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An ultrasensitive electrochemical sensor for 17β-estradiol using split aptamers
Analytica Chimica Acta xxx (xxxx) xxx
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An ultrasensitive electrochemical sensor for 17b-estradiol using split aptamers Morteza Alinezhad Nameghi a, 1, Noor Mohammad Danesh b, 1, Mohammad Ramezani a, Mona Alibolandi a, Khalil Abnous a, c, *, Seyed Mohammad Taghdisi d, e, ** a
Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran Research Institute of Sciences and New Technology, Mashhad, Iran Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran d Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran e Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b c
h i g h l i g h t s A simple electrochemical sensor was proposed for 17b-estradiol (E2) determination. In this sensing platform, split DNA aptamers for E2 were applied as recognizing agents. E2-mediated bridge assembly leads to ultrasensitive detection of E2. This aptasensor is capable of recognizing E2 within 30 min without complicated procedures. This aptasensor indicated detection limits of 0.5 pM and 0.7 pM for E2 in tap water and milk samples, respectively.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2018 Received in revised form 22 February 2019 Accepted 26 February 2019 Available online xxx
Herein, a simple electrochemical sensor is proposed for 17b-estradiol (E2) determination. In this sensing platform, split DNA aptamers for E2 were applied as recognizing agents. In the presence of E2, split aptamers are bound to E2 and establish split1-E2-split2 complex as a bridge on the surface of electrode. This physical bar leads to ultrasensitive detection of E2. This aptasensor is capable of recognizing E2 within 30 min without complicated procedures and expensive equipment. This sensing approach indicated a wide linear range with detection limits of 0.5 pM and 0.7 pM for E2 in tap water and milk samples, respectively. Simplicity and high specificity of the proposed electrochemical aptasensor make it an ideal sensing platform for ultra-low detection of other targets in complicated samples by replacement of split DNA aptamers. © 2019 Elsevier B.V. All rights reserved.
Keywords: Electrochemical sensing platform 17b-estradiol Milk sample Split DNA aptamers
1. Introduction 17b-estradiol (E2) is a common endocrine disrupting material which broadly applied in livestock for growth promotion and enhancement of milk yield [1,2]. Also, E2 can occur in surface water derived from excretions of animals and industrial effluents [3,4].
* Corresponding author. Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran. ** Corresponding author. Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran. E-mail addresses: [email protected] (K. Abnous), [email protected] (S.M. Taghdisi). 1 These authors contributed equally to the work.
Chronic exposure to E2 even at low concentration can disorganize the activity of the endocrine system and even lead to different cancers [5,6]. Therefore, there is a strong demand for analytical methods which can accurately and simply detect E2 in aquatic environments and foodstuffs to preserve the health of human beings. The major analytical methods employed for detection of E2 are based on immunoassay and instrumental analytical approaches, like high-performance liquid chromatography, liquid chromatography and liquid chromatography-mass spectrometry. Most of the above analytical approaches need complex pretreatments and high-skilled stuff. Also, most of them are timeconsuming and produce organic solvent wastes that are dangerous for the environment [7e9]. Furthermore,
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Please cite this article as: M.A. Nameghi et al., An ultrasensitive electrochemical sensor for 17b-estradiol using split aptamers, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.02.062
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immunological techniques are commonly expensive and relatively unstable [8,10]. Aptamers, generated from the approach called “Systematic Evolution of Ligands by EXponential enrichment (SELEX)”, are synthetic single-stranded nucleic acid ligands [11,12]. Aptamers can bind to their targets with high affinity and excellent selectivity through folding into tertiary structures [13,14]. Because of their unique features in terms of low cost, chemical stability, simple modification, good thermostability and in vitro synthesis, aptamers show great potentials compared to traditional antibodies [15e18]. These attributes of aptamers have resulted in their wide applications as recognition probes for the design of different biosensors. Among different analytical approaches, the electrochemical aptasensors which integrate the high selectivity of aptamers with the inexpensive and easy to miniaturization characteristics of electrochemical techniques have obtained great attention for development of biosensors [19e22]. Aptasensors which are based on split aptamer strands have shown satisfactory results for detection of their targets [23,24]. In this work, an electrochemical aptasensor was presented for