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An electrochemical dopamine aptasensor using the modified Au electrode with spindle-shaped gold nanostructure
Accepted Manuscript An electrochemical dopamine aptasensor using the modified Au electrode with spindle-shaped gold nanostructure
Ramezan Ali Taheri, Khadijeh Eskandari, Masoud Negahdary PII: DOI: Reference:
S0026-265X(18)30578-2 doi:10.1016/j.microc.2018.08.008 MICROC 3292
To appear in:
Microchemical Journal
Received date: Revised date: Accepted date:
12 May 2018 4 August 2018 5 August 2018
Please cite this article as: Ramezan Ali Taheri, Khadijeh Eskandari, Masoud Negahdary , An electrochemical dopamine aptasensor using the modified Au electrode with spindleshaped gold nanostructure. Microc (2018), doi:10.1016/j.microc.2018.08.008
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ACCEPTED MANUSCRIPT An electrochemical dopamine aptasensor using the modified Au electrode with spindle-shaped gold nanostructure
Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran,
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Ramezan Ali Taheri1, Khadijeh Eskandari1, Masoud Negahdary2*
Iran
Young Researchers and Elite Club, Marvdasht Branch, Islamic Azad University, Marvdasht,
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Iran
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*Correspondence should be addressed to Masoud Negahdary, Young Researchers and Elite Club, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran; Tell: +989124758568; Email: [email protected],
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[email protected] .
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ACCEPTED MANUSCRIPT Abstract In this study, a novel electrochemical dopamine aptasensor was developed. Spindle-shaped gold nanostructure was used to find an optimized surface for aptamer immobilization. The used nanostructure was studied through field emission scanning electron microscopy (FESEM) and energy dispersive spectroscopy (EDS). Electrochemical measurements were based on differential
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pulse voltammetry (DPV). Methylene blue (MB) was used as a redox marker during
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experiments. In order to find real selectivity of the designed aptasensor, ascorbic acid, epinephrine, norepinephrine, catechol and uric acid were used as interfering agents in the
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ng mL-1 with the limit of detection (LOD) of 2 pg mL-1 .
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presence of dopamine. This aptasensor could detect dopamine in the linear range 25 pg mL-1 - 3
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Keywords: Dopamine, Aptamer, Electrochemical detection, Biosensor.
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1. Introduction
Dopamine (C8 H11 NO 2 ) is known as a neurotransmitter and catecholamine since 1950 [1, 2]. It is
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a small molecule with a molecular weight of 153.181 g mol-1 . This biogenic neurotransmitter is produced by the adrenal glands and certain regions of the brain [2, 3]. Dopamine plays an
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important role in the formation of nerves, organizes and controls stress responses, attention, learning, and memory strength. In general, it plays a key role in the central nervous system, the
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cardiovascular system, kidney, hormones, and motivational behaviors (such as rewards and pleasures) [4-7]. Neurotransmitters are released via vesicle fusion and then diffuse across the
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synaptic cleft before binding to their respective receptors on the post-synaptic terminal [2, 3]. The concentration of dopamine in the brain affects the behavior and feelings of humans and animals. The values below the normal range of this neurotransmitter lead to Alzheimer [8, 9], Parkinson [10, 11], attention deficit hyperactivity disorder (ADHD) [12, 13], Huntington [14], Schizophrenia [15, 16] and some other diseases. These diseases are caused by impairment in the neurons involved in the production of dopamine. The rapid and accurate detection of this neurotransmitter can lead to the diagnosis of its physiological function in the body and prevent the occurrence of these diseases in some cases. The normal concentration of dopamine is between 10-1000 nmol L−1 [17, 18]. Higher levels of dopamine are also caused by drug abuse in 2
ACCEPTED MANUSCRIPT some people [19, 20]; wherein this status, the goal is to induce happiness, excitement and pleasant feelings. Currently, dopamine is detected by various quantitative methods such as highperformance liquid chromatography (HPLC) [21-23], mass spectrometry [24, 25], fluorescence spectroscopy [26,
27],
electrochemiluminescence
[28-31],
electrophoresis [32,
33] and
electrochemical methods [34-42]. Electrochemical methods are preferable to other methods because they can provide fast, accurate, cheap, easy and portable ways to detect dopamine.
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Recently, dopamine electrochemical diagnostic has been focused on increased sensitivity,
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improved selectivity and greater biocompatibility [43, 44]. In this regard, the detection of
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dopamine has been developed by various biosensors. Redox 2e / 2H+ couple reactions, caused by dopamine oxidation in physiological conditions, provide the basis for the electrochemical 45].
Ascorbic acid
[46],
epinephrine [47],
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diagnosis of this neurotransmitter [36-40,
norepinephrine [48], catechol [49] and uric acid [50] are considered as interfering factors in the
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diagnosis of dopamine due to common characteristics with dopamine (similar oxidation potential, competitive sensitivity and common presence in biological samples). Ascorbic acid is a
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vitamin that is effective in maintaining the physiological stability of the body. The concentration of this vitamin in tissues is 100-1000 times higher than dopamine [46-51]. Therefore, in clinical
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diagnosis, using tools that are more specific in order to dopamine detection will be very useful. Given the existing conditions and the presence of interfering factors, efforts should be performed
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to design specific dopamine biosensors, and these biosensors should have high selectivity and low limit of detection (LOD). Aptamers are single-stranded, and synthetic ligands that can be
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linked to their molecular targets with a high specificity and affinity (by changing spatial conformation) [52, 53]. These attributes of the aptamers have led to their widespread use in the
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structure of optimized biosensors. Aptamers have several advantages over antibodies, such as invitro production, leading to reduced contamination and immunization [54, 55]. The 5’ and 3’ ends of aptamers can be functionalized with different molecules. The functionalized aptamers have more specific binding ability against targets. Moreover, low production cost, high stability and, multi-use possibility are other important benefits of aptamers against antibodies. Selfassembled monolayers of aptamers, obtained after the immobilization process on the surface of the electrodes, lead to increased sensitivity, increased stability, and reduced response time during sensing [56, 57]. Using nanostructures in electrochemical detection processes leads to an increase in electrocatalytic activity, an increase in the contact surface, more ability to trap molecular 3
ACCEPTED MANUSCRIPT targets and facilitating the successful transfer of electrons [58, 59]. Gold nanostructures, in addition to these properties, have very high biocompatibility and do not show the immune response in in-vivo studies [60, 61]. The published reports are showed that gold surface can create self-assembled monolayers of aptamers and this process leads to the specific detection of dopamine in the presence of ascorbic acid [62-66]. Gold nanostructures with positive electrical charge can establish the stable bond with aptamers that have negative electrical charge. In this
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research, an aptasensor was designed to quick and specific detection of dopamine using a
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modified Au electrode with the spindle-shaped gold nanostructure, which can be used to quickly
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diagnose and treat of the diseases associated with this neurotransmitter.
2. Materials and methods
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2.1. Materials
Dopamine, ascorbic acid, epinephrine, norepinephrine, catechol, uric acid, 6-Mercapto-1-hexanol
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(MCH), sodium dodecyl sulfate (SDS), dithiothreitol (DTT), methylene blue (MB), Tris-HCl,
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sulfuric acid, hydrogen peroxide, Na2 HPO 4 , NaH2 PO4 and HAuCl4 were purchased from Sigma, USA. All used solutions were prepared using the deionized distilled water. All other chemicals
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were provided based on analytical grade and were used without further purification. A 5’ end thiolated dopamine aptamer was designed and purchased from Bioneer (South Korea) (5’-(SH)-
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(CH2 )6 - GGG AAU UCC GCG UGU GCG CCG CGG AAG AGG GAA UAU AGA GGC CAG
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CAC AUA GUG AGG CCC UCC UCC C-3’).
