Effect of GO nanosheets on spectrophotometric determination of tyrosine in urine and serum using nitrosonaphthol

Article abstract of DOI:10.1016/j.ab.2018.07.024

Here, we aimed to use graphene oxide to improve the selectivity and sensitivity of Tyr determination via the reaction with 1-nitroso-2-naphthol as a selective reagent of Tyr. The reaction between Tyr and 1-nitroso-2-naphthol in absence and presence of GO was studied spectrophotometrically. Different parameters such as concentrations, temperature, incubation time were optimized. The obtained data showed that the maximum absorbance was achieved by using 2 mL of 0.03% 1-nitroso-2-naphthol at temperature 60 °C for 10 min. On the basis of calibration curve of various concentrations of Tyr in the presence of 20 μg mL^?1 GO, the limit of detection was 6.4 × 10^?6 M (1.15 μg mL^?1), where in absence of GO was 1.1 × 10^?5 M (19.9 μg mL^?1). The selectivity of Tyr in presence of other amino acids and phenols was studied with and without GO. The data obtained revealed that the selectivity of Tyr in presence of GO with respect to some amino acids and phenols was improved. The proposed method has been applied for the determination of Tyr in urine and serum samples. Therefore, GO is a powerful catalytic surface for the sensitive and selective determination of Try in biological fluids.

Full text of DOI:10.1016/j.ab.2018.07.024

Accepted Manuscript
Effect of GO nanosheets on spectrophotometric determination of tyrosine in urine and
serum using nitrosonaphthol
Hossam M. Nassef, Mohamed Hagar, Abdelhameed M. Othman
PII:
S0003-2697(18)30667-5
10.1016/j.ab.2018.07.024
YABIO 13092
DOI:
Reference:
To appear in: Analytical Biochemistry
Received Date: 28 June 2018
Revised Date: 25 July 2018
Accepted Date: 26 July 2018
Please cite this article as: H.M. Nassef, M. Hagar, A.M. Othman, Effect of GO nanosheets on
spectrophotometric determination of tyrosine in urine and serum using nitrosonaphthol, Analytical
Biochemistry (2018), doi: 10.1016/j.ab.2018.07.024.
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ACCEPTED MANUSCRIPT
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
-0.2
1.0
0.8
0.6
0.4
0.2
0.0
(a)
(b)
4.0x10-5 8.0x10-5 1.2x10-4
Tyr concentration (M)
400
500
600
700
800
Wavelength (nm)
Electronic Spectra for the reaction of NN with different concentrations of Tyr in
presence of GO (solid lines) and in absence of GO (dashed lines); Inset:
Calibration curve (a) in presence of GO and (b) in absence of GO.

ACCEPTED MANUSCRIPT
Effect of GO nanosheets on spectrophotometric
determination of tyrosine in urine and serum using
nitrosonaphthol
Hossam M. Nassef 1,2, Mohamed Hagar 1,3, and Abdelhameed M. Othman 1,4*
1 Chemistry Department, Faculty of Science at Yanbu, Taibah University, Yanbu
46423, Saudi Arabia;
2 Chemistry Department, Faculty of Science, Damietta University, Damietta 34517,
Egypt
3 Chemistry Department, Faculty of Science, Alexandria University, Alexandria
23132, Egypt
4 Environmental Biotechnology Department, Genetic Engineering and Biotechnology
Research Institute,University of Sadat City, Sadat City 32897, Egypt
* Corresponding author: ayosefaly@taibahu.edu.sa; Tel.: +966-540-673-967.
hossamnassef2002@gmail.com (H.M.N.); mohamedhaggar@gmail.com (M.H.).
Abstract
Here, we aimed to use graphene oxide to improve the selectivity and sensitivity of Tyr
determination via the reaction with 1-nitroso-2-naphthol as a selective reagent of Tyr.
