Rapid taurine and lactate biomarkers determination with disposable electrochemical detectors

Article abstract of DOI:10.1016/j.electacta.2017.04.100

Taurine and lactate are biomarkers used for the diagnosis of different pathologies. In this study, a simple analytical approach for the direct determination of taurine and lactate in tears and saliva is described. The method is based on the use of single

Full text of DOI:10.1016/j.electacta.2017.04.100

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Title: Rapid taurine and lactate biomarkers determination with
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Authors: M. Revenga-Parra, E. Martınez-Perinan, B. Moreno,
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Rapid taurine and lactate biomarkers determination with
disposable electrochemical detectors
M. Revenga-Parraa,b,c, E. Martínez-Periñána,b, B. Morenoa, F. Parientea,c, E. Lorenzoa,b,c*
.
a
Departamento de Química Analítica y Análisis Instrumental, Universidad Autónoma de Madrid,
28049 Madrid, Spain.
b IMDEA-Nanoscience. Faraday 9, Campus Cantoblanco-UAM, 28049 Madrid, Spain
cInstitute for Advanced Research in Chemical Sciences (IAdChem), Universidad Autónoma de
Madrid, 28049 Madrid, Spain
*Corresponding author. Tel.: +34 91 497 4488; Fax: +34 91 497 4931.
E-mail address: encarnacion.lorenzo@uam.es (E. Lorenzo)
Postal address: Facultad de Ciencias. c/ Francisco Tomás y Valiente, 7. Departamento de Química Analítica y
Análisis Instrumental. Universidad Autónoma de Madrid, 28049, Madrid, Spain.
1

Abstract
Taurine and lactate are biomarkers used for the diagnosis of different pathologies. In this study, a simple
analytical approach for the direct determination of taurine and lactate in tears and saliva is described. The
method is based on the use of single wall carbon nanotubes screen printed electrodes modified with films
derived from the electropolymerization of a new Schiff base metallate, as liquid chromatography
electrochemical detector. The new Schiff base metallate is the complex of nickel(II) with the ligand N,N’-
bis(2,4-dihydroxybenzylidene)-1,2-diaminobenzene ([NiII(2,4DHS)]), which is electropolymerized in alkaline
solution to give electroactive films strongly adhered on the electrode surface. These films present a potent and
stable electrocatalytic activity toward the oxidation of taurine and lactate. Hence, the resulting modified
electrodes, coupled to a chromatographic system, allow the quantification of these biomarkers directly in
biological samples without need of derivatization schemes or sample pre-treatment.
Keywords: Schiff base complexes; ; ; , biomarkers, taurine electrocatalysis, lactate electrocatalysis.
2

1. Introduction
The development of highly sensitive methods for accurate quantification of different biomarkers is receiving
considerable attention in clinical diagnosis and biomedical research. Among known biomarkers, taurine and
L-lactate are of great interest, since their presence in biological fluid or tissues are related to important diseases.
Taurine (2-aminoethane sulphonic acid) is a natural organic acid present in a great number of living organisms.
This compound contains a sulphonic acid group, rather than the typical carboxilic acid moiety characteristic
in the conventional amino acids. Because of this, taurine cannot be incorporated into the proteins and therefore
it is probably the most abundant free amino acid present in a number of mammalian tissues including retina,
skeletal, cardiac muscles and brain [1]. In healthy human adults, taurine is a product of methionine and cysteine
catabolism. In addition, it can be absorbed from foods containing proteins from skeletal muscle. Although the
best-known function of taurine is conjugation of bile acids, it also plays an important role in numerous
biological and physiological processes [2, 3]. Among other metabolic actions attributed to taurine it may be
included: modulation of the intracellular calcium ion homeostasis [4], protection against the ischemia
reperfusion injury [5], it is a natural antioxidant [6] and depicts antiatheratogenic effects [7]. Indeed, deviations
from normal taurine concentrations in tissues are correlated with several pathological disorders such as
Alzheimer’s disease, growth retardation, diabetes mellitus [8-9], epilepsy [10], sepsis [11] and several kinds
of cancer [12-14] as well as different forms of psychosis including trauma [15], depression [16] and
schizophrenia [17]. Recently, it has been found that high levels of taurine in tears also has a close relationship
with the ocular surface disease and can be used to a biomarker to prevent this pathology [18]. Thus, the rapid
and accurate determination of the level of this compound in various tissues and body fluids can be of great
interest in the early diagnosis of several important pathologies and diseases.
The main problem for the development of a method for the determination of taurine is the lack of a powerful
fluorophore or an electroactive group in its structure, that allows a direct spectrophotometric or electrochemical
assay. This implies that usually its determination requires a prior derivatization step, which is complicated,
increases the analysis time and involves a previous labor-intensive. Most of the methods described for the
determination of taurine, use chromatography with spectrometric detection [8,19-23] and capillary
electrophoresis (CE) [24-27]. However, electrochemical detectors have been less employed despite their high
sensitivity, low cost, high reproducibility and possibility of miniaturization [28-32]. This is because the taurine
3