detection of E2 which not only integrated the merit of simplicity since the sensor has been composed of only split aptamers, but also enabled ultrasensitive detection of E2 because of application of electrochemical technique and split DNA aptamers of E2. Immobilization of split aptamers on the surface of gold electrode increased the sensitivity of the electrochemical aptasensor significantly compared to immobilization of intact E2 aptamer on the electrode [25]. This could be attributed to the development of a bridge on the surface of electrode in the presence of E2 which dramatically inhibits the access of redox probe, [Fe(CN)6]3-/4-, to the surface of electrode. Also, compared to the split DNA aptamersbased colorimetric sensor [26], the developed electrochemical aptasensor showed much better detection limit. This could be ascribed to the high sensitivity feature of electrochemical sensor and also E2-mediated bridge assembly on the electrode surface. 2. Materials and methods 2.1. Materials The E2 split aptamers [26] with the following sequences were synthesized by Microsynth (Switzerland): 50 -ThiolTTTTTTTTTTTTTTTGCTTCCAGCTTATTGAATTACACGCAGAGGGTA-30 (split1) and 50 GCGGCTCTGCGCATTCAATTGCTGCGCGCTGAAGCGCGGAAGCTTTTTTTTTTTT-Thiol-30 (split2). 17b-estradiol (E2), dibutyl phthalate (DBP), bovine serum albumin (BSA), bisphenol A (BPA), Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), progesterone (P4), potassium hexacyanoferrate(III) (K3[Fe(CN)6]), estriol (E3), 6-mercaptohexanol (MCH), atrazine, Potassium hexacyanoferrate(II) trihydrate (K4[Fe(CN)6]$3H2O) and testosterone were purchased from Sigma-Aldrich (USA). 2.2. Apparatus and electrochemical assessments The electrochemical experiments were conducted by a mSTAT 400 portable Biopotentiostat/Galvanostat (DropSens, Spain) equipped with DropView8400 software using disposable screenprinted gold electrodes (SPGEs, DRP-C220BT, Spain). Electrochemical experiments were performed in a solution containing 0.1 M KCl and [Fe(CN)6]3-/4- (1:1, 3 mM) as a redox couple. The electrochemical signals were measured with cyclic voltammetry (CV) by scanning from 0.45 V to 0.8 V at a scan rate of 50 mV/s and differential pulse voltammetry (DPV) by scanning from 0.05 V to 0.24 V, pulse time of 25 ms and pulse potential of 10 mV.
Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were conducted on a TESCAN MIRA3 microscope (Czech Republic) and a JPK Nanowizard II microscope (Germany), respectively. 2.3. Fabrication of the aptasensor and E2 analysis 10 mL containing split1 (250 nM) and split2 (250 nM) pretreated with TCEP (5 mM) was dropped onto the surface of gold electrode to conduct a 12 h assembly reaction at room temperature in a moisture-saturated environment. After being washed with TrisHCl (pH 7.4) to eliminate the unbound split aptamers, the electrode was incubated with 10 mL MCH (0.5 mM) for 1 h at room temperature for blocking reaction. Subsequently, the electrodes were rinsed with Tris-HCl. After that, 10 mL of different concentrations of E2 (0e15 nM) were placed on the surfaces of prepared electrodes for 30 min at room temperature, followed by washing with Tris-HCl. Finally, DPV technique was used to measure the current signal. 2.4. Analysis of the selectivity of the aptasensor The specificity of the proposed approach was examined by measuring other endocrine disrupting chemicals and structured E2 analogues, such as estriol (E3), progesterone (P4), testosterone, dibutyl phthalate (DBP), bovine serum albumin (BSA), atrazine and bisphenol A (BPA) (7 nM the concentration of each material). 2.5. Milk and serum samples analysis Milk and serum samples were diluted 10 times with 10 mM phosphate buffer saline (PBS, pH 7.4) and spiked with different quantities of E2 (0e15 nM). Thereafter, the proposed analytical method was used to detect the concentrations of E2 in the spiked milk and serum samples as mentioned above. 3. Results and discussion 3.1. Design strategy of the developed aptasensor Herein, by combining split DNA aptamers with gold electrode, a new electrochemical aptasensor was proposed for the simple, rapid and ultrasensitive detection of E2 (Scheme 1). The biosensor is simply prepared through the immobilization of Thiol-modified split aptamers on the surface of gold electrode based on Au-S bond. In the presence of E2, the split1 and split2 bind to the E2 and form a physical barrier (split1-E2-split2 complex, Fig. 1, lane 4) on the surface of electrode which significantly prohibits the access of [Fe(CN)6]3-/4- to the electrode surface, leading to a weak current. In the absence of E2, no bridge is formed on the surface of electrode (Fig. 1, lane 3), resulting in more approach of the redox probe to the electrode surface and improvement of the signal response. 3.2. Optimization of the incubation time of E2 To examine the effect of incubation time of E2 on the response of the designed sensing platform, the split DNA aptamers-modified electrodes were treated with 100 pM E2 with varying incubation times from 7 to 60 min. The maximum relative electrochemical signal was obtained at 30 min (Fig. 2). So, 30 min incubation time of E2 was chosen for the next experiments.
