Sensors and Actuators B 235 (2016) 723–731

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Gold nanoparticles-based biosensing of Leishmania major kDNA

genome: Visual and spectrophotometric detections
N. Sattarahmady a,b,∗ , A. Movahedpour a,c , H. Heli a , G.R. Hatam d,e
a
Nanomedicine and Nanobiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
b
Department of Medical Physics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
c
Department of Nanomedicine, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
d
Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
e
Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Precise and sensitive identification methods of Leishmania are required for efficient treatment of leishma-
Received 20 December 2015 niasis. Detection of Leishmania species approves the efficiency of the applied therapy. Current diagnosis
Received in revised form 28 April 2016 methods, such as culture and microscopy methods, are time-consuming and have limited sensitivity, and
Accepted 4 May 2016
are laborious when encountering lots of samples. Although molecular methods can identify Leishmania
Available online 7 May 2016
species, their diagnosis depends on individual responsiveness. Therefore, rapid, sensitive and low-cost
methods are required for identification of Leishmania species. In this study, a new method was developed
Keywords:
based on hybridization of gold nanoparticles linked a specific single stranded DNA probe from non-
Gold nanoparticles
Leishmania major
protein coding region (AB678349.1) of Leishmania major minicircle kinetoplast DNA (kDNA). Dispersion
Genosensor or aggregation of the gold nanoparticles-probe conjugates in the presence or absence of a complementary
DNA biosensor DNA sequence leads to an obvious and sensitive change in the UV–vis spectra and the solution color. The
Hybridization assay results indicated that this method is useful for diagnosis of Leishmania major from other non-Leishmania
species with a detection limit of 7.0 pg ␮L−1 . The efficacy of the present PCR-free assay was evaluated to
identify genomic DNA extracted from Leishmania major and clinical samples.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Leishmaniasis is one of the most critical epidemic-prone dis-
eases according to the World Health Organization (WHO) [1].
Leishmaniasis is a protozoan parasitic disease, transmitted by the
bite of sandflies, and by reusing infected needles and transportation
of the infected blood [2]. Leishmaniasis clinical manifestations are
classified into three forms: visceral leishmaniasis (VL), cutaneous
leishmaniasis (CL), and mucocutaneous leishmaniasis (MCL) [3,4].
Infective parasites are hosted in the macrophages and produce a
broad spectrum of ulcerative lesions in the skin (in the case of CL)
or inflammation in the mucosa (in the case of MCL and VL) [5]. 30
different species of Leishmania genus cause leishmaniasis approx-
imately yearly with an incidence rate of 1–1.5 million cases of CL
[6]. Although CL lesion is a self-healing disease but recovery takes
∗ Corresponding author at: Nanomedicine and Nanobiology Research Center,
Department of Medical Physics, School of Medicine, Shiraz University of Medical
Sciences, Shiraz, Iran.
E-mail addresses: sattarahmady@yahoo.com, nsattar@sums.ac.ir
(N. Sattarahmady).
a long time and usually leaves a disfiguring scar [4,7]. Two cate-
gories of CL, Leishmania tropica induces anthroponotic CL in cities,
and Leishmania major causes zoonotic CL in rural areas [8].
Up to now, a variety of diagnostic methods have been devel-
oped for diagnosis and identification of Leishmania. Traditionally,
Leishmania in clinical samples is detected microscopically, and also
by culturing the stained tissue specimens. Both methods are insen-
sitive. In addition, molecular methods are unable to differentiate
between the infections of lesions caused by Leishmania tropica and
Leishmania major [9]. Beside, recognition of parasite species gives
applicable and useful knowledge for appropriate clinical treat-
ment, epidemiological and taxonomic purposes in endemic areas.
Also, species determination can be effective in information esti-
mation of compromised patients [10,11]. However, isoenzymes
electrophoresis, a gold standard method of classification of Leish-
mania species [12], is slow, culture-dependent, laborious, and
expensive, and requires 15 different enzymatic inquiries [13]. Con-
ventional PCR, RT-PCR, RFLP and nested PCR improve the reliability
diagnosis and produce quantitative results; however, the technical
complexity, equipped laboratory, and product detection reduce its
usefulness [14]. Other techniques, such as ELISA using monoclonal