The reaction between Tyr and 1-nitroso-2-naphthol in absence and presence of GO
was studied spectrophotometrically. Different parameters such as concentrations,
temperature, incubation time were optimized. The obtained data showed that the
maximum absorbance was achieved by using 2 mL of 0.03 % 1-nitroso-2-naphthol at
temperature 60 °C for 10 min. On the basis of calibration curve of various
concentrations of Tyr in the presence of 20 µg mL-1 GO, the limit of detection was 6.4
x 10-6 M (1.15 µg mL-1), where in absence of GO was 1.1 x 10-5 M (19.9 µg mL-1).
The selectivity of Tyr in presence of other amino acids and phenols was studied with
and without GO. The data obtained revealed that the selectivity of Tyr in presence of
GO with respect to some amino acids and phenols was improved. The proposed
method has been applied for the determination of Tyr in urine and serum samples.
Therefore, GO is a powerful catalytic surface for the sensitive and selective
determination of Try in biological fluids.
Keywords: Graphene oxide; Spectrophotometric determination; Tyrosine; Amino
acid; Selectivity; Sensitivity.
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1. Introduction
Graphene oxide (GO) is an interesting nano-material, used in many
applications. Recently, using of graphene and graphene oxide in biosensing and
analytical application showed a great attention due to its unique properties of high
surface area, biocompatibility, good water dispersibility and high reactivity of the
oxygenated moieties present on its surface. This oxygenated lattice allows the
interaction of non-covalent bonds with amines and phenyls functional groups through
electrostatic interaction, π – π stacking and hydrogen bonding [1-5].
Tyrosine (Tyr) is one of the most important amino acid, which assists the
maintenance of a positive nitrogen in the body [6]. Tyr amino acid is involved in
several diseases such as alkaptonuria, albinism, mental illness, lung disease, liver
disease, and tyrosinemia. L-Tyrosine excretion in urine increased in patients suffering
from diabetes mellitus. Also, it can be used for early detection and screening for a
variety of cancer. So, the detection of Tyr in biological fluids as urine, serum and
saliva may help in the early detection of human cancers, and in follow up of the
treatment process of cancer diseases [7-10]. Various analytical methods have been
reported and designed for determination of Tyr amino acid including
spectrophotmetric [11-14], spectroflurometric [15-19], and electrochemical methods
[20-25]. Due to the presence of Tyr with other amino acids in biological fluids and in
pharmaceutical preparations, so the selective determination of Tyr is one of the
interesting issues for analysts.
In this study, we aimed to use GO nanosheets as one of the promising nano-
materials for improvement of the sensitivity and selectivity of Tyr determination
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during its reaction with NN reagent, which was firstly reported as a selective reagent
for determination of Tyr amino acid [26]. GO nanosheets is an excellent adsorbent for
amino acids and phenols on its surface. So, the idea of this work depends on
adsorbing of Tyr and other interfering amino acids or phenols on GO surface,
allowing the NN reagent to capture and react with Tyr amino acid as a selective
reagent to form colored product 1,2-benzo-8-alanyl-3-phenoxazone (BAPZ). This
product is not adsorbed on the GO surface and has the advantage of absorbing peak at
495 nm which is totally far from the absorption region of NN reagent at 379 nm. The
data obtained showed a significant effect of GO nanosheet in terms of enhancing the
sensitivity and selectivity, indicating that the GO may act as a catalyst for
improvement of the reaction process. Also, GO act as a micro-adsorbent for Tyr
amino acid and other interfering materials on its surface. By adjusting the reaction
conditions between Tyr and NN reagent the selectivity could be enhanced by holding
other interfering materials from the medium. This method was used for the
determination of Tyr in urine and serum samples.