Please cite this article as: M.A. Nameghi et al., An ultrasensitive electrochemical sensor for 17b-estradiol using split aptamers, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.02.062
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Scheme 1. Schematic illustration of the electrochemical aptasensor for the sensing of 17b-estradiol (E2) based on split DNA aptamers.
current obviously decreased (pink curve, b curve) which mostly occurred by the electrostatic repulsion between the phosphate skeletons of split DNA aptamers and the negatively charged redox marker. Thus, this repulsive effect hindered the approaching of redox agent onto the electrode surface. Additionally, the capture of split aptamers on the gold electrode surface was further confirmed by SEM (Fig. S1) and AFM (Fig. S2) images which showed the change of morphology of electrode and its roughness enhancement following the addition of split DNA aptamers, respectively. After addition of E2, the electrochemical signal was further diminished (black curve, d curve) since split1-E2-split2 complex as a physical barrier was formed on the electrode and further hindered the electron transfer. While, in the presence of BPA (nontarget), the current signal of split DNA aptamersmodified electrode did not significantly change (blue curve, c curve), verifying the main character of E2 for the bridge formation on the electrode surface.
3.4. Sensor sensitivity
Fig. 1. Analysis of the formation of split1-E2-split complex (physical barrier) in the presence of target using agarose gel electrophoresis. Lane 1: Split1, lane 2: Split2, lane 3: Split1 þ Split2 (no target), lane 4: Split1 þ Split2 þ E2 (split1-E2-split2 complex).
3.3. Characterization of the electrode surface and feasibility of the aptasensor The CV was utilized as an effective tool for analysis of the electrochemical properties of the modified electrode and aptasensor performance (Fig. 3). The unmodified electrode indicated the maximum CV current (green curve, a curve) which can be ascribed to the excellent electron transfer between the unmodified gold electrode and [Fe(CN)6]3-/4-. When, split aptamers (split1 and split2) were attached onto the gold electrode, the redox
Linear range and detection limit are important indicators of the performance of an analytical method. The analytical performance of the developed sensing approach was investigated by applying a series of tap water samples containing E2 with various concentrations (Fig. 4A). Fig. 4B exhibits that the relative electrochemical signal is linearly correlated with the logarithmic concentrations of E2 from 1.2 to 100 pM and 100 pM to 7 nM with a limit of detection of 0.5 pM (S/N ¼ 3). The measured detection limit was much less than the maximum residue of E2 in surface water (1.47 pM) and drinking water (294 pM) determined by the National Environmental Protection Agency of the United States and Japan, respectively [1]. Also, it is worthy to note that LOD of the developed aptasensor is superior compared to other aptamer-based analytical methods used for detection of E2 (Table 1). Among the indicated methods, a photoelectrochemical aptasensor which was based on TiO2-BiVO4 hetero-structure [27] showed better detection limit compared to the developed aptasensor. However, the preparation of this photoelectrochemical aptasensor is complicated and needs several steps while the preparation of our aptasensor is simple and requires only one step (immobilization of split aptamers on the electrode surface), leading to decrease of the time and cost of preparation of the presented aptasensor.
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Fig. 2. Influence of the E2 incubation time on the relative electrochemical response (I0 I) of the split DNA aptamers-modified electrode. I0 and I are the current signals before and after addition of E2.
Fig. 3. Electrochemical characterization of the electrode modification and the aptasensor function. CV profiles of: bare electrode (green curve, a curve), split DNA aptamersmodified electrode (pink curve, b curve), split DNA aptamers-modified electrode þ BPA (lack of bridge) (blue curve, c curve), split DNA aptamers-modified electrode þ E2 (bridge assembly) (black curve, d curve). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.5. Specificity of the aptasensor Selectivity is an important factor to evaluate the performance of a sensor. In order to confirm the selectivity of the analytical methods, the influence of potentially interfering materials like E3, P4, DBP, BSA, testosterone, atrazine and BPA were studied and compared with the response of the aptasensor towards E2. As displayed in Fig. 4C, only E2 led to a remarkable change of the
relative electrochemical response of the sensor in comparison with other substances, showing the high specificity of the proposed aptasensor. 3.6. Detection of E2 in milk and serum samples To investigate the potential application of the designed electrochemical sensor in complicated biological samples, the provided
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Fig. 4. (A) DPV peaks of the aptasensor correspond to different concentrations of E2 in tap water samples. (B) The calibration plot of the aptasensor for tap water samples (I0-I). I0 and I are the currents before and after addition of E2, respectively. (C) Relative signal response (I0-I) of the proposed analytical method in the presence of various substances (the concentration of each material was 7 nM). I0 and I are the currents before and after addition of each substance, respectively. (D) The calibration plot of the sensing platform for milk samples.
