Biosensors and Bioelectronics 129 (2019) 182–188

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

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

A sensitive electrochemical genosensor for highly specific detection of T

thalassemia gene
Mohammad-Bagher Gholivand , Arezoo Akbari
⁎

Department of Analytical Chemistry, Razi University, Kermanshah, Iran

ARTICLE INFO ABSTRACT

Keywords: Beta thalassemias (βth) are the result of mutations in the β-globin gene. In this report, an electrochemical
β-Thalassemia genosensor was made to detect the sequences anent with the β-globin gene. This biosensor is based on im-
Aminothiophenol mobilizing 20-mer single stranded oligonucleotide (probe) on the Au nanoparticles- poly (4-aminothiophenol)/
Graphene oxide reduced graphene oxide/glassy carbon electrode (AuNPs-PAT/rGO/ GCE) and hybridizing this oligonucleotide
Electrochemical biosensor
along with its complementary sequence (target). The vastness of the probe and target sequences hybridization
was studied through differential pulse voltammetry (DPV) along with electrochemical impedance spectroscopy
(EIS) using the [Fe(CN)6]3−/4− (1:1) as a hybridization index. The biosensor indicated great efficiency with
significant sensitivity along with favorable selectivity. The DPV and EIS responses with the intended con-
centrations of the sequence were linear varying from 1.0 pM to 400.0 pM (Ip ν log C) and 0.5–400.0 pM (∆Rct ν
log C) with a limit of detection of 0.06 pM and 0.035 pM, at the signal to noise ratio of 3σ. The biosensor of the
DNA indicated proper discrimination capability to mismatched two-base, three-base, and non-complementary
sequences.

1. Introduction biosensors. Nanomaterial based platforms find wide use in many elec-
trochemical, electroanalytical and bioelectrochemical applications.
The growing demand for rapid, simple, inexpensive and portable Nanoscale materials such as metal nanoparticles (Zare and Shabani,
testing methods instead of the expensive and time-consuming methods 2016; Oliveira et al., 2018; Sharma et al., 2018), metal oxides (Low
for specific sequences detection in nucleic acids has encouraged re- et al., 2017; Jiang et al., 2018), carbon allotropes (Frias et al., 2017;
search in the field of DNA sensors or genosensors. DNA sequence Jaiswal et al., 2018; Mohammadian and Faridbod, 2018) and con-
polymerization (PCR) and DNA hybridization (FISH) are commonly ductive polymers (Wang et al., 2015; da Silva et al., 2017; Moon et al.,
used methods for specific sequences detection in nucleic acids that not 2018) offer some outstanding prospects for designing new bioelectronic
only have the above-mentioned drawbacks but also are laborious. A devices exhibiting novel functions. Due to excellent properties such as
variety of DNA sensors as the most attractive alternative have been conductivity, simple synthesis, good aqueous dispersibility, large sur-
proposed for the detection of DNA sequences including optical (Scarano face area, and good biocompatibility, graphene oxide (GO) and reduced
et al., 2010), piezoelectric (Lucarelli et al., 2008) and electrochemical graphene oxide (rGO) have found applicability in the field of biosensing
(Sadik et al., 2009) transduction. Electrochemical gene sensors show (Lu et al., 2009; Chen et al., 2012) rGO alone or its composite with
attractive features such as high sensitivity, fast response and cost-ef- other materials (Zhou et al., 2009; Chen et al., 2011; Cai et al., 2014;
fectiveness needed for the preliminary detection of diseases, pre- Shamsipur et al., 2016). Reports indicate that the presence rGO has
ventative therapy of genetic disorders, and the treatment of bacterial increased the surface area and speed of electron transfer process. AuNPs
and viral infections (Kashish et al., 2015). Electrochemical DNA geno- offer great nanoplatforms to interact with biological systems and thus
sensors consist of a DNA probe immobilized onto the electrode surface. have been applied in genosensor construction (Shi et al., 2014; Tiwari
This probe is bound to a specific target DNA sequence, generating an et al., 2015; Zhao et al., 2015). The incorporation of AuNPs into rGO
electrical signal (Dolatabadi et al., 2011). increases the surface area, and adsorption of immobilized ssDNA probe
Signal amplification in DNA based sensors is usually achieved molecules results in amplified signals in analyte detection. To increase
through various surface modifications. Advancements in nanomaterials the uniformity of the dispersed metal particles at the electrode surface,
science offer amazing opportunities for making new, sensitive a conducting polymer with entrapping ability of metal nanoparticles

