Smartphone assisted naked eye detection of mercury (II) ion using horseradish peroxidase inhibitive assays

Article abstract of DOI:10.2174/0929866525666171214111503

Background: Mercury (II) ion, Hg²⁺ is among the most common pollutants with the ability to affect the environment. The implications of their elevation in the environment are mainly due to the industrialization and urbanization process. Current

Full text of DOI:10.2174/0929866525666171214111503

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90
Protein & Peptide Letters, 2018, 25, 90-95
LETTER ARTICLE
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Smartphone Assisted Naked Eye Detection of Mercury (II) Ion using
Horseradish Peroxidase Inhibitive Assays
Editor-in-Chief:
Ben M. Dunn
BENTHAM
S
C
I
E
N
C
E
Nur Khaliesah Jamadon, Nurfatini Busairi and Amir Syahir*
Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia (UPM),
43400 Serdang, Selangor, Malaysia
Abstract: Background: Mercury (II) ion, Hg2+ is among the most common pollutants with the
ability to affect the environment. The implications of their elevation in the environment are mainly
due to the industrialization and urbanization process. Current methods of Hg2+ detection primarily
depend on sophisticated and expensive instruments. Hence, an alternative and practical way of de-
tecting Hg2+ ions is needed to go beyond these limitations. Here, we report a detection method that
was developed using an inhibitive enzymatic reaction that can be monitored through a smartphone.
Horseradish peroxidase (HRP) converted 4-aminoantipyrene (4-AAP) into a red colored product
which visible with naked eye. A colorless product, on the other hand, was produced indicating the
presence of Hg2+ that inhibit the reaction.
Objectives: The aim of this study is to develop a colorimetric sensor to detect Hg2+ in water sources
using HRP inhibitive assay. The system can be incorporated with a mobile app to make it practical
for a prompt in-situ analysis.
Methods: HRP enzyme was pre-incubated with different concentration of Hg2+ at 37°C for 1 hour
prior to the addition of chromogen. The mix of PBS buffer, 4-AAP and phenol which act as a
chromogen was then added to the HRP enzyme and was incubated for 20 minutes. Alcohol was
added to stop the enzymatic reaction, and the change of colour were observed and analyse using
UV-Vis spectrophotometer at 520 nm wavelength. The results were then analysed using GraphPad
PRISM 4 for a non-linear regression analysis, and using Mathematica (Wolfram) 10.0 software for
a hierarchical cluster analysis. The samples from spectroscopy measurement were directly used for
dynamic light scattering (DLS) evaluation to evaluate the changes in HRP size due to Hg2+ mal-
functionation. Finally, molecular dynamic simulations comparing normal and malfunctioned HRP
were carried out to investigate structural changes of the HRP using YASARA software.
Results: Naked eye detection and data from UV-Vis spectroscopy showed good selectivity of Hg2+
over other metal ions as a distinctive color of Hg2+ is observed at 0.5 ppm with the IC50 of 0.290
ppm. The mechanism of Hg2+ inhibition towards HRP was further validated using a dynamic light
scattering (DLS) and molecular dynamics (MD) simulation to ensure that there is a conformational
change in HRP size due to the presence of Hg2+ ions. The naked eye detection can be quantitatively
determined using a smartphone app namely ColorAssist, suggesting that the detection signal does
not require expensive instruments to be quantified.
A R T I C L E H I S T O R Y
Received: June 8, 2017
Revised: August 31, 2017
Accepted: September 29, 2017
DOI:
10.2174/0929866525666171214111503
Conclusion: A naked-eye colorimetric sensor for mercury ions detection was developed. The col-
our change due to the presence of Hg2+ can be easily distinguished using an app via a smartphone.
Thus, without resorting to any expensive instruments that are mostly laboratory bound, Hg2+ can be
easily detected at IC50 value of 0.29 ppm. This is a promising alternative and practical method to
detect Hg2+ in the environment.
Keywords: Mercury detection, Horseradish peroxidase, inhibitive assay, naked eye detection, colorimetric assay, smartphone.
1. INTRODUCTION
tacit concerns [1]. Among these metals, Hg2+ ion is consid-
ered as the most detrimental. Exposure to as low as 1 ppm
level can cause neurotoxicity [2], nephrotoxicity [3] and
permanent damage to the vital organs [4]. In most countries,
mercury pollution is one of the most serious pollutions due
to urbanisation and industrialisation. Widely transported in
its elemental form, Hg0 is the byproduct that is unintention-
ally released into the air, water and soil. This byproduct is
Rapid industrial progress has resulted in the increase of
heavy metal concentration in the environment, which raised
*Address correspondence to this author at the Department of Biochemistry,
Faculty of Biotechnology and Biomolecular Sciences, University Putra
Malaysia (UPM), 43400 Serdang, Selangor, Malaysia;
E-mail: amirsyahir@upm.edu.my
1875-5305/18 $58.00+.00
© 2018 Bentham Science Publishers

