Biosensors and Bioelectronics 80 (2016) 532–537
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A novel electrochemical aptasensor based on Y-shape structure of dual-aptamer-complementary strand conjugate for ultrasensitive detection of myoglobin Seyed Mohammad Taghdisi a, Noor Mohammad Danesh b,c, Mohammad Ramezani b, Ahmad Sarreshtehdar Emrani d,n, Khalil Abnous e,n a
Targeted Drug Delivery Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Research Institute of Sciences and New Technology, Mashhad, Iran d Cardiovascular Research Center, Ghaem hospital, Mashhad University of Medical Sciences, Mashhad, Iran e Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b c
art ic l e i nf o
a b s t r a c t
Article history: Received 17 December 2015 Received in revised form 28 January 2016 Accepted 10 February 2016 Available online 11 February 2016
Monitoring of myoglobin (Mb) in human blood serum is highly in demand for early diagnosis of acute myocardial infarction (AMI). Here, a novel electrochemical aptasensor was developed for ultrasensitive and selective detection of Mb, based on Y-shape structure of dual-aptamer (DApt)-complementary strand of aptamer (CS) conjugate, gold electrode and exonuclease I (Exo I). The designed aptasensor obtains features of gold, such as high electrochemical conductivity and large surface area, property of Y-shape structure of DApt-CS conjugate to function as a gate and obstacle for the access of redox probe to the surface of electrode, as well as high specificity and sensitivity of aptamer toward its target and Exo I as an enzyme which specifically degrades the 3′-end of single-stranded DNA (ssDNA). In the absence of Mb, the Y-shape structure remains intact. So, a weak electrochemical signal is observed. Upon addition of target, the DApt leave the CS and bind to Mb, leading to disassembly of Y-shape structure and following the addition of Exo I, a strong electrochemical signal could be recorded. The fabricated aptasensor showed high selectivity toward Mb with a limit of detection (LOD) as low as 27 pM. Besides, the developed aptasensor was effectively applied to detect Mb in human serum. & 2016 Elsevier B.V. All rights reserved.
Keywords: Electrochemical aptasensor Y-shape structure Exonuclease I Myoglobin
1. Introduction Acute myocardial infarction (AMI) is one of the most common reasons of morbidity and mortality in the world (Lee et al., 2015; Wang et al., 2014a). The sensitive detection of cardiac markers is important in early diagnosis of AMI (Wang et al., 2014b; Yang et al., 2015). Myoglobin (Mb) is one of the best indicators of AMI (Kim et al., 2014; Wang et al., 2015; Yang et al., 2015). In AMI the concentration of Mb could increase up to 600 ng/mL while its normal range is 6–100 ng/mL (Kumar et al., 2015; Stone et al., 1977; Wang et al., 2014a). Surface plasmon resonance (SPR), liquid chromatography, colorimetric methods, mass spectrometry, fluorescent and luminescence methods have been used for detection of Mb. Most of these approaches are laborious and need expensive instruments and n
Corresponding authors. E-mail addresses: [email protected] (A.S. Emrani), [email protected] (K. Abnous). http://dx.doi.org/10.1016/j.bios.2016.02.029 0956-5663/& 2016 Elsevier B.V. All rights reserved.
trained operators (Wang et al., 2014a, 2014b; Yang et al., 2015). Utilization of aptamers in analytical approaches is growing so fast. Aptamers are short single-chain DNA or RNA molecules, selected via an in vitro process known as the systematic evolution of ligands by exponential enrichment (SELEX) (Du et al., 2015; Xiang et al., 2015). They are able to bind to a broad range of targets from small organic molecules to whole cells with high affinity and specificity based on three-dimensional sequence dependent structures (Dong et al., 2014; Zhou et al., 2014b). Aptamers possess outstanding features over traditional antibodies, including no or low toxicity and immunogenicity, low cost, small size, high reproducibility, ease of synthesis and modification and high thermal stability (Eissa et al., 2015; Lian et al., 2015; Zhao et al., 2015a, 2014b). Due to these significant advantages, aptamers have been widely used for the construction of various sensing platforms (Emrani et al., 2015; Mohammad Danesh et al., 2016; Ramezani et al., 2015). Gold has been widely applied in analytical techniques, owing to its good biocompatibility, unique electronic and optical characteristics and large surface area (Abnous et al., 2016; Liu et al.,
S.M. Taghdisi et al. / Biosensors and Bioelectronics 80 (2016) 532–537
2014; Luo et al., 2015). Generally, Gold is utilized to modify the surface of electrodes, because of its high affinity of binding to thiol-modified molecules and high electro-transfer capability (Zhao et al., 2015b). Among the diverse sensing methods, electrochemical aptasensors have several advantages, including high sensitivity, rapid response, low cost and simplicity (Bai et al., 2013; Taghdisi et al., 2015; Zhou et al., 2014a). Moreover, relative to optical aptasensors, electrochemical aptasensors require less quantity of target for recognition and do not need fluorescent labeling (Mokhtarzadeh et al., 2015). In this work, a novel electrochemical aptasensor was designed for detection of Mb, based on complementary strand of aptamer, Y-shape structure of dual-aptamer (DApt)-complementary strand of aptamer (CS) conjugate, gold electrode and exonuclease I (Exo I). In this study, a ssDNA aptamer that binds to Mb with high affinity (Wang et al., 2014a, 2014b), was applied as molecular recognition element.