determination, in most of these cases, is based on its direct oxidation at the electrode surface, which requires
high overpotentials and gives rise to reaction products that causes electrode fouling. Both things adversely
affect the sensitivity and reproducibility of the determination. The use of suitable modified electrodes can
overcome these drawbacks as it will be described below.
L-Lactate is a metabolite originated in the anaerobic metabolism and its presence causes an increase in the
proton concentration inside the cells. In situations where the rate of lactate production is high enough that
proton buffering capacity of cells may be exceeded, the cellular pH values decrease and this may result in cell
acidosis [33]. Lactate levels in plasma reflect the equilibrium between the lactate production and lactate
clearance systems [34]. Any disorder that causes an imbalance in this equilibrium may lead to lactic acidosis.
This may occur in some conditions involving the anaerobic metabolism, such as an excessive exercise,
hemorrhagic shock or pulmonary embolism [34]. Lactate levels also increase in several pathological conditions
including: respiratory failure [35], liver disease, cardiogenic [36] or endotoxic [37] shocks, renal failure [38]
and tissue hypoxia [39] and metastases in cancer [40]. Accordingly, high lactate levels may act as signal alarm
for the diagnosis of the patient conditions in operating rooms, intensive care units or point of care stations [41].
Hence, the need of devices capable to determine lactate with rapid response, minimal or without sample
preparation and low cost in samples where other potentially interfering compounds are present is evident.
Disposable and sensitive electrochemical detectors in HPLC systems, based on modified electrodes, could be
a good alternative to develop methods meeting the above-mentioned characteristics, such as those based on
metal/metal oxide modified electrodes [42-44]. Recently the incorporation of carbon nanostructures, such as
graphene nanosheets and mostly carbon nanotubes, has improved the performance of these kind of modified
electrode in the determination of carbohydrates, amino acids and related compounds [45-48].
In this work, we have developed a very effective taurine and lactate determination method based on the use of
a new modified screen printed carbon electrode as electrochemical detector in HPLC systems. The screen
printed electrode containing single wall carbon nanotubes was modified with electropolymerized films of a
new Schiff base nickel(II) complex, formed with the ligand N,N’-bis(2,4-dihydroxybenzylidene)-1,2-
diaminobenzene and NiII. The resulting Ni based modified electrode depicts a potent and persistent
electrocatalytic activity towards the oxidation of taurine and lactate in alkaline medium allowing, coupled to
4