http://dx.doi.org/10.1016/j.snb.2016.05.023
0925-4005/© 2016 Elsevier B.V. All rights reserved.
724 N. Sattarahmady et al. / Sensors and Actuators B 235 (2016) 723–731
antibodies and molecular biology methods such as Southern, Dot,
Spot, or Squash Blots suffer from lack of specificity, sensitivity and
rapidity for the isolates [15,16]. Therefore, development of sensi-
tive, fast, reliable and economical laboratory tests is required for
identification of Leishmania species.
Kinetoplast is a mitochondria-like organelle and includes a net-
work of maxi and mini circular DNA molecules. Maxicircles DNA
has a 20–30 kb containing 50–100 copies encoding proteins of
energy production and ribosomal RNAs. Minicircles DNA contains
thousands of smaller molecules concatenated in 0.5–2.7 kb net-
work, have functions in editing of maxicircle mRNA transcripts, and
represent some unknown functions [17,18]. Conserved sequence
region of 100–300 bp and variable sequence regions with limited
homology exist in minicircles [19,20]. Minicircle DNA sequences
undergo rapid evolutionary change which is used in differentiation
of Leishmania species [21].
Recent researches on nanotechnology provide new and pre-
cise tools for sensing and detection [22–27]. Along this line,
functionalization of DNA with nanomaterials such as gold nanopar-
ticles (AuNPs) leads to the design of oligonucleotide probes
(AuNPs-probe) in efficient molecular recognition [22,28–32]. The
mechanism is based on hybridization of AuNPs-probe with a
target DNA sequence, and distance dependent surface plasmon
resonance (SPR) of AuNPs. Aggregation of functionalized probes
induced a color change [28]. This procedure was applied for differ-
ent molecules and pathogens [29–33]. As for Leishmania detection,
AuNPs has been conjugated with four oligonucleotide probes from
kinetoplastid minicircle DNA of Leishmania species for the detection
of Leishmania species [34]. In another study, a visual oligochro-
matographic dipstick test for PCR products [35] has been developed
for the detection of Leishmania species based on hybridization of a
short sequence of 18S rRNA gene as target in CL, MCL, or VL patients
[14,36].
The aim of the present study was to design an AuNPs-probe
assay based on kinetoplast DNA (kDNA) sequences from Leishmania
major for diagnosis and differentiation of Leishmania major from
Leishmania tropica and Leishmania infantum species based on visual
and spectrophotometry detections.
2. Materials and methods
2.1. Materials
The Leishmania major (MRHO/IR/75/ER), Leishmania
tropica (MHOM/IR/02/Mash10) and Leishmania infantum
(MCAN/IR/96/LON49) species were provided from Tehran Univer-
sity of Medical Sciences (Iran). Chloroauric acid and tri-sodium
citrate were purchased from Sigma (USA). Thiolated oligonu-
cleotide probe was prepared with SbS Genetech Co. (China). PCR
primers used in this study were selected based on sequence
of Leishmania major minicircle kDNA [37], and purchased from
Sinaclon (Iran). YTA genomic DNA extraction mini kit for tissue
was purchased from Yekta Tajhiz Azma (Iran). All other chemicals
were purchased from Merck (Germany).
2.2. Genomic DNA extraction
DNA was extracted from the promastigote cultures of the
reference strains of Leishmania major (MCAN/IR/97/LON490) as
positive control, and Leishmania infantum (MCAN/IR/96/LON49)
and Leishmania tropica (MHOM/IR/89/ARA2) as negative controls.
DNA extraction was performed using YTA genomic DNA kit and
stored at −20 ◦ C until use. The amount of genomic DNA was deter-
mined using a Thermo Scientific NanoDrop 2000c (USA).
2.3. Clinical samples
Biopsy specimens of 9 Leishmaniasis patients were collected
after sterilization of the lesion area on slides, stained by Giemsa and
microscopically characterized. Then, the smears were cut out and
DNA extraction performed using YTA genomic DNA kit according to
the manufacturer’s instructions. The extracted DNA samples were
dissolved in 50 ␮L deionized distilled water and stored at −20 ◦ C,
after measurement of their optical densities at 260/280 nm. Some
of clinical samples were followed by PCR for strain diagnosis after
validation by the assay presented in this study.
2.4. Preparation of AuNPs
AuNPs were synthesized by citrate reduction approach as
reported elsewhere [38–40]. Briefly, sodium citrate (25 mL,
38.8 mmol L−1 ) was added to a heated chloroauric acid solution
(250 mL, 1 mm mol L−1 ) in a round bottom flask and the mixture
was refluxed for 15 min. The obtained AuNPs solution had a peak in
UV–vis spectra with a maximum peak at 520 nm, confirming appro-
priate size and size distribution of the nanoparticles. The AuNPs
solution was stored in a cool and dark place. The size and morphol-
ogy of AuNPs were also characterized by field emission electron
microscopy (FSEM, using a Zeiss, Sigma-IGMA/VP, Germany), and
transmission electron microscopy (TEM, using a Zeiss, EM10C,
Germany).
2.5. Probe design
A thiolated 24 base oligonucleotide was designed as the probe
using NCBI BLAST nucleotide search tool and Primer3 based on a
submitted sequence in GenBank (AB678349.1) of Leishmania major
minicircle kDNA (Iranian Standard strain MCAN/IR/97/LON490, iso-
late: IranJWmaj) [41]. The probe sequence was checked for any
possible homology and share sequences with any non-Leishmania
major species using the BLAST of NCBI. Moreover, the melting tem-
perature of the probe was ensured to be within a narrow range
using Oligo software. Then, the probe sequence was checked for
potential self-dimer and formation of secondary structures using
mfold software (http://mfold.rna.albany.edu) [42] which may oth-
erwise hinder the assay.
2.6. Preparation of AuNPs-probe
The probe was initially re-suspended with dithiothreitol (DTT)
solution (0.1 mmol L−1 , pH 8.3) at room temperature for 60 min
to ensure disulfide cleavage completely to react −SH group with
AuNPs. Then, it was desalted via ethanol precipitation, and cen-
trifuged for washing. This process was repeated three times. The
AuNPs-probe was prepared as previously reported [39,40] with
minor modifications. Briefly, 3.12 ␮mol alkyl thiolated oligonu-
cleotide probe and 14.7 nmol L−1 AuNPs solution were shaken
overnight at room temperature in the dark. The solution was
brought to a final concentration of 9 mmol L−1 in sodium phos-
phate buffer, pH 7.0, and then, sodium dodecyl sulfate (SDS) (5 ␮L,
0.1%) was added and shaken for 30 min at room temperature. After
that, 2 mol L−1 NaCl in 1 mmol L−1 phosphate buffer saline (PBS), pH
7.0 was added during 48 h at room temperature to reach the final
NaCl concentration of 0.1 mol L−1 . The solution was centrifuged at
13000 rcf for 20 min, and the precipitate was washed with 300 ␮L
of 10 mmol L−1 PBS, pH 7.0 containing 0.15 mmol L−1 NaCl and 1 ␮L
0.1% SDS, and re-suspended in 300 ␮L of the same buffer, and stored
in the dark at room temperature until use. This AuNPs-probe had
the same color as AuNPs with no visible aggregates.
 N. Sattarahmady et al. / Sensors and Actuators B 235 (2016) 723–731 725

2.7. AuNPs-probe hybridization assay

To optimize the condition of hybridization and best observation

of color changes, various amounts of HCl and sodium phosphate
buffer were mixed with AuNPs-probe to obtain the best color
changes. Different amounts of target DNA (9.1, 10.6, 15.1, 18.6, 21.4,
30.2 and 38.5 ng) in 10 mmol L−1 sodium phosphate buffer, pH 5.0
reached the final volume of 10 ␮L and then they were heated to
93 ◦ C for 5 min. After immediate ice cooling of the samples, 15 ␮L
of the AuNPs-probe solution was added to the target DNA solu-
tions and immediately incubated at 63 ◦ C for 12 min. Blank control
sample was prepared in a similar manner using an equivalent vol-
ume of 10 mmol L−1 sodium phosphate buffer, pH 5.0 (instead of
target DNA). Also, a sample was prepared in the same way using a
non-complementary sequence as non-target DNA with a weight
of 23.38 ng in the final volume of 10 ␮L. After addition of 5 ␮L
0.2 mol L−1 HCl, the absorbance values of the solutions were mea-
sured spectrophotometrically, and the color of the solutions was
also recorded by a digital camera.