2. Materials and methods 2.1. Materials All the sequences were purchased from Bioneer (Table 1, South Korea). Plasma from human, C reactive protein (CRP), bovine serum albumin (BSA), human immunoglobulin G (IgG), human serum albumin (HSA), hemoglobin, potassium hexacyanoferrate(III) (K3[Fe(CN)6]), Potassium hexacyanoferrate(II) trihydrate (K4[Fe(CN)6].3H2O), 6-mercaptohexanol (MCH) and Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were provided by Sigma-Aldrich (USA). Human myoglobin (Mb) was ordered from ProsPec (Germany). Exonuclease I (Exo I) was obtained from Thermo Scientific (USA).
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2.4. Effect of the concentration of Exo I on the electrochemical signal Increasing concentrations of Exo I (0–20 U) were placed on the surface of DApt-CS-modified electrodes treated with 40 nM Mb for 45 min. After incubation for 1.5 h at 37 °C, the surface of modified electrodes was washed thoroughly with the hybridization buffer. Next, the electrochemical signals were measured using DPV by scanning the potential from 0 V to 0.3 V with the pulse time of 25 ms and pulse potential of 30 mV. 2.5. Optimization of incubation time of Exo I The DApt-CS-modified electrodes, treated with 40 nM Mb for 45 min, were incubated with 10 U Exo I in Tris–HCl buffer (20 mM Tris–HCl, 2 mM MgCl2, pH 7.4). The electrodes were incubated at 37 °C from 0 to 3 h, followed by rinsing with the hybridization buffer and the electrochemical signals were recorded using DPV. 2.6. Detection performance of the designed electrochemical aptasensor The interaction of Mb with the fabricated aptasensor was investigated by electrochemical measurement. The DApt-CS-modified electrode was immersed in phosphate buffer saline (10 mM, pH 7.4) containing 40 nM Mb for 45 min at room temperature. Then, the electrode was rinsed with PBS followed by incubation with 10 U Exo I for 1.5 h at 37 °C. Finally, the electrode was washed with PBS and the electrochemical signals were recorded by CV. CV measurements were carried out in 3 mM K4[Fe(CN)6] and K3[Fe(CN)6] (redox probe) solution containing 0.1 M KCl, scanning from 0.5 V to 0.8 V at a scan rate of 50 mV/s. 2.7. Quantitative detection of Mb using electrochemical measurement
2.2. Apparatus A mstat 400 portable Biopotentiostat/Galvanostat (DropSens, Spain) was used for the measurement of differential pulse voltammetry (DPV) and cyclic voltammetry (CV). Screen-printed gold electrodes (SPGEs) were provided by DropSens (Spain). The Data were analyzed by DropView8400 software.
The DApt-CS-modified electrodes were immersed in a range of Mb concentrations (0–80 nM) in 10 mM PBS (pH 7.4) for 45 min at room temperature. After that, the electrodes were washed with PBS and 10 U Exo I was added on the surface of modified electrodes for 1.5 h at 37 °C. Then, the electrodes were rinsed with PBS and the electrochemical signals were measured using DPV.
