Highly sensitive electrochemical aptasensor for immunoglobulin E detection based on sandwich assay using enzyme-linked aptamer

Accepted Manuscript Highly sensitive electrochemical aptasensor for immunoglobulin E detection based on sandwich assay using enzyme linked aptamer Abdollah Salimi, Somayeh Khezrian, Rahman Hallaj, Asaad Vaziry PII: DOI: Reference:

S0003-2697(14)00352-2 http://dx.doi.org/10.1016/j.ab.2014.08.019 YABIO 11839

To appear in:

Analytical Biochemistry

Received Date: Revised Date: Accepted Date:

5 April 2014 18 August 2014 19 August 2014

Please cite this article as: A. Salimi, S. Khezrian, R. Hallaj, A. Vaziry, Highly sensitive electrochemical aptasensor for immunoglobulin E detection based on sandwich assay using enzyme linked aptamer, Analytical Biochemistry (2014), doi: http://dx.doi.org/10.1016/j.ab.2014.08.019

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Highly sensitive electrochemical aptasensor for immunoglobulin E detection based on sandwich assay using enzyme linked aptamer Abdollah Salimia,b*, Somayeh Khezriana, Rahman Hallaja,b, Asaad Vaziryc

a

Department of Chemistry, University of Kurdistan, 66177-15175, Sanandaj, Iran

b

Research Center for Nanotechnology, University of Kurdistan, 66177-15175, Sanandaj, Iran

c

Department of Animal Science, Faculty of Agricultural Science, University of Kurdistan, 66177-15175, Sanandaj, Iran

*Corresponding author Tel.: +98 871 6624001, Fax: +98 871 6624008. E-mail: [email protected],[email protected] (A.Salimi)

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Abstract Here, we described the fabrication of an electrochemical immunoglobulin-E(IgE) aptasensor using enzyme linked aptmer in sandwich assay method and thionine as redox probe. In this protocol,5′amine-terminated IgE aptamer and thionine were covalently attached on glassy carbon electrode modified with carbon nanotubes/ionic liquid/chitosan nanocomposite. Furthermore, another IgE aptamer was modified with biotin and enzyme horseradish peroxidase(HRP) which attached to the aptamer via biotin-streptavidin interaction. Electrochemical impedance spectroscopy(EIS) and cyclic voltammetry were performed at each stage of the chemical modification process in order to confirm the resulting surface changes. The presence of IgE induces the formation of a double aptamer sandwich structure on the electrode and the electrocatalytic reduction current of thionine in the presence of hydrogen peroxide was measured as sensor response. Under optimized condition and using differential pulse voltammetry as measuring technique the proposed aptasensor showed low detection limit( 6 pM) and high sensitivity 1.88µAnM-1. This aptasensor also exhibit good stability and high selectivity for IgE detection without interfering effect of some other proteins such as bovine serum albumin(BSA) and lysozyme. The application of the aptasensor for IgE detection in human serum sample is also investigated. The proposed protocol is quite promising as alternative sandwich approach for various protein assays. Key words: Electrochemical Aptasensor, Enzyme linked Sandwich assay, IgE, Carbon nanotubes, Chitosan, Ionic liquid.

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1. Introduction The detection and quantification of proteins have increased attention in biomedical fields, including disease diagnosis and basic discovery research. Aptamers are single-stranded DNA or RNA sequences with high affinity to a variety of targets ranging proteins, small molecules and other nucleic acids and they have been used as powerful molecular recognition not only in drug design and delivery but also in analytical bioassay. They are selected through SELEX (systematic evolution of ligands by exponential enrichment) processes from a random sequence bank [1]. Chemical stability, structure feasibility, small in size, high specificity, flexibility in design and ease of chemical modification of aptamers, make them as attractive bio-recognition elements [2,3] in compared to antibodies as traditional recognition elements. A variety of aptamer-based detection approaches such as, surface plasmon resonance [4,5], absorbance [6] fluorescence [7,8] chemiluminescence [9], quartz crystal microbalance [10] electrochemistry [2, 12-15] and electrochemiluminescence [16], have been developed for detection of different proteins and biomolecules. Among various detection techniques, electrochemical methods have attracted attention because they provide simple, sensitive and selective platform for molecular detection with rapid response. The direct detection of proteins on a surface layer is advantage of an electrochemical aptasensor. Therefore, the application of new transducers to increase the immobilized aptamer and improve sensitivity and dynamic range, and allowing regeneration of the sensor surface are recent trends in development of electrochemical aptasensors and enhancing their sensitivities[13]. Combinations of aptamers with different nanomaterials such as carbon nanotubes, gold naoparticles, quantum dot and graphene have been recently used for improving the performance of electrochemical aptasensors [1721]. Furthermore, the covalent attachments of aptamers onto the electrode surface improve the stability and sensitivity of aptasensors due to increasing the surface loading of capture probe [22]. Among 3

