Electrochemical aptasensor for ultrasensitive detection of PCB77 using thionine-functionalized MoS2-rGO nanohybrid

Journal Pre-proof

Electrochemical aptasensor for ultrasensitive detection of PCB77 using thionine-functionalized MoS2 -rGO nanohybrid Ali Mohammadi Methodology and Investigation , Esmaeil Heydari-Bafrooei , Mohammad Mehdi Foroughi , Marziyeh Mohammadi PII: DOI: Reference:

S0026-265X(19)33149-2 https://doi.org/10.1016/j.microc.2020.104747 MICROC 104747

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

5 November 2019 28 January 2020 18 February 2020

Please cite this article as: Ali Mohammadi Methodology and Investigation , Esmaeil Heydari-Bafrooei , Mohammad Mehdi Foroughi , Marziyeh Mohammadi , Electrochemical aptasensor for ultrasensitive detection of PCB77 using thionine-functionalized MoS2 -rGO nanohybrid, Microchemical Journal (2020), doi: https://doi.org/10.1016/j.microc.2020.104747

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

Highlights    

MoS2-rGO/Thi nanohybrid was synthesized. An ultrasensitive electrochemical aptasensor was developed based on the MoS2rGO/Thi for detecting PCB77. The aptasensor displayed low detection limit of 80 ag mL-1 for PCB77. The biosensor was used for PCB77 determination in water and serum samples.

1

Electrochemical aptasensor for ultrasensitive detection of PCB77 using thionine-functionalized MoS2-rGO nanohybrid Ali Mohammadi1, Esmaeil Heydari-Bafrooei*2, Mohammad Mehdi Foroughi1, Marziyeh Mohammadi2

1. Department of Chemistry, Kerman Branch, Islamic Azad University, Kerman, Iran 2. Department of Chemistry, Faculty of Science, Vali-e-Asr University of Rafsanjan, 77188– 97111, Iran Tel.: +983431312433; Email: [email protected]; [email protected] Abstract

The authors describe a PCB77 aptasensor based on the use of thionine (Thi) functionalized MoS2-rGO as a platform for aptamer immobilization. Differential pulse voltammetry and cyclic voltammetry were used to monitor the electrochemical properties of proposed aptasensor and investigated the importance of MoS2-rGO/Thi as an aptasensing platform in the measurement of PCB77, a type of polychlorinated biphenyl, as a model molecule. The electrochemical response is based on a decrease of DPV response of Thi before and after the interaction of aptamer with the target (PCB77). Based on the calibration curve, the very low detection limit of 80 ag mL-1 and a very wide linear range of 0.3 fg mL-1-0.1 ng mL-1 were obtained, which is the widest linear range and lowest detection limit among the previous methods reported in the literature. The reproducibility of this aptasensor was 4.2%. Moreover, the effective determination of the PCB 77 by our MoS2-rGO/Thi-based sensor in the complex environmental water and soil samples was realized, which displays a good application possibility.

2

Keywords: Polychlorinated biphenyls; Environmental; Aptasensor; Graphene; Label-free detection

1. Introduction

Polychlorinated biphenyls (PCBs) are a notorious group of man-made organic chemicals, which fall in the category of persistent organic pollutants (POPs). PCBs show long-term stability in the environment, strong resistance against the chemical, biological, and radiation degradation, steady accumulation in the organisms, and high toxicity [1,2]. They enter the body through eating food, swallowing dust, and breathing the air contaminated with PCBs. Therefore, PCBs are a serious threat to the environment, life safety, and human health. Day-today consumption level of PCBs is 6 μg/kg [3]. Environmental Protection Agency (EPA) authorizes a maximum amount of 0.5 g Kg-1 in the drinking [4]. This value for foods (except fish), fish, and food-packaging is 0.2–3.0, 2, and 10 mg Kg-1, respectively [5]. Therefore, according to these values, monitoring of PCBs in the environmental and food samples requires a very sensitive method to assess the risk of human and animal contact. Globally accepted technique for the determination of PCBs is gas chromatography [6,7]. However, due to its complex sample preparation and complicated instrumentation, this technique is timeconsuming, difficult, expensive, and need a skilled operator. Consequently, it is of utmost importance to develop a simple, economical, convenient, and fast method for the detection of the PCBs in food and environmental samples. In this view, there is cumulative attention in the development of biosensors able to monitor PCBs. In biosensors, the concentration of the analyte detects by monitoring a physical signal that produced by an affinity reaction that occurs between a biological molecule (biorecognition element or bioreceptor) and target (PCBs).