2.3. Preparation of DApt-CS-modified electrode
2.8. Investigation of the selectivity of the electrochemical aptasensor
CS (1 mM final concentration) was pretreated with 10 mM TCEP in immobilization buffer (1 mM EDTA, 100 mM NaCl, 10 mM Tris– HCl, pH 7.4) at room temperature for 1 h. Then, 9 mL of the above solution was placed on the surface of gold electrode for 12 h at room temperature under 100% humidity. After that, 4.5 mL of each aptamer sequence (Apt1 and Apt2, 1 mM final concentration of each sequence) in hybridization buffer (1 mM EDTA, 10 mM Tris– HCl, pH 7.4) was dropped on the surface of SPGE for 1.5 h at room temperature. Next, the surface of electrode was incubated with 1 mM MCH solution (10 ml) for 1 h to block the untreated sites of SPGE, followed by rinsing the surface of electrode with the hybridization buffer.
The selectivity was analyzed in the presence of 40 nM Mb, CRP, IgG, hemoglobin, HSA and BSA.
3. Results and discussion 3.1. Detection mechanism of the electrochemical aptasensor The designed electrochemical aptasensor was based on dual aptamer (DApt), Y-shape structure of DApt-CS conjugate, digestion of CS by Exo I and target-induced disassembly of Y-shape structure. In this work, Exo I was applied as an enzyme which
Table 1 Oligonucleotide sequences used in this study. Complimentary strands have been shown with the same color.
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Scheme 1. Schematic illustration of Myoglobin (Mb) detection based on electrochemical assay. In the absence of Mb, the Y-shape structure of DApt-CS conjugate is intact and redox probe does not have access to the surface of electrode, resulting in a weak electrochemical signal (a). In the presence of target, DApt (Apt1 and Apt2) bind to Mb and leave the CS. Exo I digests and degrades CS, resulting in the free access of redox probe to the surface of electrode and a strong electrochemical signal (b).
selectively digests ssDNA from its 3′-terminus, owing to its good selectivity, low cost and buffer compatibility (Zhao et al., 2014; Zheng et al., 2012). The principle of the developed electrochemical aptasensor has been illustrated in Scheme 1. In the absence of Mb, the Y-shape structure of DApt-CS conjugate remains intact and the CS is shielded from digestion by Exo I, resulting in the low access of redox probe to the surface of modified electrode and a weak electrochemical signal. Upon the addition of Mb, a conformational change happens. DApt leave the CS and the surface of electrode and Apt/target conjugate forms, as it has been shown that aptamer binds to its corresponding target with a higher binding constant in comparison with its complementary strand (Song et al., 2016; Wu et al., 2015; Yang et al., 2014). When the electrode is incubated with Exo I, the CS (as a ssDNA) is degraded, leading to more access of redox probe to the surface of electrode and the increase of electrochemical signal. 3.2. Optimum concentration of Exo I To obtain the optimum concentration of Exo I for the efficient reaction with CS, increasing amounts of Exo I were dropped on the surface of DApt-CS-modified electrodes treated with 40 nM Mb. As indicated in Fig. 1(a), 10 U Exo I could induce the maximum electrochemical signal, confirming the maximum activity of Exo I occurred at this concentration. 3.3. Optimum incubation time of Exo I Enzymatic reaction time is a significant factor to be optimized in sensors. The DApt-CS-modified electrodes treated with 40 nM Mb were incubated with 10 U Exo I and the electrochemical signal was assessed up to 3 h. The electrochemical signal enhanced rapidly as the incubation time enhanced and reached to maximum in 1.5 h (Fig. 1(b)), indicating the required time for efficient digestion of CS by Exo I.
Fig. 1. Optimization of the parameters involved in the electrochemical signal of electrode. (a) Relative electrochemical signal of DApt-CS-modified electrode in the presence of various concentrations of Exo I. (b) Relative electrochemical signal of DApt-CS-modified electrode as a function of Exo I incubation time.