Smart Phone Assisted Naked Eye Detection of Mercury (II) Ion
Protein & Peptide Letters, 2018, Vol. 25, No. 1 91
eventually oxidised to become Hg2+. Like other heavy met-
als, Hg2+ can be emitted in the form of free particles into the
air or can be transferred into the water via leaching from
industrial areas. Due to its non-biodegradable properties, it
cannot be biologically detoxified and can accumulate in the
human body through the food chain [5].
from MERCK. 4-aminoantipyrine (4-AAP), Sodium dihy-
drogen phosphate (NaH2PO4), and Horseradish peroxidase
(HRP) were obtained from Sigma Aldrich. Phenol was sup-
plied from R&M. Di-sodium hydrogen phosphate
(Na2HPO4) was acquired from Systerm, whereas 99.8%
ethanol was acquired from Reindemann.
The consumption of contaminated food is a major path-
way of human exposure to mercury compared to inhalation
and dermal contact [6]. Due to its bio accumulative proper-
ties, Hg2+ in fish product is a main cause of mercury poison-
ing. The outbreak of Minamata disease in 1956 caused by the
Hg2+ poisoning due to the release of methylmercury in
wastewater by the industrial factory is a classic example for
the detrimental effects of mercury as it enters human body
through contaminated fish products [7].
2.1. General Preparation
All of the reagents were of analytical grade and used
without further purification. Distilled water and double
deionised milliQ water were used throughout the experiment.
Phosphate buffer stock solution (20 mM, pH 7.2) was pre-
pared by dissolving 0.7 g Na2HPO4 and 0.6 g NaH2PO4 in
500 mL in distilled water, respectively. The phosphate buffer
solution was then filtered to remove impurities.
To date, many techniques have been developed for mer-
cury ion analysis including atomic absorption spectrometry,
inductively coupled plasma (ICP) techniques (coupled with
mass spectrometry or atomic emission spectroscopy), anodic
stripping voltammetry and X-ray fluorescence spectrometry
[8-10]. These techniques provide accuracy and precision in
determining the level of heavy metals in the environment.
However, these techniques are generally expensive, time-
consuming and can be hardly used for in-situ analysis [11].
Additionally, they require tedious sample preparation proce-
dure, expensive apparatus that are mostly laboratory bound
and highly trained personnel to operate. Thus, there is a need
to develop sensitive and rapid determination of heavy metals
in the environment for in-situ analysis through the develop-
ment of biosensors.
4-aminoantipyrine (4-AAP, 30 mM) stock solution was
prepared by dissolving 0.61 g in 100 mL distilled water.
Phenol stock solution 1.5% (v/v) was prepared by mixing 1.5
mL phenol with 98.5 mL distilled water. Meanwhile, H2O2
stock solution (4 mM) was daily prepared by an appropriate
dilution of 30% H2O2 in distilled water. HRP stock solution
was prepared by dissolving 1.0 mg enzyme in 1.0 mL PBS
buffer (1 mg/ml). From the stock solution, HRP was diluted
to 0.06 mg/ml using PBS buffer. All metal ions were used as
sample and were prepared by diluting the metal ions stock
solutions to 0.01, 0.1, 1, 5, 10, 50, and 100 ppm in distilled
water. These solutions were freshly prepared prior further
use.
2.2. HRP Enzyme Assays
In recent years, biosensors have received attention among
researchers for detecting heavy metals in the environment
[12]. Bio recognition element typically relies on enzymes,
whole cells, antibodies, or nucleic acids that could selec-
tively react with the target analytes. Meanwhile, signal
transduction converts the biological response resulted from
the interaction with the target analyte into quantifiable signal
[13]. Colourimetric approach is one of the most practical
methods for in-situ analysis. It directly provides coloured
signal proportional to the respective biological activity typi-
cally enzymatic reaction. In the presence of heavy metal, the
enzyme will be potentially inhibited, thus decrease the col-
oured signal. Due to its distinctive colour response that can
directly be observed by the naked eye, colourimetric sensors
have been extensively studied in sensing organic pollutants
and metal ions [14-16]. Colorimetric sensor have been suc-
cessfully used to detect Pb2+ in milk [6], Cu2+ for cell
signaling [17] and As3+, Cr6+ and 3,5-dichlorophenol in wa-
ter sources by using Prussian blue [14]. HRP has been used
to develop an amperometric enzyme electrode for peroxide
determination [18]. For detection of heavy metals, HRP bio-
sensor has, so far, only been reported for the detection of
mercury [19].
The activity of the enzyme was assayed using the follow-
ing reagents, which act as chromogen. These include 0.6 mL
PBS buffer, 0.15 mL 4-AAP and 0.10 mL phenol [20]. Prior
adding the chromogen, HRP enzyme can be either pre-
incubated or without pre-incubation with metal ions at 37°C.
In the case of pre-incubation, 0.05 mL HRP was pre-
incubated with 0.100 mL of metal ions for 1 hour at 37°C in
the incubator shaker (Protech model-903). After pre-
incubated, the chromogen solutions that were mixed before-
hand were added to each vial containing the sample. The
mixture was then again incubated at 37°C for 20 minutes. On
the other hand, in the case without pre-incubation, the chro-
mogen solutions was mixed directly with HRP and metal
ions and incubated at 37°C for 20 minutes. After 20 minutes
incubation period, 1.0 mL of 99.8% ethanol was added to
each vial to stop the reaction of enzyme. Absorbance reading
was taken at 540 nm on UV-Vis spectrophotometer (WPA
Biowave). The production colour was also observed.
2.3. Dynamic Light Scattering (DLS) Evaluation
0.10 mL of mercury using various concentrations (0.02,
0.2, 2 ppm) was incubated with 0.10 mL of HRP enzyme
(0.06 mg/l) in the eppendorf tube for 1 hour at 37°C in the
incubator shaker. After 1 hour incubation, total volume of
0.20 mL of the mixture was transferred into 96-wells mi-
croplates prior inserting into the dynamic light scattering
system (DLS, Malvern Zetasizer). The DLS system was set
to 37°C throughout the analysis.
2. MATERIALS AND METHODS
30% hydrogen peroxide (H2O2) and all metal standard
solutions; arsenic (V) (H3AsO4), cadmium (II) (Cd(NO3)2),
chromium (III) (Cr(NO3)3), cobalt (II) (Co(NO3)2), lead (II)
(Pb(NO3)2), mercury (II) (Hg(NO3)2), Nickel (II) (Ni(NO3)2),
Silver (II) (Ag(NO3)2), Zinc (II) (Zn(NO3)2) were acquired