2. Experimental
2.1. Material and Instruments
Graphene oxide was obtained from Sigma Co. (Munich, Germany). The particle
size of the GO was determined by Scanning Electron Microscopy (SEM-FEI, FEG
Quants 250, Hillsboro, OR, USA, magnification 20–1,000,000× high resolution). All
spectrophotometric measurements were carried out at 25 ± 1 °C using a UV-vis
spectrophotometer model (UV-1800 SHIMADZU, Nakagyo-KU, Kyoto, Japan).
Fourier Transform Infrared Spectroscopy (FTIR) was carried out using a Nicolet iS 10
Thermo scientific (Waltham, MA, USA). A bench-top sonicator of Model 1510 was
used to disperse graphene oxide in water (Branson, Danbury, CT, USA).The
Tyrosine, Catechol, Cysteine, Phenol, Pyrogallol, Resorcinol, Tryptophan, β-
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Naphthol, Sodium Hydroxide, Sodium Nitrite, Nitric acid, and Sulfuric acid were
delivered from Sigma Co. (Munich, Germany).
2.2. Methods
2.2.1. Prepaaration and characterization of Nitrosonaphthol (NN)
NN was prepared using the reported method [27, 28], (more details are presented
in supplementary data).
2.2.2. Preparation of BAPZ
BAPZ was prepared using the reported method [29, 30], (more details are
presented in supplementary data)
2.2.3. Using of GO for signal improvement of BAPZ
The reaction between NN and Tyrosine was fully characterized in presence and
absence of GO, using spectrophotometer (UV-1800 SHIMADZU, Nakagyo-KU,
Kyoto, Japan). UV-vis spectra were recorded in an aqueous solution of GO (0.2 µg mL-
1), Tyr (5x10-5 M), NN (0.03 % W/V), BAPZ and BAPZ-GO. BAPZ in absence of GO
showed a clear peak with maximum absorption wavelength at λmax 468 nm.
2.2.4. Effect of Tyrosine Concentration in absence and presence of GO
The great enhancement in the peak absorption, has been utilized to prepare a
calibration curve in absence and presence of GO, that can be used for Tyr
determination. A stock solution of 1x10-3 M of Tyr was prepared in pure water with
adding drops of 1 M NaOH till complete dissolution of Tyr. In absence of GO, 2 mL
of an aqueous solution of (0.03 % W/V) NN reagent was added to the corresponding
volumes (0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mL) of 1x10-3 M Tyr, followed by addition of
300 µL conc. HNO3, then the final volume was completed to 10 mL with distilled
o
water. The produced solution was heated at 60 C for 10 min. The electronic spectra
of the product, BAPZ, have been measured at different concentrations of Tyr (2x10-5-
1.2x10-4 M), the blank solution was containing NN reagent and HNO3. Warring: when
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nitric acid is added to these reaction mixtures, the major reaction could be competed
with other reactions such as nitration reaction [31].
In presence of GO, the same procedure has been used with addition of 200 µL of
(1mg mL-1) GO to the solution mixture of each concentration of Tyr. Then, the
electronic spectra were recorded. The blank solution was containing NN, GO and
HNO3.
2.2.5. Reaction of different interferents with NN in absence and presence of GO
The reaction of NN with different interferents such as Catechol, Cystine, Phenol,
Pyrogallol, Resorcinol, and Tryptophan has been studied in absence and presence of
GO. 2 mL (0.03 % W/V) NN was added to 5 mL of each interferent (to final
concentration 5x10-5 M), followed by addition of 300 µL conc. HNO3, then the final
volume was completed to 10 mL. The produced solution was heated for 10 min at 60
oC. Then, the electronic spectra were measured. The same method has been
completely repeated in presence of GO.
2.2.6. Effect of interferents on the determination of Tyrosine in absence and presence
of GO
The influence of the interferents on the BAPZ signal has been investigated in
absence and presence of GO. 2 mL (0.03 % W/V) NN was added to 0.6 mL (1x10-3
M) Tyr, then 5 mL (1x10-4 M) of each interferent was added, followed by addition of
300 µL conc. HNO3, then the final volume was completed to 10 mL. The produced
o
solution was heated for 10 min at 60 C. Then, the electronic spectra were recorded.