Table 1 Comparison of the designed aptasensor with other reported E2 sensing platforms. Method
LOD
Linear range
Reference
An aptasensor based on Poly(N-isopropylacrylamide) microgel-based etalons A Fluorescent aptasensor based on gold nanoparticles quenching the fluorescence of Rhodamine B A FRET-based turn-on fluorescent aptasensor A fluorescent aptasensor based on Ru complex and quantum dots TiO2-BiVO4 heterostructure to enhance photoelectrochemical efficiency for sensitive aptasensing A Colorimetric aptasensor using nanoparticles assembled by aptamer and cationic polymer A Colorimetric aptasensor using split aptamers and gold nanoparticles An electrochemical sensor using split aptamers
3.2 pM 480 pM 350 pM 37 nM 22 fM 1.57 nM ~367 pM 0.5 pM
3.2e640 pM 0.48e200 nM 0.35e35000 nM 0.08e0.4 mM 0.1e250 pM Not reported ~0.367e367000 nM 1.5e100 pM and 100e7000 pM
[3] [10] [1] [9] [27] [5] [26] The present work
Table 2 Recovery of E2 from milk samples (n ¼ 4). Data are mean ± standard deviation (SD). Milk samples
Added E2 (pM)
Found (pM)
Recovery (%)
RSD (%, n ¼ 4)
1 2 3 4
10 50 300 1500
9 44.7 285 1525.5
90 89.4 95 101.7
5.2 6.4 1.8 2
sensing approach was utilized to detect E2 in diluted milk samples spiked with E2. Fig. 4D indicates that the relative electrochemical signals from the milk samples spiked with E2 are similar with those
in tap water samples. The sensor showed a detection limit of 0.7 pM with linear ranges of 3e300 pM and 300 pM-9 nM. Also, the recoveries of the developed aptasensor for milk samples were found within the range from 89.4% to 101.7% and relative standard deviations (RSDs) varied from 1.8% to 6.4% (Table 2). The results validated the reliability and accuracy of the proposed method for E2 detection in milk samples. Moreover, the recoveries of the sensing approach for serum samples ranged from 92.1% to 102% (Table S1). The satisfactory recoveries of the presented sensing approach indicated that the aptasensor possessed good accuracy in real samples.
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4. Conclusion In summary, a simple and rapid electrochemical sensor was proposed for E2 detection in real samples based on split DNA aptamers. This sensing platform was initiated upon the immobilization of split aptamers on the gold electrode surface and formation of split1-E2-split2 complex as a physical barrier in the presence of target, resulting in ultrasensitive detection of E2 in tap water (0.5 pM) and milk (0.7 pM) samples. The sensor showed excellent selectivity towards E2 and could distinguish it from other endocrine disrupting materials. Overall, the designed electrochemical aptasensor can be readily expanded to the ultra-low detection of a broad range of analytes in real samples by changing of the split DNA aptamers. Conflict of interest There is no conflict of interest about this article. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment Financial support of this study was provided by Mashhad University of Medical Sciences. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.02.062. References [1] G. Zhang, T. Li, J. Zhang, A. Chen, A simple FRET-based turn-on fluorescent aptasensor for 17b-estradiol determination in environmental water, urine and milk samples, Sensor. Actuator. B Chem. 273 (2018) 1648e1653. [2] X. Du, L. Dai, D. Jiang, H. Li, N. Hao, T. You, H. Mao, K. Wang, Gold nanrods plasmon-enhanced photoelectrochemical aptasensing based on hematite/Ndoped graphene films for ultrasensitive analysis of 17b-estradiol, Biosens. Bioelectron. 91 (2017) 706e713. [3] Y. Jiang, M.G. Colazo, M.J. Serpe, Poly(N-isopropylacrylamide) microgel-based etalons for the label-free quantitation of estradiol-17b in aqueous solutions and milk samples, Anal. Bioanal. Chem. 410 (2018) 4397e4407. [4] L. Fan, G. Zhao, H. Shi, M. Liu, A simple and label-free aptasensor based on nickel hexacyanoferrate nanoparticles as signal probe for highly sensitive detection of 17b-estradiol, Biosens. Bioelectron. 68 (2015) 303e309. [5] D. Zhang, W. Zhang, J. Ye, S. Zhan, B. Xia, J. Lv, H. Xu, G. Du, L. Wang, A labelfree colorimetric biosensor for 17b-estradiol detection using nanoparticles assembled by aptamer and cationic polymer, Aust. J. Chem. 69 (2016) 12e19. [6] W. Na, J.W. Park, J.H. An, J. Jang, Size-controllable ultrathin carboxylated polypyrrole nanotube transducer for extremely sensitive 17b-estradiol FETtype biosensors, J. Mater. Chem. B 4 (2016) 5025e5034. [7] J.A. Rather, E.A. Khudaish, P. Kannan, Graphene-amplified femtosensitive aptasensing of estradiol, an endocrine disruptor, Analyst 143 (2018) 1835e1845.