There are various materials which can be used as solid supports for immobilization of biorecognition elements in biosensors [8-12]. Among them, graphene and graphene-based

3

nanomaterials have attracted the attention of researchers due to the intrinsic properties, such as high surface-to-volume ratio, high mechanical strength, outstanding chemical and thermal stability, and high electronic conductivity for the electron transfer reactions [13-15]. One of the extraordinary advantages of graphene is combined with other nanostructures; this makes possible to use the distinctive, unique and complementary features of both modifiers on the electrode for improving the figure of merits of the biosensor [17,18].

Molybdenum disulfide (MoS2) as a layered material has attracted the attention of researchers in the fields of optoelectronics and nanoelectronics [19,20]. MoS2 is a unique material due to the ultra-large specific surface area, and high ability in a surface modification which can interact with plentiful of nanostructures and organic molecules to produce new nanocomposite. In addition to these benefits, its exceptional electronic and electrochemical properties, suggest that it can be useful in the field of biosensing. Previous studies showed that a combination of graphene with MoS2 can be significantly improved the sensitivity of electrochemical detections [21-27].

In this paper, considering the advantages of the rGO and MoS2, we designed an aptasensor using Thionine (Thi)-functionalized MoS2-rGO nanocomposite as a substrate for immobilization of aptamer. Thi as a signal indicator is on the surface of the electrode, and due to the electron transfer between Thi and the electrode, the current peaks of Thi could be easily seen. After the interaction of surface-confined aptamer with PCB, a complex structure of aptamer-PCB was produced, which would significantly hinder the charge transfer on the electrode and lead to a decrease in the signal of Thi. The sensing mechanism of MoS2-rGO based aptasensor for PCB77 determination is shown in Figure 1. The very sensitive determination of PCB with high selectivity was attained by evaluating the decrease of Thi electrochemical signal. Moreover, the offered aptamer-based sensor was applied for detection

4

of PCB in water and soil samples. However, no more paper has been published in the aspect of thionine functionalized MoS2-rGO nanosheets for electrochemical (bio)sensing.

2. Material and methods

2.1. Materials and apparatus

Graphite powder (99.8%) was purchased from Sigma-Aldrich. Ascorbic acid, sodium molybdate, thiourea, ethanol, sodium hydroxide and citric acid and PCBs were purchased from Merck (Darmstadt, Germany). Thiol-terminated 40-mer PCB77 aptamer (5′-SH-(CH2)6-GGC GGG GCT ACG AAG TAG TGA TTT TTT CCG ATG GCC CGT G-3′) was acquired from Bioneer Corporation. The stock solution of the aptamer was ready in phosphate buffer (pH 7.4) and was kept in the freezer before use. X-ray diffraction analysis (XRD) was carried out using a Philips Xʼpert MPD X-ray diffractometer with CuKα radiation (λ = 1.51418 Å) generated at a voltage of 40 kV. The morphologies of nanocomposites were studied by a field emission scanning electron microscope (FE-SEM). Sonication in the synthesis procedure was performed by using A SonoSwiss SW 3H ultrasonic bath (Ramsen, Switzerland) with the power of 280 W at 38 kHz.

2.2. Hydrothermal synthesis of MoS2 and MoS2-rGO hybrid nanomaterials

The pristine nano-MoS2 and MoS2-rGO were synthesized according to the modified procedure reported previously by our group [28,29]. The detailed synthetic procedures of MoS2 and MoS2-rGO given in supplementary information.

2.3. Synthesis of MoS2-rGO/Thi hybrid nanomaterials

5

A solution containing 1 mg mL−1 MoS2/rGO was prepared by ultrasonic agitation and mixed with 1 mL 2 mg mL−1 Thi solution. Then vigorous stirring of the mixed solution was performed for one day at room temperature. The prepared solution centrifuged and washed with DI water several times to separate un-bound Thi molecules; subsequently, MoS2-rGO/Thi nanohybrid was achieved.