different nanomaterials, carbon nanotubes have been used extensively as transducers for achievement the sensitivity of electrochemical aptasensor due to their large surface to volume ratio and high mechanical strength [19, 23]. However, irreversible aggregation of CNTs through π− π staking and van der Waals interaction decrease the homogeneity of CNTs which directly immobilized onto electrode surface and dropped fabrication reproducibility [24]. To prevent the aggregation of CNTs, ionic liquids (IL) has been used as dispersing agents for CNTs or as binder for formation of IL-CNTs paste, due to the cation–π interactions of ILs with CNTs [25]. Chitosan (Chit) a polysaccharide with reactive amino and hydroxyl functional groups has many advantages such as excellent membrane forming ability, high permeability toward water, good adhesion and high biocompatibility has been used as an immobilizing support for biofabrication [26,27]. Therefore, the combination of Chit/IL as binder with different nanomaterials such as TiO2graphene [26], CNTs-gold nanoparticles[28], Fe3O4-graphene [ 29] and multiwalled carbon nanotubes [30] have been used as effective support for immobilizing of hemoglobin, cholesterol oxidase, ssDNA probe or aptamer and designing of effective biosensors, gensensors and aptasensors. The specificity and low detection limit of double-antibody sandwich assay has ensured their widespread using. This approach is used for detection of large molecules, since steric hindrance renders it unlikely that a small molecule will simultaneously bend to the antibodies [31]. Furthermore, it is difficult for a small molecule to possess different binding sites that enable simultaneous reaction with two antibodies [32]. In addition, the majority of current aptamer based protein assays that adapted single aptamer binding configuration is insufficiently sensitive [33]. In order to improve the sensitivity and specificity of aptasensors sandwich based assay have been employed as the major analytical techniques for sensitive and selective detection of proteins as well as small biomolecules where enzyme-linked immunosorbent assay (ELISA) has become the standard approach [32]. This strategy 4

offers the advantages of high sensitivity and simple operation for biosensor fabrication when compared to the strategy using only one recognition element to capture and label the target molecules. In this approach two aptamer are required, the capture aptamer and the reporter aptamer, which both of them are specific to protein. For fabrication of an electrochemical aptasensor, first aptamer is immobilized onto electrode surface and interact with the target proteins. The sandwich structure results when the second enzyme tagged aptamer react with the captured target protein. Therefore, the development of target specific aptamer in a sandwich structure can be used for detection of various molecules such as cocaine, ATP and 2,4,6-trinitrotouluene(TNT) [11, 32-34]. Here, the sandwich assay stategy was applied for measuring of Immunoglobulin E (IgE) as the target protein using voltammetry as detection approach. First an amine terminated aptamer capture and thionine ( redox mediator) covalently attached to amine- function group of chitosan via amide bond formation

using Phtaloyl chloride (Ph) as linker. In the presence of IgE, the sandwich assay

aptasensing process completed when a biotinated aptamer which conjugated to streptavidine-HRP is added. HRB has been used as label for signal amplification and catalyzing the oxidation of thionine by H2O2 [35] . The oxidized form of thionine was reduced at MWCNTs/IL/Chitosan modified glassy carbon electrode and differential pulse voltammetry (DPV) was used as measuring technique. The fabrication process, stability and selectivity

of aptasensor were investigated by cyclic voltammetry

and electrochemical impedance spectroscopy techniques. The analytical characteristics of aptasensor was evaluated and compared with other IgE aptasensors. 2. Experimental 2.1. Materials and reagents

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DNA aptamers was custom-synthesized by Bioneer Co (South Korea) and their sequences given as follows: 5′-NH2-GCGCGGGGCACGTTTATCCGTCCCTCCTAGTGGCGTGCCCCGCGC-3′ 5′-biotin-GCGCGGGGCACGTTTATCCGTCCCTCCTAGTGGCGTGCCCCGCGC-3′. IgE purified from human myeloma (Abcam, US.). Multiwall carbon nanotubes with purity 95% (10– 20 nm diameter), 1 µm length and surface specific area of 480 m2/g were obtained from Nanolab (Brighton,MA). Ionic liquid, 1-buthyl-methylpyrolydinium bis (trifluro-methyl sulfonyl) imide [C4mpyr][NTf2], chitosan and Bovine serum albumin (BSA), lysozime and streptavidin-labeled horseradish peroxidase were purchased from Sigma. H2O2, thionine (TH), phathayol chloride, K3 Fe(CN)6 and K4Fe(CN)6 and all other reagents with analytical grade were purchased from Merck or Fluka and used without further purification 2.2.Apparatus and procedures Cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) measurements were performed on an AUTOLAB modular electrochemical system (ECO Chemie, Ultecht, The Netherlands) equipped with a PGSTAT 101 module and driven by GPES and FRA software's (ECO Chemie) in conjunction with a conventional three-electrode system and a personal computer for data storage and processing. A modified glassy carbon electrode employed as the working electrode and a platinum wire as the counter electrode. All potentials were referred to an Ag/AgCl/KCl (3 M) electrode. The parameters for DPV measurements were as follows: 50 ms modulation time, 0.5 s interval time, 25 mV modulation amplitude, and 5 mV potential steps. The impedance spectra were recorded within the frequency range of 0.1-10 kHz with 5 mV amplitude in

