determination of cdna encoding bcr/abl fusion gene in...
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Determination of cDNA encoding BCR/ABL fusion gene in patients with chronicmyelogenous leukemia using a novel FRET-based quantum dots-DNA nanosensor
Mojtaba Shamsipur, Vahid Nasirian, Ali Barati, Kamran Mansouri, Asad VaisiRaygani, Soheila Kashanian
PII: S0003-2670(17)30230-1
DOI: 10.1016/j.aca.2017.02.015
Reference: ACA 235075
To appear in: Analytica Chimica Acta
Received Date: 3 November 2016
Revised Date: 31 January 2017
Accepted Date: 13 February 2017
Please cite this article as: M. Shamsipur, V. Nasirian, A. Barati, K. Mansouri, A. Vaisi Raygani,S. Kashanian, Determination of cDNA encoding BCR/ABL fusion gene in patients with chronicmyelogenous leukemia using a novel FRET-based quantum dots-DNA nanosensor, Analytica ChimicaActa (2017), doi: 10.1016/j.aca.2017.02.015.
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Determination of cDNA encoding BCR/ABL fusion gene in
patients with chronic myelogenous leukemia using a novel FRET-
based quantum dots-DNA nanosensor
Mojtaba Shamsipur,a,* Vahid Nasirian,a Ali Barati,a Kamran Mansouri,b Asad Vaisi
Raygani,c Soheila Kashaniana a Department of Chemistry, Razi University, Kermanshah 6714967346, Iran b Medical Biology Research Center, Kermanshah University of Medical Sciences,
Kermanshah, Iran c Department of Biochemistry School of Medicine Kermanshah University of Medical
Sciences, Kermanshah 6714869914, Iran
ABSTRACT
In the present study, we developed a sensitive method based on fluorescence resonance
energy transfer (FRET) for the determination of the BCR/ABL fusion gene, which is used as
a biomarker to confirm the clinical diagnosis of both chronic myelogenous leukemia (CML)
and acute lymphocytic leukemia (ALL). For this purpose, CdTe quantum dots (QDs) were
conjugated to amino-modified 18-mer oligonucleotide ((N)DNA) to form the QDs-(N)DNA
nanosensor. In the presence of methylene blue (MB) as an intercalator, the hybridization of
QDs-(N)DNA with the target BCR/ABL fusion gene (complementary DNA), brings the MB
(acceptor) at close proximity of the QDs (donor), leading to FRET upon photoexcitation of
the QDs. The enhancement in the emission intensity of MB was used to follow up the
hybridization, which was linearly proportional to concentration of the target complementary
DNA in a range from 1.0 × 10-9 to 1.25 × 10-7 M. The detection limit of the proposed method
was obtained to be 1.5 × 10-10 M. Finally, the feasibility and selectivity of the proposed
nanosensor was evaluated by the analysis of derived nucleotides from both mismatched
sequences and clinical samples of patients with leukemia as real samples.
Keywords: BCR/ABL; chronic myelogenous leukemia (CML); FRET; CdTe QDs;
Fluorescence.
________________________________________________
* Corresponding author.
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E-mail addresses: [email protected] (M. Shamsipur).
1. Introduction
Chronic myelogenous leukemia (CML) is a clonal myeloproliferative sickness caused
by the neoplastic process of the primitive hemopoietic stem cell [1,2]. The diagnosis of CML
is faced with some troubles because patients with CML do not show any distinguishable
symptoms in their initial phase for 3–5 years [2]. The chimeric oncogene breakpoint cluster
region and the cellular abl (BCR/ABL) is a conventional gene that exists in most CML
patients and encodes a cytoplasmatic hybrid protein [3-5]. Therefore, the BCR/ABL has a
powerful role in the pathogenesis of CML [6]. In fact, the detection of BCR/ABL gene will
supply an early diagnosis and improve detecting of minimal remaining leukemia cells in the
CML patients, particularly after the bone marrow transplantation (BMT). In the last years,
many studies have been performed on the generation of the chimeric oncogene BCR/ABL
that leads to the development the CML monitoring techniques [3]. These techniques included
chromosome analysis, real-time quantitative reverse transcription [7], fluorescence in situ
hybridization [8], surface plasmon resonance imaging (SPRi) [9], chemiluminescence
imaging [10], flow cytometry [11], and electrochemical analysis [2,12-19]. However, these
expensive methods are associated with some limitations such as having less diagnostic
precision and being time-consuming. Therefore, it is very important to develop simple, rapid,
and effective techniques for diagnostics and pathogenics of genetic disease.