a chromatographic system, the quantification of these biomarkers in biological samples without derivatization
schemes.
2. Experimental section
2.1 Reagents and Apparatus
1,2-diaminobenzene, 2,4-dihydroxybenzaldehyde, taurine, L-(+)-lactic acid lithium salt 97%,
acetonitrile, ethanol and sodium hydroxide were obtained from Sigma-Aldrich Co. and were used as
received. Ni(NO3)2 and acetone was obtained from Scharlau. Water was purified with a Millipore
Milli-Q system and all the experiments were carried out at room temperature. Stock solutions
containing taurine or lactate were freshly prepared daily in 0.1 M NaOH aqueous solution.
All electrochemical measurements were carried out using an Autolab potentiostat/galvanostat type PGSTAT
302N (Eco Chemie, Netherlands) and the software package GPES 4.9. Integrated single walled carbon
nanotubes modified screen printed electrodes (SWCNTSPEs) from Dropsens, including a silver
pseudoreference electrode and a carbon counter electrode were used. A wall jet flow cell from Dropsens
(model FLWCL) was employed for on-line analysis. The supporting electrolyte was 0.1 M NaOH.
HPLC experiments were performed with a modular chromatograph (Jasco) coupled with an AUTOLAB
potentiostast. Optimum chromatographic conditions were achieved using a Dionex AminoPac PA10 anion-
exchange analytical column (4x250 mm) from Thermo Fisher with a mobile phase of 0.1 M NaOH plus 3%
acetonitrile as organic modifier. The HPLC system was run isocratically at 0.75 mL min-1 flow rate.
2.2 Preparation of NiII-(N,N’-bis(2,4-dihydroxybenzylidene)-1,2-diaminobenzene) ([NiII(2,4DHS)])
The ligand, N,N’-bis(2,4-dihydroxybenzylidene)-1,2-diaminobenzene (2,4DHS), was synthetized according
to the usual synthesis procedure of tetradentate Schiff base ligands [49]. 1,2-diaminobenzene (0.1080 g; 1.00
mmol) was mixed with 2,4-dihydroxybenzaldehyde (0.2762 g; 2.00 mmol) in refluxing ethanol for 3 h. The
precipitate obtained was filtered and washed several times with ethanol and ether. Finally, the product was
recrystallized by ethanol (0.2446 g, 70%). IR (KBr): νC=N 1610 cm−1; νAr-O-H 1308 cm−1; νC=C 1481 cm−1; νAr-H
740 cm−1; UV–vis in acetone: λmax 362 nm.
5

2.0 mM solutions of the Ni complex [NiII(2,4DHS)] were prepared by addition of solid Ni(NO3)2
crystals into a 2.0 mM solution of 2,4DHS in acetone under stirring. The rapid change in the color of
the solution from brightly yellow to deep orange evidenced the complex formation.
2.3 Preparation of [NiII(2,4DHS)]/SWCNT modified screen printed electrode
SWCNTSPEs were modified with [NiII(2,4DHS)] complex by immersing the electrode in 0.1 M
NaOH solution containing 0.1 mM [NiII(2,4DHS)] and cycling the potential between +0.2 V and +0.8
V at a scan rate of 0.1 V s-1. The resulting modified electrode was then conditioned in 0.1 M NaOH
solution by potential cycling from +0.2 V to +0.8 V at a scan rate of 0.1 V s-1 for about 10 min (50
cycles of potential scans).
2.4 Determination of biomarkers in real samples
For taurine and lactate quantification in healthy volunteers’ tears and saliva samples, respectively, standard
addition method was used. Samples (0.10 mL of tears or saliva) were spiked with suitable amounts of the
analyte, diluted in 0.50 mL of 0.1 M NaOH and injected into the HPLC system for analysis.
3. Results and discussion
3.1 Spectrophotometric characterization of [NiII(2,4DHS)]
[NiII(2,4DHS)] was prepared by mixing Ni(NO3)2 crystals with a solution of 2,4DHS in acetone as described
in experimental section. The complex formation was corroborated by UV-vis spectroscopy. As can be observed
in Fig. 1, the absorbance spectrum of 2,4DHS shows a maximum at 362 nm, which corresponds to the π→π*
transition of the electrons present in the azomethine group [50]. When [NiII(2,4DHS)] complex was formed, a
red shift of this absorption band is observed up to 382 nm. In addition, a new broad absorption band due to the
metal to ligand charge transfer transition appears. The stoichiometry and the formation constant of the complex
were determined using the spectral data obtained from solutions containing a constant concentration of ligand
and different concentrations of metal. From these results (data not shown), it was found a 1:1 stoichiometric
ratio and a formation constant of 6.0x107.
Figure 1
3.2 Electropolymerization of [NiII(2,4DHS)] on SWCNTSPE
6