⁎
Corresponding author.
E-mail address: [email protected] (M.-B. Gholivand).

https://doi.org/10.1016/j.bios.2019.01.017
Received 12 October 2018; Received in revised form 3 January 2019; Accepted 4 January 2019
Available online 15 January 2019
0956-5663/ © 2019 Published by Elsevier B.V.
M.-B. Gholivand, A. Akbari Biosensors and Bioelectronics 129 (2019) 182–188

into its matrix has been advised (Shin and Huh, 2012; Wilson et al., common cell was utilized along with saturated calomel electrode (SCE),
2012). 4-Aminothiophenol (AT) as one of this class compound with Pt wire and modified electrode as reference, counter and working
nanoparticles assembling ability via covalent or electrostatic interac- electrodes. The solutions pH was adjusted by a JENWAY-3510 pH meter
tions, has attracted significant attention. The presence of thiol group in equipped with a combined glass electrode. The scanning electron mi-
AT structure assembled the AuNPs and resulted in unique morphology. croscopy (SEM) (Philips XL 30) was used for morphology study.
Thalassemia is a genetic blood disorder passed down through fa-
milies (inherited). Due to a disease-causing variant in one or more of 2.3. Producing modified electrode
the globin genes, a disruption in the normal ratio of alpha globin to beta
globin production is created and results in a microcytic anemia of Alumina (1.0 and 0.05 µm) was used to polish bare GCE and then
varying degree. There are two primary types of Thalassemia disease: was completely cleaned and made free adsorbed particle by distilled
Alpha Thalassemia disease and Beta Thalassemia disease. Beta water washing and ethanol container bath ultrasonicating for 10 min-
Thalassemia Major (also called Cooley's Anemia) is a serious illness. Its utes. 5 μL of 1 mg mL−1 aqueous solution GO was dropped on the GCE
symptoms appear in the first two years of life and include paleness of surface. After drying, GO film was reduced electrochemically in a N2-
the skin, poor appetite, irritability, and failure to grow. The increasing saturated phosphate buffer (0.1 M, pH 7.4) by cyclic voltammetry (CV)
interest in biosensors and especially genosensors during recent years, at potential window of −1.5–0.0 V using scan rate of 100 mV s−1
and reporting only one genosensor for Thalassemia detection (Hamidi- throughout 40 cycles. The rGO/ GC electrode was washed using water
Asl et al., 2016) encourage us to fabricate a simple, portable equipment and submerged in a solution 0.2 mM NaAuCl4, 0.5 M H2SO4 and 10 mM
with lower costs, compared to the more classical techniques. AT. CV technique was applied for electrochemical deposition using a
In the present study a DNA biosensor based on one-step electro- repetitive potential scan between −0.5 - + 1.7 V (versus SCE) at a scan
ploymerization of 4-aminothiophenol monomer and Au nanoparticles rate of 50 mV s−1 for 50 cycles (Fig. S1). Poly (4-aminothiophenol)
on reduced graphene oxide /Glassy carbon electrode (rGO /GCE) that (PAT) film containing Au nanoparticles were made on the surface of
was utilized as a tool for sensing ultra-traces of β-globin gene. Au na- rGO/ GC electrode simultaneously.
noparticles were electrochemically disturbed into the matrix of con-
ductive polymer, assembled on the surface of rGO /GCE electrode. The 2.4. Working electrode activation
thiolated strand of human β-thalassemia gene was attached to AuNPs
covalently through a self-assembly approach as the probe to detect β- The working electrode surface was activated by cyclic voltammetry
globin gene (antisense strand of human β-thalassemia gene). in 0.50 M HClO4 without stirring at the potential range 0.0–0.8 V and
scan rate of 100 mV s−1 for 20 cycles.
2. Experimental
2.5. Immobilizing the probe on the working electrode
2.1. Reagents and materials
The immobilizing the pDNA on the activated AuNPs-PAT/rGO/ GCE
A 20-mer oligonucleotide related to human β-thalassemia gene was carried out by dropping of 5 μL of 0.1 μM pDNA solution on its
sense-strand (IVSII-1)) and its complementary (CIVSII-1) oligonucleo- surface and incubating for 2 h at ambient temperature. pDNA along the
tide related to antisense strand of human β-thalassemia gene were thiol groups at 5-end were covalently bonded to the AuNPs presented in
utilized as probe and target DNA. DNA oligonucleotides along with the the modified electrode body through Au–S binding. To remove un-
sequences that follow were achieved from Fazabiotech Co. (Tehran, specific adsorbed DNA probe, the pDNA/AuNPs-PAT/rGO/ GC elec-
Iran): trode was washed using deionized water. Ultimately, to block the free
sites and reduce the non-specific bindings, the electrode was submerged
Probe DNA (pDNA): 5′-SH-ACTTCAGGATGAGTCTATGG-3′ (IVSII-1) into the BSA solution (0.25%) for 15 min.
Complementary DNA (cDNA): 5′-CCATAGACTCATCCTGAAGT-3′
(CIVSII-1) 2.6. Hybridization
Non-complementary sequences (ncS): 5′-AATCTCATGGCCGATTC
GTT-3′ The hybridizing process was done by casting 10 μL cDNA with dif-
Double-base mismatched DNA (dbmDNA): 5′-CGATTGACTCATCCT ferent concentrations on the pDNA/Au NPs-PAT/rGO/ GCE surface,
GAAGT-3′ and keeping it for 35 min at the ambient temperature, and then to re-
Three-base mismatched DNA (tbmDNA): 5′-CGATTGACACATCCTG move the non-specifically adsorbed DNA, the hybridized electrode was
AAGT-3′ rinsed using PBS (pH 7.4).