2.2. Apparatus
Electrochemical
measurements
were
performed
by
a
μ-Autolab
potentiostat/galvanostat
equipped with NOVA 1.8 software (Netherlands). A three-electrode system was used during analyte detection; where an Au (2 mm of diameter) (Metrohm, Switzerland) or modified Au electrode with spindle-shaped gold nanostructure, a platinum wire (Azar electrode, Iran) and An Ag/AgCl, 3 mol L−1 KCl (Azar electrode, Iran) were used as working, counter and reference electrodes respectively. Field emission scanning electron microscopy (FESEM) was followed via 4
ACCEPTED MANUSCRIPT a Zeiss, Sigma-IGMA/VP (Germany) equipped with energy-dispersive X-ray spectroscopy (EDS). In order to find the best aptamer immobilization time, a digital multimeter (Victor, China) and several screen-printed electrodes (PalmSens, Netherlands) were used. Immobilization temperature (4 o C) was provided by a refrigerator and binding temperature (37 o C) was provided
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by an incubation oven.
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2.3. Synthesis of spindle-shaped gold nanostructure
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The synthesis solution (10 mL) was contained 500 mmol L−1 H2 SO4 , 20 mmol L−1 HAuCl4 and 36 mmol L−1 SDS [67] (as the structure and shape-directing agent). Then, this solution was
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chronoamperometry method at -0.3 V for 5 minutes.
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poured into an electrochemical cell and the electrodeposition process was performed using the
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2.4. Preparing poly crystal Au electrode and the modified form with spindle-shaped gold
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nanostructure
First, before any electrochemical study, the polycrystal gold electrode was washed completely
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with distilled water and then polished using 0.3 μm-alumina (Al2 O3 ) and polishing pad until it reached the mirror-like surface. Then, in order to remove the alumina molecules and other
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possible contamination molecules from the surface, the polycrystal electrode was immersed in distilled water and ethanol 1:3 (v/v) for 5 minutes; ultimately it was washed again with distilled
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water. This process was performed before all electrochemical studies, electrodeposition and immobilization.
Following
the
electrodeposition
process
under
the
mentioned
conditions, a layer of gold nanostructure was deposited on the surface of the polycrystal gold electrode. Then, the modified Au electrode with gold nanostructure was evaluated for morphological features and determination the diameter and purity of nanostructure by FESEM and EDS.
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ACCEPTED MANUSCRIPT 2.5. Aptamer immobilization Before using the aptamer stock, dilution process was performed using Tris-HCl solution (containing 0.5 M NaCl and 5 mM MgCl2 , pH 7.4) to find the 10 μM concentration. The thiolated aptamer was inactive for the immobilization process and their bonds were in oxidized protected form (S-S). Breaking disulfide bonds and provide a reduced form (S-H) was performed
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by 10 μL DTT solution (10 mmol L−1 sodium acetate, pH 5.2 and 500 mmol L−1 DTT) during 20
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minutes [68]. Then, DTT and thiol free fragments were extracted via ethyl acetate (three times, each one 100 μL). In this step, the aptamer was ready for use. Because S-H bonds were unstable,
immobilization processes were followed at 4
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10 μL of prepared aptamer was dripped on the surface of Au electrode immediately. All aptamer C. Finding the optimum time for aptamer
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immobilization on the surface of the electrode was performed by measuring the open circuit potential (OCP). This experiment was performed using an Au screen printed electrode and a
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digital multimeter. The measurements continued until potential changes were stabilized and this moment (175 minutes) was considered as the optimal time for immobilization of aptamer on the
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surface of Au electrode in all experiments.
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2.6. Electrochemical measurements
After any aptamer immobilization, in order to find a well-aligned array of aptamer on the surface
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of modified Au electrode with the spindle-shaped gold nanostructure, 10 μL MCH (1.0 mmol L−1 ) was dropped on the surface of it; also, this process led to removing unbound aptamer
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strands. After 30 minutes, the aptasensor was prepared; then, it was washed with deionized distilled water. In this step, the aptasensor was ready to use. All electrochemical measurements were performed at room temperature (25 o C); also, all binding processes between aptasensor and analyte were performed at the physiological temperature (37 o C). Finding the optimum binding time between aptasensor and analyte was followed by DPV technique using a concentration of dopamine (50 pg mL-1 ) at various times (5-50 minutes). The fixed current change occurred after 45 minutes and this time was considered as the optimum binding time during all electrochemical measurements. In this research, the potential range in the presence of 20 μM MB as redox marker was between 0- _ 400 mv versus Ag/AgCl, 3 mol L−1 KCl. It should be noted that the 6
ACCEPTED MANUSCRIPT electrolyte in all electrochemical measurements was 0.01 M phosphate buffer saline (PBS) prepared through Na2 HPO 4 , NaH2 PO4 (pH 7.4). 2.7. Human sampling In order to evaluate the actual performance of the designed aptasensor against real samples, 10 human serum samples (3 ml) were considered. The filled informed consent was received from all
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the participants. Before analysis, the samples were investigated by HPLC method in a clinic and
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divided into two groups including healthy (dopamine concentration < 30 pg mL-1 ) and patient
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(dopamine concentration > 30 pg mL-1 ) [69]. Before sampling, all healthy and patient
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participants were fasting at least for 10 hours.
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3. Results and discussion
3.1. Characterization of spindle-shaped gold nanostructure
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FESEM was used to investigate the morphology of spindle-shaped gold nanostructure on the
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surface of the Au electrode. The bonding process (Au-S) between spindle-shaped gold nanostructure and aptamer has let to provide the facilitated and controllable assembly of the
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aptamer. Hybridization between thiolated aptamer and gold molecules depends on surface coverage [70]. The distributions of spindle-shaped gold nanostructure as an increased sensing
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interface are shown in Figure 1 (a-c). Using SDS as the structure and shape-directing agent has led to the special morphology of gold nanostructure that provided a stable surface for aptamer
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immobilization. The diameter of spindle-shaped gold nanostructure was 80-200 nm. Figure 1 (d) showed the EDS spectrum of the deposited gold nanostructure on the surface. The result of this analysis showed that the used nanostructure had high purity.
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(a)
(c)
(d)
Figure 1. a, b and c: FESEM images of modified Au electrode with spindle -shaped gold nanostructure at different magnification; d: EDS study to find the purity of synthesized spindle-shaped gold nanostructure
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ACCEPTED MANUSCRIPT 3.2. Finding the optimized time for aptamer immobilization and dopamine binding The aptamer immobilization was performed by measuring the OCP. In this experiment, the potential changes (mV) against time (second) were recorded (Figure 1 supplementary material (S)). The potential value based on the negative charge of the nucleic acid structure was significantly increased within 175 minutes and then the potential value reached at a fixed level.
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During this time, a self-assembled monolayer of aptamer had covered the surface of the modified
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Au electrode with the spindle-shaped gold nanostructure. Previous reports confirmed that the
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stability of aptamers on the surface of gold nanostructures is very high compared to free aptamers; also, the immobilized aptamers on the surface of gold nanostructures have high resistance against nuclease [71]. In the next experiment, finding the optimized binding time was
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followed. Here, the DPVs were recorded in the presence of a fixed concentration of dopamine (50 pg mL-1 ) during 0-50 minutes (37 o C). The fixed current was found at 40 minutes and this
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time was considered in all experiments as the best binding time (Figure 2S).