The same procedure has been completely repeated in presence of GO.
2.3 Application in real samples
2.3.1 Determination of Tyr in urine
Urine sample were collected from 3 healthy adult volunteers. To each 10 mL
urine sample, 1 mL of 4 M HCl was added, followed by addition of 1 mL acetonitrile.
o
The solution mixture was left for overnight in the refrigerator at 4 C. The mixture
was filtered and centrifuged at 5000 rpm for 10 min. 1 ml of each urine sample
solution spiked with Tyr was added to 2 mL (0.03 % W/V) NN followed by addition
of 1 ml of GO solution (20 µg/ml) and 300 µL conc. HNO3, then the final volume was
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completed to 10 mL. The produced solution was heated for 10 min at 60 oC. Then, the
electronic spectra were measured, and the Tyr quantity was calculated from the
calibration curve.
2.3.2 Determination of Tyr in serum solution
The application of the proposed method for determination of Tyr in serum was
performed by tacking 4.0 ml plasma of a real health three volunteers followed by
addition of 3.0 ml of citric acid solution (5 %). The solution was centrifuged for 10.0
min at 5000 rpm to remove proteins, then the supernatant serum solution was placed
in 10 mL volumetric flask. 2 mL (0.03 % W/V) NN was added to serum sample
followed by addition of 1 ml of GO solution (20 µg/ml) and 300 µL conc. HNO3, then
the final volume was completed to 10 mL with distilled water. The produced solution
o
was heated for 10 min at 60 C. Then, the electronic spectra were measured, and the
Tyr quantity was calculated from the calibration curve.
3. Results and discussion
The reaction of Tyr with 1-nitroso-2-naphthol (NN) had been used for
determination of Tyr before [26]. This reported method was chosen to study the effect
of GO nanosheets on the sensitivity and selectivity for Tyr amino acid. However, haze
white color was formed during the reaction with time, this fine precipitate is not easy
to be separated by centrifugation, making the method not accurate nor precise for the
determination of Tyr [26]. In this work we aim to use GO nanosheets as a catalyst and
micro-separating material for increasing the sensitivity and selectivity of Tyr
determination.
3.1. SEM of GO
Morphological studies of the GO were performed by SEM. The SEM
micrographs are shown in Figure 1. The graph showed that, the nanosheets of GO are
clear and regular in shape.
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Figure 1. SEM analysis of GO.
3.2. Preparation and reactions of NN-Reagent with amino acids and phenols
Two different groups of biological interest compounds, L-amino acids and
phenolic compounds, have been subjected to react with 1-nitroso-2-naphthol in the
presence of nitric acid to produce colored compounds. Although, the reaction
mechanism is not identical for all, the specificity of the reaction can best be described
by treating each group separately.
3.2.1. Synthesis of 1-nitroso-2-naphthol
Basic solution of 2-naphthol reacted effectively with nitrous acid solution (sodium
nitrite and Sulfuric acid) to afford 1-nitroso-2-naphthol. The compound was
confirmed by reported melting point and spectra data (IR and UV-visible) [27].
3.2.2. Reaction of 1-nitroso-2-naphthol with L-amino acids and phenols
In the presence of nitric acid, Tyr (phenolic amino acid) reacts with 1-nitroso-
2-naphthol to produce 1,2-benzo-8-alanyl-3-phenoxazone (BAPZ) (2), pink-purple
water-soluble compound. The reaction mixture was concentrated to one third then left
to cool. The precipitated product was filtered and recrystallized from ethanol-water.
BAPZ was confirmed by its IR spectra [29, 32, 33]. IR spectra showed abroad band at
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3550-2600 cm-1 corresponding to NH2 and OH groups. C=O showed a peak at 1632
cm-1 (scheme 1).