Meanwhile, the fluorescence resonance energy transfer (FRET)-based nanosensors
have been extensively considered to overcome some of the limitations in various fields such
as studding cleavage [20], nucleic acid detections [21], hybridization [22], structure,
functioning, and interactions of proteins [23]. FRET occurs when the electronic excitation
energy of a donor chromophore is transferred to a nearby acceptor molecule through dipole–
dipole interaction between the donor–acceptor pair [24]. A relatively larger distance between
the excitation and emission wavelengths in FRET, compared with a single fluorophore, can
remarkably reduce the crosstalk between the excitation light and the resulting fluorescence
signals, which is very necessary for an efficient fluorescence analytical method [25,26].
In this regard, semiconductor quantum dots (QDs) as a class of fluorescent nanomaterials,
with sizes smaller than the excitation Bohr radius [27], have attracted many attentions as
fluorescent probes owing to their specific optical properties such as narrow size-tunable
photoluminescence (PL) spectra, broad absorption spectra, intense quantum yields, and
extreme resistance against photobleaching, as compared to the conventional molecular dyes
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[28,29]. In addition, QDs can be easily capped with various water-soluble functional groups
[30,31] to form QD-bioconjugates via streptavidin–biotin interaction [32], glycosidic bonding
[33], electrostatic interaction [34], and metal-thiol bonding [35]. Benefited from these
advantages, QDs have been extensively employed as excellent donors in the FRET-based
nanosensors to enhance the amplification of their signals in feasible ways for the detection of
specific analytes such as nutrients [28, 36], explosives [37], proteolysis [38], enzymes [39],
and pH sensing [40].
In the present study, our goal was to develop a high sensitive, simple, and low-coast
FRET-based QDs-DNA nanosensor for the determination of the BCR/ABL fusion gene in
patients with CML (Type b3a2). We developed the nonosensor via a carbodiimide coupling
reaction between an amino-modified 18-mer oligonucleotides ((N)DNA) and terminal
carboxylic groups of thioglycolic acid-capped CdTe (TGA-CdTe) QDs. By addition of 18-
mer oligonucleotides target complementary DNA (cDNA) to the solution containing the
QDs-(N)DNA and methylene blue (MB), the double strands structures of DNA will be
formed between the probe and target sequences and, consequently, MB as an intercalator is
brought to a close proximity to the QDs. By the excitation of QDs, FRET between the QDs (a
donor) and MB (as acceptor) was observed and, accordingly, the enhancement in the
emission intensity of MB can be calibrated to the target cDNA concentration (see Scheme 1).
As real samples, the clinical samples confirmed to be positive CML by PCR were conducted.
In addition, the specificity and efficiency of the nanosensor were investigated using mismatch
and noncomplementary sequences. It is worth mentioning that, to the best of knowledge,
there is no previous literature report on the preparation of a FRET based nonobiosensor for
the detection of the BCR/ABL fusion gene in patients with CML.