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0.1 M KCl solution containing 2.5 mM [Fe(CN)6]4-/3-. All electrochemical measurements were performed at room temperature.

2.3. Preparation of TH/MWCNTs/IL/Chit/GC electrode The nanocomposite was prepared by direct mixing of 0.2 mg chitosan, 5 mg of MWCNTs and 10 µl IL, followed by grounding in an agate mortar until a homogeneous paste obtained [30]. The MWCNTs/IL/Chit modified GC electrode was fabricated by rubbing some of this paste on the glassy carbon (GC) electrode surface to form a homogeneous layer. For covalent attachment of thionine onto nanocomposite, the GC-Chit-IL-MWCNTs modified electrode immersed in saturated solution of pphthaloyl chloride in toluene for 5 h. The electrode was then washed with toluene to eliminate the excess of p-phthaloyl chloride. Finally the covalent attachment of thionine was performed by immersing of the electrode in 2 mM of thionine in acetonitrile solution for 10 h at room temperature. The modified electrode was washed with acetonitrile and double distilled water thoroughly and it was stored in PBS (pH 7.0) at 4oC before using. 2.5. Aptasensor fabrication and IgE detection The glassy carbon electrode modified with nanocomposite and thionine (GC/MWCNTs/IL/Chit/TH) was immersed into a saturated solution of p-phthaloyl chloride in toluene for 5 h. The excess of pphthaloyl chloride were eliminated by washing the electrode several times with toluene. Subsequently, the 5′-NH2-IgE aptamer (primary aptamer) was covalently attached to

the electrode surface by the

amide link. For this purpose, the 5′-NH2 aptamer solution in 0.05 M PBS (10 µl, 10 µM) was added to the electrode surface and incubated for 12 h at room temperature. Then, the aptamer modified electrode was rinsed thoroughly with PBS in order to remove any unbounded aptamers followed by placing a 10 µl aliquot of 0.1 M PBS (pH 7.4) containing 1% BSA for 30 min on the electrode surface 7

to reduce non-specific binding. The surface of the primary aptamer-modified electrode was treated with 0.05 M PBS containing different concentrations of IgE for 15 min. Finally 10 µL of a pre-mixed solution containing 10 µM of biotinated-aptamer and 60 µg mL-1 of streptavidin-HRP (horseradish peroxidase) was dropped on the electrode surface and incubated at temperature of 37 oC for 1h to bind labeled aptamer to the second IgE binding site. The electrocatalytic reduction peak current of H2O2 was monitored as signal response for detaction of IgE using differential pulse voltammetry as measuring technique. The aptasensor fabrication process and IgE measuring is shown in Scheme 1. Here Scheme 1 3. Results and discussion 3.1. Characterization of TH/MWCNT/IL/Chit/GC electrode The typical SEM image of GCE modified with MWCNTs/IL/Chit is shown in figure 1. As can be observed, a relatively homogeneous film of the prepared nanocomposite with unique structure formed on the electrode surface. It may be suggested that MWCNTs and Chit have been uniformly integrated with ionic liquid. In order to investigate the electrochemical properties of GC electrode modified with MWCNTs/IL/Chit nanocomposite and thionine as redox probe, cyclic voltammograms of TH/MWCNT/IL/Chit/GC electrode at different scan rates were recorded (Fig. 2A). As illustrated, a '

well defined redox couple with E° = -0.21 V vs. Ag/AgCl electrode is observed for covalently attached thionine. The variation of peak currents vs. scan rates is shown in the inset of Fig. 2A. As can be seen the peak currents linearly increased with increase in the scan rate from 10 to 1000 mV s-1 as expected for redox surface controlled process. Furthermore, the peak-to-peak potential separation was about 70 mV for sweep rates below 100 mVs-1, reflecting facile charge transfer kinetics over this range of sweep rate. At sweep rates>1000 mVs-1 peak separations began to increase, indicating the charge 8

transfer kinetics limitation. Based on the Laviron theory the electron transfer rate constant (ks) and charge transfer coefficient (α) can be determined by measuring the variation of peak potential with scan rate [36]. Using the equation Ep= K-2.303 (RT/αnF) log(υ), K= Eo'-2.303 (RT/αnF) log (RTks/nF), charge transfer coefficient for proposed redox couple was calculated. Through introducing this value in the following Eq. (1), an apparent surface electron transfer rate constant (ks) was estimated: log  = log 1 − + 1 −  − log 