2.8. Colorimetric detection of genomic DNA

The hybridization assay was applied to detect Leishmania

genomic DNA. To determine the limits of detection and quanti-
tation of genomic DNA using AuNPs-probe, 10 ␮L of the genomic
DNA of the extracted Leishmania major samples (6.2, 10.4, 11.4, 13.2,
15.6, 18.2 and 20.7 ng) were assayed. Blank control samples includ-
ing 10 mmol L−1 sodium phosphate buffer, pH 5.0 were employed.
Samples containing 61.5 ng genomic DNA of Leishmania tropica and
86 ng genomic DNA of Leishmania infantum were employed as neg-
ative controls. The above solutions were incubated at 93 ◦ C for
10 min, ice cooling and immediately incubated at 63 ◦ C for 12 min.
After this thermal cycle, the solutions were subjected to acid addi-
tion and then examined both visually and spectrophotometrically.
2.9. Colorimetric detection of clinical samples
For hybridization assay, mixtures of 5 ␮L of the extracted
genomic DNA from clinical samples and 5 ␮L of 10 mmol L−1 PBS,
pH 5.0 were heated at 93 ◦ C for 10 min. Then the samples were ice
cooled, 15 ␮L of AuNPs-probe were added and incubated at 63 ◦ C
for 12 min. Then, 5 ␮L of 0.1 mol L−1 HCl was added and the results
evaluated visually and spectrophotometrically.
3. Results
3.1. Characterization of AuNPs
Characterization of the synthesized AuNPs with a mean diame-
ter of 16 ± 3 (n = 50) nm including SEM and TEM images and UV–vis
spectra was presented in Supplementary material.
3.2. Immobilization and hybridization of the probe
oligonucleotide
The probe oligonucleotide (5 -SH-(CH2 )6 -T10 -

AACCCACTAAAGCGTCACCCAACA-3 ) was selected as a part of
a submitted nucleotide sequence in GenBank (AB678349.1)
of Leishmania major minicircle kDNA (Iranian Standard strain
MCAN/IR/97/LON490, isolate: IranJWmaj) [41] based on the
results of bioinformatic evaluations.
Fig. 1 presents UV–vis spectra (A) and visual color changes
(B) of AuNPs, and AuNPs-probe solutions, before and after acid
addition. Both AuNPs and AuNPs-probe solutions had a maximum
absorbance at ∼520 nm, before acid addition. Addition of HCl solu-
tion to AuNPs and AuNPs-probe solutions induced a red shift in the
Fig. 1. UV–vis spectra (A) and visual color changes (B) of AuNPs (a, b), and AuNPs-
probe solutions (c, d), before and after addition of 5 ␮L HCl solution 0.2 M.
spectra with a maximum at ∼560 and 590 nm, respectively. Visual
colors of the AuNPs and AuNPs-probe solutions showed turning the
red color to blue purple after acid addition.
To evaluate the distinguishability of AuNPs-probe to detect the
complementary sequence in Leishmania major kDNA, the hybridiza-
tion assay was performed in the presence of non-complementary
and different amounts of complementary sequences. Hybridization
of AuNPs-probe with complementary sequence produces double
stranded DNA, which increases the stability of AuNPs-probe toward
aggregation. At non-hybridization conditions, AuNPs-probe is
aggregated upon acid addition. Fig. 2 shows UV–vis spectra (A) and
visual color changes (B) after acid addition to AuNPs-probe solution,
in the absence (blank control), presence of non-complementary
sequence, and presence of different concentrations of comple-
mentary sequence. After acid addition, AuNPs-probe solution
showed a ∼70 nm shift in the absorption maximum, and its color
turned to purple. Similar results were obtained in the presence
of non-complementary sequence; 80 nm shift in the maximum of
absorbance was observed, and the color of the solution was simi-
lar to the case of AuNPs-probe solution. The results arose from the
instability of AuNPs and their aggregation in the non-hybridization
condition upon acid addition (acid induction of aggregation). The
non-complementary sequence is unable to hybridize with AuNPs-
probe, and cannot prevent AuNPs aggregation. Contrary, in the
presence of target sequence, AuNPs-probe was hybridized, and the
double stranded structure formed on the AuNPs surface prevented
their aggregation upon acid addition. Shift in the maximum of the
absorption peak, and its intensity depended on the concentration
of target sequence, and the color and its intensity was distinguish-
able with or without the target sequence with the naked eye. The
limit of quantitation of the complementary sequence by AuNPs-
probe hybridization assay was estimated as 0.35 ng ␮L−1 , based
on the lowest amount of complementary sequence needed for a
visual change in the solution color. On the other hand, Fig. 3 shows
a calibration curve for the spectrophotometric determination of
complementary sequence by AuNPs-probe. The calibration sensi-
726 N. Sattarahmady et al. / Sensors and Actuators B 235 (2016) 723–731
Fig. 2. UV–vis spectra (A) and visual color changes (B) after HCl (5 ␮L, 0.2 M) addition to AuNPs-probe solution, in the absence (blank control; i), presence of 23.38 ng non-
complementary sequence (h), and presence of different concentrations of complementary sequence of 9.1 (a), 10.6 (b), 15.1 (c), 18.6 (d), 21.40 (e), 30.20 (f) and 38.5 (g) ng
in 10 mmol L−1 sodium phosphate buffer, pH 5.0.
Fig. 3. A calibration curve for the spectrophotometric determination of Leishmania
major target sequence via AuNPs-probe hybridization assay.
tivity was 1.23 OD ␮L ng−1 , the relative standard deviation (RSD)
was 5.19%, and the detection limit was obtained based on 3SD/b cri-
terion (SD and b are the standard deviation of the blank control and
the slope of the calibration curve, respectively) to be 11.9 pg ␮L−1 .
3.3. Detection of genomic DNA
In order to evaluate the usefulness of AuNPs-probe hybridiza-
tion assay for genomic DNA detection, DNA extracts of standard
cultures of Leishmania major (as positive sample) and Leishmania
infantum and Leishmania tropica (as negative samples) were pre-
pared. Fig. 4A shows the color of AuNPs probe in the presence
of different concentrations of positive, negative and blank con-
trol samples, after acid addition. Based on the images, the color of
AuNPs-probe solution in the presence of 10.4, 11.4, 13.2, 15.6, 18.2,
and 20.7 ng genome of Leishmania major remained red. However,
AuNPs-probe solution containing blank control, Leishmania infan-
tum, Leishmania tropica genomes and the 6.2 ng of Leishmania major
genomic DNA turned purple, after acid addition. Therefore, the
quantitation limit of Leishmania major genomic DNA by this assay
was 0.34 ng ␮L−1 . Fig. 4B shows UV–vis spectra of AuNPs-probe in
the presence of different amounts of genomic DNA of Leishmania
major (6.2, 10.4, 11.4, 13.2, 15.6, 18.2, and 20.7 ng), and in the pres-
ence of Leishmania infantum (61.5 ng) and Leishmania tropica (86 ng)
genomic DNA samples, after acid addition. UV–vis spectra of the
samples containing 10.4, 11.4, 13.2, 15.6, 18.2, and 20.7 ng of Leish-
mania major genomic DNA showed a maximum peak at 528 nm, and
the blank control, and Leishmania infantum and Leishmania trop-
ica genomic DNA samples showed a wide absorbance spectrum
with a bathochromic shift up to 600 nm. Therefore, hybridization of
AuNPs-probe with the genomic DNA is confirmed visually and by
UV–vis spectra, and there is a dependency of the optical density of
the absorbance spectra on the genomic DNA amounts. Fig. 4C rep-
resents a calibration curve for the spectrophotometric detection
of Leishmania major genomic DNA by AuNPs-probe hybridization
assay. A calibration sensitivity of 2.12 OD ␮L ng−1 and a detection
limit of 7.0 pg ␮L−1 were obtained. The value of detection limit
obtained by the AuNPs-probe hybridization assay was >30 times
lower than a visual detection method of Leishmania species using
four oligonucleotide probes from kinetoplastid minicircle DNA [34].
3.4. Analysis of patient samples
In order to confirm the applicability of the AuNPs-probe
hybridization assay for analysis of clinical samples, DNA was
extracted from nine leishmaniasis patients and directly (without
PCR amplification) evaluated by AuNPs-probe hybridization assay.
Gel documentations of the samples are shown in Fig. 5. The results
indicated that 6 samples were of Leishmania major, and three of
 N. Sattarahmady et al. / Sensors and Actuators B 235 (2016) 723–731 727