(Scheme 1)
2. Experimental
2.1. Materials and apparatus
CdCl2.2.5 H2O, sodium borohydride (NaBH4), tellurium powder (99.997%), TGA and
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from
Sigma chemical company (St. Louis, Mo, http://www.sigmaaldrich.com). Other usual
chemicals were obtained from Merk (Darmstadt, Germany, http://www.merck.de). Phosphate
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buffers (20 mM) and other solutions were prepared with ultrapure Milli-Q water (resistance
=18 M Ω cm-1). The tested 18 mp-oligonucleotides in this project were synthesized by Faza
Biotech Company (Iran, http://www.fazabiotech.com) according to specific sequences of
BCR/ABL gene as follows:
Amino-modified probe: 5′-NH2(CH2)6 AGA GTT CAA AAG CCC TTC-3′
Target complementary DNA (18-base sequence): 5′-GAA GGG CTT TTG AAC TCT-3′
Single-base mismatch: 5′-GAA GGG CAT TTG AAC TCT-3′
Non-complementary: 5'-CTT CCC GAA AAC TTG AGA-3'
All fluorescence measurements were performed by a Varian Cary Eclipse
spectrofluorometer equipped with a micro quartz cell (1 cm×1 cm) in fast scan mode. Both
excitation and emission slits were set at 5 nm. UV-vis spectra were recorded on an Agilent
8453 diode array spectrophotometer over the range of 220-800 nm. The background UV-vis
spectrum was corrected with the blank phosphate buffer. The transmission electron
microscopy (TEM) images were recorded using an EM-10C Zeiss transmission electron
microscope (Zeiss, Germany) with accelerating voltages of 160 kV. Samples for recording
TEM images were prepared by drop casting solution on carbon coated copper grids and dried
at room temperature. In addition, a Malvern Dynamic Light Scattering (DLS) apparatus (UK,
http://www.Malvern.com) was used to investigate the size distribution of the synthesized
QDs.
2.2. Preparation of TGA capped CdTe QDs
The water-soluble CdTe QDs were synthesized according to the reported procedure of
our group [41]. Briefly, 0.1 g of Te powder was reduced by 0.280 g of NaBH4 in 7 mL
deionized water under stirring and nitrogen atmosphere. When the violet color of the solution
was removed, the solution was ultra-filtered to remove the superfluous precipitate of NaBH4.
200 mL nitrogen-saturated solution containing 0.358 g CdCl2.2.5, H2O and 0.2 mL TGA as a
stabilizing agent was added into the fresh prepared oxygen-free NaHTe aqueous solution (pH
10.0). The mixture (Cd2+, NaHTe and TGA) was transferred to an autoclave and heated in an
oven at 90 °C for 3 h. Then, the obtained solution was three times washed with ethanol to
remove excess Cd2+ and TGA, and subsequently centrifuged at 4000 rpm for 15 min. The
obtained precipitate was dispersed in 250 mL ultrapure water as QDs mother solution and
kept at 4 °C in dark. The TGA-CdTe solution was quite stable in the phosphate buffer (pH
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7.4) with no considerable changes in its optical characterization under such storage
conditions for 3 months.
2.3. Attachment of (N)DNA onto QDs
The surface carboxylic groups of functionalized QDs can be coupled to (N)DNA via
an amide linkage, which is performed by EDC. For this purpose, 30 µL of a QDs solution
(1.5×10-5 M) was added to 200 µL aqueous solution of EDC (0.1 mM) and incubated at pH
8.0 for 15 min. Then, 0.02 µmol of (N)DNA was added to the activated QDs under stirring.
The obtained solution was incubated at 37 °C for 60 min, and then stored at 4 °C in dark. The
formation of QDs-(N)DNA can be confirmed by gel electrophoresis based on the difference
between velocity shift assays of free QDs and the QDs-(N)DNA conjugates [42]. To
investigate the mobility of the particles, 10 µL of QDs-(N)DNA was loaded in 1% agarose
gel and run at 6 V cm−1 using tris-acetate-ethylenediamine tetraacetic acid (TAE, 0.1×) as a
mobile buffer. In addition, 10 µL of the TGA-QDs solution as the control test was loaded in
another lane. After electrophoresis for 40 min, a CCD captured the digital images of the gel
under UV.
2.4. Preparation of cDNA
In general, cDNA was synthesized from a messenger RNA (mRNA) template through
a catalyzed reaction by the enzyme reverse transcriptase. In this study, to prepare cDNA of
BCR/ABL genome, DNA of positive venous blood cells was extracted based on a previously
described method [42]. Accordingly, cDNA was synthesized with an equal amount (1 µg) of
the total RNA using M-MuLV Reverse Transcriptase according to the manufacturer's
recommendations (Promega, Madison, WI) and following a PCR amplification procedure.