 1 − ∆  − 1  2.303

The α and ks were estimated as 0.51 and 7.3 s-1, respectively, suggesting that MWCNT/IL/Chit nanocomposite has high ability for promoting electron between thionine and electrode surface. The stability of redox system was also investigated by recording consequence cyclic voltammograms of modified electrode. The results indicate that only 3% decrease in peak current of thionine was observed after 200 repetitive cycles (Fig. 2B), which indicate the high stability of TH redox system. In addition, the modified electrode response was 96% of its initial activity after it has been kept at 4 o

C for10 days. The high stability is related to the chemical and mechanical stability of the MWCNTs-

IL-Chit nanocomposite and covalent attachment of the aptamer onto the nanocomposite surface, which indicates the promising properties of nanocomposite as a useful platformfor aptasensor fabrication. The surface coverage concentration (Γ) of immobilized TH was estimated by integrating the area under the cathodic peak. According to the equation Γ= Q/nFA and assuming A=0.15cm2, the surface concentration of immobilized TH was 9.67(±0.80)×10-10 mol cm-2, indicating high loading capacity of nanocomposite for immobilization redox probe. Here Figs. 1 and 2 3.2. Characterization of IgE aptasensor

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The preparation of the sensing interface as a key step has great importance on the aptasensor response. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were widely used to confirm each step of the sensing interference modification. Thus, the ability of the aptamer film to insulate the electrode surface at different stages of the aptasensor preparation was investigated by recording cyclic voltammograms of modified electrode in the presence of reversible [Fe(CN)6]4-/3- redox system. As shown in Fig. 3A voltammogam "a ", at the bare GC electrode a pair of well-defined reversible redox peaks with high peak currents was observed. After nonocomposite immobilized on to electrode surface the peak current of [Fe(CN)6]4-/3- redox couple was increased due to increasing of electrode surface area. After immobilization of aptamer probe on the electrode surface, the peak current clearly decreased while the peak-to-peak potential separation increased which accounted for an increased resistance to the charge-transfer process across aptamer probe attached on the electrode and reducing the effective surface area and available active sites for electron transfer process (Fig. 3A, voltammogram “b”). When the aptasensor was incubated with solutions containing 2 ng ml-1 IgE and biotinated aptamer which conjugated to streptavidine-HRP, it could be found that the voltammetric peak response was further decreased and peak potential separations increased, respectively, due to the more

insulating property of IgE and biotinated aptamer /conjugated to

streptavidine-HRP layer on the electrode surface which hindered the electron transfer process (Fig. 3A, voltammogram “d” and

e). The aptasensor preparation process was also conducted by

electrochemical impedance spectroscopy, which the diameter of the semicircle of the Nyquest plots indicated the magnitude of the electron transfer resistance. As can be seen in Fig. 3B, prior to the immobilization of the aptamer capture probe, the Rct of the modified electrode was small due to low resistance of nanocomposite for redox probe. After covalently attached of aptamer capture probe on the electrode surface, the Rct increased due to the blocking and repulsive property of negatively

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charged of aptamer for the redox probe with negatively charged [Fe(CN)6]4-/3- [37]. After incubating of aptasensor with IgE and streptavidine-HRP-biotin aptamer, the Rct was further increased due to the hindrance and repulsive effect on the electrode response. Therefore, on the basis of the CV and EIS results, the modification of the electrode and construction of sensing interface for IgE was achieved. Here Fig. 3 3.3. Optimization of experimental conditions To obtain an optimal electrochemical response of the proposed aptasensor, the experimental conditions, such as pH effect, H2O2 concentration and incubation time were optimized. As shown in Fig. 4A, the peak currents increase with increasing of pH values from 2 to 7 and decreases when its increases further. Therefore, pH 7 was selected as optimized value for IgE sensing. The effect of H2O2 concentration on the aptasensor response was also investigated. As shown in Fig. 4B, the peak current of the aptasensor increased with increasing H2O2 concentration from 0.5 to 2.5 mM, and then started to level off. Thus, 2.5 mM of H2O2 was chosen as optimum concentration for the determination of IgE. The effect of biotinated-aptamer and streptavidin-HRP concentrations was also investigated. When 10, 20, 40, 60, 80 and 100 µL of a pre-mixed solution containing 60 µg mL-1 streptavidin-HRP (horseradish peroxidase) and 10 µM of biotinated aptamer were used to bind labeled aptamer to the second IgE binding site, the highest aptasensor response was obtained with adding 20 µL of biotinated-aptamer and SA-HRP. The effect of incubation time on the current response of the sensor was also studied. As shown in Fig. 4C, the current response intensified with the increase of the incubation time and tended to level off after about 20 min, indicating that this incubation time was efficient for the self-assembly of supramolecular complex on the sensor interface. The effect of incubation time between 5′,-biotinated aptamer and streptavidin-horseradish peroxidase ( SA-HRP)