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Fig. 2. Proof of concept for the fabricated aptasensor. CV profiles of: bare electrode (red curve), CS-modified electrode (black curve), DApt-CS-modified electrode (blue curve), DApt-CS-modified electrodeþ Exo I (gray curve), DApt-CS-modified electrodeþ Mb (pink curve) and DApt-CS-modified electrodeþ Mbþ Exo I (green curve). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.4. Feasibility of the designed aptasensor for detection of Mb CV measurements were exploited to specify the formation and function of the developed aptasensor. As shown in Fig. 2, the maximum redox peak belonged to the bare electrode (red curve) because of its high capability of electron transfer. The electrochemical signal of electrode significantly decreased when CS was dropped on the surface of electrode (black curve), which confirmed the immobilization of CS on the surface of electrode. The electron transfer of redox probe [Fe(CN)6]3 /4 was dramatically prohibited by the negatively charged phosphate groups of the immobilized CS through electrostatic repulsive force to the negatively charged redox probe (Chen et al., 2014; Zhao et al., 2015b). The electrochemical signal was reduced even more (blue curve) when the DApt were added on the surface of electrode, indicating the formation of DApt-CS conjugate (Y-shape structure) which contained more negative phosphate groups and created a physical barrier for the access of redox probe to the surface of modified electrode. Addition of 10 U Exo I for 1.5 h, did not enhance the electrochemical signal (gray curve), which could be attributed to the protection of all captured sequences (CS and DApt) from digestion against Exo I. None of captured sequences contained free 3′-ends for degradation by Exo I due to formation of Y-shape structure. In the presence of Mb, the electrochemical signal rose (pink curve), confirming the release of DApt from CS, formation of Apt/Mb conjugate and disassembly of Y-shape structure, resulting in further access of redox probe to the surface of electrode. Upon addition of 10 U Exo I for 1.5 h to the DApt-CSmodified electrode treated with 40 nM Mb, the peak current significantly increased (green curve), indicating digestion of CS by Exo I and additional access of redox probe to the surface of electrode. Also, it confirmed reclining position or lying flat of CS on the surface of SPGE. 3.5. Quantification of Mb Fig. 3(a) exhibits the DPV peaks of electrode at different concentrations of Mb. The DPV peak increased and arrived to plateau at concentration of 40 nM Mb. The designed electrochemical aptasensor displayed a well linear range (100 pM to 40 nM) toward Mb (Fig. 3(b)). The limit of detection (LOD) was determined to be
Fig. 3. (a) DPV peaks of the DApt-CS-modified electrode in the presence of various concentrations of Mb in PBS (from bottom to top 0, 0.1, 0.3, 1, 3, 10, 20, 40 and 80 nM). (b) Mb standard curve in PBS. (c) Relative electrochemical signal of DAptCS-modified electrode in the presence of various materials.
27 pM (0.45 ng/mL) regarding the three times of the standard deviation of the blank/slope. Reported detection limits of Mb in other studies were as following: 25 mg/500 mg for ultra-performance liquid chromatography-elec-
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Table 2 Recovery and relative standard deviation (RSD) of Mb in human serum samples. Serum samples
Found (nM)
Added Mb (nM)
Total found (nM)
Recovery (%) RSD (%, n¼ 3)
1 2 3 4
2 2 2 2
2 5 15 30
3.82 6.7 15.65 32.8
95.5 95.7 92.1 102.5
5.6 3.9 7.4 3.2
trospray ionization mass spectrometry (Giaretta et al., 2013), 460 pg/ mL for two-dimensional liquid chromatography-electrospray ionization mass spectrometry (Mayr et al., 2006), linear range of 0.91–182 mg/mL for colorimetric sensing (Zhang et al., 2011), 16 ng/mL for fluorescent sensing (Darain et al., 2009), 87.6 ng/mL for SPR (Osman et al., 2013), 2.25 mg/mL for electrochemical biosensor based on biomimetic material (Moreira et al., 2013) and 50 pM for an optical aptasensor using personal glucose meter (Wang et al., 2014a). In comparison with the designed electrochemical aptasensor, most of these approaches are pricey, time-consuming and have higher LODs. 3.6. Evaluation of the selectivity of the designed aptasensor In addition to sensitivity, specificity is an important factor for sensing platforms. The relative electrochemical signal of the DAptCS-modified electrode toward Mb was significantly more than other materials (Fig. 3(c)), including HSA, BSA, hemoglobin, IgG and CRP. These results demonstrated high selectivity of the developed aptasensor toward Mb. 3.7. Detection of Mb in human serum To evaluate the applicability of the proposed assay, the designed electrochemical aptasensor was applied to measure Mb in human serum. Known concentrations of Mb were spiked into serum. The serum samples were diluted 10 times, followed by measuring the concentration of Mb using the designed sensing method. The recoveries for Mb in human serum samples were in the range of 92.1–102.5% with relative standard deviations between 3.2% and 7.4% (Table 2), suggesting the developed aptasensor could be utilized to precisely detect Mb in complex biological samples.
4. Conclusion In conclusion, a novel electrochemical aptasensor was designed for ultrasensitive and selective detection of Mb, based on DApt, Exo I, Y-shape structure of DApt-CS conjugate and target-induced disassembly of Y-shape structure. The limit of detection for Mb was determined as low as 27 pM. In addition, the developed aptasensor could precisely detect Mb in human serum.
Conflict of interest There is no conflict of interest about this article.