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3.3. Aptasensor production
In order to produce the aptasensor, modification of the Au electrode with the spindle-shaped gold
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nanostructure, finding the optimized time for aptamer immobilization, finding the optimized time for dopamine binding and the treatment with MCH were performed, respectively. The
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summarized design steps of the aptasensor are shown in Figure 2.
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Figure 2. Schematic illustration of the designed dopamine aptasensor
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First, the polished Au electrode was immersed in a solution containing spindle-shaped gold nanostructure; then the electrodeposition process was addressed using the chronoamperometry
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technique. During the electrodeposition process, HAuCl4 molecules created a layer of spindleshaped gold nanostructure on the surface of an Au electrode (working electrode). To find the
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specified morphology of gold nanostructure the SDS molecules were used as the structure and shape-directing agent. Prepared nano gold surface for aptamer immobilization had the highavailable electrochemically active surface area. The used thiolated RNA aptamer had dopaminebinding sites that led to capture and detection of the dopamine with high affinity. The covalently bonding between the thiolated aptamer and Au electrode occurred by Au-S interaction. In the next step, MCH was used to prevent the nonspecific attachment of aptamer molecules on the surface of a gold electrode; this process led to finding the self-assembled monolayer of aptamer with well-aligned and highly ordered architecture. Generally, aptamers have the negative charge based on the presence of sugar-phosphate backbone in their structures. In the designed 10
ACCEPTED MANUSCRIPT aptasensor, the produced negative charge was at its maximum value, when there was not any dopamine molecule. Along with increasing the concentration of dopamine, the numbers of free aptamers were reduced. Thus, the produced negative charge was also reduced alternately. In other words, in the presence of dopamine molecules, the electron transfer process between MB as the redox agent and the electrode was changed. When the dopamine was existed on the surface of aptasensor, the conformation of aptamer strands was changed and the free negative
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charge of aptamer was reduced. In this regard, the electron transfer route was changed along with
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the concentration of dopamine. In addition, in the presence of dopamine, the MB molecules were
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found only in distances away from aptamer strands; thus, the active locations of aptamer strands were occupied with dopamine molecules. The whole of the mentioned mechanism has led to
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increasing the currents of DPVs along with the increase of the dopamine concentrations. Figure 3 (a) shows the recorded DPVs in various concentrations of dopamine including: without (no-
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dopamine), 25, 50, 100, 150, 250, 500, 750, 1000, 2000 and 3000 pg mL-1 respectively. Figure 3 (b) shows the logarithmic calibration curve of the dopamine aptasensor (I (μA) = 54.878 log (C, pg mL-1 ) - 82.34, R² = 0.9907). The results showed that the LOD was estimated about 2 pg mL-1
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(LOD= 3σ whereas σ= standard deviation of the blank signal; signal-to-noise (S/N) = 3).
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Moreover, the limit of quantitation (LOQ) was estimated as 7 pg mL-1 .
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ACCEPTED MANUSCRIPT Figure 3. a): DPVs (a-k) in various concentrations of dopamine (without, 25, 50, 100, 150, 250, 500, 750, 1000, 2000 and 3000 pg mL-1 respectively); b): calibration curve of the designed aptasensor, LOD= 2 pg mL-1 and LOQ= 7 pg mL-1 .
The various aptamer-based methods of dopamine detection (in particular electrochemical
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methods) have been collected in Table 1 and the several important analytical features including
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the used detection technique, the type of nanostructures, detection range, the value of LOD and
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investigation of real samples were considered. It should be noted that the other methods (except
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aptasensing) for the dopamine detection have been stated in Table 1S.
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Detection technique
Nanostructure
CV, square wave voltammetry
Graphene oxide/nile
4
Detection
samples
range
Serum
Nonadiabatic tapered optical
LOD
Reference
1 nM
[72]
0 - 0.1 μM
1 nM
[73]
10 nM - 0.2 mM
Serum, Plasma
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Fluorescent
Graphene oxide
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3
Fluorescent
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2
Real
blue/gold nanoparticles
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(SWV)
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Row
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Table 1. Common main aptasensors for the detection of dopamine
Graphene oxide
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1-500 μM
--------
[74]
----------
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0 - 1 μM
37 nM
[75]
Serum
0.5-50 nM
0.2 nM
[76]
19 nM
[77]
62 nM
[78]
fiber (NATOF)
5
DPV, EIS
Gold nanocompositesPrussian blue/carbon nanotubes 0.03– 0.21
6
Fluorescent
Quantum dots
------
7
CV
-------
Serum
13
μM
0.1- 1 μM
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CV, DPV, Electrochemical
Graphene-DNA composite
Serum
0.1 - 100 μM
30 nM
[79]
Gold nanoflower
-------
25–250 μM
25000 nM
[80]
1000 n M
[81]
0.22 nM
[82]
1000 nM
[83]
130 nM
[84]
1.8 nM
[85]
impedance spectroscopy (EIS) 9
DPV
1.0×10−7 M 10
Enzymatic spectroscopy
--------
Serum
to 5.0×10−11
Gold-platinum nanoparticles
Serum
12
CV
-----------
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13
CV, DPV
14
CV, EIS
Gold nanoparticles
Serum
15
EIS
Gold nanoparticles
16
CV, DPV
Gold nanoparticles
17
EIS
Gold nanostars
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EIS, CV
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19
EIS, DPV
20
DPV
21
UV-vis spectroscopy
22
Ubiquitous glucose meter
23
100 nM - 5 μM
0.5 - 20 μM
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Graphene Oxide-gold
1 - 30 nM
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CV, DPV, EIS
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Serum
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Nanoparticle
5 nM - 0.5 μM
Serum
5 - 75 μM
3360 nM
[86]
Serum
5 - 300 nM
2.1 nM
[87]
Urine,
1 - 100 ng
Plasma
L−1
0.0019 nM
[88]
--------
0.25 - 66 μM
74 nM
[89]
0.000078 nM
[90]
10 nM
[91]
0.036 nM
[92]
30 nM
[93]
390000 nM
[94]
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1 pM - 160 Serum
Carbon nanoparticles
Serum
Gold nanoparticles
---------
Gold nanoparticles
Serum
Chemiluminescence
Gold nanoparticles
Urine
24
Fluorescent
Carbon dots, nano-graphite
Urine
0.1 - 5 nM
0.055 nM
[95]
25
CV, DPV, EIS
Silver nanoparticles
Serum
3 - 110 nM
0.7 nM
[37]
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Graphene oxide
nM 30 nM - 6.0 μM 5.4×10−7 M5.4×10−6 M 0.08-100 μM 2.5×10−13 2×10−9 M
25 pg mL-1 26
DPV
Gold nanostructure
Serum
(0.163 nM)-
2 pg mL-1
3 ng mL-1
(0.01 nM)
(20 nM)
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This work
ACCEPTED MANUSCRIPT The analytical parameters of the designed dopamine aptasensor in this research were comparable to or better than the results reported for determination of this catecholamine (Table 1 and Table 1S). In fact, the used specified morphology of spindle-shaped gold nanostructure provided a larger surface for successful aptamer immobilization. This nanostructure was used for the first time in an aptasensor for dopamine detection. Also, simple, timesaving and cheap establishment were some important achievements of the designed aptasensor. Moreover, the found LOD in this
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research was lower than the ones reported via most of dopamine aptasensors. The diagnostic
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sensitivity and specifity of developed dopamine aptasensor were 100% and 80%, respectively. In
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addition, positive predictive value (PPV), negative predictive value (NPV) and the accuracy were 83.33%, 100% and 90%, respectively. In a research, Talemi et al. introduced a dopamine
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aptasensor that showed a lower LOD (0.0019 nM) in comparison with our research [88]. The mentioned LOD was obtained when the graphite electrode used as the working electrode, while
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the LOD was 4570 nM when the gold electrode considered as working electrode. One of the weaknesses of the Talemi et al. research was that a few real samples have been evaluated. In
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addition, some of the other interfering factors including epinephrine [47], norepinephrine [48], catechol [49] and uric acid have not been investigated to find the more accurate selectivity of the
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proposed aptasensor. DPV has some technical advantages compared to EIS used by Talemi et al., which consists of analytical performance in less time and with more sensitivity. In other
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research, Wang et al. designed an aptasensor for dopamine detection using a nanocomposite containing graphene oxide [90]. As we know, gold has good biocompatibility in biomedical
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investigations compared to other materials like carbon. We used more real samples compare to Wang et al. In addition, the values of the chosen healthy samples were borderline which shows
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the good performance of the designed aptasensor in our research. Other disadvantages of Wang et al. work were including an incomplete investigation of interfering factors, longer detection time (60 minutes) and not checking the stability and reproducibility performances.