2.4. Immobilization of MoS2-rGO/Thi/AuNP on the electrode

To the preparation of the substrate, the polishing of the glassy carbon electrode (GCE, 2.0 mm diameter, Metrohm) was carried out with 0.1 and 0.05 mm alumina powder on a Metrohm polishing cloth. The electrode extensively washed with DI water and then sonicated in DI water and absolute ethanol to remove adherent Al2O3 from the surface. Conclusively, the electrode was dried at the room temperature. MoS2-rGO/Thi suspension (0.10%) was prepared by dissolving 0.005 g MoS2-rGO/Thi in 5.0 mL DI water by the aid of sonication for 1 h. 10 µL of MoS2-rGO/Thi suspension was directly cast on the electrode. The as-prepared MoS2rGO/Thi films were washed with DI water and dried at the room temperature. The electrodeposition of AuNP was carried out by employing a constant potential of -0.2 V in a solution containing 1.0 mmol L−1 HAuCl4 and 0.1 mol L−1 KNO3 for 3 min.

2.5. Immobilization of aptamer on MoS2-rGO/Thi/AuNP modified GCE

Immobilization of the aptamer on the MoS2-rGO/Thi/AuNP modified GCE was performed by immersion of the modified electrode in 0.2 mol L-1 aptamer solution (prepared in 0.1 mol L-1 phosphate buffer, pH=7.4) for 4 h, and next, the electrode was immersed in phosphate buffer (0.1 mol L-1, pH=7.4) and DI water to remove excess aptamer molecules. Finally, to block nonspecific adsorption and achieving a well-aligned aptamer monolayer, the aptasensor was incubated in an aqueous solution of 6-mercapto-1-hexanol (1.0 mmol L-1) for 30 min.

6

2.6. PCB77 detection

After preparing the aptamer-functionalized MoS2-rGO/Thi/AuNP, the aptasensor was dipped in Tris buffer (pH=7.4) containing different concentrations of PCB77 for 15 min. Then, to eliminate non-bonded PCB77, the electrodes were washed with phosphate buffer (0.1 mol L-1, pH=7.4) and lastly, differential pulse voltammograms of the aptasensor before and after interaction with PCB77 were acquired. The changes in the peak currents related to PCB77 concentration.

2.7. Electrochemical measurements

Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were carried by Autolab PGSTAT204 with NOVA 2.1 software in an electrochemical cell containing three-electrode configuration. All electrochemical measurements were performed in phosphate buffer (0.1 mol L-1, pH=7.4). The DPV and CV scan were taken from -0.35 to 0.05 V(vs. Ag/AgCl). The DP voltammograms were acquired in a pulse width of 50 ms and a pulse amplitude of 50 mV.

2.8. Real samples preparation

The water samples were gathered using a standard polyethylene water sampler. Tap water was obtained from our laboratory in the Vali-e-Asr University of Rafsanjan (Iran). The river water was sampled from Zayandehroud River near Khajou Bridge in Isfahan (Iran). The samples were then filtered several times using Whatman No. 45 filter paper to eliminate the particles and suspended substances.

For the preparation of soil samples, we worked according to the procedure of sporring et al. [30]. Surface soil samples were collected from an agricultural site near the Vali-e-Asr

7

University of Rafsanjan. The samples air-dried at 35 °C and mildly crushed with a pestle and mortar so that all the material passed easily across a sieve of 1 mm aperture size. The sieved samples were extra dried at 35 °C till their weight is fixed. 30 mL n-hexane/acetone (1:1, v/v) was added to 5 g of the soil sample. Then, sonication of the mixture was performed for 1 h and followed by centrifugation at 4000 rpm for 20 minutes, and removal of the supernatant. The filtrates were led in two separate directions: (1) a certain quantity of the filtrate was added into 0.1 mol L−1 phosphate buffer at pH 7.4 for aptasensing determination of the PCB77; and (2) another part of the the filtrate (with certain amount) was transferred to a rotary evaporator at 60 °C for approximately drying and then passed through a florisil column and eluted with 50 mL n-hexane. After reduction of the volume of the obtained fraction to 800 μl and adding 100μl 10 ng μl-1 dichlorbinyl, the sample is ready for GC-MS analysis.