Fig. 4. (A) Visual and (B) UV–vis spectra of different amounts of Leishmania major genomic DNA (10.4 (b), 11.4 (c), 13.2 (d), 15.6 (e), 15.7 (e), 18.2 (f) and 20.7 (d) ng) as positive
samples, Leishmania major (6.2 ng; a), Leishmania infantum (61.5 ng; h), Leishmania tropica (86 ng; i) genomic DNA as negative samples and, after AuNPs-probes hybridization
assay. Blank control (j) contains only AuNPs-probe without any genomic DNA. (C) A calibration curve for the spectrophotometric determination of Leishmania major genomic
DNA.

Leishmania tropica, based on the standard bands of Leishmania
major and Leishmania tropica, respectively. Fig. 6 shows photo-
graphic images (A) and UV–vis spectra (B) of the clinical samples
treated by the AuNPs-probe hybridization assay, after acid addi-
tion. It is observed that the colors of the 6 samples of Leishmania
major remained red, while the colors of three samples of Leish-
mania tropica, and the blank control turned from red to purple.
The results were also confirmed by UV–vis spectra. UV–vis spectra
(Fig. 6B) of the samples containing Leishmania major genomic DNA
showed a maximum peak at ∼528 nm, and the samples containing
Leishmania tropica genomic DNA and blank control showed a wide
absorbance spectra with maxima at ∼580–590 nm. Based on these
results, AuNPs-probe hybridization assay confirmed the gel docu-
mentation of PCR products of these samples. Therefore, detection
of genomic DNA of Leishmania major in clinical samples is achiev-
able without using sophisticated devices and PCR amplification;
this is an efficient and rapid alternative method for conventional
culture-based detections of Leishmania major.
728 N. Sattarahmady et al. / Sensors and Actuators B 235 (2016) 723–731

Fig. 5. PCR result obtained from patient samples. Ladder (a), Control sample of Leishmania major (lane b); Control sample of Leishmania tropica (lane c); clinical samples
(lanes d–i); clinical samples (lanes j–l) that are agreement with the bands of standard species of Leishmania major and Leishmania tropica, respectively.

Fig. 6. (A) Photographic images of extracted DNA from clinical samples of leishmaniasis patients after AuNPs-probe hybridization assay, positive samples that contain
Leishmania major genomic DNA (samples a–f), negative samples or samples contain other Leishmania strains (samples g–i) and blank control (j). (B) UV–vis spectra of clinical
samples, samples a–f (blue lines), samples g–i (red lines) and blank control j (green line). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

Table 1
A comparison of different methods for the detection of Leishmania major genomic DNA.