The concentration of the synthesized cDNA was obtained as 1206.0 ng µL-1 by nanodrop.
Two different volumes of the cDNA sequences (1.0 and 1.5 µL) were spiked separately into
two sample vials contains 1 mL phosphate (100 mM), NaCl (10 mM), and MgCl2 (3 mM) at
pH 7.4, and sequentially stored at 4 °C.
2.5. Detection of target cDNA
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The hybridization experiments were studied in the buffer solutions containing 100
mM phosphate (pH 7.4), 10 mM NaCl and 3 mM MgCl2 with no detectable background
signal. The hybridization reactions were performed by the combination of 50 µL of
conjugated QDs-(N)DNA, 20 µL MB (1.5×10-5 M) and the desired concentrations of target
cDNA in a 1.0 mL micro tube. The total volume of the mixtures was fixed at 200 µL, and
were shaken for 20 min at 50 °C and, subsequently, cooled at 4 °C. The hybridization
solutions were transferred to a microquartz cuvette to record emission spectra of MB with the
excitation wavelength 488 nm.
3. Results and discussion
3.1. Characterization of MB and QDs
Fig. 1a shows the TEM image of the prepared CdTe QDs that confirms a good mono-
dispersity and spherical morphology of these nanoparticles with a particle size of about 3.5
nm. In addition, the DLS analysis supported the TEM results and showed that the distribution
of average size of CdTe QDs particles is relatively narrow with a mean size of 10 nm (Fig.
1a). The obtained size of the particles in DLS is usually greater than the obtained results from
TEM because the hydrodynamic radius of the particles is estimated in DLS. In fact, TEM
probes the electron rich area of a particle and gives the particle size without this solvation
layer. Therefore, no significant difference was obtained from the size of nanoparticles in
colloidal suspension and when dried in TEM. Whereas, DLS gives the particle size along
with the around solvation layer with higher size than that measured by TEM.
The quantum yield (QY) of the synthesized QDs was determined to be 0.24, relative to
the QY of the fluorescein dye in a sodium borate buffer (pH 9.5, λx = 490 nm, and QY =
0.93) [43]. The normalized photoluminescence (PL) spectra of CdTe QDs is presented in Fig.
1b, The QDs show a narrow and symmetric emission spectra at the maximum wavelength
599 nm with full width at half maximum (FWHM) about 70 nm, excited at 488 nm, and a
broad absorption spectrum from 200 to 550 nm. Such narrow emission spectrum can
considerably reduce the possible cross-talk between the emission spectral of the donor QDs
and the acceptor [44]. In addition, the broad absorption spectrum of CdTe QDs provides
flexible choices for suitable excitation wavelength to minimize the background emission
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from the acceptor [45]. On the other hand, MB shows an absorption spectrum from 550 to
668 nm, and emission spectra with maximum intensity at 678 nm (Fig. 1b).
Meanwhile, Fig. 1b shows an acceptable spectral overlapping between the absorption
spectrum of MB and the emission spectrum of the QDs. This spectral overlapping is suitable
for archiving maximum FRET. In addition, the broad absorption spectrum of QDs provides
flexibility in choice of a suitable wavelength for QDs excitation to minimize the background
emission interference and cross-talk between the CdTe QDs and MB.
(Fig. 1)
3.2. Calculation of Förster radius between CdTe QDs and MB
The experimental FRET efficiency (E) was calculated by Eq. 1:
(1)
where FD and FDA are the fluorescence intensities of the free donor (QD) and the donor in the
presence of acceptor (MB), r is the distance between the donor and acceptor and R0 is the
Förster radius for which the energy transfer efficiency is diminished to 50% of the maximal
as calculated using Eq. 2 [46]:
(2)
where κ is an orientation factor, which is regarded as 2/3 for a randomly orientated donor–
acceptor pair, n is the refractive index of the medium, which is 1.33 for water, Qd is the QY
of the donor in the absence of acceptors that obtained as 24%, J is the spectral integral as a
function of wavelength, expressing the spectral overlap between the emission spectrum of the
donor and the absorption spectrum of the acceptor, which is obtained from Eq. 3:
(3)
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where FD(λ) is the dimensionless emission intensity; λ is the wavelength; ε (λ) is the molar
absorption coefficient of MB at λ. The calculated overlap integral is around 33.194×10-17 M-
1 cm-1 nm4. Thus, according to Eq. 2, the Förster radius was found to be 6.2 Å.