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Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
An ultrasensitive electrochemical sensor for 17b-estradiol using split aptamers Morteza Alinezhad Nameghi a, 1, Noor Mohammad Danesh b, 1, Mohammad Ramezani a, Mona Alibolandi a, Khalil Abnous a, c, *, Seyed Mohammad Taghdisi d, e, ** a
Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran Research Institute of Sciences and New Technology, Mashhad, Iran Department of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran d Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran e Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b c
h i g h l i g h t s A simple electrochemical sensor was proposed for 17b-estradiol (E2) determination. In this sensing platform, split DNA aptamers for E2 were applied as recognizing agents. E2-mediated bridge assembly leads to ultrasensitive detection of E2. This aptasensor is capable of recognizing E2 within 30 min without complicated procedures. This aptasensor indicated detection limits of 0.5 pM and 0.7 pM for E2 in tap water and milk samples, respectively.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2018 Received in revised form 22 February 2019 Accepted 26 February 2019 Available online xxx
Herein, a simple electrochemical sensor is proposed for 17b-estradiol (E2) determination. In this sensing platform, split DNA aptamers for E2 were applied as recognizing agents. In the presence of E2, split aptamers are bound to E2 and establish split1-E2-split2 complex as a bridge on the surface of electrode. This physical bar leads to ultrasensitive detection of E2. This aptasensor is capable of recognizing E2 within 30 min without complicated procedures and expensive equipment. This sensing approach indicated a wide linear range with detection limits of 0.5 pM and 0.7 pM for E2 in tap water and milk samples, respectively. Simplicity and high specificity of the proposed electrochemical aptasensor make it an ideal sensing platform for ultra-low detection of other targets in complicated samples by replacement of split DNA aptamers. © 2019 Elsevier B.V. All rights reserved.
Keywords: Electrochemical sensing platform 17b-estradiol Milk sample Split DNA aptamers
1. Introduction 17b-estradiol (E2) is a common endocrine disrupting material which broadly applied in livestock for growth promotion and enhancement of milk yield [1,2]. Also, E2 can occur in surface water derived from excretions of animals and industrial effluents [3,4].
* Corresponding author. Pharmaceutical Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran. ** Corresponding author. Targeted Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran. E-mail addresses: [email protected] (K. Abnous), [email protected] (S.M. Taghdisi). 1 These authors contributed equally to the work.
Chronic exposure to E2 even at low concentration can disorganize the activity of the endocrine system and even lead to different cancers [5,6]. Therefore, there is a strong demand for analytical methods which can accurately and simply detect E2 in aquatic environments and foodstuffs to preserve the health of human beings. The major analytical methods employed for detection of E2 are based on immunoassay and instrumental analytical approaches, like high-performance liquid chromatography, liquid chromatography and liquid chromatography-mass spectrometry. Most of the above analytical approaches need complex pretreatments and high-skilled stuff. Also, most of them are timeconsuming and produce organic solvent wastes that are dangerous for the environment [7e9]. Furthermore,
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Please cite this article as: M.A. Nameghi et al., An ultrasensitive electrochemical sensor for 17b-estradiol using split aptamers, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.02.062
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immunological techniques are commonly expensive and relatively unstable [8,10]. Aptamers, generated from the approach called “Systematic Evolution of Ligands by EXponential enrichment (SELEX)”, are synthetic single-stranded nucleic acid ligands [11,12]. Aptamers can bind to their targets with high affinity and excellent selectivity through folding into tertiary structures [13,14]. Because of their unique features in terms of low cost, chemical stability, simple modification, good thermostability and in vitro synthesis, aptamers show great potentials compared to traditional antibodies [15e18]. These attributes of aptamers have resulted in their wide applications as recognition probes for the design of different biosensors. Among different analytical approaches, the electrochemical aptasensors which integrate the high selectivity of aptamers with the inexpensive and easy to miniaturization characteristics of electrochemical techniques have obtained great attention for development of biosensors [19e22]. Aptasensors which are based on split aptamer strands have shown satisfactory results for detection of their targets [23,24]. In this work, an electrochemical aptasensor was presented for detection of E2 which not only integrated