[NiII(2,4DHS)] was electropolymerized onto single walled carbon nanotubes modified screen printed
electrodes (SWCNTSPEs) by cycling the potential from +0.2 to +0.8 V at 0.1 V s-1 in a solution 0.1 mM
[NiII(2,4DHS)] in 0.1 M NaOH. Figure 2A shows the electroactive film growth pattern obtained under
continuous potential cycling. The anodic and cathodic peaks observed are due to the Ni2+/Ni3+ redox couple
present in the polymeric film. The current increases gradually with the consecutive potential cycling suggesting
the growth of the film. During the
electropolymerization process, the charge measured under the anodic voltammetric wave increases linearly
with the continuous potential cycling during the first 50 cycles and then remains practically constant (inset
Fig. 2A). Thus, 50 potential cycles were applied as optimal in the electrode modification step.
Figure 2
If after several potential cycles, the electrode is removed from the electrochemical cell, rinsed with water and
placed in a 0.1 M NaOH containing no nickel-complex, the voltammetric response from +0.2 to +0.8 V depicts
the typical response of the Ni2+/Ni3+ redox couple, with well-defined anodic (at +0.39 V) and cathodic peaks
(at +0.59 V) (see Fig. 2B). This fact confirms that the electrode surface is covered with a stable [NiII(2,4DHS)]
electroactive film. A significant peak-to-peak separation (ΔEp) of 120 mV is observed. This behavior has
been observed with other Ni based complex deposited on electrode surface [51] and suggests that counter-ion
transport (OH-) is the limiting step. The change of the Ni redox-state inside the film should involve the
diffusional transport of the counter-ion into/out-of the film necessary to maintain electroneutrality [52]. A
larger hysteresis in the voltammetry is indeed expected when the counter-ion diffusion becomes rate limiting.
In fact, as one would expect when the charge transfer is limited by the diffusion of OH- ions towards the film,
peak currents present a linear dependence with the square root of the potential scan rate over the range of 0.01-
0.40 V s-1 (Fig. 3B). If the potential is cycled at scan rates higher than 0.50 V s-1, the peak-to-peak separation
increases (Fig. 3C). This electrochemical behavior is indicative of serious limitations in the charge transfer
kinetics under these conditions.
Figure 3
On the other hand, the peak current density of Ni2+ electrooxidation increases in an approximately linear way
with the number of cycles. Thus, we used the peak current density (Ip) at 0.10 V s-1 as a measurement of the
7

film thickness. For 50 electrodeposition cycles, the value of Ip was found to be 1.3  0.1 mA cm-2. This value
is about 2 times higher than that obtained when a screen-printed carbon electrode without carbon nanotubes
was modified with the [NiII(2,4DHS)] following the same procedure. In addition, the reproducibility of Ip
(evaluated from the response of 10 different electrodes) is much better than that obtained without carbon
nanotubes. A relative standard deviation of 7% was calculated compared to 40% without nanotubes.
For HPLC electrochemical detector application, one of the properties to be evaluated for modified electrodes
is their stability. In order to check the stability of the developed modified electrode, the potential was
continuously cycled from +0.2 to +0.8 V at 0.1 V s-1 and the anodic Ip for 50 electrodeposition cycles was
evaluated after each cycle. Figure 2B (inset) depicts the anodic Ip as a function of the number of potential
cycles. As can be seen, after a few conditioning scans, the Ip remains practically constant, suggesting a strong
adherence of the film to the electrode surface and therefore a great stability of the modified electrode. In fact,
the electrochemical response of the [NiII(2,4DHS)]/SWCNTSPE remains constant for several weeks, if it is
stored under dry conditions.
3.3 Electrocatalytic propierties of [NiII(2,4DHS)]/SWCNTSPE
Nickel metal centers have shown high electrocatalytic activity in basic media towards the oxidation of organic
compounds containing hydroxyl groups and sulfur derivatives [53]. Hence, [NiII(2,4DHS)]/SWCNTSPE can
be valuable in designing new electrocatalytic and sensing platforms. With this purpose in mind, the
electrocatalytic activity of [NiII(2,4DHS)]/SWCNTSPE towards the oxidation of two important biomarkers,
taurine and lactate, was evaluated (Fig. 4). In solutions where no biomarker was added, the well-defined
voltammetric response of the Ni2+/Ni3+ redox couple is evident (Fig. 4A and 4B, curve a). Upon addition of
taurine, a dramatic increase in the anodic peak current concomitant with a decrease in the cathodic current is
observed (Fig. 4A, curve b). This behavior is characteristic of an electrocatalytic effect and indicates that the
film redox centers confined at the electrode surface participate in the oxidation of taurine, which causes a
decrease in the number of Ni(III) centers and the consequent decrease in the cathodic current in the backward
scan.
In the case of lactate, although it was also observed a strong electrocatalytic effect it is slightly different. As
can be seen in Fig. 4B (curve b), in the presence of lactate there is also a dramatic increase in the anodic peak
8