3.3. Characterization of the QDs-(N)DNA
The formation of QDs-(N)DNA can be confirmed by gel electrophoresis based on the
difference between the velocity shift assays of free QDs and the QDs-(N)DNA conjugates
[47]. The charge-to-mass ratio of the particles is the main parameter for the definition of the
velocity in the gel electrophoresis if other conditions such as the running buffer, the strength
of the electric field, and the shape of the materials were fixed [48]. By the attachment of
DNA to QDs, the charge-to-mass of QDs will be changed and, therefore, show a different
velocity in the gel electrophoresis.
Fig. 2 shows the shift in mobility of the TGA-QDs and the conjugated QDs-(N)DNA
in the gel electropherogram, from a volume loading 5 µL of each solution. As seen, the TGA-
QDs is immigrated faster than the conjugated QDs-(N)DNA due to the successful grafting of
(N)DNA onto TGA-QDs [48, 49]. The conjugation of (N)DNA did not interfere with the
optical properties or QY of the QDs, and the difference between the absorption and emission
intensity of QDs and QDs-(N)DNA was insignificant.
(Fig. 2)
3.4. Optimization of the nanosensor at different conditions
The efficiency of FRET process (E) is defined as the number of quanta transferred to
the acceptor per total absorbed quanta by the donor [50, 51]. Some factors such as DNA/QDs
molar ratio, the MB concentration and the hybridization time are the most effective factors
affecting E, which should be optimized to obtain maximum FRET, when the developed probe
exited at 488 nm.
The number of attached (N)DNA to QDs surface ((N)DNA/QDs) is the main factor that
must be optimized for a desirable limitation of detection due to its significant effect on the
fluorescence quantum yield and FRET efficiency to obtain [52].
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Fig. 3b shows the obtained results from study the FRET signal as a function of MB
concentration at fixed amount of QDs-(N)DNA and target cDNA. As can be seen, the
intensity of the FRET signal was enhanced by increasing the MB concentration in the range
0.0−1.5×10-5 M of MB, which exposed the considerable effect of MB concentration on the
FRET process. The hybridization time is another important factor that was optimized. As
shows in Fig. 3c, the maximum intensity of the hybridization assay at a steady state, was
obtained after 20 min, which was adjusted for other experiment steps. According to the
obtained results, the noticeably effected of consuming time on hybridization process and the
fluorescence intensity of MB was approved.
For this purpose, different mole ratios of (N)DNA/QDs were investigated to obtain an
optimum of FRET signal at the constant MB and cDNA target concentrations. Maximum
transferred FRET signal was achieved at mole ratio 45:1 of (N)DNA/QDs (Fig. 3a). At ratios
below this value, the FRET signal was negligible due to insufficient ways to transfer energy
from QDs to near MB dyes and by increasing DNA/QDs ratio further than 45:1, the FRET
signal was levelled off at a constant intensity.
(Fig. 3)
3.5. Detection of target cDNA sequence
The determination of the BCR/ABL gene was carried out through hybridization
between conjugated probe (N)DNA 18-mer on the QDs surface and the target sequence
DNA. The MB could intercalate between the formed dsDNA and, therefore, it was brought
within a distance on the order of the Förster radius. MB specifically intercalates with guanine
bases in the DNA sequence, and is widely used as a chemical indicator to detect the
accomplished hybridization in fluorescence and electrochemical DNA biosensors [53-58]. As
a result, the FRET performed from QDs as the donor to MB as the acceptor upon the
excitation of the QDs. All experiments were performed in buffered solutions.