All stock solution of oligonucleotides (100 µM) was made ready 2.7. Electrochemical detection
using deionized water and was kept frozen.
All chemicals had the analytical grade and were utilized as received. The sensing ability of the pDNA/AuNPs-PAT/rGO/ GC electrode
Graphene oxide, sodium phosphate dibasic, bovine serum albumin toward cDNA was evaluated by EIS and differential pulse voltammetric
(BSA), 4-aminothiophenol (AT), sodium tetrachloroaurate (III) dihy- (DPV) techniques based on “signal on” and “signal off” strategies, re-
drate (NaAuCl4·3H2O), potassium hexacyanoferrate (II) trihydrate spectively. In the impedimetric determination, after hybridization of
(K4Fe(CN)6·3H2O), H2SO4, potassium hexacyanoferrate(III) (K3Fe 10 μL of the cDNA with different concentrations at the pDNA/AuNPs-
(CN)6),sodium phosphate monobasic, and HClO4 were gained from PAT/rGO/ GCE surface and incubation for 35 min, the electrode was
Sigma-Aldrich (Madrid, Spain). All the specimens were developed in washed using phosphate buffer and was utilized for recording of the EIS
0.1 M phosphate buffer (PBS, pH=7.4) and kept at 4 °C prior to ap- responses of 0.1 M phosphate buffer solution (PBS, pH 7.4) containing
plication. Ultrapure water was gained from a system of Mill-Q water 5.0 mM of [Fe(CN)6]3−/4− couple (1:1) as external probe. The obtained
purification. results showed that the charge transfer resistance (Rct) is raised by in-
creasing the accumulated cDNA at the electrode surface showing s a
2.2. Apparatus “signal on” strategy signifying that the hybridization is occurring at the
genosensor surface. The difference between responses of the pDNA/
Electrochemical tests were done with the help of an Autolab (Eco AuNPs-PAT/rGO/ GCE before and after hybridization with cDNA (ΔRct
Chemie BV, Netherlands) being controlled using NOVA software1.8. A = Rct(cDNA)-Rct(pDNA)) was used as the measurement signal.

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Scheme 1. Fabrication and detection process of the DNA biosensor.