OH
OH
O
N
H2SO4, NaNO2
1
R2
COOH
NH2
R
R1
NH2
HO
HO
COOH
O
NH2
R
R1
HO
N
N
O
N
O
N
3
RNH2 = L-Tryptophan,
L-phenylalanine,
L-alanine,
4
O
2
R1 = H, R2 = H
R1 = OH, R2 = H
R1 = H, R2 = OH
R1 = OH, R2 = OH
L-arginine,
L-leucine,
L-cysteine,
L-asparagine,
L-lysin,
L-histidine
Scheme 1: Reaction of 1-nitroso-2-naphthol with L-amino acids and phenols
8

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80
60
40
20
0
(A)
(B)
1451
1258
(C-O)
Asym(C O)Sym
1618
(N=O)
1606
(C=C)
-
98
91
84
77
80
60
40
20
0
(C)
1230
(C O)Sym
(C-O)Asym
(C=C)
1632
(C=O)
1329
1587
(C=N)
1562
-
3500
3000
2500
2000
1500
1000
500
Wavenumbers (cm-1)
Fig. 2 FT-IR specta of (A) 1-Nitroso-2-Naphthole (NN), (B) Tyr, and (C) BAPZ.
On the other hand, amino acids under investigation, do not have a phenolic part,
proceed in the same reaction condition to afford aliphatic azo-compounds (3) [34].
These azo compounds showed maximum absorbance at the UV range 435-465 nm,
while cysteine showed two absorbance peaks at 400 and 550 nm, this could be
attributed to its ability for the cyclization of its α-mercapto-group to afford thiadiazole
derivative (scheme 2).
SH
HOOC
HOOC
O
N
S
N
HS
N
OH
N
N
OH
+
OH
HOOC
NH2
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Scheme 2: Proposed mechanism for the reaction of 1-nitroso-2-naphthol with L-
cysteine
Similarly, the reaction of phenolic derivatives, except pyrogallol, with 1-
nitroso-2-naphthol in presence of nitric acid, produced relatively stable pink-purple
water-soluble compounds that began to fade within a few hours [35] . The hydroxyl
group, and its position of the phenolic compounds, both appear to be critical. The
reaction proceeds smoothly for all phenolic compounds to afford 9H-Benzo[a]
phenoxazine (4) derivatives except for pyrogallol where no color has been appeared.
On the other hand, reaction of resorcinol and 1-nitroso-2-naphthol in the presence of
nitric acid proceeded effectively to produce the highest intense reddish violet color
which has absorption maximum at 538 nm.
3.3. Using of GO for signal improvement of BAPZ
The reaction of Tyr amino acid with NN in absence of GO showed a clear peak
with maximum absorption wavelength at λmax 468 nm, which is totally far away from
the absorption band of NN and/or Tyr. NN and Tyr spectra showed maximum
wavelengths at 379 and 273 nm, respectively. Where GO spectral absorption band was
found at 223 nm. In presence of GO, The recorded spectrum of BAPZ-GO showed a
fantastic increase in absorbance ( 220 %) at 495 nm with a 27 nm red shift, compared
to BAPZ in absence of GO (Fig. 3). This red shift in λmax of BAPZ in presence of GO is
an evidence of its adsorption on GO surface [36].
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0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
(NN+GO)
(Tyr)
(NN+Tyr+GO)
(NN)
(NN+Tyr)
(GO)
300
400
500
600
700
800
Wave length (nm)
Fig. 3 Electronic Spectra for Tyr, NN, GO, NN+GO, BAPZ, and BAPZ-GO.
The obtained data indicated that, GO could act as a catalyst for the reaction
between Tyr and NN, with an excellent increase in absorption with a red shift of 27
nm. Moreover, the colored product BAPZ was not adsorbed on GO surface. The
desorption of BAPZ from the GO surface could be attributed to the involvement of
the free phenolic hydroxyl group, responsible for H-bonding with active groups
present on GO surface, in BAPZ formation. This advantage lead to the possible use of
GO as an optical sensor for determination of Tyr amino acid.