The corresponding emission spectra of this FRET-based nanosensor for different
concentrations of the target cDNA are shown in Fig. 4. As seen, by increasing the
concentration of the target cDNA, the emission intensity of MB is gradually increased, while
the QDs emission is quenched. These observations indicated the suitability of the modified
CdTe with oligonucleotide and MB for energy transfer from the excited donor to the
unexcited acceptor in the FRET system. The emission intensity of MB at 700 nm was used
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for the quantitative determination of the target cDNA concentration. This signal was linearly
proportional to the target cDNA concentration over the range of 1.0×10−9−1.25×10−7 M (R2 =
0.991). By further increasing the target cDNA, MB emission signal was levelled off at
constant amount due to the reduction of the available sites for more hybridizations. It is
interesting to note that, in the absence of target DNA, the MB fluorescence is only slightly
detectable for the QD-(N)DNA/MB system, which may suggest too weak ability of MB for
binding to a single sequence DNA, under the experimental conditions used. The imit of
detection (LOD) of the method was obtained as 1.5×10−10 M based on equation LOD = 3S0/K
(n=6), where K is the slope of the calibration curve.
(Fig. 4)
A comparison between the figures of the merit of our procedure and the previously
reported electrochemical biosensors for the detection of BCR/ABL is summarized in Table 1.
It is worth mentioning that, although the sensitivity of some of the reported electrochemical
sensors is higher than that of our developed FRET-based nanosensors for determination of
BCR/ABL fusion gene, they often suffer from some limitations including problem with
determination of amount of DNA immobilized on the electrode surface, expensive and long
procedures in synthesis of intercalators, and limited number of truly selective probes for
hybridization process. Compared with many other developed DNA detection systems that
requires half an hour to over 1 h for complication of hybridization [2, 12, 13, 15, 16, 17, 18],
our method is much faster and is completed in 20 min, which is a result of fast hybridization
of DNA in the proposed FRET system. Moreover, as it can be seen from “Remarks” column
of Table 1, in some of previous electrochemical methods some important criteria such as
application to real samples, results of recovery tests and relative standard deviations have not
been reported.
(Table 1)
3.6. Detection of single-base mutants and non-complementary sequences
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In order to evaluate the specificity and selectivity of the nanosensor, one-base mismatched
sequence and non-complementary sequences were examined with QDs-(N)DNA in the
presence of MB under the optimum experimental conditions. The obtained results were
compared with the fluorescence intensities of MB in the presence of the target cDNA
sequence at similar concentration (1.25×10-7 M) (Fig. 5). The results clearly showed that the
emission intensity of MB for the non-complementary sequence is very low. In addition, for
one-base mismatched target sequence, the emission intensities of MB were only 30% of that
observed for target cDNA sequence. The obtained results clearly confirmed the strong
intercalation of MB within the double-stranded DNAs with sequence specificity against the
mismatches or non-complementary strands in the FRET assay, as reported before [61-63].
These results confirmed the high selectivity of the proposed nanosensor with very low
interface of the unspecific targets. Accordingly, the removal of these DNAs becomes
unnecessary, which is considered as a distinct advantage for this FRET-based method.
3.7. Real sample assay
In genetics, cDNA is the single stranded DNA whose introns have been removed and
is recognized as a convenient way instead of RNA in various fields such as working with the
coding sequence, gene cloning, and the creation of a cDNA library. Because, the
degradability of RNA by omnipresent RNases can easily led to occurrence of mutations, in
this study, to investigate the practical applicability of the FRET nanosensor for the actual real
samples, we used the cDNA of clinical samples, with confirmed positive CML by PCR, as
analyte. The prepared FRET-based nanosensor was then applied to the quantitative
determination of BCR/ABL fusion gene (target cDNA) of the positive CML patients
according to the descripted method. The obtained results are reported in Table 2. The results
illustrated that this FRET-based nanosensors possesses a high potential for the detection of
BCR/ABL fusion gene in positive CML patients.