3. Result and discussion

XPS analysis was applied to characterize the chemical compositions of the nanocomposite. Figure 2 (A) presents the high-resolution XPS spectra of the S 2p region for the MoS2-rGO, Thi, and MoS2-rGO/Thi. As shown by the dotted lines, an apparent change in S 2p binding energy can be seen for the nanohybrid. According to the XPS spectra of MoS2-rGO nanohybrid, two promonent bands were detected at 162.1 and 163.8 eV, that can be assigned to S 2p3/2 and 2p1/2 orbitals, respectively. These two peaks correspond to divalent sulfide ions (S-2) of MoS2, which are in agreement with the results of the previous report [28]. For the thionine, the peaks of S 2p3/2 and S 2p1/2 orbitals are located at 164.3 and 166.0 respectively, which is in good accordance with a previous report [26]. The signals of MoS2-rGO emerges in MoS2-rGO/Thi nanocomposite precisely in the same binding energy. However, the S 2p peaks of the thionine observe in the XPS of the nanocomposite but in higher binding energies. These observations imply that there is an interaction between thionine and MoS2 nanosheets in the nanohybrid.

8

MoS2 sheets donate electrons to thionine, and this leads to a decrease in binding energy [26]. The S edge of the MoS2 sheets, which can be modified by using thiol chemistry, is the most probable binding site for thionine. Moreover, positively charged thionine can be merely close to the negatively charged S edge of MoS2 [26].

Figure 2(B) shows the XRD patterns of rGO, MoS2, MoS2-rGO, and MoS2-rGO/Thi. In the XRD patterns of the MoS2, the peaks at 14.2°, 33.1°, 40.4°, and 59.7° are indexed as (002), (100), (103), and (110) crystallographic planes of MoS2, respectively (JCPDS#37-1492). The as-synthesized MoS2 nanoflower has high purity because of the lack of any additional peak related to impurities. The intensity of the diffraction peak at 214.3 (002) is lower when compared to pure MoS2 (JCPDS#37-1492) that's because of the petals of MoS2 nanoflowers comprised of a series of layers of nanosheets. Furthermore, the slight shift in the position of the (002) peak in comparison with standard hexagonal phase MoS 2 (JCPDS#37-1492) is because of the lattice distortion of the synthesized MoS 2 nanoflowers [28]. For MoS2-rGO, the main diffraction peaks of MoS2 were observed and the new peak at 25.7°, as a (002) plane of hexagonal graphite structure indicative of the presence of rGO. The result indicated the existence of rGO and the formation of the MoS2-rGO nanohybrids. As shown in XRD pattern of the MoS2-rGO/Thi nanohybrid, the peak related to (002) plane of rGO has shifted to a higher 2θ value (27.4°), which implies an increment in the ordering of graphene sheets and limited return of the graphitic structure due to the π–π interactions with thionine [31]. The XRD results prove the being of π–π interactions between thionine and graphene nanosheets in the MoS2rGO/Thi, as others have stated for rGO-Thi nanocomposite [31]. The π–π stacking interactions lead to thionine molecules that intercalated to graphene sheets bind to both the top and bottom sheets of the GO. Furthermore, The patterns of the MoS2-rGO/Thi show all diffraction peaks of MoS2. No peaks of by-products were seen, which implies the success of our synthetic scheme to produce the pure phase of MoS2-rGO/Thi hybrid platform. Based on the results

9

obtained by XRD and XPS, in addition to the fact that thionine interacts with graphene sheets through a strong π–π interaction, there is also a strong interaction between it and MoS2 in this nanocomposite. Actually, thionine was anchor to MoS 2-rGO in two way: interaction with rGO and with MoS2. Figure 2 (C) shows the Raman spectrum of MoS2-rGO/Thi in the range 0−2000 cm−1. The observed locations of the peaks in the present study and those of Thi–MoS2, Thi-rGO, and polycrystalline thionine, as reported by Sun et al. [32], Sousa et al. [33], and Hutchinson et al. [34], respectively, are found to be approximately equal. The asymmetric stretching vibration of C−N bond at 1405 and 1438 cm−1, bending vibration of C−S bond at 1119 cm−1 and the vibration of the NH2 group at 1530 cm−1 are observed in the Raman spectrum of MoS2rGO/Thi. Again, the band around 482 and 1631 cm−1 corresponds to the C−C bending vibration and C=C, respectively, and the peaks at 609 and 1031 cm−1 is assigned to the vibration of C−H bond. The peaks at about 374, and 404 cm−1 are the characteristic peaks of MoS2. These peaks 1 are due to the in-plane E2g displacement of Mo and S atoms and out-of-plane A1g symmetric

vibration of only S atoms along the C axis, respectively [29]. In addition, two peaks at 1350 cm-1 and 1590 cm-1 are D and G characteristic peaks of rGO which correspond to the vibration of sp2-bonded carbon atoms and the defect-induced vibration, respectively. The Raman data verifies the presence of rGO, MoS2, and Thi in the nanocomposite.