Moreover, the DPV was utilized instead of the above sensing 0.189 V in 0.1 M PBS (pH 7.4) accompanied with 5 mM [Fe(CN)6]3-/4-
strategy. Throughout these experiments, [Fe(CN)6]3−/4− was used as a as an electrochemical redox probe are shown in Fig. 2. Randles
cheap external probe with proper electrochemical behavior. To design equivalent circuit (Fig. 2A, inset) was used during modifications. The
the “signal off” determination by DPV approach, 10.0 μL of cDNA with bare GCE (curve a) showed a tiny semicircle whose charge transfer
different concentration was dropped on to the surface of the pDNA resistance was 436 Ω. After modifying the GCE by rGO, EIS spectrum
modified electrode and incubated for 35 min. The washed electrode was showed a straight line indicating the role of rGO in facilitating the
immersed in 0.1 M PBS (pH=7.4) containing 5.0 mM of [Fe(CN)6]3−/ transfer of electron at the electrode surface (curve b). Further mod-
4−
couple (1:1) and its DP voltammogram under pulse amplitude of ification of rGO/GCE with AuNPs-PAT, increased the charge transfer
50 mV and potential window of 0.0–0.4 V was recorded. The reduction resistance relative to rGO/GCE (curve c), which might be because of
in peak current (Ip) was dependent on the cDNA concentration. worse conductivity of the polymer film. After immobilizing the pDNA at
Regeneration of the biosensor was carried out in NaOH solution the AuNPs-PAT/rGO/GC electrode surface, there was an increase in the
(0.1 M for 10 min at room temperature) by regeneration and rehy- Rct value (193 Ω) which could be ascribed to the repellence of redox
bridization cycles (Fig. S2a and S2b). probe from approaching electrode surface by negative-charged phos-
The DNA biosensor fabrication and detection processes were sche- phate skeletons of DNA (curve d). Finally after blocking the free sites of
matically shown in Scheme 1. pDNA/AuNPs-PAT/rGO/GCE by immersing it in BSA solution the Rct of
the resulted electrode was further increased (246 Ω, curve e) which is
3. Results and discussion due to reduction in effective surface area. Similar outcomes were also
achieved using cyclic voltammetry (Fig. 2B).
3.1. Field emission gun scanning electron microscope (FE-SEM) study The IR spectra of GO, rGO, AuNPs-PAT/rGO and pDNA/AuNPs-
PAT/rGO were recorded and the results have been reported in sup-
To study the surface morphology, the outcome SEM image of the plementary file (Fig. S3).
modified electrodes (Fig. 1) was utilized.
As it is clear from Fig. 1A, SEM image of rGO indicted a planar 3.2. Genosensor parameters optimization
sheet-like structure, showing that rGO had been readily exfoliated into
separate sheets on the surface of electrode. When AT was electro In the study, to evaluate the effect of parameters such as target in-
polymerized on rGO surface (Fig. 1B), as expected, polymeric film PAT cubation time and pDNA concentration on the genosensor response,
was shaped on the surface. rGO modifications by PAT and AuNPs are DPV method was utilized. The decline in the peak current of the probe
seen in Fig. 1C. As shown, the Au nanoparticles are distributed uni- modified electrode before and after cDNA hybridization (ΔI) was used
formly as -SH functional groups shown in PAT assist to anchor the Au as the response signal.
nanoparticles. Moreover, the target incubation time is a vital effective factor in the
EIS as a suitable and sensitive tool was utilized to study the electron genosensor response (Liu et al., 2010; Shamsipur et al., 2016). There-
transfer process occurring at electrodes-solution interface. The diameter fore, the effect of incubation time of cDNA (20.0 pM) on the sensor
of the semicircle segment of impedance spectra at high frequencies, response was examined with the findings, which is indicated in Fig. 3A.
correspond to the electron transfer resistance, (Rct) and its linear part at As seen, the current response boosted drastically with an increase in
lower frequencies, depicts the diffusion-limiting step of the electro- incubation time up to 35 min and leveled off after 35 min, which shows
chemical process. The typical Nyquist plots of bare GC bare(a), rGO/GC that the hybridization reaction was mostly finished after 35 min. We
(b), AuNPs-PAT/rGO/GC(c), pDNA/AuNPs-PAT/rGO/GC (d) and BSA/ chose 35 min as the best DNA hybridization time.
pDNA/AuNPs-PAT/rGO/GC (e) electrodes which have been recorded at To sidestep the non-specific adsorption of the cDNA, and providing
frequencies ranging from 0.01 Hz to 10 kHz, and formal potential of false positive signal, optimization of the pDNA concentration on the

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Fig. 1. SEM images of rGO/GCE (A), PAT/rGO/GCE (B), and AuNPs -PAT/rGO/GCE (C).