3.4 Effect of reagent (NN) concentration, temperature, and reaction time
The effect of NN concentration on the absorbance of BAPZ was achieved by
taking different volumes 1 to 5 mL of 0.03 % W/V NN. The obtained data showed
that, the maximum absorbance of BAPZ was obtained at 2 mL of the reagent. The
change in absorbance was studied at different temperatures from 25 – 80 °C. The
maximum absorbance was achieved at 58 – 62 °C, therefore, 60 °C was used as the
optimum temperature during all the experiments. The effect of reaction time between
Tyr amino acid and NN was also investigated at 5, 10, 15, and 20 min. The obtained
results, showed that after 10 min., the absorbance was reached to its steady state,
indicating that the reaction reached its equilibrium point. Different volumes of conc.
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nitric acid (100-500 µL) were added to accelerate the reaction [26]. The maximum
absorbance of the product was obtained with 300 µL of conc. HNO3, and was used as
an optimum volume during all experiments.
3. 5. Effect of Tyrosine Concentration in absence and presence of GO
The great enhancement in the absorption peak, has been utilized to prepare a
calibration curve in absence and presence of GO for Tyr determination. The test
solutions were prepared by adding different concentartions ranged from 2x10-5 to 1.2
x10-4 M of Tyr, and 2 mL of NN reagent solution (0.03 %). The mixture was heated in
a water bath at 60 °C for five min., then 300 µl of conc. HNO3 was added, and the
mixture was heated for more five min. The developed red colour was measured at 468
nm. The same calibration curve was measured at 495 nm in presence of 20 µg mL-1 of
GO. The limit of detection (LOD) was calculated as (3.3σ/S), where σ is the standard
deviation of the intercepts and S is the slope of the calibration curve. The data
obtained from Fig 4, showed that the limit of detection was improved from 1.1 x 10-5
M to 6.4 x 10-6 M in absence and presence of GO nanosheets, respectively.
0.8
1.0
(a)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
-0.1
-0.2
0.8
0.6
0.4
0.2
0.0
(b)
4.0x10-5 8.0x10-5 1.2x10-4
Tyr concentration (M)
400
500
600
700
800
Wavelength (nm)
Fig. 4 Electronic Spectra for the reaction of NN with different concentrations of
Tyr (2x10-5 – 1.2 x 10-4 M) in presence of GO (solid lines) and in absence of GO
(dashed lines); Inset: Calibration curve (a) in presence of GO and (b) in absence
of GO.
3. 6. Effect of interferents on the determination of Tyr in absence and presence of GO
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The influence of interfering compounds (Tryptophan, L-phenylalanine, L-
alanine, L-arginine, L-leucine, L-cysteine, L-asparagine, L-lysin, L-histidine amino
acids, and phenol, pyrogalol, catechol and resorcinol) on the BAPZ signal has been
investigated in absence and presence of 20 µg mL-1 GO. A 2 mL (0.03 % W/V) of NN
reagent was added to 0.5 mL (1x10-3 M) Tyr, then 5 mL (1x10-4 M) of each
interferent was added, followed by addition of 300 µL conc. HNO3, then the final
volume was completed to 10 mL.
The data obtained (Fig. 5) showed that, in absence of Tyr and GO, most of
interferents interact with NN forming the corresponding NN-amino acid with no
significant peaks in the absorption region of Tyr. Where, their absorbance strengths at
495 nm were completely negligible with respect to BAPZ signal for the same
concentration. This behavior may be attributed to the absence of phenolic hydroxyl
group in all of the amino acids under interest, except Tyr. However, in case of Cys,
there were an absorption peaks at λmax 400 and 550 nm, this could be explained in the
term of high conjugated pattern of the proposed product (scheme 2). In absence of Tyr
and in presence of GO, the signal of the corresponding NN-interfered amino acids
was increased by around 3-7 %, indicating that GO has no significant effect on the
reaction between most of the interferents and NN.