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was also investigated. The aptasensor response increased with increasing incubation time and almost saturated after 20 min, and this value was also used as optimum incubation time between biotinated aptamer and SA-HRP. Here Fig.4 3.4. Analytical characterization of IgE aptasensor The amine terminated aptamer / biotinated aptamer-SA-HRP and the target

IgE combined on the

electrode surface for completing of the aptasesing process. The application of the proposed aptasensor for sensitive quantification of target protein based on the electrocatalytic performance of HRP labeled complex, has been carried out by DPV. Fig.5 shows the aptasensor response in the presence different concentration of IgE. As illustrated the peak current increase with increasing of IgE concentration, consistent with the formation of large quantity of the HRP-labeled aptamer fragment/IgE complex on the electrode surface modified with amine- terminated aptamer . These results indicate that the HRP attached to the aptasensor surface retains high catalytic activity toward hydrogen peroxide reduction and also the thionine is a suitable electron transfer mediator for electrocatalytic process. The following mechanism indicates the aptasensor response based on enzymatic catalytic reduction of H2O2: HRP red + H% O% → HRP ox + H% O 2 Thionie red + HRP ox → HRP red + Thionine ox

(3)

Thionine ox + 2e- → Thionine red

(4)

Therefore, thionine is recycled in the system, leading to increase of the reduction current. These results indicate, HRP is used as label for signal amplification and catalyzing the oxidation of thionine by H2O2 and the oxidation product of thionine was reduced at MWCNTs/IL/Chitosan modified glassy 12

carbon electrode. The increase of the reduction current was related to the amount of biotinated aptamer-SA-HPR on the electrode, further related to the concentration of IgE. The plot of catalytic current vs. IgE concentration is shown in inset of Fig.5. As can be seen, the bioelectrocatalytic current of the aptasensor increased with increasing IgE concentration. For concentration range 50 pM -2 nM the regression equation was I(µA)=1.8811[IgE] (nM) +2.8394 (µA) and the correlation coefficient was 0.9906The detection limit was calculated as 6 pM (based on S/N = 3). Therefore, the proposed aptasensing sandwich assay can successfully detect the IgE with high sensitivity and low detection limit. The analytical parameters for proposed aptasensor are comparable or better than reported values for other IgE aptasensors with different transduction methods such as, electrochemical impedance spectroscopy, fluorescence, luminescence and chemiluminescence, field effect transistor and voltammetry ( Table 1). Since, in compared to condition with higher concentration of IgE, at lower concentration of IgE the abundant of available active sites of aptamer on the electrode surface is more, the calibration plots with different slopes are observed and aptasensor sensitivity declines when it has been used to determine higher concentrations of IgE. The reproducibility of aptasensor fabrication process was investigated by preparing 5 independently aptasensor and measuring of aptasensors response in the presence 5 nM of IgE under optimized experimental conditions. The obtained RSD was 4.5% , which indicate acceptable reproducibility of the proposed system. Here Fig. 5 3.5. Selectivity of IgE aptasensor and real sample analysis Although aptamer has high specificity to binding with the target protein, the non-specific adsorption of other proteins coexisting in the complex sample may possibly cause an interfering signal. Hence, the detection selectivity of the aptasensor is an important criterion for its actual application. To 13

evaluate the selectivity of the aptasensor, control experiments were performed using some potential interfering proteins including BSA and lysozyme. Since lysozyme has a very high basic charge (pI 9.1) and its nonspecifically bending to nucleic acids has been known, it can be used as a good candidate for control experiment. The concentration of the BSA for control experiment was chosen very high (1%), as value of serum concentration level. Fig. 6 exhibits different current response signals of the proposed sensing system after adding of 0.2 nM IgE, 40 nM Lysozyme and 1% BSA . Here Fig. 6 As illustrated the change of TH peak current after BSA and lysozyme addition is negligible, while a significant increase in peak current observed in the presence low concentration of IgE (0.2 nM). The histogram of the aptasensor signals after incubation withIgE, BSA and lysozyme is also shown in Fig.6D. These observations indicate that the IgE-aptamer binding event is based on the specific recognition between them but not on the other factors, such as nonspecific protein adsorption. Therefore, the proposed strategy has a sufficient specificity to IgE against other proteins and the proposed aptasensor can be used for selective detection of IgE. The applicability of proposed IgE aptasensor was evaluated by applying it to the analysis of IgE concentration in a human serum sample using DPV as measuring technique and standard addition method was adopted for this purpose. The obtained result was compared with the determined value by standard ELISA method. The observed value for IgE in serum sample was 1.10 (±0.04) nM (the average of three replicate measurements) which is very close to that obtained by standard ELISA method (1.06 nM). As can be seen a good agreement between results obtained by two methods was observed. So, the introduced modified electrode and proposed strategy might be a promising system for detecting IgE in real samples.