Fig. 2. A) Impedance spectra (Nyquist plots) and B) CVs of bare GC (a), rGO/GC (b), AuNPs-PAT/rGO/GC(c), pDNA/AuNPs-PAT/rGO/GC (d) and BSA/ pDNA/
AuNPs-PAT/rGO/GC (e) recorded in the solution of 0.1 M phosphate buffer of pH 7.4 containing 5.0 mM of [Fe(CN)6]3−/4− couple (1:1).

performance of genosensor was carried out. The probe density was outcomes were summarized in Fig. 3B. As it is clear, the maximum
controlled by changing the concentrating pDNA in the range of 5 nM to immobilization and hence the best response (ΔI) was achieved at the
0.3 µM, and its effect on the genosensor response was examined and the pDNA concentration of 0.1 µM and thus this amount of pDNA was used

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Fig. 3. Effect of incubation time of cDNA (20 pM) (A) and pDNA concentration (B) on DPV responses of the solution of 0.1 M phosphate buffer(pH 7.4) of containing
5.0 mM of [Fe(CN)6]3−/4−.

= 0.9974) (Fig. 5b). The limit of detection (LOD) of the proposed

“signal off” genosensor for the analysis of the concentrating target DNA
was 0.06 pM, at the S/N ratio 3σ, in which σ is the relative standard
deviating a blank solution (n = 10).
To monitor the target DNA according to a “signal on” response, the
EIS approach was also utilized. The pDNA modified electrode loaded
with various concentration of cDNA after 35 min of incubation was
submerged in foregoing supporting electrolyte containing Fe(CN)6]3−/
4−
and the variation in the Rct value was tracked. Rct values changed
with the variety in cDNA amounts (Fig. 5c). This implies the hy-
bridization is happening at the genosensor because more negatively
charged phosphate backbones are collected. Subsequently, the Rct value
expands following the development of double stranded DNA. The dif-
ferent between the Rct of the pDNA/AuNPs-PAT/rGO/GCE before and
Fig. 4. Effect of pH of solution on the response of genosensor to 20.0 pM cDNA after hybridization with cDNA (ΔRct = Rct(cDNA)- Rct(pDNA)) was
on DPV responses the solution of 0.1 M phosphate buffer of containing 5.0 mM utilized as the measurement signal. The linearity between the analytical
of [Fe(CN)6]3−/4−. signal (ΔRct) with the logarithmic value cDNA ranging from 0.5 to
400 pM follows the following equation (Fig. 5d):

R (Ohm) = 1316.1 logC[cDNA]/(pM) + 582.56 (R2 = 0.9976)

throughout the experiment.
The calculated detection limit (S/N = 3σ, n = 10) was 0.035 pM.
The influence of pH as the other parameter on the performance of
The results showed that the proposed sensor is able to detect the
the prepared genosensor was also studied. Fig. 4 indicates the effect of
cDNA with a wide linear range and a very low LOD successfully.
pH ranging from 5.4 to 9.4 on the genosensor signal to 20.0 pM of
cDNA. The maximum sensitivity was achieved at pH 7.4 (Fig. 4). Thus,
pH 7.4 was selected as the optimum pH for further uses. 3.4. Stability, reproducibility, repeatability and selectivity study

3.3. Analytical performance of genosensor To examine the genosensor stability, it was submerged in phosphate
buffer of pH 7.4 for about 10 days and kept in fridge at 4 °C, after which
Under optimal conditions, the sensitivity of the pDNA/AuNPs-PAT/ the peak current reduced only to 5.2% of its initial current, which
rGO/GCE DPV toward the various concentration of cDNA was eval- shows an acceptable stability. When the modified electrode was kept at
uated using DPV and EIS. In this investigation, 0.1 M PBS with pH the room temperature (34 °C) for 15 days under the identical condi-
= 7.4 as supporting electrolyte and 5.0 mM of [Fe(CN)6]3−/4− as ex- tions, just 14.6% of the peak current reduced. The good stability could
ternal redox probe were used. Fig. 5a reveals the DP voltammograms s be because the DNA sequences were tightly attached to the surface of
of the foregoing sensor to cDNA with different concentrations. As it was pDNA modified electrode.
expected, the peak current was linearly reduced by increasing cDNA The genosensor reproducibility was evaluated utilizing five in-
concentrations that vividly indicates a “signal off” process. Incubation dividual genosensors prepared under similar conditions for monitoring
of cDNA at pDNA/AuNPs-PAT/rGO/GC electrode surface increases the of 20.0 pM of cDNA. The outcomes showed a relative standard devia-
number of [pDNA]-[cDNA] conjugates and prevents the diffusion of the tion (RSD) of 3.8% for DPV signals indicated. The outcome of the five
external probe toward to electrode surface whose results are reducing replicate determinations of cDNA solution (20 pM) using one electrode
in peak current. The ΔI was linear with the logarithm of the cDNA under the optimal conditions ended in an RSD 2.1%. The obtained re-
concentrating in the range from 1.0 pM to 400.0 pM and followed up sults show that the repeatability and reproducibility of the sensor are
from the regression equation of I (μA) = -8 logC [cDNA]/(pM) + 30.0 (R2 acceptable.