Acknowledgment Financial support of this study was provided by Mashhad University of Medical Sciences.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A novel electrochemical aptasensor based on Y-shape structure of dual-aptamer-complementary strand conjugate for ultrasensitive detection of myoglobin Seyed Mohammad Taghdisi a, Noor Mohammad Danesh b,c, Mohammad Ramezani b, Ahmad Sarreshtehdar Emrani d,n, Khalil Abnous e,n a
Targeted Drug Delivery Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Research Institute of Sciences and New Technology, Mashhad, Iran d Cardiovascular Research Center, Ghaem hospital, Mashhad University of Medical Sciences, Mashhad, Iran e Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b c
art ic l e i nf o
a b s t r a c t
Article history: Received 17 December 2015 Received in revised form 28 January 2016 Accepted 10 February 2016 Available online 11 February 2016
Monitoring of myoglobin (Mb) in human blood serum is highly in demand for early diagnosis of acute myocardial infarction (AMI). Here, a novel electrochemical aptasensor was developed for ultrasensitive and selective detection of Mb, based on Y-shape structure of dual-aptamer (DApt)-complementary strand of aptamer (CS) conjugate, gold electrode and exonuclease I (Exo I). The designed aptasensor obtains features of gold, such as high electrochemical conductivity and large surface area, property of Y-shape structure of DApt-CS conjugate to function as a gate and obstacle for the access of redox probe to the surface of electrode, as well as high specificity and sensitivity of aptamer toward its target and Exo I as an enzyme which specifically degrades the 3′-end of single-stranded DNA (ssDNA). In the absence of Mb, the Y-shape structure remains intact. So, a weak electrochemical signal is observed. Upon addition of target, the DApt leave the CS and bind to Mb, leading to disassembly of Y-shape structure and following the addition of Exo I, a strong electrochemical signal could be recorded. The fabricated aptasensor showed high selectivity toward Mb with a limit of detection (LOD) as low as 27 pM. Besides, the developed aptasensor was effectively applied to detect Mb in human serum. & 2016 Elsevier B.V. All rights reserved.
Keywords: Electrochemical aptasensor Y-shape structure Exonuclease I Myoglobin
1. Introduction Acute myocardial infarction (AMI) is one of the most common reasons of morbidity and mortality in the world (Lee et al., 2015; Wang et al., 2014a). The sensitive detection of cardiac markers is important in early diagnosis of AMI (Wang et al., 2014b; Yang et al., 2015). Myoglobin (Mb) is one of the best indicators of AMI (Kim et al., 2014; Wang et al., 2015; Yang et al., 2015). In AMI the concentration of Mb could increase up to 600 ng/mL while its normal range is 6–100 ng/mL (Kumar et al., 2015; Stone et al., 1977; Wang et al., 2014a). Surface plasmon resonance (SPR), liquid chromatography, colorimetric methods, mass spectrometry, fluorescent and luminescence methods have been used for detection of Mb. Most of these approaches are laborious and need expensive instruments and n
Corresponding authors. E-mail addresses: [email protected] (A.S. Emrani), [email protected] (K. Abnous). http://dx.doi.org/10.1016/j.bios.2016.02.029 0956-5663/& 2016 Elsevier B.V. All rights reserved.
trained operators (Wang et al., 2014a, 2014b; Yang et al., 2015). Utilization of aptamers in analytical approaches is growing so fast. Aptamers are short single-chain DNA or RNA molecules, selected via an in vitro process known as the systematic evolution of ligands by exponential enrichment (SELEX) (Du et al., 2015; Xiang et al., 2015). They are able to bind to a broad range of targets from small organic molecules to whole cells with high affinity and specificity based on three-dimensional sequence dependent structures (Dong et al., 2014; Zhou et al., 2014b). Aptamers possess outstanding features over traditional antibodies, including no or low toxicity and immunogenicity, low cost, small size, high reproducibility, ease of synthesis and modification and high thermal stability (Eissa et al., 2015; Lian et al., 2015; Zhao et al., 2015a, 2014b). Due to these significant advantages, aptamers have been widely used for the construction of various sensing platforms (Emrani et al., 2015; Mohammad Danesh et al., 2016; Ramezani et al., 2015). Gold has been widely applied in analytical techniques, owing to its good biocompatibility, unique electronic and optical characteristics and large surface area (Abnous et al., 2016; Liu et al.,
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2014; Luo et al., 2015). Generally, Gold is utilized to modify the surface of electrodes, because of its high affinity of binding to thiol-modified molecules and high electro-transfer capability (Zhao et al., 2015b). Among the diverse sensing methods, electrochemical aptasensors have several advantages, including high sensitivity, rapid response, low cost and simplicity (Bai et al., 2013; Taghdisi et al., 2015; Zhou et al., 2014a). Moreover, relative to optical aptasensors, electrochemical aptasensors require less quantity of target for recognition and do not need fluorescent labeling (Mokhtarzadeh et al., 2015). In this work, a novel electrochemical aptasensor was designed for detection of Mb, based on complementary strand of aptamer, Y-shape structure of dual-aptamer (DApt)-complementary strand of aptamer (CS) conjugate, gold electrode and exonuclease I (Exo I). In this study, a ssDNA aptamer that binds to Mb with high affinity (Wang et al., 2014a, 2014b), was applied as molecular recognition element.