Detection method Linear range LOD Reference

AuNPs-probe-visual – 11.5 ng ␮L−1 [34]

Electrochemical biosensing 2 pg ␮L−1 –2 ␮g ␮L−1 2 pg ␮L−1 [64]
RT-PCR – 10 fg [65]
Direct Boil-LAMP – 1.0 × 103 parasites mL−1 [66]
Magnetic beads-cadmium selenite quantum dots – 103 cells mL−1 [67]
AuNPs-probe-visual – 0.34 ng ␮L−1 a This work
AuNPs-probe-spectrophotometric 0.04–0.086 ng ␮L−1 7.0 pg ␮L−1 This work

LOD: Limit of detection.

a
Limit of quantitation (LOQ) value.
 N. Sattarahmady et al. / Sensors and Actuators B 235 (2016) 723–731
Scheme 1. The basic biosensing mechanism of the biosensor.
4. Discussion
With the genetic knowledge about pathogenic microbes and
genetic diseases, DNA biosensors have become more significant in
diagnostics and forensics [43–45]. A DNA biosensor works based
on interactions between two complementary DNA sequences of
Watson-Crick base pairing [46]. Combination of DNA base pairing
character with surface plasmon property of metal nanostructures
resulted in highly sensitive, fast, elegant and easy-to-use optical
biosensors. Changes in the surface plasmon properties of metal
nanostructures depend on the particle size, and induce a color
change employed for sensing [47,48]. Plasmonic property of AuNPs
depends on size, surface function and interval distance between the
particles [49]. Presence or absence of complementary sequence of
AuNPs-probe DNA results in dispersion or aggregation of AuNPs
and produces a specific color. London-van der Waals attractive
forces between the AuNPs induce aggregation and change of the
solution color to blue. On the other hand, electrostatic interactions
between DNA and AuNPs induce particle dispersion and retain the
solution color to red [50]. The basic biosensing mechanism of the
biosensor is presented in Scheme 1.
The strategy of colorimetric detection of DNA using AuNPs [33]
has been applied for identification of different pathogens in bio-
logical fluids including Escherichia coli [51], mycobacterium [52],
pseudomonas syringae [53], HBV [54], HCV [55] and brucella [39,40].
AuNPs has a stable red color for more than one month. Addition of
HCl to the AuNPs solution induces a purple color.
The primary aim of this study was to develop a specific probe for
Leishmania major pathogens. Bioinformatic analyses of nucleotide
database indicated that the designed probe has a significant homol-
ogy to Leishmania major versus any non-Leishmania major species
with an E-value of 0.001.
Up to now, different techniques have been applied for species
differentiation in Leishmania species such as PCR-RFLP, RT-PCR
and isoenzyme analysis [55]. These techniques have shortcomings
such as long time and complicated equipment requirement. For
these assays, several DNA sequences were selected as the target
sequences from intrachromosal DNA, rRNA gene [56], ITS regions
[57], GP63 [58] or extrachromosomal DNA, and the repetitive kDNA
minicircles [59]. Minicircles kDNA is a better target for identifi-
729
cation of Leishmania parasites, compared to intrachromosal DNA,
because the copy number of intrachromosal DNA (less than 200) is
lower than that of kDNA minicircles (tens of thousands) [60–62].
The procedures presented in this study include the stages of
denaturation of target DNA at 93 ◦ C for 5 min, immediate ice cool-
ing, adding the AuNPs-probe and annealing at 63 ◦ C for 10 min;
the addition of HCl and the results was visualized within 1 min
with naked eye. However, the AuNPs-probe in the presence of non-
complementary sequence followed aggregation by acid through a
non-cross linking process [63]. All of the stages of the procedure
were carried out in a single tube preventing contaminations. Also,
change in the AuNPs-probe absorption spectra could be recorded
for quantitative analyses of the results.
The proposed assay was applied for serial dilutions of the
extracted genome of Leishmania major and a limit of quantita-
tion of 0.34 ng ␮L−1 was attained with naked eye. In addition, in
Table 1, a comparison between different methods for the detection
of Leishmania is presented. Nine leishmaniasis samples were tested
by both AuNPs-probe hybridization and PCR assays. There was a
100% agreement (6/9) for Leishmania major and (3/9) for Leishmania
tropica between the AuNPs-probe hybridization assay and PCR for
Leishmania major positive samples. These results suggest that the
proposed assay has comparable efficiency to conventional methods
and RT-PCR. The results of this assay were repeatable, and those
of Leishmania major detection are deduced only after 15 min with
parallel testing for a lot of samples.
5. Conclusion
An AuNPs-probe based on a sequence from non-protein cod-
ing region (AB678349.1) of Leishmania major minicircle kDNA was
employed as a DNA biosensor for visually and spectrophotometri-
cally detections of Leishmania major. The biosensor was applied for
complementary sequence and extracted genomic DNA with a high
sensitivity. Extracted genomic DNA from other Leishmania species
was applied for confirmation of the specificity of this biosensor. The