(Table 2)
4. Conclusion
In this study, for the first time, we developed a FRET-based nanosensor to detect
BCR/ABL fusion gene in CML positive patients. The proposed nanosensor provided specific
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detection of target BCR/ABL fusion gene through a sensitive and rapid procedure, without
any need for excess operations, with a good linear relationship between the FRET signal and
the concentration of target cDNA in a broad range of concentration. This label-free system
could be recommended as a simple and specific platform to eliminate the urgent necessities
and problems in the quantification detection of the BCR/ABL gene in the CML positive
patients.
Acknowledgements
The authors acknowledge the support of this work by Research Councils of Razi
University and Kermanshah University of Medical Sciences and Iran's Scientific Elite
Federation.
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Figure legends
Scheme 1. Schematic representation of the designed nanosensor for the detection of
BCR/ABL fusion gene based on a FRET system using the CdTe QDs-DNA as a donor and
the MB as an acceptor.
Fig. 1. TEM image (a) and DLS histogram (b) of the synthesized TGA-CdTe QDsand PL
emission and absorption spectra of TGA-CdTe QDs and MB (c). The inset of (c) shows
photograph images of synthesized TGA-CdTe QDs under room light (left) and UV light with
365 nm (right).
Fig. 2. Agarose gel (1.0 %) electrophoresis of TGA-CdTe QDs (lanes 1, 2) and the QDs-
(N)DNA conjugates obtained through EDC activation (lanes 3, 4). Tris-acetate-
ethylenediamine tetraacetic acid (TAE, 0.1×) was applied as a mobile buffer and run at
voltage gradient 60 V cm−1.
Fig. 3. Fluorescence intensity of MB resulted from FRET between the QDs-(N)DNA and MB
as a function of (N)DNA/QDs at constants MB and cDNA concentration (a), MB
concentration at constants QDs-(N)DNA (33 pM) and target DNA (120 nM) concentrations
(b) and hybridization time at constant amounts of DNA (50 µL) cDNA (100 nM) and MB
(1.5×10-5 M) (c). λex= 488.
Fig. 4. The emission spectra from the solution containing 50 µL of QDs-(N)DNA conjugate,
20 µL MB (1.5×10-5 M) at different concentrations of cDNA: (I) 0, (II) 1, (III) 15, (IV) 35,
(V) 60, (VI) 90, (VII) 110, and (VIII) 125 nM. Inset shows the calibration curve of F/F0 vs.
concentration of target cDNA, where F0 is the fluorescence intensity of MB at 700 nm and F
is the emission intensity of MB in the presence of increasing concentrations of target cDNA.
Fig. 5. Variations of MB emission intensity for different DNA sequences, (a)
uncomplementary sequence, (b) one-base mismatched sequence and (c) full complementary
sequence (DNA concentration: 12.5×10-8 M).
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Table 1 A comparison between the Figures of merit of this study and different articles for BCR/ABL fusion gene determination.
Method Detection system LR (M)
LOD (M)
Remarks Ref.
DPV1 GCE2 1.2×10−7‒6.7×10−7 5.9×10−8
RSD3: 6.18%, pH: 4.8, BE4: Modified DNA, Indicator: MB5, Selectivity: Good, Hybridization temp.: 45 °C, Hybridization time: 35 min, Response time: 5 min, RS6: NR7, Recovery: NR
[2]
DPV
Modified GCE by suspension of graphene in the presence of chitosan,polyaniline layer and AuNPs
1.0×10-11‒1.0×10-9 2.1×10-12
RSD: 4.2%-5.4%, pH: 7.5, BE: functional hairpin DNA, Indicator: streptavidin-alkali phosphatase, Hybridization time: 120 min, Response time: 17 min, Hybridization temp.: 25 °C, RS: cDNA from K562 cells, Recovery: NR