92 Protein & Peptide Letters, 2018, Vol. 25, No. 1
Jamadon et al.
Selectivity is an important parameter in evaluating the
performance of a biosensor [24]. To determine the selectivity
of assay system, various heavy metals ion were used to study
the inhibition effects on HRP. These metal ions are Ag2+,
As5+, Cd2+, Co2+, Cr3+, Cu2+, Hg2+, Ni2+, Pb2+, and Zn2+. The
metal ions were added without being pre-incubated to show
the direct effects on HRP enzyme. At 1.0 ppm concentration,
Hg2+ gave the lowest absorbance reading (0.39) and the least
intense red colour formation (Figure 2). The results obtained
also showed the same tendency as in previous study using
HRP to detect various types of Hg [19]. This is followed by
2.4. Molecular Dynamics (MD) Simulation
MD simulation was carried out using YASARA soft-
ware. The starting atomic coordinate of native HRP was ob-
tained from Protein Data Bank (PDB ID: 1HCH). Then, the
cysteine side chain residues 11-91, 44-49, 97-301 and 177-
209 of native HRP were modified to alanine for generating
the initial structure of mutant HRP. Each protein, native or
modified HRP, was centred in a cubic box and solvated with
water molecules. The temperature was kept close to 310 K.
The production MD period of 15 ns at constant temperature
was performed on both native and mutant HRP.
other metals; Zn2+ < As5+ < Cd2+ < Cr3+ < Co2+ < Ag2+
Ni2+.
<
2.5. Statistical Analysis
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Half inhibition concentration (IC50) of each heavy metal
was determined using Graphpad PRISM 4 for non-linear
regression analysis software available at the internet,
whereas the hierarchical cluster analyses of HRP inhibition
along with other enzymes (for comparison) were generated
using algorithm in Methematica 10.0 software (Wolfram) via
cosine and eucledian similarity distances.
3. RESULTS
3.1. Naked Eye Colour Determination
In this study, the naked eye detection for heavy metal
ions was developed using the reaction of HRP inhibitive
assay [21]. In the presence of HRP, both chromogens (phe-
nol and 4-AAP) reacted with H2O2 substrate to produce an
intensely red coloured quinone imine dye [22]. The col-
ourimetric response can be used to directly determine HRP
enzymatic activity (Figure 1).
Control Ag(2+) As(5+) Cd(2+) Co(2+) Cr(3+) Cu(2+) Hg(2+) Ni(2+) Pb(2+) Zn(2+)
Figure 2. The absorbance reading for 1.0 ppm of various heavy
metals ion with HRP enzyme. Error bars are standard deviations,
while n = 3. Control is without the presence of any heavy metal ion.
Broader concentration range of Hg2+ was tested to deter-
mine concentration dependency on the detection. When
compared with control, the absorbance reading was signifi-
cantly reduced as concentration of Hg2+ increases. At 0.1
ppm, however, the visual colour observed was similar to that
of control. In contrast, at 0.5 and 1.0 ppm of Hg2+, the prod-
uct was noticeably pale, while at 5.0 and 10.0 ppm, the col-
our has fully turned into colourless indicating the full inhibi-
tion HRP activity. The calculated IC50 value is 0.290 ppm
(Figure 3).
It is expected that HRP is inhibited by the presence of
heavy metals ions since heavy metals deteriorate protein via
many mechanisms [23]. Thus, colourless (or paler) product
was produced in the presence of heavy metals ion. For mer-
cury (II) ions, a noticeable paler coloured solution was
formed when reacted with more than 0.1 ppm concentration.
Hg2+
Figure 1. Schematic diagram of the enzymatic reaction. 4-AAP was
oxidised to produce red coloured quinoneimine. In the presence of
Hg2+, the HRP was inhibited, thus keeping the solution colourless.
Figure 3. The sigmoidal curve obtained to calculate IC50 of mer-
cury by using Graphpad PRISM software. Error bars shown are the
standard deviation, and n =3.