3.4. Investigation the reproducibility, regeneration and stability In the next step, to perform reproducibility experiment, the aptasensor was refabricated several times (n=6) and related DPVs were recorded (Figure 3S). Refabricating was performed using piranha solution (3:1 mixture of concentrated sulfuric acid (H2 SO4 ) with hydrogen peroxide 15
ACCEPTED MANUSCRIPT (H2 O 2 )) for 3 minutes. After this time, the modified Au electrode with spindle-shaped gold nanostructure was washed with deionized water and aptamer immobilization on the surface of it was reperformed. The results showed the successful reproducibility performance for this dopamine aptasensor. The relative standard deviation (RSD) was 8.03% (n=6). The regeneration study was performed using a fixed concentration of dopamine (50 pg mL-1 ). In this experiment, five binding-rebinding cycles were repeated. A DPV was recorded for each binding and
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rebinding (Figure 4S). Because in the bound form the dopamine molecules were captured by the
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specified aptamer; to find unfold aptamer and releasing dopamine, rebinding process was
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performed via immersing the aptasensor in hot water (95 o C) for 5 minutes. Previous reports confirmed that the used procedure could provide rebinding process and the released analyte [96,
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97]. Subsequently, aptasensor was cooled down at room temperature and these cycles repeated. This experiment showed that the aptasensor has good regeneration ability (RSD 10.09%, n=5). In
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the next experiment, the stability of dopamine aptasensor was investigated during 23 continues days. In order to perform this evaluation, the aptasensor was bound with 50 pg mL-1 dopamine
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and DPVs were recorded for each considered day separately. The aptasensor was stable (based on the current changes) until the day 15 and could maintain 91% of its initial activity. Thus, the
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optimum stability of this aptasensor was 15 days (Figure 4).
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Figure 4. Stability of the dopamine aptasensor during 23 days
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3.5. Investigation interferences effects on the response of dopamine aptasensor The selectivity of the designed dopamine aptasensor was investigated in the presence of almost
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all interfering agents (similar oxidation potential, competitive sensitivity and common presence in biological samples) including ascorbic acid,
uric acid, catechol, norepinephrine and
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epinephrine. The results of this experiment showed that the aptasensor was highly selective in the presence of the high concentrations of interferences (Figure 5 (a-e)). The significant increments in the current were found in the presence of dopamine (≈ 2.7 μA: 25 pg mL-1 and ≈ 10 μA: 50 pg mL-1 ). It should be noted that the used concentrations of dopamine were very much lower than the concentrations of interferences (10 ng mL-1 and 100 pg mL-1 ). The current changes of interferences based on the without signal were as: ascorbic acid I ≈ 1.1 μA, 1.15 μA and 1.8 μA, uric acid I ≈ 1.1 μA, 1.25 μA and 1.26 μA, catechol I ≈ 1.5 μA, 1.75 μA and ≈ 1.8 μA, norepinephrine I ≈ 1.65 μA, 1.7 μA and ≈ 1.73 μA, epinephrine I ≈ 1.7 μA, 1.8 μA and 1.82 μA for the without, concentrations of 10 ng mL-1 and 100 pg mL-1 , respectively. 17
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ACCEPTED MANUSCRIPT Figure 5. DPVs in the presence of various interferences; a): ascorbic acid; b): uric acid; c): catechol; d): norepinephrine; e): epinephrine
3.6. Real samples
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In order to evaluate the performance of aptasensor in the presence of real samples, 10 human
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serum samples (divided into two groups: healthy and patient) were investigated. Firstly, the samples were investigated by HPLC as a highly sensitive analytical method for the detection of
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dopamine. Then, samples were investigated by aptasensor and the found results in both methods were compared (Table 2). The samples containing dopamine level lower than 30 pg mL-1 were
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considered as the healthy group. The results showed that the aptasensor could detect dopamine in the serum of real samples sensitively. One very important point that found in the results was
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sample No.5 that reported as healthy (concentration= 27 pg mL-1 ) by HPLC method, but the
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(concentration= 32 pg mL-1 ) [69].
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aptasensing result for this sample showed that this sample should be considered as a patient
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Table 2. Comparison the results of aptasensor with HPLC method Sample
Sample type
Aptasensor result
Healthy
26 pg mL-1
25 pg mL-1
Healthy
25 pg mL-1
27 pg mL-1
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HPLC result
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Healthy
29 pg mL-1
28 pg mL-1
4
Healthy
25 pg mL-1
25 pg mL-1
5
Healthy
27 pg mL-1
32 pg mL-1
6
Patient
96 pg mL-1
101 pg mL-1
7
Patient
127 pg mL-1
125 pg mL-1
8
Patient
189 pg mL-1
176 pg mL-1
9
Patient
54 pg mL-1
54.5 pg mL-1
1 2
19
ACCEPTED MANUSCRIPT 10
73 pg mL-1
Patient
74 pg mL-1
4. Conclusion Here, a new facile bioelectrochemical method to the quantitative detection of dopamine was
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introduced. A specified morphology of spindle-shaped gold nanostructure led to the increment of
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the surface area of a gold electrode for aptamer immobilization. Some important advantages of this signal-on aptasensor compared to other related works were including fast response (40 min
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for binding with dopamine), LOD 0.01 nM, sensitivity 100%, specifity 80%, PPV 83.33% and NPV 100%. In order to find other practical features, the reproducibility (RSD: 8.03%, n=6),
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regeneration (RSD: 10.09%, n=5) and stability (91%) performances were evaluated. Finally, dopamine was detected in the presence of the main interfering agents and also 10 real clinical
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samples were investigated. The aptasensor was less prone to investigated interferences and this shows that it can be used for analysis real human serum samples with high selectivity. This
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aptasensor can be used in the accurate and immediate diagnosis of some diseases of the central
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nervous system such as Alzheimer, Parkinson, Schizophrenia, and Huntington.
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Conflict of Interest: The authors declare that they have no conflict of interest.
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ACCEPTED MANUSCRIPT Highlights
Gold nanostructures with special morphology could play an important role in improving the diagnostic function of this aptasensor.