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4. Conclusion In conclusion, a sensitive and selective aptamer/IgE/ HRP linked aptamer sandwich assay is developed for IgE detection. The aptasensor was constructed by covalent attachment of

anti-IgE

aptamer onto GC electrode modified with IL-MWCNTs-IL nanocomposite. The other anti-IgE aptamer modified with biotin and labelled with streptavidin-horseradish peroxidase has been used for completing of sandwich assay. The IgE detection has been achieved by reaction of H2O2 and thionine which catalyzed by HRP using differential pulse voltammetry as measuring technique. The cathodic peak current of thionine was increased with increasing of IgE concentration at range up to 20 nM and calculated detection limit was 6 pM. Furthermore, other proteins such as lysozyme and bovine serum albomine haven’t been interfered with the detection of IgE, therefore, the proposed method provided a new electrochemical aptasensing approach for the detection of IgE. The application of the proposed aptasensor for IgE detection in human serum as real sample was also evaluated. Obviously, this sandwich structure system proposed in this work may be used broadly for the cases of some other proteins or molecules with two aptamer-binding sites, such as thrombin and platelet-derived growth factor (PDGF)-BB. Acknowledgments This research was supported by the Iranian Nanotechnology Initiative and the Research Office of the University of Kurdistan.

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[24] D. Li, M.B. Muller, S. Gilje, R.B. Kaner, G.G. Wallace, Processable aqueous dispersions of graphene nanosheets, Nature Nanotech. 3 (2008) 101-105. [25] N. Maleki, A. Safavi, F. Tajabadi, High-Performance Carbon Composite Electrode Based on an Ionic Liquid as a Binder, Anal. Chem. 78 (2006) 3820-3826. [26] J-Y. Sun, K-J. Huang, S-F. Zhao, Y. Fan, Z-W. Wu, Direct electrochemistry and electrocatalysis of hemoglobin on chitosan-room temperature ionic liquid-TiO2-graphene nanocomposite film modified electrode, Bioelectrochem. 82 (2011) 125-130. [27] M. Zhang, A. Smith, W. Gorski, Carbon Nanotube−Chitosan System for Electrochemical Sensing Based on Dehydrogenase Enzymes, Anal. Chem. 76 (2004) 5045-5050. [ 28] A.I. Gopalan, K-P. Lee, D. Ragupathy, Development of a stable cholesterol biosensor based on multi-walled carbon nanotubes–gold nanoparticles composite covered with a layer of chitosan–roomtemperature ionic liquid network, Biosens. Bioelectron. 24 (2009) 2211-2217. [29] H. Teymourian, A. Salimi, S. Khezrian, Fe3O4 magnetic nanoparticles/reduced graphene oxide nanosheets as a novel electrochemical and bioeletrochemical sensing platform, Biosens. Bioelectron. 49 (2013) 1-8. [30] S. Khezrian, A. Salimi, H. Teymourian, R. Hallaj, Label-free electrochemical IgE aptasensor based on covalent attachment of aptamer onto multiwalled carbon nanotubes/ionic liquid/chitosan nanocomposite modified electrode, Biosens. Bioelectron.43 ( 2013) 218-225. [31] X. Zuo, Y. Xiao, W. Plaxco, High Specificity, Electrochemical Sandwich Assays Based on Single Aptamer Sequences and Suitable for the Direct Detection of Small-Molecule Targets in Blood and Other Complex Matrices, J. Am. Chem. Soc. 131 (2009) 6944-6945. [32] M.Y. Ho, N. D,Souza, P. Migliorato, Electrochemical Aptamer-Based Sandwich Assays for the Detection of Explosives. Anal. Chem. 84(2012) 4245-4247. [33] S.J. Xiao, P.P. Hu, X.D. Wu, Y.L. Zou, L.Q. Chen, L. Peng, J. Ling, S.J. Zhen, L. Zhan, Y.F. Li, C.Z. Huang, Sensitive Discrimination and Detection of Prion Disease-Associated Isoform with a DualAptamer Strategy by Developing a Sandwich Structure of Magnetic Microparticles and Quantum Dots, Anal. Chem, 82 (2010) 9736-9742. [34] D.W. Zhang, C.J. Sun, F.T. Zhang, L. Xu, Y.L. Zhou, X.X. Zhang, An electrochemical aptasensor based on enzyme linked aptamer assay, Biosens. Bioelectron. 31(2012) 363-368. [35] R. Hu, W. Wen, Q. Wang, H. Xiang, X. Zhang, H. Gu, S. Wang, Novel electrochemical aptamer biosensor based on an enzyme–gold nanoparticle dual label for the ultrasensitive detection of epithelial tumour marker MUC1, Biosens. Bioelectron. 53 (2014) 384-389.