Smart Phone Assisted Naked Eye Detection of Mercury (II) Ion
Protein & Peptide Letters, 2018, Vol. 25, No. 1 93
The IC50 values for other metal ions have been also de-
termined (Table 1). As5+, Cr3+ and Cu2+ were chosen since
they are the common metal ions found polluting Malaysia
water sources. An inhibition profile based on IC50 values for
heavy metal ions was evaluated. Comparison was made be-
tween the inhibitive HRP assay and other types of inhibitive
assay namely AChE from Periopthalmodon schlosseri [25],
AChE from Pangasius Hypothalmus [26], urease (free en-
zyme and immobilized enzyme) [27], Daphnia Magna, Rain-
bow Trout and standard methods of Microtox™ [28], papain
[29], bromelain [30] and coriandrum assays [31]. Data were
analysed using hierarchical cluster analysis via similarities
calculated using Euclidean distance (dE) and cosine distance
(dC). The dE clusters scattered data in a high dimensional
Euclidean space (Figure 4A), while the dC considers the an-
gle between unit vectors over absolute differences in magni-
tude (Figure 4B), which suggested the pattern of inhibition.
(A)
(B)
Table 1. IC50 of Hg2+ and other metal ions.
Heavy Metals
IC50
Hg2+
As5+
Cr3+
Cu2+
0.29
6.18
6.218
14.28
Figure 4. Hierarchical cluster analyses of inhibition profiles in
detecting the tested heavy metal ions using (A) Euclidean distance
and (B) Cosine distance. This clustering analysis was calculated by
using Wolfram Mathematica 10 software.
In dE clustering, it was found that the inhibitive HRP as-
say was assigned far apart from other assays, which demon-
strated sensitivity distinctness of HRP assay towards the
tested metal ions. Nevertheless, the developed detection sys-
tem is cheaper and easier to be prepared than other assay
systems. This means that some modifications need to be
made to improve its sensitivity. Meanwhile, dC showed simi-
larity of inhibition mechanism. It was found that the inhibi-
tive HRP assay was closely clustered with bromelain assay,
suggesting similar inhibition pattern.
7
6
5
4
3
2
1
0
Previous study has mentioned that Hg can inhibit protein
or enzyme by reacting the sulfhydryl group or disulphide
bridges [19]. Sulfhydryl groups are considered as the main
target of mercury as they are the most reactive nucleophilic
sites for protein amino acid side chains. Hg can also react
with the disulphide bridges of an enzyme, which eventually
lead to the enzyme deterioration. In order to investigate and
validate the mechanism of HRP inhibition by Hg, dynamic
light scattering (DLS) and molecular dynamics (MD) simula-
tion were conducted.
HRP milliQ
(control)
HRP + 0.01 ppm HRP + 0.1 ppm Hg HRP + 1 ppm Hg
Hg
Figure 5. The hydrodynamic radius of HRP enzyme in a function
of mercury concentration estimated using DLS.
A constant increase in hydrodynamic radius was propor-
tionally observed with Hg2+ concentration (Figure 5). This
suggests the breakage of HRP disulphide bonds that hold the
integrity of the enzyme structure. The reaction has caused
HRP enzyme structure to ‘open up’ and dawdled the
brownian motion detectable in the DLS measurement.
Further study to investigate the structural changes of
HRP by Hg2+ was conducted by using a MD simulation
(YASARA). Conformational change was observed in the
structure upon deletion of disulphide bridges. Using the
known x-ray crystallographic structure of resting form of