current. However, at the reverse scan no reduction peak is observed. A decrease in current is also evident at
potential above +0.65 V probably due to mass transport effects. At bare SWCNTSPE (insets in Fig. 4A and
4B) no oxidation peaks were observed neither for taurine nor for lactate.
The above results confirm that electropolymerization of [NiII(2,4DHS)] onto SWCNTSPE gives rise to films
with a great density of redox centers, which results in a high electrocatalytic activity towards the oxidation of
taurine and lactate.
Figure 4
It is well known that carbon nanotubes, besides increase the effective electroactive area, may play a key role
on avoiding electrode fouling. To asses if this is the case, similar experiments to those described above were
carried out with screen printed carbon electrodes without carbon nanotubes. In this case, on successive taurine
measurements, a significant shift (±100 mV) in the catalytic peak potential is observed. This effect, which is
not observed when carbon nanotubes are present, confirms the antifouling effect of CNTs.
The electrocatalytic responses for taurine and lactate at different scan rates (v: from 0.002 to 0.1 V s-1) confirm
that, in both cases, the plot of ICAT/v1/2 vs v shows the characteristic shape of an EC electrocatalytic process. In
addition, the catalytic current increases linearly with the scan rate indicating that the process is controlled by
the diffusion of the biomarker (Fig. 5).
Figure 5
The catalytic constants were estimated by chronoamperometry following the method of Galus [54]. This
procedure is based on the equation:
ICAT


exp( )
 1/ 2
  1/ 2 erf ( 1/ 2 ) 



IRES

Where ICAT and IRES are the currents at the modified electrode and in the presence and in the absence of
biomarkers, respectively.  is the argument of the error function described as:  = kC*t, where C* is the bulk
concentration of the biomarker in the solution and k is the catalytic rate constant. If  exceeds 2, the error
function is almost equal to one and the above equation can be simplified to:
9

ICAT
1/ 2
 1/ 2 1/ 2


kC*t

IRES
Where t is the elapsed time in seconds. From this equation, the catalytic rate constant as a function of the
biomarker concentration can be estimated by plotting ICAT/IRES vs t1/2. A linear dependence with the square root
of time was obtained for each concentration of taurine and lactate studied. From the slopes of these plots,
values of kCAT for taurine and lactate were found to be (6.2±0.5) x102 and (4.4±0.7) x102 M-1 s-1, respectively.
3.4 [NiII(2,4DHS)]/SWCNTSPE as amperometric detector in flow systems
Considering the high electrocatalytic and reproducible response of the as prepared
[NiII(2,4DHS)]/SWCNTSPE towards the oxidation of taurine and lactate, it can be employed as
electrochemical detector in a FIA or HPLC system for the determination of both biomarkers in complex
matrixes.
The operational potential for the [NiII(2,4DHS)]/SWCNTSPE towards the oxidation of taurine was evaluated
from the hydrodynamic curve (Fig. S1). The maximum response was found to be around +0.6 V, which agrees
well with the electrocatalytic behavior depicted in Fig.4. At potentials, higher than +0.6V the current response
exhibits a noticeable decrease probably due to the competition reaction of the excess of hydroxide ions present
at the electrode surface, with the subsequent oxygen evolution [55-57]. Thus, a value of +0.55 V, at which the
catalytic /background current ratio is the highest, was chosen as operational potential for the following flow-
through measurements. Similar behavior was observed for lactate.
Taurine and lactate FIA responses showed very good peak shapes (Fig. S2). Moreover, peak currents are
dependent on the biomarker concentration in solution in a wide linear range, confirming the utility of the
[NiII(2,4DHS)]/SWCNTSPE as a amperometric detector for both biomarkers. The detection and quantification
limits, obtained according their usual definition, were found to be of 0.38 µM and 7.6 µM for taurine and
lactate, respectively. Relative standard deviations less than 7.5% were obtained for the determination of 100
µM of each analyte with three different modified electrodes, which can be considered a good reproducibility
(Table S1).
3.5 [NiII(2,4DHS)]/SWCNTSPE - HPLC for taurine and lactate determination in real samples.
10