This aptasensor could detect dopamine in the linear range 25 pg mL -1- 3 ng mL-1 with the limit of detection (LOD) of 2 pg mL-1
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Ramezan Ali Taheri, Khadijeh Eskandari, Masoud Negahdary PII: DOI: Reference:
S0026-265X(18)30578-2 doi:10.1016/j.microc.2018.08.008 MICROC 3292
To appear in:
Microchemical Journal
Received date: Revised date: Accepted date:
12 May 2018 4 August 2018 5 August 2018
Please cite this article as: Ramezan Ali Taheri, Khadijeh Eskandari, Masoud Negahdary , An electrochemical dopamine aptasensor using the modified Au electrode with spindleshaped gold nanostructure. Microc (2018), doi:10.1016/j.microc.2018.08.008
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ACCEPTED MANUSCRIPT An electrochemical dopamine aptasensor using the modified Au electrode with spindle-shaped gold nanostructure
Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran,
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Ramezan Ali Taheri1, Khadijeh Eskandari1, Masoud Negahdary2*
Iran
Young Researchers and Elite Club, Marvdasht Branch, Islamic Azad University, Marvdasht,
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Iran
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*Correspondence should be addressed to Masoud Negahdary, Young Researchers and Elite Club, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran; Tell: +989124758568; Email: [email protected],
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[email protected] .
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ACCEPTED MANUSCRIPT Abstract In this study, a novel electrochemical dopamine aptasensor was developed. Spindle-shaped gold nanostructure was used to find an optimized surface for aptamer immobilization. The used nanostructure was studied through field emission scanning electron microscopy (FESEM) and energy dispersive spectroscopy (EDS). Electrochemical measurements were based on differential
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pulse voltammetry (DPV). Methylene blue (MB) was used as a redox marker during
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experiments. In order to find real selectivity of the designed aptasensor, ascorbic acid, epinephrine, norepinephrine, catechol and uric acid were used as interfering agents in the
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ng mL-1 with the limit of detection (LOD) of 2 pg mL-1 .
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presence of dopamine. This aptasensor could detect dopamine in the linear range 25 pg mL-1 - 3
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Keywords: Dopamine, Aptamer, Electrochemical detection, Biosensor.
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1. Introduction
Dopamine (C8 H11 NO 2 ) is known as a neurotransmitter and catecholamine since 1950 [1, 2]. It is
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a small molecule with a molecular weight of 153.181 g mol-1 . This biogenic neurotransmitter is produced by the adrenal glands and certain regions of the brain [2, 3]. Dopamine plays an
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important role in the formation of nerves, organizes and controls stress responses, attention, learning, and memory strength. In general, it plays a key role in the central nervous system, the
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cardiovascular system, kidney, hormones, and motivational behaviors (such as rewards and pleasures) [4-7]. Neurotransmitters are released via vesicle fusion and then diffuse across the
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synaptic cleft before binding to their respective receptors on the post-synaptic terminal [2, 3]. The concentration of dopamine in the brain affects the behavior and feelings of humans and animals. The values below the normal range of this neurotransmitter lead to Alzheimer [8, 9], Parkinson [10, 11], attention deficit hyperactivity disorder (ADHD) [12, 13], Huntington [14], Schizophrenia [15, 16] and some other diseases. These diseases are caused by impairment in the neurons involved in the production of dopamine. The rapid and accurate detection of this neurotransmitter can lead to the diagnosis of its physiological function in the body and prevent the occurrence of these diseases in some cases. The normal concentration of dopamine is between 10-1000 nmol L−1 [17, 18]. Higher levels of dopamine are also caused by drug abuse in 2
ACCEPTED MANUSCRIPT some people [19, 20]; wherein this status, the goal is to induce happiness, excitement and pleasant feelings. Currently, dopamine is detected by various quantitative methods such as highperformance liquid chromatography (HPLC) [21-23], mass spectrometry [24, 25], fluorescence spectroscopy [26,
27],
electrochemiluminescence
[28-31],
electrophoresis [32,
33] and
electrochemical methods [34-42]. Electrochemical methods are preferable to other methods because they can provide fast, accurate, cheap, easy and portable ways to detect dopamine.
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Recently, dopamine electrochemical diagnostic has been focused on increased sensitivity,
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improved selectivity and greater biocompatibility [43, 44]. In this regard, the detection of
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dopamine has been developed by various biosensors. Redox 2e / 2H+ couple reactions, caused by dopamine oxidation in physiological conditions, provide the basis for the electrochemical 45].
Ascorbic acid
[46],
epinephrine [47],
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diagnosis of this neurotransmitter [36-40,
norepinephrine [48], catechol [49] and uric acid [50] are considered as interfering factors in the
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diagnosis of dopamine due to common characteristics with dopamine (similar oxidation potential, competitive sensitivity and common presence in biological samples). Ascorbic acid is a
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vitamin that is effective in maintaining the physiological stability of the body. The concentration of this vitamin in tissues is 100-1000 times higher than dopamine [46-51]. Therefore, in clinical
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diagnosis, using tools that are more specific in order to dopamine detection will be very useful. Given the existing conditions and the presence of interfering factors, efforts should be performed
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to design specific dopamine biosensors, and these biosensors should have high selectivity and low limit of detection (LOD). Aptamers are single-stranded, and synthetic ligands that can be
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linked to their molecular targets with a high specificity and affinity (by changing spatial conformation) [52, 53]. These attributes of the aptamers have led to their widespread use in the
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structure of optimized biosensors. Aptamers have several advantages over antibodies, such as invitro production, leading to reduced contamination and immunization [54, 55]. The 5’ and 3’ ends of aptamers can be functionalized with different molecules. The functionalized aptamers have more specific binding ability against targets. Moreover, low production cost, high stability and, multi-use possibility are other important benefits of aptamers against antibodies. Selfassembled monolayers of aptamers, obtained after the immobilization process on the surface of the electrodes, lead to increased sensitivity, increased stability, and reduced response time during sensing [56, 57]. Using nanostructures in electrochemical detection processes leads to an increase in electrocatalytic activity, an increase in the contact surface, more ability to trap molecular 3
ACCEPTED MANUSCRIPT targets and facilitating the successful transfer of electrons [58, 59]. Gold nanostructures, in addition to these properties, have very high biocompatibility and do not show the immune response in in-vivo studies [60, 61]. The published reports are showed that gold surface can create self-assembled monolayers of aptamers and this process leads to the specific detection of dopamine in the presence of ascorbic acid [62-66]. Gold nanostructures with positive electrical charge can establish the stable bond with aptamers that have negative electrical charge. In this
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research, an aptasensor was designed to quick and specific detection of dopamine using a
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modified Au electrode with the spindle-shaped gold nanostructure, which can be used to quickly
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diagnose and treat of the diseases associated with this neurotransmitter.
2. Materials and methods
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2.1. Materials
Dopamine, ascorbic acid, epinephrine, norepinephrine, catechol, uric acid, 6-Mercapto-1-hexanol
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(MCH), sodium dodecyl sulfate (SDS), dithiothreitol (DTT), methylene blue (MB), Tris-HCl,
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sulfuric acid, hydrogen peroxide, Na2 HPO 4 , NaH2 PO4 and HAuCl4 were purchased from Sigma, USA. All used solutions were prepared using the deionized distilled water. All other chemicals
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were provided based on analytical grade and were used without further purification. A 5’ end thiolated dopamine aptamer was designed and purchased from Bioneer (South Korea) (5’-(SH)-
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(CH2 )6 - GGG AAU UCC GCG UGU GCG CCG CGG AAG AGG GAA UAU AGA GGC CAG
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CAC AUA GUG AGG CCC UCC UCC C-3’).