Figure S1 highlights the Raman spectrum of the MoS2 and MoS2-rGO/Thi in the range 3051 490 cm-1. The characteristic peaks of MoS2 (E2g and A1g ) were seen in this spectrum. Notably,

the interval between these two peak positions can be applied to approximately evaluate the number of MoS2 nanosheet layers. As the layer number increases, a redshift happens for the 1 E2g mode, whereas the A1g mode experiences a blueshift [35]. This phenomenon is due to the

suppression of the vibration modes as a result of the increase in interlayer van der Waals force

10

and long-range Coulombic interlayer interactions in multilayer MoS2 as the layer number increases [36]. Furthermore, surface reconstruction leads also to the frequency changes in the 1 E2g and A1g modes. As can be seen in Figure S1, by compositing of MoS2, rGO and Thi in 1 MoS2-rGO/Thi nanocomposite, a redshift in the A1g mode and blueshift in the E2g mode

happened, representing a decrease in the number of layers of MoS2in the MoS2-rGO/Thi.

The morphologies of MoS2-rGO and MoS2-rGO/Thi nanohybrids are presented in Figure 3 (AC). The SEM image of MoS2-rGO (A) showed a petal-like arrangement where rGO successfully decorated with MoS2 nanoflowers. It is detected that the nanopetals are very thin and covered each other. Figure 3 (B and C) shows the SEM and TEM images of the MoS2rGO/Thi nanohybrid. Layered configurations with nanometer sizes can be seen, demonstrating that MoS2 exists mostly in the shape of MoS2 sheets in the MoS2-rGO nanohybrid, which is dissimilar from the raw bulk MoS2. Thionine molecules can not be seen by TEM and SEM techniques because they are very small.

Figure 3 (D-F) displays the topography of the thin layer of MoS2-rGO/Thi and MoS2rGO/Thi/AuNP on the surface of the GCE analyzed by SEM. The SEM image of bare GCE (D) exhibited a rough surface. As shown in Figure 3E, the MoS2-rGO/Thi layer was homogeneously distributed over the electrode. Electrodeposition of the AuNP on the MoS2rGO/Thi-modified electrode surface appeared as spots, as seen in Figure 3F. The diameter of the AuNP was about 30 nm. The results from SEM implied that the MoS2-rGO/Thi had been effectively immobilized on the surface of the GCE to form nanocomposite films. Thus AuNP with granular morphology was successfully immobilized on the surface of the MoS2-rGO/Thimodified electrode, and they were evenly dispersed in the MoS2-rGO/Thi matrix with minimal aggregation.

11

Thi was effectively anchored to MoS2-rGO nanohybrid can be further verified by using CV characterization. Figure 4A displays the cyclic voltammograms of different modified electrodes in phosphate buffer (0.1 mol L-1, pH=7.4) at a scan rate of 0.1 V s-1. The bare (Figure 4A, curve a) and MoS2-rGO-modified GCE (Figure 4A, curve b) show no redox peaks in the potential range of -0.35-0.0 V, but the background current increase at MoS2-rGO modified GCE. However, the MoS2-rGO/Thi modified GCE (Figure 4A, curve c) exhibits a distinct redox peak at E1/2 = -0.17 V. According to the electrochemical behavior of thionine as discussed in previous works [23,26], the presence of this pair of redox peaks shows that Thi has been efficaciously linked to MoS2-rGO nanohybrid and preserved its electrochemical behavior. After immobilization of AuNP on the surface of the MoS2-rGO/Thi modified GCE, the peak current increased severely (Figure 4A, curve c). The highly conductive AuNP on the MoS2rGO nanocomposites acts as a channel for transport of the electron, which leads to enhancement in the conductivity of the MoS2-rGO. The presence of AuNP on the surface of the electrode, in addition to enhancing the electrochemical signal of Thi, is required to attach the thiolatedaptamer to the electrode surface.