[12]
DPV 4-Aminobenzene
sulfonic acid/ GCE 1.0×10-12 ‒1.1×10-11 9.4×10-13
RSD: 6.29%, pH: 7.0, BE: locked nucleic acid, Indicator: MB, Hybridization time: 30 min, Response time: 5 min, Hybridization temp: 42.8 °C, RS: PCR products from K562 cells, Recovery: NR
[19]
DPV u electrode 1.0×10-9–16.0×10-9 1.2×10-10 RSD: 5.13%, pH: 8.0, BE: hairpin locked nucleic acid, Indicator: MB, Hybridization time: 60 min, Hybridization temperature: 58.8 °C, RS: PCR products
[13]
DPV Modified GCE 6.2×10-8 ‒3.1×10-7 5.3×10-9 RSD: NR, pH: 7.0, BE: hairpin DNA, Indicator : Nitroacridone, Hybridization time: 30 min , Hybridization temperature: 45°C, RS: PCR products
[15]
DPV GCE modified by poly eriochrome black T and AuNPs
1.0×10-12 ‒1.0×10-8 1.0×10-13
RSD: NR, pH: 7.4, BE: thiol modified DNA and biotin labeled DNA, Indicator: HRP, Hybridization time: 45 min, Response time: 25 min, Hybridization temp.: 50.0 °C, RS: PCR products from K562 cells, Recovery: NR
[16]
SPRI8 Gold islands array 1.0×10-10‒5.0×10-7 1.0 ×10-10 RSD: 12.6%, pH: 7.4, BE: thiol modified DNA, Hybridization time: 20 min, Hybridization temp.: 25 °C, RS: PCR products from K562 cells
[9]
CLI9
Bis-three-way junction nanostructure and cascade DNA machineries
1.0×10-13‒1.0×10-7
RSD: 12.6%, pH: 7.4, BE: thiol modified DNA, Indicator: Hybridization time: 20 min, Hybridization temperature: 25 °C, RS: PCR products from K562 cells
[10]
CV10 EIS11
Polyaniline-AuNps 6.9×10-18‒6.9×10-13 6.94×10-18
RSD: 1.3%‒2.1%, pH: 8.6, BE: DNA, Indicator: polymerase/nicking enzyme machinery, Hybridization time: 90 min, Hybridization temp.: 25°C, RS: Spiked DNA in human serum, Recovery: 96.0%‒ 98.2%
[17]
DPV Au electrode
modified by AuNPs 1.0×10-9–1.0×10-7 1.0×10-10
RSD: 6.01%, pH: 7.4, BE: thiolated-hairpin locked nucleic acid, Indicator: Benzoate binuclear copper (II) complex, Hybridization time: 60 min, Response time: 15 min, Hybridization temp. 37 °C, RS: PCR products
[18]
FRET CdTe QDs 1.0×10-9–1.25×10-7 1.5×10-10 RSD: 5.2%, pH: 7.4, BE: Amine modified DNA, Indicator: MB, Hybridization time: 20 min, Hybridization temp.: 50 °C, RS: cDNA products from PCR
This work
1 Differential pulse voltammetry. 2 Glassy carbon electrode. 3 Relative standard deviation. 4 Biorecognition element. 5 Methylene blue. 6 Real sample. 7 Not reported. 8 Surface plasmon resonance imaging. 9 Chemiluminescence imaging. 10 Cyclic voltammetry. 11 Electrochemical impedance spectroscopy.
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Table 2 The obtained results from cDNA analysis of real sample by the developed FRET nanosensor
Sample Added cDNA
(M)
Detected cDNA
(M)
Recovery
(%)
RSD
(%)
1 10.00× 10-8 8.90 (± 0.5)×10-8 89 5.6
2 25.0× 10-9 22.50 (± 0.7)×10-9 90 3.1
3 50.00× 10-9 46.50 (± 1.2)×10-9 93 2.6
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Scheme 1
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Fig. 1
a)
b)
c)
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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- A sensitive FRET-based nanosensor for detection of BCR/ABL fusion gene in leukemia patients.
- Conjugation of CdTe QDs to amino-modified 18-mer oligonucleotide to form QDs-(N)DNA nanosensor.
- Hybridization of QDs-(N)DNA with the target BCR/ABL in the presence of methylene blue (MB) as intercalator.
- Occurrence of FRET from QDs (donors) to MB (acceptor) upon photoexcitaion of CdTe in hybrid.
- Linear relationship of enhanced emission of MB with amount of target DNA from 1.0×10-9 to 1.25×10-7 M.