the merit of simplicity since the sensor has been composed of only split aptamers, but also enabled ultrasensitive detection of E2 because of application of electrochemical technique and split DNA aptamers of E2. Immobilization of split aptamers on the surface of gold electrode increased the sensitivity of the electrochemical aptasensor significantly compared to immobilization of intact E2 aptamer on the electrode [25]. This could be attributed to the development of a bridge on the surface of electrode in the presence of E2 which dramatically inhibits the access of redox probe, [Fe(CN)6]3-/4-, to the surface of electrode. Also, compared to the split DNA aptamersbased colorimetric sensor [26], the developed electrochemical aptasensor showed much better detection limit. This could be ascribed to the high sensitivity feature of electrochemical sensor and also E2-mediated bridge assembly on the electrode surface. 2. Materials and methods 2.1. Materials The E2 split aptamers [26] with the following sequences were synthesized by Microsynth (Switzerland): 50 -ThiolTTTTTTTTTTTTTTTGCTTCCAGCTTATTGAATTACACGCAGAGGGTA-30 (split1) and 50 GCGGCTCTGCGCATTCAATTGCTGCGCGCTGAAGCGCGGAAGCTTTTTTTTTTTT-Thiol-30 (split2). 17b-estradiol (E2), dibutyl phthalate (DBP), bovine serum albumin (BSA), bisphenol A (BPA), Tris(2-carboxyethyl) phosphine hydrochloride (TCEP), progesterone (P4), potassium hexacyanoferrate(III) (K3[Fe(CN)6]), estriol (E3), 6-mercaptohexanol (MCH), atrazine, Potassium hexacyanoferrate(II) trihydrate (K4[Fe(CN)6]$3H2O) and testosterone were purchased from Sigma-Aldrich (USA). 2.2. Apparatus and electrochemical assessments The electrochemical experiments were conducted by a mSTAT 400 portable Biopotentiostat/Galvanostat (DropSens, Spain) equipped with DropView8400 software using disposable screenprinted gold electrodes (SPGEs, DRP-C220BT, Spain). Electrochemical experiments were performed in a solution containing 0.1 M KCl and [Fe(CN)6]3-/4- (1:1, 3 mM) as a redox couple. The electrochemical signals were measured with cyclic voltammetry (CV) by scanning from 0.45 V to 0.8 V at a scan rate of 50 mV/s and differential pulse voltammetry (DPV) by scanning from 0.05 V to 0.24 V, pulse time of 25 ms and pulse potential of 10 mV.
Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were conducted on a TESCAN MIRA3 microscope (Czech Republic) and a JPK Nanowizard II microscope (Germany), respectively. 2.3. Fabrication of the aptasensor and E2 analysis 10 mL containing split1 (250 nM) and split2 (250 nM) pretreated with TCEP (5 mM) was dropped onto the surface of gold electrode to conduct a 12 h assembly reaction at room temperature in a moisture-saturated environment. After being washed with TrisHCl (pH 7.4) to eliminate the unbound split aptamers, the electrode was incubated with 10 mL MCH (0.5 mM) for 1 h at room temperature for blocking reaction. Subsequently, the electrodes were rinsed with Tris-HCl. After that, 10 mL of different concentrations of E2 (0e15 nM) were placed on the surfaces of prepared electrodes for 30 min at room temperature, followed by washing with Tris-HCl. Finally, DPV technique was used to measure the current signal. 2.4. Analysis of the selectivity of the aptasensor The specificity of the proposed approach was examined by measuring other endocrine disrupting chemicals and structured E2 analogues, such as estriol (E3), progesterone (P4), testosterone, dibutyl phthalate (DBP), bovine serum albumin (BSA), atrazine and bisphenol A (BPA) (7 nM the concentration of each material). 2.5. Milk and serum samples analysis Milk and serum samples were diluted 10 times with 10 mM phosphate buffer saline (PBS, pH 7.4) and spiked with different quantities of E2 (0e15 nM). Thereafter, the proposed analytical method was used to detect the concentrations of E2 in the spiked milk and serum samples as mentioned above. 3. Results and discussion 3.1. Design strategy of the developed aptasensor Herein, by combining split DNA aptamers with gold electrode, a new electrochemical aptasensor was proposed for the simple, rapid and ultrasensitive detection of E2 (Scheme 1). The biosensor is simply prepared through the immobilization of Thiol-modified split aptamers on the surface of gold electrode based on Au-S bond. In the presence of E2, the split1 and split2 bind to the E2 and form a physical barrier (split1-E2-split2 complex, Fig. 1, lane 4) on the surface of electrode which significantly prohibits the access of [Fe(CN)6]3-/4- to the electrode surface, leading to a weak current. In the absence of E2, no bridge is formed on the surface of electrode (Fig. 1, lane 3), resulting in more approach of the redox probe to the electrode surface and improvement of the signal response. 3.2. Optimization of the incubation time of E2 To examine the effect of incubation time of E2 on the response of the designed sensing platform, the split DNA aptamers-modified electrodes were treated with 100 pM E2 with varying incubation times from 7 to 60 min. The maximum relative electrochemical signal was obtained at 30 min (Fig. 2). So, 30 min incubation time of E2 was chosen for the next experiments.
