development of valine’s (MSUD biomarker) elec­
trochemical sensor was described using silver nanoparticles capped with
saffron as carbon’s paste electrode modifier, having improved analytical
properties. The proposed sensor was the first to be reported. The
modified electrode had a broad linear range ranging from 0.258 to
11.94 ng L− 1 of valine mass concentrations in the case of buffer solu­
tions. The assay proved to be simple, rapid, and sensitive with a low
detection limit of 0.085 and 0.097 ng L− 1 for buffer and urine samples,
respectively. Due to their different oxidation potentials, the chemicals
with which valine could interact did not affect its determination.
However, there was variation in measurements making it difficult to
accurately determine valine. This procedure, though, could be used to
confirm the presence of valine at mass concentrations greater than
0.258 ng L− 1 in aqueous samples and 0.293 ng L− 1 in urine samples.
Thus, the proposed electrochemical sensor could be a promising tool
for the reliable and sensitive detection of valine in clinical samples and
therefore for the monitoring of MSUD. Methods currently used for the
detection of valine rely on the use of mass detectors, which are expen­
sive and cannot be adapted for in situ analysis. However, electro­
chemical methods benefit from the portable nature of the electrodes
used. In conclusion, the proposed approach can be effectively applied to
the analysis of real samples in complex matrices such as urine samples,
having been successfully applied to them, and therefore can be used in
the clinical diagnosis and monitoring of MSUD.
The authors wish to thank L. Papadopoulou for her precious help in
acquiring SEM images. “This research is co-financed by Greece and the
European Union (European Social Fund- ESF) through the Operational
Programme “Human Resources Development, Education and Lifelong
Learning” in the context of the project “Reinforcement of Postdoctoral
Researchers—2nd Cycle” (MIS-5033021), implemented by the State
Scholarships Foundation (ІΚΥ)”.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biosx.2022.100275.
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