2.2. Apparatus
Electrochemical
measurements
were
performed
by
a
μ-Autolab
potentiostat/galvanostat
equipped with NOVA 1.8 software (Netherlands). A three-electrode system was used during analyte detection; where an Au (2 mm of diameter) (Metrohm, Switzerland) or modified Au electrode with spindle-shaped gold nanostructure, a platinum wire (Azar electrode, Iran) and An Ag/AgCl, 3 mol L−1 KCl (Azar electrode, Iran) were used as working, counter and reference electrodes respectively. Field emission scanning electron microscopy (FESEM) was followed via 4
ACCEPTED MANUSCRIPT a Zeiss, Sigma-IGMA/VP (Germany) equipped with energy-dispersive X-ray spectroscopy (EDS). In order to find the best aptamer immobilization time, a digital multimeter (Victor, China) and several screen-printed electrodes (PalmSens, Netherlands) were used. Immobilization temperature (4 o C) was provided by a refrigerator and binding temperature (37 o C) was provided
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by an incubation oven.
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2.3. Synthesis of spindle-shaped gold nanostructure
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The synthesis solution (10 mL) was contained 500 mmol L−1 H2 SO4 , 20 mmol L−1 HAuCl4 and 36 mmol L−1 SDS [67] (as the structure and shape-directing agent). Then, this solution was
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chronoamperometry method at -0.3 V for 5 minutes.
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poured into an electrochemical cell and the electrodeposition process was performed using the
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2.4. Preparing poly crystal Au electrode and the modified form with spindle-shaped gold
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nanostructure
First, before any electrochemical study, the polycrystal gold electrode was washed completely
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with distilled water and then polished using 0.3 μm-alumina (Al2 O3 ) and polishing pad until it reached the mirror-like surface. Then, in order to remove the alumina molecules and other
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possible contamination molecules from the surface, the polycrystal electrode was immersed in distilled water and ethanol 1:3 (v/v) for 5 minutes; ultimately it was washed again with distilled
aptamer
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water. This process was performed before all electrochemical studies, electrodeposition and immobilization.
Following
the
electrodeposition
process
under
the
mentioned
conditions, a layer of gold nanostructure was deposited on the surface of the polycrystal gold electrode. Then, the modified Au electrode with gold nanostructure was evaluated for morphological features and determination the diameter and purity of nanostructure by FESEM and EDS.
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ACCEPTED MANUSCRIPT 2.5. Aptamer immobilization Before using the aptamer stock, dilution process was performed using Tris-HCl solution (containing 0.5 M NaCl and 5 mM MgCl2 , pH 7.4) to find the 10 μM concentration. The thiolated aptamer was inactive for the immobilization process and their bonds were in oxidized protected form (S-S). Breaking disulfide bonds and provide a reduced form (S-H) was performed
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by 10 μL DTT solution (10 mmol L−1 sodium acetate, pH 5.2 and 500 mmol L−1 DTT) during 20
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minutes [68]. Then, DTT and thiol free fragments were extracted via ethyl acetate (three times, each one 100 μL). In this step, the aptamer was ready for use. Because S-H bonds were unstable,
immobilization processes were followed at 4
o
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10 μL of prepared aptamer was dripped on the surface of Au electrode immediately. All aptamer C. Finding the optimum time for aptamer
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immobilization on the surface of the electrode was performed by measuring the open circuit potential (OCP). This experiment was performed using an Au screen printed electrode and a
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digital multimeter. The measurements continued until potential changes were stabilized and this moment (175 minutes) was considered as the optimal time for immobilization of aptamer on the
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surface of Au electrode in all experiments.
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2.6. Electrochemical measurements
After any aptamer immobilization, in order to find a well-aligned array of aptamer on the surface
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of modified Au electrode with the spindle-shaped gold nanostructure, 10 μL MCH (1.0 mmol L−1 ) was dropped on the surface of it; also, this process led to removing unbound aptamer
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strands. After 30 minutes, the aptasensor was prepared; then, it was washed with deionized distilled water. In this step, the aptasensor was ready to use. All electrochemical measurements were performed at room temperature (25 o C); also, all binding processes between aptasensor and analyte were performed at the physiological temperature (37 o C). Finding the optimum binding time between aptasensor and analyte was followed by DPV technique using a concentration of dopamine (50 pg mL-1 ) at various times (5-50 minutes). The fixed current change occurred after 45 minutes and this time was considered as the optimum binding time during all electrochemical measurements. In this research, the potential range in the presence of 20 μM MB as redox marker was between 0- _ 400 mv versus Ag/AgCl, 3 mol L−1 KCl. It should be noted that the 6
ACCEPTED MANUSCRIPT electrolyte in all electrochemical measurements was 0.01 M phosphate buffer saline (PBS) prepared through Na2 HPO 4 , NaH2 PO4 (pH 7.4). 2.7. Human sampling In order to evaluate the actual performance of the designed aptasensor against real samples, 10 human serum samples (3 ml) were considered. The filled informed consent was received from all
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the participants. Before analysis, the samples were investigated by HPLC method in a clinic and
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divided into two groups including healthy (dopamine concentration < 30 pg mL-1 ) and patient
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(dopamine concentration > 30 pg mL-1 ) [69]. Before sampling, all healthy and patient
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participants were fasting at least for 10 hours.
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3. Results and discussion
3.1. Characterization of spindle-shaped gold nanostructure
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FESEM was used to investigate the morphology of spindle-shaped gold nanostructure on the
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surface of the Au electrode. The bonding process (Au-S) between spindle-shaped gold nanostructure and aptamer has let to provide the facilitated and controllable assembly of the
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aptamer. Hybridization between thiolated aptamer and gold molecules depends on surface coverage [70]. The distributions of spindle-shaped gold nanostructure as an increased sensing
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interface are shown in Figure 1 (a-c). Using SDS as the structure and shape-directing agent has led to the special morphology of gold nanostructure that provided a stable surface for aptamer
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immobilization. The diameter of spindle-shaped gold nanostructure was 80-200 nm. Figure 1 (d) showed the EDS spectrum of the deposited gold nanostructure on the surface. The result of this analysis showed that the used nanostructure had high purity.
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(a)
(c)
(d)
Figure 1. a, b and c: FESEM images of modified Au electrode with spindle -shaped gold nanostructure at different magnification; d: EDS study to find the purity of synthesized spindle-shaped gold nanostructure
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ACCEPTED MANUSCRIPT 3.2. Finding the optimized time for aptamer immobilization and dopamine binding The aptamer immobilization was performed by measuring the OCP. In this experiment, the potential changes (mV) against time (second) were recorded (Figure 1 supplementary material (S)). The potential value based on the negative charge of the nucleic acid structure was significantly increased within 175 minutes and then the potential value reached at a fixed level.
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During this time, a self-assembled monolayer of aptamer had covered the surface of the modified
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Au electrode with the spindle-shaped gold nanostructure. Previous reports confirmed that the
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stability of aptamers on the surface of gold nanostructures is very high compared to free aptamers; also, the immobilized aptamers on the surface of gold nanostructures have high resistance against nuclease [71]. In the next experiment, finding the optimized binding time was
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followed. Here, the DPVs were recorded in the presence of a fixed concentration of dopamine (50 pg mL-1 ) during 0-50 minutes (37 o C). The fixed current was found at 40 minutes and this
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time was considered in all experiments as the best binding time (Figure 2S).