In the last years, the importance of monitoring biomarkers in non-blood media is increasing in parallel with
the demand for non-invasive analysis. Taurine and lactate are biomarkers, related to several pathologies, that
can be present in abnormal levels in urine, saliva and tears, besides in blood. Therefore, the development of
non-invasive analytical method for the determination of these biomarkers is a deal great of interest. Hence, we
have developed a method for direct determination of taurine and lactate in tears and saliva, using the
[NiII(2,4DHS)]/SWCNTSPE as amperometric detector in a HPLC system. We have optimized all the
parameters, such as the influence of the mobile phase composition and flow rate, which is a crucial step on a
good chromatographic separation. The optimization has been evaluated in terms of electrochemical signal and
retention times. Best results were obtained when a 0.1 M NaOH solution containing acetonitrile (3%) as
organic modifier and a flow rate of 0.75 mL min-1 were used. Fig. 6 shows the chromatogram of a mixture of
taurine and lactate. As can be seen, two separated peaks, symmetric with minimal peak tailing, corresponding
to the two analytes are observed. A summary of the analytical figures of merit obtained is presented in Table
1.
Figure 6
The detection limit, calculated from 3:1 signal-to-noise ratio of the standard deviation of the lowest
concentration signal, was found to be of 0.97 µM and 4.74 µM for taurine and lactate, respectively. After
injecting five replicates of a solution containing the two compounds, relative standard deviation values
between 1.3-4.0% were obtained, demonstrating the good repeatability of [NiII(2,4DHS)]/SWCNTSPE and no
evidence of fouling or deactivation. The reproducibility of the method was evaluated from chromatograms
obtained for a mixture of 20 µM taurine and 50 µM lactate, using three different [NiII(2,4DHS)]/SWCNTSPEs
as detectors. A relative standard deviation lower than 9% was calculated. These values demonstrate that the
method is reliable and valuable to quantify both biomarkers.
The method developed compared to other reported in the bibliography for the determination of lactate or
taurine shows in general improved detection limits (see Table 2S).
The applicability of the developed method was demonstrated by the direct determination of taurine and lactate
in tears and saliva samples, respectively, from healthy volunteers. Prior to the determination, 0.10 mL of the
sample were diluted in 0.50 mL of 0.1 M NaOH. A typical chromatogram for a sample of tears or saliva is
11

shown in Fig. 7A and 7B, respectively. Peaks at 8.8 min (Fig. 7A) and 5.1 min (Fig. 7B) were identified as
taurine and lactate, respectively, by adding analyte standard solutions into the respective sample.
Figure 7
A recovery study of taurine and lactate was carried out to evaluate the accuracy of the method. For this purpose,
the samples were spiked with a known amount of each analyte and analyzed in triplicate. The results are
presented in Table 2. The good recoveries indicate that the developed method is adequate and accurate,
providing a suitable detection method for the analysis of these biomarkers related to different pathologies in
biological samples.
4. Conclusions
We have developed a simple analytical method for direct determination of two important biomarkers, taurine
and lactate, in tears and saliva, respectively. A new Schiff base metallate is prepared just by addition of solid
Ni(NO3)2 crystals into a solution of N,N’-bis(2,4-dihydroxybenzylidene)-1,2-diaminobenzene (2,4DHS), in
acetone. The nickel complex is electropolymerized onto the single walled carbon nanotubes modified screen
printed electrodes. The electrocatalytic effect of the resulting modified electrodes towards the oxidation of
taurine and lactate is exploited to develop a chromatographic method to determine these biomarkers, using the
developed modified electrodes as electrochemical detectors. The method represents a significant advance that
provides an alternative and attractive solution to an important clinical problem, since determination of these
biomarkers in biological fluids by non-invasive methods helps the rapid diagnosis of several pathologies.
Acknowledgments
The authors acknowledge Ministerio de Economía y Competitividad (project No. CTQ2014-53334-C2-1-R
and CTQ2015-71955-REDT) and Comunidad de Madrid (NANOAVANSENS Program) for financial support.
E.L. gratefully acknowledges the Fulbright scholarship-Salvador de Madariaga program from the Ministerio
de Economía y Competitividad.
12