Cys
0.3
0.2
Tyr
Tryp
0.1
0.0
PhAlan
Leuc
Hist
-0.1
Lys
Alan
Asp
Arg
-0.2
400
500
600
700
800
Wavelength (nm)
Fig. 5 Electronic Spectra for the reaction of NN with (5x10-5 M) amino acids.
On the other hand, in presence of Tyr and in absence of GO, the reaction of
NN with Tyr to form BAPZ is more preferable than the reaction of NN with other
13

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interfering amino acids, due to the selectivity of NN toward Tyr, showing a
tremendous decrease in percent of interference, especially with Cys (Fig. 6 A). While,
in presence of Tyr and GO, the interference was decreased to about 0-1 %, due to the
action of GO as a powerful catalyst for the reaction between NN and Tyr. Moreover,
GO may help in holding the other interfering materials on its surface [2, 5],
encouraging the NN to capture Tyr from GO surface forming BAPZ, which is not
adsorbed on GO. Phenolic compounds showed a moderate interfering effect in
absence of Tyr and GO, due to the presence of hydroxyl groups, readily to react with
NN. Similarly, as previously described in amino acids, GO was used to reduce the
interference effect (Fig. 6 B). Therefore, It could be concluded that, graphene oxide
nanosheets had increased the selectivity of the proposed method for the determination
of Tyr using NN in presence of different interfering amino acids and phenols.
0.4
NN+Cys+Tyr+GO
(A)
80
60
0.3
40
20
0.2
0
NN+Cys+Tyr
0.1
0.0
NN+Cys
-0.1
400
500
600
700
800
Wavelength (nm)
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0.6
0.4
0.2
0.0
60
(B)
40
20
NN+Ph+Tyr+GO
0
NN+Ph+Tyr
NN+Ph+GO
NN+Ph
400
500
600
700
800
Wavelength (nm)
Fig. 6 Electronic Spectra for the reaction of NN with (5x10-5 M) of interferents
(A) Cys and (B) Ph, in absence and presence of (6x10-5 M) Tyr and 20 µg mL-1of
GO; Inset: Corresponding interference percent.
3.7 Application in real samples
The proposed method has been applied for the determination of Tyr in urine
and serum samples of 3 healthy adult volunteers, using NN reagent in presence of (20
µg/mL) GO as shown in table 1. 1 mL urine and serum samples after treatment, were
spiked with Tyr to final concentrations of 20, 60, 100 x 10-6 M in measured solution.
The actual Tyr amount found in urine samples were 21.2 ± 0.42, 18.7 ± 0.17 and 22.1
± 0.10, respectively. In serum samples, the actual Tyr found were 26.5 ± 0.51, 28.3 ±
0.27 and 27.9 ± 0.15, respectively. The data obtained by the Liquid Chromatography
standard method of British Pharmacopoeia [37] showed a good agreement with the
results of the proposed method. The obtained data reveals that the proposed method
can be used as a sensitive and selective method for the determination of Tyr in
biological fluids.
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Table 1 Determination of Tyr in urine and serum samples
Sample
no.
Added
(x10-6 M)
Total Found
(x10-6 M)
Actual Found Reference method [37]
(x10-6 M)
B.P. (LC)
(Actual found x10-6 M)
20.8 ± 0.36
Urine
20
60
41.2 ± 0.82
78.7 ± 0.70
122.1 ± 0.56
46.5 ± 0.90
88.3 ± 0.83
127.9 ± 0.65
21.2 ± 0.42
18.7 ± 0.17
22.1 ± 0.10
26.5 ± 0.51
28.3 ± 0.27
27.9 ± 0.15
19.2 ± 0.25
100
20
21.3 ±0.15
Serum
25.8 ± 0.48
60
27.3 ± 0.35
100
26.8 ± 0.18
3.8 Comparison with other methods
The present work was compared with other reported methods [24, 38-40] for
the determination of Tyr as shown in Table 2. Although the present method has a
moderate sensitivity in comparison with other methods [38-40], it has the advantages
of application in real samples and intensive interference study compared with the
reported methods [38, 39]. On the other hand, the reported method [40] needs more
than 24 hours for sensor preparation, making this method more tedious and time
consuming than the present study. The selectivity and LOD of the present work are
better than the reported one based on filtered multi-walled carbon nanotubes on
Teflon net [24].