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Fig. 5. (a) DPV responses of the pDNA/AuNPs-PAT/rGO/GCE recorded in 0.1 M phosphate buffer (pH 7.4) and 5.0 mM [Fe(CN)6]3−/4−, after incubation in the
different concentrations of cDNA. (b) Calibration curve of DPV peak current versus logarithm of cDNA concentration. (c) Nyquist plots of the pDNA/AuNPs-PAT/
rGO/GCE recorded in 0.1 M phosphate buffer (pH 7.4) and 5.0 mM [Fe(CN)6]3−/4−, after incubation in the different concentrations of cDNA. (d) The calibration
curve of ΔRct versus logarithm of cDNA concentration.

The specificity of the pDNA/AuNPs-PAT/rGO/GC genosensor to- the desired amount of cDNA was spiked to the serum samples to deliver
wards different target DNA sequences including complementary, non- the mentioned concentration shown in Table 1. The standard addition
complementary two-base, three-base mismatched was investigated method was employed to record analytical signals via EIS. The accuracy
using DPV technique and their voltammograms are presented in Fig. 6. of the used procedure was evaluated by calculating the recoveries of the
It is obvious that after incubation of the genosensor with com- cDNA in the real samples that varied from 91.4% to 104.0%. The pre-
plementary target (cDNA) there was a marked decrease in peak in- cision of the proposed method was evaluated using calculating the re-
tensity revealing the process of hybridization. Incubation of non-com- lative standard deviations ranged from 4.2% to 5.8%. The results reveal
plementary and three-base mismatched showed no significant change the acceptable application of the extended genosensor for the assay of
in the voltammogram of pDNA/AuNPs-PAT/rGO/GCE, suggesting that the β-thalassemia gene in human's serum samples.
no hybridization is taking place.
While, incubated of two bases mismatched on the pDNA/AuNPs-
PAT/rGO/GCE, there was a light decrease in peak current in contrast 4. Conclusion
with that of the pDNA. This might be because of the partial hy-
bridization of pDNA. These outcomes uncover the selectivity and spe- We have presented an efficient DNA electrochemical genosensor for
cificity of the proposed genosensor towards different target DNA se- over-sensitive detecting β-thalassemia gene. We produced the DNA
quences. biosensor using immobilizing thiol tagged probe DNA on AuNPs-PAT
modified rGO/GCE. AuNPs-PAT/rGO shows a large surface area for
3.5. Real sample analysis immobilizing probe DNA. [Fe(CN)6]3−/4− was selected as a proper
electrochemical probe for the “signal off” (DPV) and “signal on” (EIS)
Serum samples were collected from normal persons, and stored approaches. The proposed biosensor is greatly selective, sensitive and
frozen until assay. 2 mL of methanol was added to 1.5 mL of serum. remains significantly activity (86% of the initial activity) following 15
After vortexing of the serum samples for 5 min, the precipitated pro- days of use at the ambient temperature. Therefore, the biosensor de-
teins were separated by centrifugation for 10 min at 14000 rpm and veloped in this study could be a critical device for specifying the ex-
filtrated through a 0.45-μm milli-pore filter. Finally, the treated serum istence of low concentrations of β-thalassemia gene. This genosensor is
samples were diluted to 10 mL with PBS (0.1 M, pH=7.4). Afterward, the second work in specific detection of thalassemia gene.

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Appendix A. Supporting information

Supplementary data associated with this article can be found in the

online version at doi:10.1016/j.bios.2019.01.017.

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The authors gratefully acknowledge the support of this work (with Zhao, H., Jia, X., Wang, B., Wang, N., Li, X., Ni, R., Ren, J., 2015. Biosens. Bioelectron. 65,
grant number 57021) by the Research center of Razi University, Iran, 23–30.
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97/303200 for financial support.

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