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3.3. Aptasensor production
In order to produce the aptasensor, modification of the Au electrode with the spindle-shaped gold
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nanostructure, finding the optimized time for aptamer immobilization, finding the optimized time for dopamine binding and the treatment with MCH were performed, respectively. The
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summarized design steps of the aptasensor are shown in Figure 2.
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Figure 2. Schematic illustration of the designed dopamine aptasensor
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First, the polished Au electrode was immersed in a solution containing spindle-shaped gold nanostructure; then the electrodeposition process was addressed using the chronoamperometry
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technique. During the electrodeposition process, HAuCl4 molecules created a layer of spindleshaped gold nanostructure on the surface of an Au electrode (working electrode). To find the
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specified morphology of gold nanostructure the SDS molecules were used as the structure and shape-directing agent. Prepared nano gold surface for aptamer immobilization had the highavailable electrochemically active surface area. The used thiolated RNA aptamer had dopaminebinding sites that led to capture and detection of the dopamine with high affinity. The covalently bonding between the thiolated aptamer and Au electrode occurred by Au-S interaction. In the next step, MCH was used to prevent the nonspecific attachment of aptamer molecules on the surface of a gold electrode; this process led to finding the self-assembled monolayer of aptamer with well-aligned and highly ordered architecture. Generally, aptamers have the negative charge based on the presence of sugar-phosphate backbone in their structures. In the designed 10
ACCEPTED MANUSCRIPT aptasensor, the produced negative charge was at its maximum value, when there was not any dopamine molecule. Along with increasing the concentration of dopamine, the numbers of free aptamers were reduced. Thus, the produced negative charge was also reduced alternately. In other words, in the presence of dopamine molecules, the electron transfer process between MB as the redox agent and the electrode was changed. When the dopamine was existed on the surface of aptasensor, the conformation of aptamer strands was changed and the free negative
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charge of aptamer was reduced. In this regard, the electron transfer route was changed along with
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the concentration of dopamine. In addition, in the presence of dopamine, the MB molecules were
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found only in distances away from aptamer strands; thus, the active locations of aptamer strands were occupied with dopamine molecules. The whole of the mentioned mechanism has led to
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increasing the currents of DPVs along with the increase of the dopamine concentrations. Figure 3 (a) shows the recorded DPVs in various concentrations of dopamine including: without (no-
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dopamine), 25, 50, 100, 150, 250, 500, 750, 1000, 2000 and 3000 pg mL-1 respectively. Figure 3 (b) shows the logarithmic calibration curve of the dopamine aptasensor (I (μA) = 54.878 log (C, pg mL-1 ) - 82.34, R² = 0.9907). The results showed that the LOD was estimated about 2 pg mL-1
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(LOD= 3σ whereas σ= standard deviation of the blank signal; signal-to-noise (S/N) = 3).
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Moreover, the limit of quantitation (LOQ) was estimated as 7 pg mL-1 .
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ACCEPTED MANUSCRIPT Figure 3. a): DPVs (a-k) in various concentrations of dopamine (without, 25, 50, 100, 150, 250, 500, 750, 1000, 2000 and 3000 pg mL-1 respectively); b): calibration curve of the designed aptasensor, LOD= 2 pg mL-1 and LOQ= 7 pg mL-1 .
The various aptamer-based methods of dopamine detection (in particular electrochemical
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methods) have been collected in Table 1 and the several important analytical features including
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the used detection technique, the type of nanostructures, detection range, the value of LOD and
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investigation of real samples were considered. It should be noted that the other methods (except
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aptasensing) for the dopamine detection have been stated in Table 1S.
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Detection technique
Nanostructure
CV, square wave voltammetry
Graphene oxide/nile
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Detection
samples
range
Serum
Nonadiabatic tapered optical
LOD
Reference
1 nM
[72]
0 - 0.1 μM
1 nM
[73]
10 nM - 0.2 mM
Serum, Plasma
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Fluorescent
Graphene oxide
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3
Fluorescent
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2
Real
blue/gold nanoparticles
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(SWV)
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Row
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Table 1. Common main aptasensors for the detection of dopamine
Graphene oxide
-------
1-500 μM
--------
[74]
----------
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0 - 1 μM
37 nM
[75]
Serum
0.5-50 nM
0.2 nM
[76]
19 nM
[77]
62 nM
[78]
fiber (NATOF)
5
DPV, EIS
Gold nanocompositesPrussian blue/carbon nanotubes 0.03– 0.21
6
Fluorescent
Quantum dots
------
7
CV
-------
Serum
13
μM
0.1- 1 μM
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CV, DPV, Electrochemical
Graphene-DNA composite
Serum
0.1 - 100 μM
30 nM
[79]
Gold nanoflower
-------
25–250 μM
25000 nM
[80]
1000 n M
[81]
0.22 nM
[82]
1000 nM
[83]
130 nM
[84]
1.8 nM
[85]
impedance spectroscopy (EIS) 9
DPV
1.0×10−7 M 10
Enzymatic spectroscopy
--------
Serum
to 5.0×10−11
Gold-platinum nanoparticles
Serum
12
CV
-----------
---------
13
CV, DPV
14
CV, EIS
Gold nanoparticles
Serum
15
EIS
Gold nanoparticles
16
CV, DPV
Gold nanoparticles
17
EIS
Gold nanostars
18
EIS, CV
----------
19
EIS, DPV
20
DPV
21
UV-vis spectroscopy
22
Ubiquitous glucose meter
23
100 nM - 5 μM
0.5 - 20 μM
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Graphene Oxide-gold
1 - 30 nM
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CV, DPV, EIS
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Serum
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Nanoparticle
5 nM - 0.5 μM
Serum
5 - 75 μM
3360 nM
[86]
Serum
5 - 300 nM
2.1 nM
[87]
Urine,
1 - 100 ng
Plasma
L−1
0.0019 nM
[88]
--------
0.25 - 66 μM
74 nM
[89]
0.000078 nM
[90]
10 nM
[91]
0.036 nM
[92]
30 nM
[93]
390000 nM
[94]
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1 pM - 160 Serum
Carbon nanoparticles
Serum
Gold nanoparticles
---------
Gold nanoparticles
Serum
Chemiluminescence
Gold nanoparticles
Urine
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Fluorescent
Carbon dots, nano-graphite
Urine
0.1 - 5 nM
0.055 nM
[95]
25
CV, DPV, EIS
Silver nanoparticles
Serum
3 - 110 nM
0.7 nM
[37]
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Graphene oxide
nM 30 nM - 6.0 μM 5.4×10−7 M5.4×10−6 M 0.08-100 μM 2.5×10−13 2×10−9 M
25 pg mL-1 26
DPV
Gold nanostructure
Serum
(0.163 nM)-
2 pg mL-1
3 ng mL-1
(0.01 nM)
(20 nM)
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This work
ACCEPTED MANUSCRIPT The analytical parameters of the designed dopamine aptasensor in this research were comparable to or better than the results reported for determination of this catecholamine (Table 1 and Table 1S). In fact, the used specified morphology of spindle-shaped gold nanostructure provided a larger surface for successful aptamer immobilization. This nanostructure was used for the first time in an aptasensor for dopamine detection. Also, simple, timesaving and cheap establishment were some important achievements of the designed aptasensor. Moreover, the found LOD in this
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research was lower than the ones reported via most of dopamine aptasensors. The diagnostic
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sensitivity and specifity of developed dopamine aptasensor were 100% and 80%, respectively. In
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addition, positive predictive value (PPV), negative predictive value (NPV) and the accuracy were 83.33%, 100% and 90%, respectively. In a research, Talemi et al. introduced a dopamine
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aptasensor that showed a lower LOD (0.0019 nM) in comparison with our research [88]. The mentioned LOD was obtained when the graphite electrode used as the working electrode, while
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the LOD was 4570 nM when the gold electrode considered as working electrode. One of the weaknesses of the Talemi et al. research was that a few real samples have been evaluated. In
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addition, some of the other interfering factors including epinephrine [47], norepinephrine [48], catechol [49] and uric acid have not been investigated to find the more accurate selectivity of the
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proposed aptasensor. DPV has some technical advantages compared to EIS used by Talemi et al., which consists of analytical performance in less time and with more sensitivity. In other
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research, Wang et al. designed an aptasensor for dopamine detection using a nanocomposite containing graphene oxide [90]. As we know, gold has good biocompatibility in biomedical
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investigations compared to other materials like carbon. We used more real samples compare to Wang et al. In addition, the values of the chosen healthy samples were borderline which shows
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the good performance of the designed aptasensor in our research. Other disadvantages of Wang et al. work were including an incomplete investigation of interfering factors, longer detection time (60 minutes) and not checking the stability and reproducibility performances.