2. Materials and methods 2.1. Materials All the sequences were purchased from Bioneer (Table 1, South Korea). Plasma from human, C reactive protein (CRP), bovine serum albumin (BSA), human immunoglobulin G (IgG), human serum albumin (HSA), hemoglobin, potassium hexacyanoferrate(III) (K3[Fe(CN)6]), Potassium hexacyanoferrate(II) trihydrate (K4[Fe(CN)6].3H2O), 6-mercaptohexanol (MCH) and Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were provided by Sigma-Aldrich (USA). Human myoglobin (Mb) was ordered from ProsPec (Germany). Exonuclease I (Exo I) was obtained from Thermo Scientific (USA).
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2.4. Effect of the concentration of Exo I on the electrochemical signal Increasing concentrations of Exo I (0–20 U) were placed on the surface of DApt-CS-modified electrodes treated with 40 nM Mb for 45 min. After incubation for 1.5 h at 37 °C, the surface of modified electrodes was washed thoroughly with the hybridization buffer. Next, the electrochemical signals were measured using DPV by scanning the potential from 0 V to 0.3 V with the pulse time of 25 ms and pulse potential of 30 mV. 2.5. Optimization of incubation time of Exo I The DApt-CS-modified electrodes, treated with 40 nM Mb for 45 min, were incubated with 10 U Exo I in Tris–HCl buffer (20 mM Tris–HCl, 2 mM MgCl2, pH 7.4). The electrodes were incubated at 37 °C from 0 to 3 h, followed by rinsing with the hybridization buffer and the electrochemical signals were recorded using DPV. 2.6. Detection performance of the designed electrochemical aptasensor The interaction of Mb with the fabricated aptasensor was investigated by electrochemical measurement. The DApt-CS-modified electrode was immersed in phosphate buffer saline (10 mM, pH 7.4) containing 40 nM Mb for 45 min at room temperature. Then, the electrode was rinsed with PBS followed by incubation with 10 U Exo I for 1.5 h at 37 °C. Finally, the electrode was washed with PBS and the electrochemical signals were recorded by CV. CV measurements were carried out in 3 mM K4[Fe(CN)6] and K3[Fe(CN)6] (redox probe) solution containing 0.1 M KCl, scanning from 0.5 V to 0.8 V at a scan rate of 50 mV/s. 2.7. Quantitative detection of Mb using electrochemical measurement
2.2. Apparatus A mstat 400 portable Biopotentiostat/Galvanostat (DropSens, Spain) was used for the measurement of differential pulse voltammetry (DPV) and cyclic voltammetry (CV). Screen-printed gold electrodes (SPGEs) were provided by DropSens (Spain). The Data were analyzed by DropView8400 software.
The DApt-CS-modified electrodes were immersed in a range of Mb concentrations (0–80 nM) in 10 mM PBS (pH 7.4) for 45 min at room temperature. After that, the electrodes were washed with PBS and 10 U Exo I was added on the surface of modified electrodes for 1.5 h at 37 °C. Then, the electrodes were rinsed with PBS and the electrochemical signals were measured using DPV.
2.3. Preparation of DApt-CS-modified electrode
2.8. Investigation of the selectivity of the electrochemical aptasensor
CS (1 mM final concentration) was pretreated with 10 mM TCEP in immobilization buffer (1 mM EDTA, 100 mM NaCl, 10 mM Tris– HCl, pH 7.4) at room temperature for 1 h. Then, 9 mL of the above solution was placed on the surface of gold electrode for 12 h at room temperature under 100% humidity. After that, 4.5 mL of each aptamer sequence (Apt1 and Apt2, 1 mM final concentration of each sequence) in hybridization buffer (1 mM EDTA, 10 mM Tris– HCl, pH 7.4) was dropped on the surface of SPGE for 1.5 h at room temperature. Next, the surface of electrode was incubated with 1 mM MCH solution (10 ml) for 1 h to block the untreated sites of SPGE, followed by rinsing the surface of electrode with the hybridization buffer.