results showed that this method has a high specificity and sensitiv-
ity to determine Leishmania major in clinical samples. The present
assay is a fast, reliable and economical method for epidemiological,
taxonomic and clinical purposes.
730 N. Sattarahmady et al. / Sensors and Actuators B 235 (2016) 723–731
Acknowledgements
The paper has been extracted from the A. Movahedpour M.Sc.
thesis supported by the Research Council of Shiraz University
of Medical Sciences (7448) and Nanomedicine and Nanobiology
Research Center. We would also like to thank the Iran National Sci-
ence Foundation (INSF) for supporting this research, and Prof. A.
Khamesipour for his valuable assistances.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.snb.2016.05.023.
References
[1] S. Kumari, M. Samant, P.T. Khare, P. Misra, S. Dutta, B.K. Kolli, S. Sharma, K.P.
Chang, A. Dube, Photodynamic vaccination of hamsters with inducible
suicidal mutants of Leishmania amazonensis elicits immunity against visceral
leishmaniasis, Eur. J. Immunol. 39 (2009) 178–191.
[2] H.W. Murray, J.D. Berman, C.R. Davies, N.G. Saravia, Advances in
leishmaniasis, Lancet 366 (2005) 1561–1577.
[3] G.De. Muylder, K.K.H. Ang, S. Chen, M.R. Arkin, J.C. Engel, J.H. McKerrow, A
screen against Leishmania intracellular amastigotes: comparison to a
promastigote screen and identification of a host cell-specific hit, PLoS Negl.
Trop. Dis. 5 (2011) 1253.
[4] J.A.R. Postigo, Leishmaniasis in the world health organization eastern
mediterranean region, Int. J. Antimicrob. Agents 36 (2010) 62–65.
[5] S.M. Salman, N.G. Rubeiz, A.G. Kibbi, Cutaneous leishmaniasis: clinical
features and diagnosis, Clin. Dermatol. 17 (1999) 291–296.
[6] P. Desjeux, Leishmaniasis: current situation and new perspectives, Comp.
Immunol. Microbiol. Infect. Dis. 27 (2004) 305–318.
[7] B.L. Herwaldt, Leishmaniasis, Lancet 354 (1999) 1191–1199.
[8] M. Mohebali, E. Javadian, M.R. Yaghoobi-Ershadi, A.A. Akhavan, H. Hajjaran,
M.R. Abaei, Characterization of Leishmania infection in rodents from endemic
areas of the Islamic Republic of Iran, East Mediterr. Health J. 10 (2004)
591–599.
[9] C.K. Myint, Y. Asato, Y. Yamamoto, H. Kato, A.M. Bhutto, F.R. Soomro, M.Z.
Memon, J. Matsumoto, J.D. Marco, M. Oshiro, K. Katakura, Y. Hashiguchi, H.
Uezato, Polymorphisms of cytochrome b gene in Leishmania parasites and
their relation to types of cutaneous leishmaniasis lesions in Pakistan, J.
Dermatol. 35 (2008) 76–85.
[10] G. Wortmann, L. Hochberg, H.H. Houng, C. Sweeney, M. Zapor, N. Aronson, P.
Weina, C.F. Ockenhouse, Rapid identification of Leishmania complexes by a
real-time PCR assay, Am. J. Trop. Med. Hyg. 73 (2005) 999–1004.
[11] M. Jirku, E. Zemanova, A. Al-Jawabreh, G. Sch nian, J. Lukes, Development of a
direct species-specific PCR assay for differential diagnosis of Leishmania
tropica, Diagn. Microbiol. Infect. Dis. 55 (2006) 75–79.
[12] J.A. Rioux, G. Lanotte, E. Serre, F. Pratlong, P. Bastien, J. Perieres, Taxonomy of
Leishmania. Use of isoenzymes. Suggestions for new classification, Ann.
Parasitol. Hum. Comp. 65 (1990) 111–125.
[13] I.B. Abda, F. De Monbrison, N. Bousslimi, K. Aoun, A. Bouratbine, S. Picot,
Advantages and limits of real-time PCR assay and PCR-restriction fragment
length polymorphism for the identification of cutaneous Leishmania species
in Tunisia, Trans. R. Soc. Trop. Med. Hyg. 105 (2011) 17–22.
[14] C. Carson, R.J. Quinnell, J. Holden, L.M. Garcez, S. Deborggraeve, O. Courtenay,
Comparison of Leishmania oligoC-TesT PCR with conventional and real-time
PCR for diagnosis of canine Leishmania infection, J. Clin. Microbial. 48 (2010)
3325–3330.
[15] J.J. Shaw, R. Lainson, L. Ryan, R.R. Braga, D. McMahon-Pratt, J.R. David,
Leishmaniasis in Brazil: xXIII. The identification of Leishmania braziliensis
braziliensis in wild-caught neotropical sandflies using monoclonal antibodies,
Trans. R. Soc. Trop. Med. Hyg. 81 (1987) 69–72.
[16] R.S. Pacheco, U.G. Lopes, C.M. Morel, G. Grimaldi Jr., H. Momen, Schizodeme
analysis of Leishmania isolates and comparison with some phenotypic
techniques, in: J.A. Rioux (Ed.), Leishmania Taxonomie Et Phylogenese
Applications Eco-epidemiologiques, CNRS: INSERM, Montpellier, France,
1986, pp. 57–65.
[17] L. Simpson, Kinetoplast DNA in trypanosomatid flagellates, Int. Rev. Cytol. 99
(1986) 119–179.
[18] D.A. Maslov, A.A. Kolesnikov, G.N. Zaitseva, Conservative and divergent base
sequence regions in the maxicircle kinetoplast DNA of several
trypanosomatid flagellates, Mol. Biochem. Parasitol. 12 (1984) 351–364.
[19] M. Barrois, G. Riou, F. Galibert, Complete nucleotide sequence of minicircle
kinetoplast DNA from Trypanosoma equiperdum, Proc. Natl. Acad. Sci. U. S. A.
78 (1981) 3323–3327.
[20] R. Singh, C. Dutta, H.K. Majumder, Analysis of sequences of two different
classes of Kinetoplast DNA minicircles of a Leishmania spp, J. Biosci. 19 (1994)
171–182.
[21] D.F. Wirth, D. McMahon-Pratt, Rapid identification of Leishmania species by
specific hybridization of kinetoplast DNA in cutaneous lesions, Proc. Natl.