Figure 4 (B) displays the DP voltammograms of the GCE coated with (a) MoS2-rGO, (b) MoS2rGO/Thi/AuNP, (c) MoS2-rGO/Thi/AuNP/APT, and (d) MoS2-rGO/Thi/AuNP/APT after interaction with PCB77 in phosphate buffer (0.1 mol L-1, pH=7.4). No peak was observed on the MoS2-rGO modified GCE (Figure 4B, curve a), while a noticeable oxidation peak can be found on MoS2-rGO/Thi/AuNP modified GCE (Figure 4B, curve b), which is caused from the oxidation of Thi. By modification of the MoS2-rGO/Thi/AuNP functionalized electrodes with aptamer, the current decreased (Figure 4B, curve c), which suggested the aptamer dramatically reduced the active sites for electron transfer. The binding of PCB77 on the aptamer modified electrode (Figure 3B, curve d), led to a further decrease in the DPV peak current because the formation of PCB77-aptamer complex insulates the electron transfer.

12

In order to improve its performance and sensitivity, all parameters related to the electrochemical assay developed by MoS2-rGO/Thi/AuNP were optimized. Optimized parameters include the load amount of MoS2-rGO/Thi nanocomposite, aptamer anti-PCB77 concentration, aptamer immobilization time, and the interaction time between the PCB77 and aptamer-confined electrode. The optimization results are presented in more detail in “Supplementary Materials” (Section S.4). The optimal load amount of the nanocomposite, concentration of aptamer, immobilization time, and interaction time are 10 L, 200 nmol L-1, 9 h, and 150 min, respectively.

The aptasensor was incubated in Tris buffer (pH=7.4) solution containing different concentrations of PCB77, and then located in the electrochemical cell with 0.1 mol L-1 phosphate buffer and DP voltammograms of the electrode was recorded in the range -0.3-0.05 V. The voltammograms were shown in inset of Figure 5(A), and the variation in the current as a function of the concentration of the PCB77 target was shown in the Figure S2. The Figures show that the DPV peak currents associated with Thi decrease with increase in the PCB77 concentration. Thus, the concentration of PCB77 can be determined. Finally, a linear plot of signal (I) versus the logarithm of PCB77 concentration was yielded (Figure 5A). The linearity is in the range of 0.3 fg mL-1-0.1 ng mL-1. The detection limit (LOD) of the method was estimated to be 80 ag mL-1 at an S/N=3. The results are very attractive because the aptasensor has the lowest LOD and the widest linearity among the best previously published methods for PCB77 detection (Table 1). Compared to other electrochemical aptasensors, the proposed MoS2-rGO/Thi/AuNP based biosensor has key advantages for biomolecule immobilization, which is attributable to its large specific surface area, good conductivity, and the synergistic effect of each component. The presence of MoS2 in this system is vital and useful. Firstly, the nano petal-like structure of MoS2 leads to more plentiful active edge sites, stronger confinement and higher specific surface area. Hence, when combining with rGO, MoS2 13

nanoflowers shows improved electrochemical activity, which is significantly favorable for electrochemical determination. Also, compared to other noble metal nanoparticles, MoS 2 nanoparticles are economically efficient and simply synthesized, which is certainly additional merit for the nanocomposite. In addition to immobilization of the thiolated aptamer, gold nanoparticles play the role of a channel for electron transfer, further enhancing the electron conductivity of the nanocomposite. Besides, the sensitivity of the proposed biosensor enhanced by attaching the electrochemical indicator (Thi) to nanocomosite on the surface of the electrode (and not in the solution or labeled to biomolecules). Therefore, the electrochemical indicator is close to the electrode and any change in the electrode surface after the recognition reaction of bioreceptor and target effects on the electrochemical properties of indicator. It seems that all these factors together have contributed to the high sensitivity of the sensor. The selectivity of the method investigated through the addition of other species that might coexist in environmental samples (Benzopyrene, p-nitrophenol, Atrazine, Biphenyl, Bisphenol A, Hydroquinone, Hg2+, Fe3+, Co2+, Cu2+, Cd2+, and Zn2+) as well as three structurally similar polychlorobiphenyls (PCB81, PCB101, and PCB126) to the test solution. Figures 5(B) displays that the presence of these species, has no significant effect on the aptasensor response. The results show good selectivity of the sensor. For the use of the aptasensor in the detection of the PCB77 in environmental samples, it is essential to test the reproducibility of the sensor. Therefore, eight aptasensors (GC/MoS2-rGO/Thi/AuNP/APT) were applied for the determination of 0.1 pg mL-1 of PCB77. The Relative Standard Deviation (RSD) of the eight signals (ΔI) was 4.2%. Storage stability of the sensor is also a critical problem through real sample analysis. After 30 days storage of aptasensor at the refrigerator temperature, the signal for 0.1 pg mL-1 PCB77 solution reached 91% of the signal on the first day. The results endorse good selectivity, reproducibility, and storage stability of the sensor.