The selectivity was analyzed in the presence of 40 nM Mb, CRP, IgG, hemoglobin, HSA and BSA.
3. Results and discussion 3.1. Detection mechanism of the electrochemical aptasensor The designed electrochemical aptasensor was based on dual aptamer (DApt), Y-shape structure of DApt-CS conjugate, digestion of CS by Exo I and target-induced disassembly of Y-shape structure. In this work, Exo I was applied as an enzyme which
Table 1 Oligonucleotide sequences used in this study. Complimentary strands have been shown with the same color.
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Scheme 1. Schematic illustration of Myoglobin (Mb) detection based on electrochemical assay. In the absence of Mb, the Y-shape structure of DApt-CS conjugate is intact and redox probe does not have access to the surface of electrode, resulting in a weak electrochemical signal (a). In the presence of target, DApt (Apt1 and Apt2) bind to Mb and leave the CS. Exo I digests and degrades CS, resulting in the free access of redox probe to the surface of electrode and a strong electrochemical signal (b).
selectively digests ssDNA from its 3′-terminus, owing to its good selectivity, low cost and buffer compatibility (Zhao et al., 2014; Zheng et al., 2012). The principle of the developed electrochemical aptasensor has been illustrated in Scheme 1. In the absence of Mb, the Y-shape structure of DApt-CS conjugate remains intact and the CS is shielded from digestion by Exo I, resulting in the low access of redox probe to the surface of modified electrode and a weak electrochemical signal. Upon the addition of Mb, a conformational change happens. DApt leave the CS and the surface of electrode and Apt/target conjugate forms, as it has been shown that aptamer binds to its corresponding target with a higher binding constant in comparison with its complementary strand (Song et al., 2016; Wu et al., 2015; Yang et al., 2014). When the electrode is incubated with Exo I, the CS (as a ssDNA) is degraded, leading to more access of redox probe to the surface of electrode and the increase of electrochemical signal. 3.2. Optimum concentration of Exo I To obtain the optimum concentration of Exo I for the efficient reaction with CS, increasing amounts of Exo I were dropped on the surface of DApt-CS-modified electrodes treated with 40 nM Mb. As indicated in Fig. 1(a), 10 U Exo I could induce the maximum electrochemical signal, confirming the maximum activity of Exo I occurred at this concentration. 3.3. Optimum incubation time of Exo I Enzymatic reaction time is a significant factor to be optimized in sensors. The DApt-CS-modified electrodes treated with 40 nM Mb were incubated with 10 U Exo I and the electrochemical signal was assessed up to 3 h. The electrochemical signal enhanced rapidly as the incubation time enhanced and reached to maximum in 1.5 h (Fig. 1(b)), indicating the required time for efficient digestion of CS by Exo I.
Fig. 1. Optimization of the parameters involved in the electrochemical signal of electrode. (a) Relative electrochemical signal of DApt-CS-modified electrode in the presence of various concentrations of Exo I. (b) Relative electrochemical signal of DApt-CS-modified electrode as a function of Exo I incubation time.