Acad. Sci. U. S. A. 79 (1982) 6999–7003.
[22] A. Rahi, N. Sattarahmady, H. Heli, Zepto-molar electrochemical detection of
Brucella genome based on gold nanoribbons covered by gold nanoblooms, Sci.
Rep. 5 (2015), 8060.
[23] A. Rahi, K. Karimian, H. Heli, Nanostructured materials in electroanalysis of
pharmaceuticals, Anal. Biochem. 497 (2016) 39–47.
[24] N. Sattarahmady, H. Heli, R. Dehdari Vais, An electrochemical acetylcholine
sensor based on lichen-like nickel oxide nanostructure, Biosens. Bioelectron.
48 (2013) 197–202.
[25] H. Heli, J. Pishahang, Cobalt oxide nanoparticles anchored to multiwalled
carbon nanotubes: synthesis and application for enhanced electrocatalytic
reaction and highly sensitive nonenzymatic detection of hydrogen peroxide,
Electrochim. Acta 123 (2014) 518–526.
[26] H. Heli, J. Pishahang, H. Barzegar Amiri, Synthesis of hexagonal CoAl-layered
double hydroxide nanoshales/carbon nanotubes composite for the
non-enzymatic detection of hydrogen peroxide, J. Electroanal. Chem. 768
(2016) 134–144.
[27] H. Heli, O. Amirizadeh, Non-enzymatic glucose biosensor based on
hyperbranched pine-like gold nanostructure, Mater. Sci. Eng. C 63 (2016)
150–154.
[28] H. Wang, R. Yang, L. Yang, W. Tan, Nucleic acid conjugated nanomaterials for
enhanced molecular recognition, ACS Nano 3 (2009) 2451–2460.
[29] C.S. Thaxton, D.G. Georganopoulou, C.A. Mirkin, Gold nanoparticle probes for
the detection of nucleic acid targets, Clin. Chim. Acta 363 (2006) 120–126.
[30] L. He, M.D. Musick, S.R. Nicewarner, F.G. SaLinas, S.J. Benkovic, M.J. Natan, C.D.
Keating, Colloidal Au-enhanced surface plasmon resonance for ultrasensitive
detection of DNA hybridization, J. Am. Chem. Soc. 122 (2000) 9071–9077.
[31] A. Vaseghi, N. Safaie, B. Bakhshinejad, A. Mohsenifar, M. Sadeghizadeh,
Detection of pseudomonas syringae pathovars by thiol-linked DNA-gold
nanoparticle probes, Sens. Actuators B 181 (2013) 644–651.
[32] P. Bakthavathsalam, V.K. Rajendran, J.A.B. Mohammed, A direct detection of
Escherichia coli genomic DNA using gold nanoprobes, J. Nanobiotechnol. 10
(8) (2012) 1–10.
[33] H.D. Hill, C.A. Mirkin, The bio-barcode assay for the detection of protein and
nucleic acid targets using DTT-induced ligand exchange, Nat. Protoc. 1 (2006)
324–336.
[34] M. Andreadou, E. Liandris, M. Gazouli, S. Taka, M. Antoniou, G.
Theodoropoulos, I. Tachtsidis, N. Goutas, D. Vlachodimitropoulos, I.
Kasampalidis, J. Ikonomopoulos, A novel non-amplification assay for the
detection of Leishmania spp. in clinical samples using gold nanoparticles, J.
Microbiol. Methods 96 (2014) 56–61.
[35] I. Renuart, P. Mertens, T. Leclipteux, One step oligochromatographic device
and method of use, EU patent WO 2004/099438A1 (2004).
[36] D.A. Maslov, A.A. Kolesnikov, G.N. Zaitseva, Conservative and divergent base
sequence regions in the maxicircle kinetoplast DNA of several
trypanosomatid flagellates, Mol. Biochem. Parasitol. 12 (1984) 351–364.
[37] K. Pandey, B.D. Pandey, A.K. Mallik, J. Acharya, K. Kato, O. Kaneko, P.E. Ferreira,
A new molecular surveillance system for leishmaniasis, Am. J. Trop. Med. Hyg.
90 (6) (2014) 1082–1086.
[38] K.C. Grabar, R.G. Freeman, M.B. Hommer, M.J. Natan, Preparation and
characterization of Au colloid monolayers, Anal. Chem. 67 (1995) 735–743.
[39] N. Sattarahmady, G.H. Tondro, M. Gholchin, H. Heli, Gold nanoparticles
biosensor of Brucella spp. genomic DNA: visual and spectrophotometric
detections, Biochem. Eng. J. 97 (2015) 1–7.
[40] N. Sattarahmady, Z. Kayani, H. Heli, Highly simple and visual colorimetric
detection of Brucella melitensis genomic DNA in clinical samples based on
gold nanoparticles, J. Iran. Chem. Soc. 12 (2015) 1569–1576.
[41] S. Khademvatan, N. Neisi, S. Maraghi, J. Saki, Diagnosis and identification of
Leishmania spp. From Giemsa-stained slides, by real-time PCR and melting
curve analysis in south-west of Iran, Ann. Trop. Med. Parasitol. 105 (2011)
559–565.
[42] M. Zuker, Mfold web server for nucleic acid folding and hybridization
prediction, Nucl. Acids Res. 33 (2003) 3406–3415.
[43] S. Cosnier, P. Mailley, Recent advances in DNA sensors, Analyst 133 (2008)
984–991.
[44] A. Sassolas, B.D. Leca-Bouvier, L.J. Blum, DNA biosensors and microarrays,
Chem. Rev. 108 (2008) 109–139.
[45] S. Hahn, S. Mergenthaler, B. Zimmermann, W. Holzgreve, Nucleic acid based
biosensors: the desires of the user, Bioelectrochemistry 67 (2005) 151–154.
[46] F.R.R. Teles, L.P. Fonseca, Trends in DNA biosensors, Talanta 77 (2008)
606–623.
[47] T. Endo, K. Kerman, N. Nagatani, Y. Takamura, E. Tamiya, Label-free detection
of peptide nucleic acid-DNA hybridization using localized surface plasmon
resonance based optical biosensor, Anal. Chem. 77 (2005) 6976–6984.
[48] D.G. Thompson, A. Enright, K. Faulds, W.E. Smith, D. Graham, Ultrasensitive
DNA detection using oligonucleotide-silver nanoparticle conjugates, Anal.
Chem. 8 (2008) 2805–2810.
[49] E. Liandris, M. Gazouli, M. Andreadou, M. comor, N. Abazovic, A. Leonardo,
Direct detection of unamplified DNA from pathogenic mycobacteria using