94 Protein & Peptide Letters, 2018, Vol. 25, No. 1
Jamadon et al.
HRP (PDB: 1HCH), two 3D models known as native and
mutant were constructed. The HRP contains four disulphide
bridges incorporated in cysteine residues 11-91, 44-49, 97-
301 and 177-209 [32]. At 15 ns, the mutant structure was
distorted compared to the native when both structures were
superimposed to one another (Figure 6). Meanwhile, the ra-
dius of gyration rg for mutant structure started to increase
from 2.5 ns onwards. It is important to note that the tendency
of size expansion found here in the MD simulation was not
to be directly compared with the DLS results that were
measured in much longer time frame.
value. The colour produced by the inhibitive assay for [Hg2+]
from 1 ppb, 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm and 10 ppm
was measured using a mobile smartphone (Figure 7). The
results obtained are rapid, thus suggesting that the colour
produced can be rapidly determined using this application
for in-situ monitoring. The additional application demon-
strates that the qualitative naked eye detection can be quanti-
fied using simple instruments to detect the level of heavy
metals in our environment. The data showed that G and B
were significantly increased at the level of 1 ppm. Interest-
ingly, when the RGB is converted to a grayscale luminosity
using the formula of GS = 0.21 R + 0.72 G + 0.07 B, we
found that the IC50 of the assay was 0.4 ppm, which was
comparable to the measurement using the spectrophotometer.
This also suggests that a comprehensive color spectrum
evaluation technique could be more accurate compared with
single wavelength estimation.
CONCLUSION
Mercury is regarded as one of the most serious pollutants
affecting environment worldwide [33]. Despite the existing
prevailing danger, there is inadequate monitoring on these
pollutants in the environment. Conventional analytical tech-
niques are often very costly, time-consuming, and require
trained personnel to operate. Due to high toxicity caused by
heavy metals, there is an obvious need to develop sensitive
and rapid determination methods for detecting heavy metals
in the environment through the development of a biosensor.
Figure 6. The superposed structure of native (red) and mutant HRP
(blue) indicates that mutant structure has slightly distorted com-
pared to native HRP. The blue arrow shows the changes occur in
the structure.
A naked-eye colourimetric sensor for mercury ions detec-
tion has been developed. The colour change due to the pres-
ence of Hg2+ can be easily distinguished using an app via a
smartphone. Thus, without resorting to any expensive in-
struments that are mostly laboratory bound, Hg2+ can be
easily detected at 0.290 ppm IC50 value. The HRP inhibition
by mercury was due to the reaction of mercury with sulfhy-
dryl group of the HRP. Further experiment using DLS
method and MD simulation suggested that the metal dis-
With the advancement in technology today, a smartphone
has become a powerful tool used for more than just commu-
nication. Colour Assist app is one of many that allow us to
translate colour to a quantitative red-green-blue (RGB)
Figure 7. The colour product formed with and without the presence of heavy metals. [Hg2+] = 1 ppb, 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm and
10 ppm for A to G, while H is a control.

Smart Phone Assisted Naked Eye Detection of Mercury (II) Ion
Protein & Peptide Letters, 2018, Vol. 25, No. 1 95
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rupted HRP functional by reacting with disulphide bonds. In
order for this naked eye detection to be used for environ-
mental monitoring, there is still some modification to be
made in future so that it could effectively detect metal ions
other than Hg2+. Field trials also need to be conducted to
determine the effectiveness of this method for in-situ moni-
toring.
[15]
[16]
[17]
[18]
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
[19]
[20]
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENT
The authors would like to thank the Universiti Putra Ma-
laysia for the funding under project no. UPM/700-2/1/GP-
IPS/2016/9508300.
[21]
[22]
[23]
[24]
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