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Fig. 2. Proof of concept for the fabricated aptasensor. CV profiles of: bare electrode (red curve), CS-modified electrode (black curve), DApt-CS-modified electrode (blue curve), DApt-CS-modified electrodeþ Exo I (gray curve), DApt-CS-modified electrodeþ Mb (pink curve) and DApt-CS-modified electrodeþ Mbþ Exo I (green curve). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.4. Feasibility of the designed aptasensor for detection of Mb CV measurements were exploited to specify the formation and function of the developed aptasensor. As shown in Fig. 2, the maximum redox peak belonged to the bare electrode (red curve) because of its high capability of electron transfer. The electrochemical signal of electrode significantly decreased when CS was dropped on the surface of electrode (black curve), which confirmed the immobilization of CS on the surface of electrode. The electron transfer of redox probe [Fe(CN)6]3 /4 was dramatically prohibited by the negatively charged phosphate groups of the immobilized CS through electrostatic repulsive force to the negatively charged redox probe (Chen et al., 2014; Zhao et al., 2015b). The electrochemical signal was reduced even more (blue curve) when the DApt were added on the surface of electrode, indicating the formation of DApt-CS conjugate (Y-shape structure) which contained more negative phosphate groups and created a physical barrier for the access of redox probe to the surface of modified electrode. Addition of 10 U Exo I for 1.5 h, did not enhance the electrochemical signal (gray curve), which could be attributed to the protection of all captured sequences (CS and DApt) from digestion against Exo I. None of captured sequences contained free 3′-ends for degradation by Exo I due to formation of Y-shape structure. In the presence of Mb, the electrochemical signal rose (pink curve), confirming the release of DApt from CS, formation of Apt/Mb conjugate and disassembly of Y-shape structure, resulting in further access of redox probe to the surface of electrode. Upon addition of 10 U Exo I for 1.5 h to the DApt-CSmodified electrode treated with 40 nM Mb, the peak current significantly increased (green curve), indicating digestion of CS by Exo I and additional access of redox probe to the surface of electrode. Also, it confirmed reclining position or lying flat of CS on the surface of SPGE. 3.5. Quantification of Mb Fig. 3(a) exhibits the DPV peaks of electrode at different concentrations of Mb. The DPV peak increased and arrived to plateau at concentration of 40 nM Mb. The designed electrochemical aptasensor displayed a well linear range (100 pM to 40 nM) toward Mb (Fig. 3(b)). The limit of detection (LOD) was determined to be
Fig. 3. (a) DPV peaks of the DApt-CS-modified electrode in the presence of various concentrations of Mb in PBS (from bottom to top 0, 0.1, 0.3, 1, 3, 10, 20, 40 and 80 nM). (b) Mb standard curve in PBS. (c) Relative electrochemical signal of DAptCS-modified electrode in the presence of various materials.
27 pM (0.45 ng/mL) regarding the three times of the standard deviation of the blank/slope. Reported detection limits of Mb in other studies were as following: 25 mg/500 mg for ultra-performance liquid chromatography-elec-
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Table 2 Recovery and relative standard deviation (RSD) of Mb in human serum samples. Serum samples
Found (nM)
Added Mb (nM)
Total found (nM)
Recovery (%) RSD (%, n¼ 3)
1 2 3 4
2 2 2 2
2 5 15 30
3.82 6.7 15.65 32.8
95.5 95.7 92.1 102.5
5.6 3.9 7.4 3.2
trospray ionization mass spectrometry (Giaretta et al., 2013), 460 pg/ mL for two-dimensional liquid chromatography-electrospray ionization mass spectrometry (Mayr et al., 2006), linear range of 0.91–182 mg/mL for colorimetric sensing (Zhang et al., 2011), 16 ng/mL for fluorescent sensing (Darain et al., 2009), 87.6 ng/mL for SPR (Osman et al., 2013), 2.25 mg/mL for electrochemical biosensor based on biomimetic material (Moreira et al., 2013) and 50 pM for an optical aptasensor using personal glucose meter (Wang et al., 2014a). In comparison with the designed electrochemical aptasensor, most of these approaches are pricey, time-consuming and have higher LODs. 3.6. Evaluation of the selectivity of the designed aptasensor In addition to sensitivity, specificity is an important factor for sensing platforms. The relative electrochemical signal of the DAptCS-modified electrode toward Mb was significantly more than other materials (Fig. 3(c)), including HSA, BSA, hemoglobin, IgG and CRP. These results demonstrated high selectivity of the developed aptasensor toward Mb. 3.7. Detection of Mb in human serum To evaluate the applicability of the proposed assay, the designed electrochemical aptasensor was applied to measure Mb in human serum. Known concentrations of Mb were spiked into serum. The serum samples were diluted 10 times, followed by measuring the concentration of Mb using the designed sensing method. The recoveries for Mb in human serum samples were in the range of 92.1–102.5% with relative standard deviations between 3.2% and 7.4% (Table 2), suggesting the developed aptasensor could be utilized to precisely detect Mb in complex biological samples.
4. Conclusion In conclusion, a novel electrochemical aptasensor was designed for ultrasensitive and selective detection of Mb, based on DApt, Exo I, Y-shape structure of DApt-CS conjugate and target-induced disassembly of Y-shape structure. The limit of detection for Mb was determined as low as 27 pM. In addition, the developed aptasensor could precisely detect Mb in human serum.
Conflict of interest There is no conflict of interest about this article.
Acknowledgment Financial support of this study was provided by Mashhad University of Medical Sciences.
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