DNA-derivatized gold nanoparticles, J. Microbiol. Methods 78 (2009) 260–264.
[50] K. Sato, K. Hosokawa, M. Maeda, Rapid aggregation of gold nanoparticles
induced by non-cross-linking DNA hybridization, J. Am. Chem. Soc. 125
(2003) 8102–8103.
 N. Sattarahmady et al. / Sensors and Actuators B 235 (2016) 723–731
[51] W. Wang, C. Chen, M. Qian, X.S. Zhao, Aptamer biosensor for protein
detectionusing gold nanoparticles, Anal. Biochem. 373 (2008) 213–219.
[52] C. Thiruppathiraja, S. Kamatchiammal, P. Adaikkappan, D.J. Santhosh, M.
Alagar, Specific detection of Mycobacterium sp. genomic DNA using
duallabeled gold nanoparticle based electrochemical biosensor, Anal.
Biochem. 417 (2011) 73–79.
[53] A. Vaseghi, N. Safaie, B. Bakhshinejad, A. Mohsenifar, M. Sadeghizadeh,
Detection of Pseudomonas syringae pathovars by thiol-linked
DNA-goldnanoparticle probes, Sens. Actuators B 181 (2013) 644–651.
[54] D. Xi, X. Luo, Q. Ning, Q. Lu, K. Yao, Z. Liu, The detection of HBV DNA with
goldnanoparticle gene probes, J. Nanjing Med. Univ. 21 (2007) 207–212.
[55] S.M. Shawkya, D. Bald, H.M.E. Azzazy, Direct detection of
unamplifiedhepatitis C virus RNA using unmodified gold nanoparticles, Clin.
Biochem. 43 (2010) 1163–1168.
[56] G.J. Van Eys, G.J. Schoone, N.C. Kroon, S.B. Ebeling, Sequence analysis of small
subunit ribosomal RNA genes and its use for detection and identification of
Leishmania parasites, Mol. Biochem. Parasitol. 51 (1992) 133–142.
[57] G. Sch nian, A. Nasereddine, N. Dinse, C. Schweynoch, H.D. Schallig, W.
Presber, C.L. Jaffe, PCR diagnosis and characterization of Leishmania in local
and imported clinical samples, Diagn. Microbiol. Infect. Dis. 47 (2003)
349–358.
[58] N. Tupperwara, V. Vineethb, S. Rathb, T. Vaidya, Development of a real-time
polymerase chain reaction assay for the quantification of Leishmania species
and the monitoring of systemic distribution of the pathogen, Diagn.
Microbiol. Infect. Dis. 61 (2008) 23–30.
[59] L. Nicolas, G. Milon, N. Prina, Rapid differentiation of Old World Leishmania
species by LightCycler polymerase chain reaction and melting curve analysis,
J. Microbiol. Methods 51 (2002) 295–299.
[60] S. Brewster, D.C. Barker, Analysis of minicircle classes in Leishmania (Viannia)
species, Trans. R. Soc. Trop. Med. Hyg. 96 (2002) 55–63.
[61] G. Anders, C.L. Eisenberger, F. Jonas, C.L. Greenblatt, Distinguishing Leishmania
tropica and Leishmania major in the Middle East using the polymerase chain
reaction with kinetoplast DNA-specific primers, Trans. R. Soc. Trop. Med. Hyg.
96 (2002) 87–92.
[62] R. Kumar, R.A. Bumb, N.A. Ansari, R.D. Mehta, P. Salotra, Cutaneous
leishmaniasis caused by Leishmania tropica in Bikaner, India: parasite
identification and characterization using molecular and immunologic tools,
Am. J. Trop. Med. Hyg. 76 (2007) 896–901.
[63] H.I. Peng, B.L. Miller, Recent advancements in optical DNA biosensors:
exploiting the plasmonic effects of metal nanoparticles, Analyst 136 (2011)
436–447.
731
[64] S. Mohan, P. Srivastava, S.N. Maheshwari, S. Sundar, R. Prakash,
Nano-structured nickel oxide based DNA biosensor for detection of visceral
leishmaniasis (Kala-azar), Analyst 136 (2011) 2845–2851.
[65] M.G. Tellevik, K.E. Muller, K.R. Lokken, A.H. Nerland, Detection of a broad
range of Leishmania species and determination of parasite load of infected
mouse by real-time PCR targeting the arginine permease gene AAP3, Acta
Trop. 137 (2014) 99–104.
[66] K. Mikita, T. Maeda, S. Yoshikawa, T. Ono, Y. Miyahira, A. Kawana, The direct
Boil-LAMP method: a simple and rapid diagnostic method for cutaneous
leishmaniasis, Parasitol. Int. 63 (2014) 785–789.
[67] M. Andreadou, E. Liandris, M. Gazouli, A. Mataragka, I. Tachtsidis, N. Goutas, D.
Vlachodimitropoulos, J. Ikonomopoulosa, Detection of Leishmania-specific
DNA and surface antigens using a combination of functionalized magnetic
beads and cadmium selenite quantum dots, J. Microbiol. Methods 123 (2016)
62–67.
Biographies
Dr. N. Sattarahmady received her Ph.D. in biophysics from Institute of Biochemistry
and Biophysics, University of Tehran, Iran. She currently is an associate professor of
biophysics at Shiraz University of Medical Sciences. Her current interests include
protein structure, design of nanostructured materials for encapsulation, drug deliv-
ery and biosensors, and nanomedicine.
Mr. A. Movahedpour is a M.Sc. student in nanomedicine at Shiraz University of Med-
ical Sciences, Shiraz, Iran. His interests include nanobiosensors and nanomedicine.
Dr. H. Heli received his PhD in electrochemistry from K.N. Toosi University of Tech-
nology, Tehran, Iran. He currently is an assistant professor of chemistry at Shiraz
University of Medical Sciences, Shiraz, Iran. His major interests are synthesis of new
nanostructured and targeted materials and their applications in electrocatalysis,
bioelectrocatalysis, and electrochemical and medical nanodevices.
Dr. G.R. Hatam received his Ph. D. in Parasitology from Tarbiat Modaress University,
Tehran, Iran. He is currently Professor in Pasasitology, Shiraz University of Medical
Sciences, Shiraz, Iran.