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Scheme 1. Schematic illustration of the electrochemical aptasensor for the sensing of 17b-estradiol (E2) based on split DNA aptamers.
current obviously decreased (pink curve, b curve) which mostly occurred by the electrostatic repulsion between the phosphate skeletons of split DNA aptamers and the negatively charged redox marker. Thus, this repulsive effect hindered the approaching of redox agent onto the electrode surface. Additionally, the capture of split aptamers on the gold electrode surface was further confirmed by SEM (Fig. S1) and AFM (Fig. S2) images which showed the change of morphology of electrode and its roughness enhancement following the addition of split DNA aptamers, respectively. After addition of E2, the electrochemical signal was further diminished (black curve, d curve) since split1-E2-split2 complex as a physical barrier was formed on the electrode and further hindered the electron transfer. While, in the presence of BPA (nontarget), the current signal of split DNA aptamersmodified electrode did not significantly change (blue curve, c curve), verifying the main character of E2 for the bridge formation on the electrode surface.
3.4. Sensor sensitivity
Fig. 1. Analysis of the formation of split1-E2-split complex (physical barrier) in the presence of target using agarose gel electrophoresis. Lane 1: Split1, lane 2: Split2, lane 3: Split1 þ Split2 (no target), lane 4: Split1 þ Split2 þ E2 (split1-E2-split2 complex).
3.3. Characterization of the electrode surface and feasibility of the aptasensor The CV was utilized as an effective tool for analysis of the electrochemical properties of the modified electrode and aptasensor performance (Fig. 3). The unmodified electrode indicated the maximum CV current (green curve, a curve) which can be ascribed to the excellent electron transfer between the unmodified gold electrode and [Fe(CN)6]3-/4-. When, split aptamers (split1 and split2) were attached onto the gold electrode, the redox
Linear range and detection limit are important indicators of the performance of an analytical method. The analytical performance of the developed sensing approach was investigated by applying a series of tap water samples containing E2 with various concentrations (Fig. 4A). Fig. 4B exhibits that the relative electrochemical signal is linearly correlated with the logarithmic concentrations of E2 from 1.2 to 100 pM and 100 pM to 7 nM with a limit of detection of 0.5 pM (S/N ¼ 3). The measured detection limit was much less than the maximum residue of E2 in surface water (1.47 pM) and drinking water (294 pM) determined by the National Environmental Protection Agency of the United States and Japan, respectively [1]. Also, it is worthy to note that LOD of the developed aptasensor is superior compared to other aptamer-based analytical methods used for detection of E2 (Table 1). Among the indicated methods, a photoelectrochemical aptasensor which was based on TiO2-BiVO4 hetero-structure [27] showed better detection limit compared to the developed aptasensor. However, the preparation of this photoelectrochemical aptasensor is complicated and needs several steps while the preparation of our aptasensor is simple and requires only one step (immobilization of split aptamers on the electrode surface), leading to decrease of the time and cost of preparation of the presented aptasensor.
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Fig. 2. Influence of the E2 incubation time on the relative electrochemical response (I0 I) of the split DNA aptamers-modified electrode. I0 and I are the current signals before and after addition of E2.
Fig. 3. Electrochemical characterization of the electrode modification and the aptasensor function. CV profiles of: bare electrode (green curve, a curve), split DNA aptamersmodified electrode (pink curve, b curve), split DNA aptamers-modified electrode þ BPA (lack of bridge) (blue curve, c curve), split DNA aptamers-modified electrode þ E2 (bridge assembly) (black curve, d curve). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.5. Specificity of the aptasensor Selectivity is an important factor to evaluate the performance of a sensor. In order to confirm the selectivity of the analytical methods, the influence of potentially interfering materials like E3, P4, DBP, BSA, testosterone, atrazine and BPA were studied and compared with the response of the aptasensor towards E2. As displayed in Fig. 4C, only E2 led to a remarkable change of the
relative electrochemical response of the sensor in comparison with other substances, showing the high specificity of the proposed aptasensor. 3.6. Detection of E2 in milk and serum samples To investigate the potential application of the designed electrochemical sensor in complicated biological samples, the provided
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Fig. 4. (A) DPV peaks of the aptasensor correspond to different concentrations of E2 in tap water samples. (B) The calibration plot of the aptasensor for tap water samples (I0-I). I0 and I are the currents before and after addition of E2, respectively. (C) Relative signal response (I0-I) of the proposed analytical method in the presence of various substances (the concentration of each material was 7 nM). I0 and I are the currents before and after addition of each substance, respectively. (D) The calibration plot of the sensing platform for milk samples.