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19

Figure captions
Fig. 1. Absorption spectra of a solution containing 30 µM 2,4DHS (line a), 2.0 mM Ni(NO3)2 (line b) and 30
µM [NiII(2,4DHS)] (structure inset) (line c) in acetone.
Fig. 2. (A) Cyclic voltammograms of 0.1 mM of [NiII(2,4DHS)] complex at SWCNTSP electrode in 0.1 M
NaOH during modification of the electrode surface. The inset shows the plot of the anodic charge vs the number
of electropolymerizing scans. (B) Cyclic voltammogram of [NiII(2,4DHS)] /SWCNTSPE obtained after 50
electropolymerizing scans in 0.1 M NaOH. The inset shows the anodic peak current density dependence on
the number of continuous cyclic potential scans for a film derived from [NiII(2,4DHS)]. Scan rate 0.1 V s-1.
Fig. 3. (A) Cyclic voltammetric response of [NiII(2,4DHS)]/SWCNTSPE in 0.1 M NaOH at different scan
rates. The inset (B) shows the dependence of the anodic and cathodic peak current on the square root of the
scan rate. The inset (C) shows the dependence of the peak potential on the logarithm of scan rate.
Fig. 4. Cyclic voltammograms of [NiII(2,4DHS)]/SWCNTSPE in 0.1 M NaOH in the absence (curve
a) or in the presence (curve b) of 5.00 mM of taurine (A) or 0.1 M of lactate (B). In the insets, cyclic
voltammograms of bare SWCNTSPE in 0.1 M NaOH in the presence of 5.00 mM of taurine (A) or
0.1 M of lactate (B). Scan rate 0.005 V s-1.
Fig. 5. Variation of the electrocatalytic current (ICAT) with the square root of the scan rate (A) and
variation of the scan rate-normalized current (ICAT /v1/2) with the scan rate (B) for
[NiII(2,4DHS)]/SWCNTSPE in 0.1 M NaOH containing taurine, as an example.
Fig. 6. Typical chromatograms obtained using [NiII(2,4DHS)]/SWCNTSPE as electrochemical
detector for standard mixture solutions of taurine and lactate at different concentrations.
Chromatographic conditions: AminoPac PA10 column; 0.1 M NaOH and 3% acetonitrile mobile
phase; 0.75 mL min-1 flow rate; 20 µL injection volume; +0.55 V detection potential.
Fig. 7. Chromatograms showing the profile of spiked tears (A) and saliva (B) sample using
[NiII(2,4DHS)]/SWCNTSPE for electrochemical detection. Other conditions as in Fig. 4.
20

1.00
0.75
0.50
0.25
0.00
OH
c
N
O
O
Ni
N
a
OH
b
350
400
450
500
550
 / nm
Figure 1
21

200
100
0
B
A
50
100
0
Scans
1.4
1.2
1.0
0.8
150
120
90
60
30
0
-100
-200
-100
0
20
40
60
80
100
0
20
40
60
80
Number of cycles
Number of cycles
0.2
0.4
0.6
0.8
1.0
1.2
0.2
0.4
0.6
0.8
1.0
1.2
E / V
E / V
Figure 2
22

800
400
0
600
300
0
A
B
0.40 V s-1
Scan rate
-300
0.01 V s-1
0.2
0.4
0.6
1/2
v1/2 / (Vs-1)
1.2
0.8
0.4
0.0
C
-400
-3
-2
-1
0
log (v / V s-1)
0.0
0.3
0.6
0.9
E / V
Figure 3
23

180
120
60
10
5
10
5
60
30
0
b
b
0
0.0
0
0.0
0.2
0.4
0.6
0.8
0.2
0.4
0.6
0.8
a
a
0
A
B
0.8
0.2
0.4
0.6
0.2
0.4
0.6
E / V
E / V
Figure 4
24

v1/2 / (mV s-1)1/2
0
2
4
6
8
10
200
160
120
80
A
B
40
30
20
10
0
20
40
60
80
100
v / mV s-1
Figure 5
25

Taurine
0.3
0.2
0.1
0.0
150 µM
20.0 µM
200 µM
Lactate
20.0 µM
0
2
4
6
8
t / min
Figure 6
26

A
50 nA
Taurine
0
3
6
9
B
50 nA
Lactate
0
2
4
6
t / min
Figure 7
Table 1. Analytical parameters for the sequential determination of taurine and lactate using the
[NiII(2,4DHS)]/SWCNTSPE as amperometric detector in HPLC
Sensitivity
Detection Limit Quantification
Limit Linear
(µM)
range
Biomarker
(µC mM-1 cm-2)
(µM)
0.97
4.74
(µM)
3.22
15.8
Taurine
Lactate
106±9
15±2
up to 160
up to 210
Table 2. Determination of biomarkers in real samples
Recovery % ± SD*
Taurine
Lactate
Sample
Tears
112±5
-
-
Saliva
94±7
*SD: Standard deviation of three determinations
27