However, using of GO showed an improvement in the selectivity and
sensitivity by deacreasing LOD, which encourage the use of GO in other
spectrophotometric techniques.
Table 2 A comparison of Tyr determination using NN in presence of GO with other
reported methods
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Used strategy
Technique
Linear range
LOD
application
Ref
MSNP- CPE
DPV
Amp
DPV
DPV
5 × 10−7 to 6 × 10−4 M
2 × 10−6 to 100 × 10−6 M
1 × 10−9 to 2 × 10−8 M
1.5 x 10-7 M Artificial urine.
1.9 x 10-7 M Milk & serum
1.5 x10-10 M Milk
[38]
[39]
[40]
[24]
MWCNT - PBP- CPE
AuNPs - GO - GCE
MWCNT – Teflon - SPE
NN
25 × 10−6 to 750 × 10−6 M 8 × 10−6 M
1.1 x 10-5 M
plasma & blood
----
Spectro-
photometry
present
work
2 x 10-5 to 1.2 x 10-4 M
6.4 ×10−6 M Urine & Serum
NN - GO
* MWCNT = Multi-walled carbon nanotubes
* CPE = Carbon paste electrode
* SPE = Screen printed electrode
* GCE = Glassy carbon electrode
* PBP = Poly bromocresol purple
* MSNP = Mesoporous silica nanoparticles
* DPV = Differential pulse voltammetry
* Amp = Amperometry
4. Conclusions
GO nanosheets have been used for enhancement of the selectivity and sensitivity of
spectrophotometric determination of Tyr in aqueous media. In presence of GO, the
signal was enhanced, indicating that GO may act as a powerful catalyst for the
reaction between NN and Tyr. The limit of detection was significantly decreased from
1.1x10-5 to 6.4x10-6 M in absence and presence of GO, respectively. The selectivity
was improved with respect to interfering amino acid and phenols, due to holding of
interfering materials on GO surface, giving the chance of NN to capture Tyr from GO
surface to form BAPZ. The proposed method has been applied for the determination
of Tyr in urine and serum samples. The actual Tyr amount found in urine samples
were 21.2 ± 0.42, 18.7 ± 0.17 and 22.1 ± 0.10, respectively, while in serum samples,
the actual Tyr found were 26.5 ± 0.51, 28.3 ± 0.27 and 27.9 ± 0.15, respectively.
These data are in good correlation with that obtained by standard method of British
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Pharmacopoeia . Therefore, the proposed method could be used as a selective and
sensitive optical sensor for Tyr and protein with free Tyr amino acids determination in
biological fluids in presence of different interfering amino acids and phenols.
Author contributions: Hossam M. Nassef and Abdelhameed M. Othman designed the
experiments, evaluated data’s analysis and wrote the paper, Mohamed Hagar shared
in the experimental part, wrote the paper. All of authors read and approved the final
manuscript.
Conflicts of Interest: All the authors declare that they have no conflict of interest.
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-
-
-
-
Determination of tyrosine amino acid (Tyr) in presence of graphene oxide
GO improves the selectivity and sensitivity of Tyr determination.
The lower limit of detection was significantly increased.
The selectivity was improved with respect to interfering amino acid and
phenols.
-
-
The method has been applied for the determination of Tyr in urine and serum
samples
The method could be used as optical sensor for Tyr determination.