3.4. Investigation the reproducibility, regeneration and stability In the next step, to perform reproducibility experiment, the aptasensor was refabricated several times (n=6) and related DPVs were recorded (Figure 3S). Refabricating was performed using piranha solution (3:1 mixture of concentrated sulfuric acid (H2 SO4 ) with hydrogen peroxide 15
ACCEPTED MANUSCRIPT (H2 O 2 )) for 3 minutes. After this time, the modified Au electrode with spindle-shaped gold nanostructure was washed with deionized water and aptamer immobilization on the surface of it was reperformed. The results showed the successful reproducibility performance for this dopamine aptasensor. The relative standard deviation (RSD) was 8.03% (n=6). The regeneration study was performed using a fixed concentration of dopamine (50 pg mL-1 ). In this experiment, five binding-rebinding cycles were repeated. A DPV was recorded for each binding and
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rebinding (Figure 4S). Because in the bound form the dopamine molecules were captured by the
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specified aptamer; to find unfold aptamer and releasing dopamine, rebinding process was
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performed via immersing the aptasensor in hot water (95 o C) for 5 minutes. Previous reports confirmed that the used procedure could provide rebinding process and the released analyte [96,
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97]. Subsequently, aptasensor was cooled down at room temperature and these cycles repeated. This experiment showed that the aptasensor has good regeneration ability (RSD 10.09%, n=5). In
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the next experiment, the stability of dopamine aptasensor was investigated during 23 continues days. In order to perform this evaluation, the aptasensor was bound with 50 pg mL-1 dopamine
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and DPVs were recorded for each considered day separately. The aptasensor was stable (based on the current changes) until the day 15 and could maintain 91% of its initial activity. Thus, the
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optimum stability of this aptasensor was 15 days (Figure 4).
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Figure 4. Stability of the dopamine aptasensor during 23 days
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3.5. Investigation interferences effects on the response of dopamine aptasensor The selectivity of the designed dopamine aptasensor was investigated in the presence of almost
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all interfering agents (similar oxidation potential, competitive sensitivity and common presence in biological samples) including ascorbic acid,
uric acid, catechol, norepinephrine and
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epinephrine. The results of this experiment showed that the aptasensor was highly selective in the presence of the high concentrations of interferences (Figure 5 (a-e)). The significant increments in the current were found in the presence of dopamine (≈ 2.7 μA: 25 pg mL-1 and ≈ 10 μA: 50 pg mL-1 ). It should be noted that the used concentrations of dopamine were very much lower than the concentrations of interferences (10 ng mL-1 and 100 pg mL-1 ). The current changes of interferences based on the without signal were as: ascorbic acid I ≈ 1.1 μA, 1.15 μA and 1.8 μA, uric acid I ≈ 1.1 μA, 1.25 μA and 1.26 μA, catechol I ≈ 1.5 μA, 1.75 μA and ≈ 1.8 μA, norepinephrine I ≈ 1.65 μA, 1.7 μA and ≈ 1.73 μA, epinephrine I ≈ 1.7 μA, 1.8 μA and 1.82 μA for the without, concentrations of 10 ng mL-1 and 100 pg mL-1 , respectively. 17
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3.6. Real samples
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In order to evaluate the performance of aptasensor in the presence of real samples, 10 human
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serum samples (divided into two groups: healthy and patient) were investigated. Firstly, the samples were investigated by HPLC as a highly sensitive analytical method for the detection of
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dopamine. Then, samples were investigated by aptasensor and the found results in both methods were compared (Table 2). The samples containing dopamine level lower than 30 pg mL-1 were
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considered as the healthy group. The results showed that the aptasensor could detect dopamine in the serum of real samples sensitively. One very important point that found in the results was
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sample No.5 that reported as healthy (concentration= 27 pg mL-1 ) by HPLC method, but the
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(concentration= 32 pg mL-1 ) [69].
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aptasensing result for this sample showed that this sample should be considered as a patient
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Table 2. Comparison the results of aptasensor with HPLC method Sample
Sample type
Aptasensor result
Healthy
26 pg mL-1
25 pg mL-1
Healthy
25 pg mL-1
27 pg mL-1
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HPLC result
3
Healthy
29 pg mL-1
28 pg mL-1
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Healthy
25 pg mL-1
25 pg mL-1
5
Healthy
27 pg mL-1
32 pg mL-1
6
Patient
96 pg mL-1
101 pg mL-1
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Patient
127 pg mL-1
125 pg mL-1
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Patient
189 pg mL-1
176 pg mL-1
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Patient
54 pg mL-1
54.5 pg mL-1
1 2
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73 pg mL-1
Patient
74 pg mL-1
4. Conclusion Here, a new facile bioelectrochemical method to the quantitative detection of dopamine was
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introduced. A specified morphology of spindle-shaped gold nanostructure led to the increment of
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the surface area of a gold electrode for aptamer immobilization. Some important advantages of this signal-on aptasensor compared to other related works were including fast response (40 min
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for binding with dopamine), LOD 0.01 nM, sensitivity 100%, specifity 80%, PPV 83.33% and NPV 100%. In order to find other practical features, the reproducibility (RSD: 8.03%, n=6),
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regeneration (RSD: 10.09%, n=5) and stability (91%) performances were evaluated. Finally, dopamine was detected in the presence of the main interfering agents and also 10 real clinical
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samples were investigated. The aptasensor was less prone to investigated interferences and this shows that it can be used for analysis real human serum samples with high selectivity. This
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aptasensor can be used in the accurate and immediate diagnosis of some diseases of the central
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nervous system such as Alzheimer, Parkinson, Schizophrenia, and Huntington.
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Conflict of Interest: The authors declare that they have no conflict of interest.
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References
[1] J.W. Kebabian, D.B. Calne, Multiple receptors for dopamine, Nature, 277 (1979) 93-96.
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ACCEPTED MANUSCRIPT Highlights
Gold nanostructures with special morphology could play an important role in improving the diagnostic function of this aptasensor.
This aptasensor could detect dopamine in the linear range 25 pg mL -1- 3 ng mL-1 with the limit of detection (LOD) of 2 pg mL-1
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