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19

Figure captions Scheme 1: Schematic outline of the principle for sandwich assay IgE aptasensing. Table 1: Comparison of Analytical Characteristics for different IgE aptameric assay methods. Fig. 1: SEM image of MWCNTs/IL/Chit nanocomposite immobilized onto GC electrode .

Fig.2. (A) CV responses of Thionine/NH2-Aptamer/MWCNTs/IL/Chit/GC electrode in PBS (0.1M, pH 7.4) at different scan rates (from inner to outer) 10, 20, 40, 50, 60, 70, 80, 90, and 100 m V s-1.Inset A is the plot of peak current vs. scan rate. (B) 1th and 200th

cyclic voltammetric response of Thionine/NH2-

Aptamer/MWCNTs/IL/Chit/GC electrode at scan rate 100 mVs-1. Fig. 3: (A) CVs and (B) Nyquist plots of 2.5 mM [Fe(CN)6]3-/4- in 0.1 M KCl recorded at bare GC, MWCNTs/IL/Chit/GC, NH2-Aptamer/MWCNTs/IL/Chit/GC, IgE(5nM)/NH2-Aptamer/MWCNTs/IL/ Chit/GC and HRP-SA-biotin-Apt/IgE/NH2-Aptamer/MWCNTs/IL/Chit/GC electrodes (scan rate in A is :0.1 V s-1; frequency range in B is 0.1 Hz to 10 kHz). Fig 4: The effect of pH (A), H2O2 concentration (B), Incubation time (C) on the aptasensor response. Fig. 5: DPVs of HRP-SA-biotin-Apt/IgE/NH2-Aptamer/MWCNTs/IL/Chit/GC electrode after incubation for 15 min with different IgE concentrations of 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 2, 4, 6, 10 and 20 in PBS (0.1 M, pH 7.4) containing 2.5 mM of H2O2. Inset is the plot of current response vs. IgE concentrations. Fig. 6: The DPVs of NH2-Aptamer/MWCNTs/IL/Chit/GC electrode before (solid line) and after incubated for 15 min with (A) 1% BSA and HRP-SA-biotin-Apt (B) 40 nM Lysozyme and HRP-SA-biotinApt (C) 0.2 nM IgE HRP-SA-biotin-Apt. Electrolyte: PBS (0.1 M, pH 7.4) containing 2.5 mM of H2O2.

Histogram of the change of aptasensor response after reaction with 0.2 nM of IgE, 1% of BSA and 40 nM of Lysozyme.

20

Table 1: Comparison of Analytical Characteristics for different IgE aptameric assay methods

Transduction Method

Limit of Detection

Quartz crystal Microbalance

0.016 nM

Metal-enhanced fluorescent

0,006 nM

Carbon nanotube Field effect transistor

16nM

Ag-nanoparticles enhanced florescent Carbon nanotube field-effect transistor Luminescent Aptazyme-linked oligonucleotide assay

0.066- 41.66 nM 50-1200 nM

0.25 nM

1000 nM

Chemiluminescence coupled to magnetic beads

4600 nM

0.0032 – 6.66 nM 0.25-20nM

0.1 nM

0.29 nM

Fluorescence anisotropy

0.016 – 1.33 nM

0.00026 nM

Graphene field-effect transistor

Micro-ELISA

Linear Range

0.1-70 nM Up to 10 nM 0.29-160 nM 0.0048 - 20 nM

0.013 nM 0.35 nM

up to 8 nM

Reference

[10] [38] [39] [8] [40] [41] [42] [43] [44] [45]

1–60 nM

[46]

AC Voltammetry

0.036 nM

0.36–54 nM

[2]

Differential Pulse Voltammetry

0.037 nM

0.5–30 nM

[30]

2.5–100 nM

[47]

Electrochemical Impedance Spectroscopy Differential Pulse Voltammetry

0.1 nM 0.006 nM

0.05-2 nM 2- 20nM

21

This study

Scheme 1: Schematic outline of the principle for sandwich assay IgE aptasensing.

22

Fig.1. SEM image of MWCNTs/IL/Chit nanocomposite immobilized onto GC electrode .