Table 1 Comparison of the designed aptasensor with other reported E2 sensing platforms. Method
LOD
Linear range
Reference
An aptasensor based on Poly(N-isopropylacrylamide) microgel-based etalons A Fluorescent aptasensor based on gold nanoparticles quenching the fluorescence of Rhodamine B A FRET-based turn-on fluorescent aptasensor A fluorescent aptasensor based on Ru complex and quantum dots TiO2-BiVO4 heterostructure to enhance photoelectrochemical efficiency for sensitive aptasensing A Colorimetric aptasensor using nanoparticles assembled by aptamer and cationic polymer A Colorimetric aptasensor using split aptamers and gold nanoparticles An electrochemical sensor using split aptamers
3.2 pM 480 pM 350 pM 37 nM 22 fM 1.57 nM ~367 pM 0.5 pM
3.2e640 pM 0.48e200 nM 0.35e35000 nM 0.08e0.4 mM 0.1e250 pM Not reported ~0.367e367000 nM 1.5e100 pM and 100e7000 pM
[3] [10] [1] [9] [27] [5] [26] The present work
Table 2 Recovery of E2 from milk samples (n ¼ 4). Data are mean ± standard deviation (SD). Milk samples
Added E2 (pM)
Found (pM)
Recovery (%)
RSD (%, n ¼ 4)
1 2 3 4
10 50 300 1500
9 44.7 285 1525.5
90 89.4 95 101.7
5.2 6.4 1.8 2
sensing approach was utilized to detect E2 in diluted milk samples spiked with E2. Fig. 4D indicates that the relative electrochemical signals from the milk samples spiked with E2 are similar with those
in tap water samples. The sensor showed a detection limit of 0.7 pM with linear ranges of 3e300 pM and 300 pM-9 nM. Also, the recoveries of the developed aptasensor for milk samples were found within the range from 89.4% to 101.7% and relative standard deviations (RSDs) varied from 1.8% to 6.4% (Table 2). The results validated the reliability and accuracy of the proposed method for E2 detection in milk samples. Moreover, the recoveries of the sensing approach for serum samples ranged from 92.1% to 102% (Table S1). The satisfactory recoveries of the presented sensing approach indicated that the aptasensor possessed good accuracy in real samples.
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4. Conclusion In summary, a simple and rapid electrochemical sensor was proposed for E2 detection in real samples based on split DNA aptamers. This sensing platform was initiated upon the immobilization of split aptamers on the gold electrode surface and formation of split1-E2-split2 complex as a physical barrier in the presence of target, resulting in ultrasensitive detection of E2 in tap water (0.5 pM) and milk (0.7 pM) samples. The sensor showed excellent selectivity towards E2 and could distinguish it from other endocrine disrupting materials. Overall, the designed electrochemical aptasensor can be readily expanded to the ultra-low detection of a broad range of analytes in real samples by changing of the split DNA aptamers. Conflict of interest There is no conflict of interest about this article. Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment Financial support of this study was provided by Mashhad University of Medical Sciences. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.02.062. References [1] G. Zhang, T. Li, J. Zhang, A. Chen, A simple FRET-based turn-on fluorescent aptasensor for 17b-estradiol determination in environmental water, urine and milk samples, Sensor. Actuator. B Chem. 273 (2018) 1648e1653. [2] X. Du, L. Dai, D. Jiang, H. Li, N. Hao, T. You, H. Mao, K. Wang, Gold nanrods plasmon-enhanced photoelectrochemical aptasensing based on hematite/Ndoped graphene films for ultrasensitive analysis of 17b-estradiol, Biosens. Bioelectron. 91 (2017) 706e713. [3] Y. Jiang, M.G. Colazo, M.J. Serpe, Poly(N-isopropylacrylamide) microgel-based etalons for the label-free quantitation of estradiol-17b in aqueous solutions and milk samples, Anal. Bioanal. Chem. 410 (2018) 4397e4407. [4] L. Fan, G. Zhao, H. Shi, M. Liu, A simple and label-free aptasensor based on nickel hexacyanoferrate nanoparticles as signal probe for highly sensitive detection of 17b-estradiol, Biosens. Bioelectron. 68 (2015) 303e309. [5] D. Zhang, W. Zhang, J. Ye, S. Zhan, B. Xia, J. Lv, H. Xu, G. Du, L. Wang, A labelfree colorimetric biosensor for 17b-estradiol detection using nanoparticles assembled by aptamer and cationic polymer, Aust. J. Chem. 69 (2016) 12e19. [6] W. Na, J.W. Park, J.H. An, J. Jang, Size-controllable ultrathin carboxylated polypyrrole nanotube transducer for extremely sensitive 17b-estradiol FETtype biosensors, J. Mater. Chem. B 4 (2016) 5025e5034. [7] J.A. Rather, E.A. Khudaish, P. Kannan, Graphene-amplified femtosensitive aptasensing of estradiol, an endocrine disruptor, Analyst 143 (2018) 1835e1845.
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Please cite this article as: M.A. Nameghi et al., An ultrasensitive electrochemical sensor for 17b-estradiol using split aptamers, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.02.062