23

A

2

8

-0.5 Current ( µ µA)

Current ( µA)

4.5

-3

y = 0.0419x + 0.3109 2 R = 0.9977

4 0 -4

y = -0.0453x - 0.584 2 R = 0.9993

-8 0

40

80 -1

Scan rate (mV s )

-5.5 -0.46

-0.36

-0.26

-0.16

-0.06

Potential(V)vs .Ag/AgCl 6.2

B

4.2

Current ( µA)

2.2

0.2

-1.8

-3.8

-5.8 -0.48

-0.38

-0.28

-0.18

-0.08

0.02

Potential(V)vs .Ag/AgCl

Fig.2. (A) CV responses of Thionine/NH2-Aptamer/MWCNTs/IL/Chit/GC electrode in PBS (0.1M, pH 7.4)

at different scan rates (from inner to outer) 10, 20, 40, 50, 60, 70, 80, 90, and 100 m V s-1. Inset A is

the plot of peak current vs. scan rate. (B) 1th and 200th cyclic voltammetric response of thionine/NH2Aptamer/MWCNTs/IL/Chit/GC electrode at scan rate 100 mVs-1.

24

A

Current ( µA)

450

100

GCE Nanocomposite/GCE -250

NH2-Aptamer/Nanocomposite/GCE IgE/TH/NH2-Aptamer/Nanocomposite/GCE Streptavidin-HRP-Biotin Aptamer/IgE/TH/NH2 Aptamer/Nanocomposite/GCE

-600 -0.45

-0.05

0.35

0.75

Potential(V)vs .Ag/AgCl

B 320

-Z''/Ω

220

GCE Nanocomposite/GCE 120

NH2-Aptamer/Nanocomposite/GCE IgE/TH/NH2-Aptamer/Nanocomposite/GCE Streptavidin-HRP-Biotin Aptamer/IgE/TH/NH2 Aptamer/Nanocomposite/GCE

20 -20

230

480

730

Ω Z'/Ω Fig.3. (A) CVs and (B) Nyquist plots of 2.5 mM [Fe(CN)6]3-/4- in 0.1 M KCl recorded at bare GC, MWCNTs/IL/Chit/GC, NH2-Aptamer/MWCNTs/IL/Chit/GC, IgE(5nM)/NH2-Aptamer/MWCNTs/IL/ Chit/GC and HRP-SA-biotin-Apt/IgE/NH2-Aptamer/MWCNTs/IL/Chit/GC electrodes (scan rate in A is :0.1 V s-1; frequency range in B is 0.1Hz to 10 kHz).

25

Current ( µ A)

(A)

3.9

3.5

3.1

2.7

2.3 1

4

7

10

13

pH

Current( µA)

(B)

3.0

2.0

1.0

0.0 0.0

1.0

2.0

3.0

4.0

Concenteration H2O 2( mM)

(C)

C urrent (µ A )

0.9

0.8

0.7

0.6 0

10

20

30

incubation timec(min)

Fig 4: The effect of pH (A), H2O2 concentration (B), Incubation time (C) on the aptasensor response.

26

-1.5

6.5

A

B

-2.5

Current (µ A)

5.5

Current( µA)

-3.5

4.5

-4.5

C

3.5

-5.5

-6.5 -0.35

Fig.

2.5 0

-0.15

Potential(V)vs.Ag/AgCl

5

10

15

20

Concenteration(nM)

5. DPVs of HRP-SA-biotin-Apt/IgE/NH2-Aptamer/MWCNTs/IL/Chit/GC electrode after

incubation for 15 min with different IgE concentrations of 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 2, 4, 6, 10 and 20 in PBS (0.1 M, pH 7.4) containing 2.5 mM of H2O2. Inset is the plot of current response vs. IgE concentrations.

27

(A)

- 1.5

C urrent ( µ A )

C u rren t ( µ A )

-1.5

-2.5

-3.5 - 0.35

-0 .2 5

- 2.5

- 3.5

-0.0 5

-0.15

(B)

-0 .35

Potential(V) vs.Ag/AgCl 1

- 0.25

- 0.05

Potential(V) vs.Ag/AgCl

D

(C ) Current ( µ A)

0.8

Current/ µ A

-0 .15

0.6 0.4 0.2

-2 .2

-3 .2

0

Lys

BSA

IgE

-4 .2 - 0.3 5

Analyte

- 0.2 5

- 0.15

Potential(V) vs.Ag/AgCl

Fig. 6. The DPVs of NH2-Aptamer/MWCNTs/IL/Chit/GC electrode before (solid line) and after incubated for 15 min with (A) 1% BSA and HRP-SA-biotin-Apt (B) 40 nM Lysozyme and HRP-SAbiotin-Apt (C) 0.2 nM IgE HRP-SA-biotin-Apt. Electrolyte: PBS (0.1 M, pH 7.4) containing 2.5 mM of H2O2.(D) Histogram of the change of aptasensor response after reaction with 0.2 nM of IgE, 1% of BSA and 40 nM of Lysozyme.

28

- 0 .0 5

“Graphical Abstract”

29