14

Recovery of the spiked PCB77 in water and soil samples was used to determine the accuracy of the method. We analyzed each sample four times to eliminate any observational bias. Table 2 shows the resultant data and compares the data achieved from the aptasensor with those obtained by standard GC-MS. As shown in Table 2, the recoveries of PCB77 detection are in the range of 96%–108% (n=5), indicating that the accuracy of the method is acceptable. Furthermore, the results from the proposed sensor are well consistent with those obtained from GC-MS method, the representative that the aptasensor is qualified for successful detection of PCB77 in water and soil samples. More importantly, compared with GC-MS, the procedure of sample analysis has significantly easy as it benefits from a lower determination time and less potential errors produced from the elaborate sample preparation of the chromatography method. Meanwhile, the results of the real sample analysis show that the aptasensor has benefits such as high selectivity, accuracy, and sensitivity for the determination of PCB77 in soil and water samples without any interferences. 4. Conclusions

In summary, we have developed a method to make the Thi-based electroactive nanohybrid, MoS2-rGO/Thi, and immobilizing anti-PCB77 aptamer on it for sensitive PCB77 detection in environmental samples. The key advantages of the method involve very low limit of detection (80 ag mL-1), extremely wide linearity (0.3 fg mL-1-0.1 ng mL-1), simple, and very selective for the monitoring of PCB77. As a result, MoS2-rGO/Thi modified electrode has revealed to be an attractive substrate for immobilization of aptamers and construction of aptamer-based devices. Therefore, this support can be suggested as a new tool for immobilization of aptamer in aptasensing assays. Moreover, though the offered method was focused on PCB77 detection, it has the potential for wider application for other analytes.

Acknowledgement

15

The authors gratefully acknowledge the support of this work by the Research Council of Valie-Asr University of Rafsanjan (VRU), Iran.

Author statement

Ali Mohammadi: Methodology and Investigation Esmaeil Heydari-Bafrooei: Conceptualization, Validation, Resources, Writing-Original draft preparation Mohammad Mehdi Foroughi: Supervision Marziyeh Mohammadi: Supervision. 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.

References

[1] C. Zani, G. Toninelli, B. Filisetti, F. Donato, Polychlorinated Biphenyls and Cancer: An Epidemiological Assessment, J. Environ. Sci. Health, Part C: Environ. Health Sci. 31 (2013) 99-144. [2] S. Manzetti, E.R. van der Spoel, D. van der Spoel, Chemical Properties, Environmental Fate, and Degradation of Seven Classes of Pollutants, Chem. Res. Toxicol. 27 (2014) 713-737. [3] American Academy of Pediatrics Committee on Environmental Health, Polychlorinated biphenyls, dibenzofurans and dibenzodioxins, Pediatric Environmental Health (2003) 491-502. [4] A. Verdian, E. Fooladi, Z. Rouhbakhsh, Recent progress in the development of recognition bioelements for polychlorinated biphenyls detection: Antibodies and aptamers, Talanta 202 (2019) 123–135. [5] Food and Drug Administration, Unavoidable Contaminants in Food for Human Consumption and Food Packaging Material: Tolerances for Polychlorinated Biphenyls (PCB), (1996).

16

[6] T. Portoles, C. Sales, M. Abalos, J. Saulo, E. Abad, Evaluation of the capabilities of atmospheric pressure chemical ionization source coupled to tandem mass spectrometry for the determination of dioxin-like polychlorobiphenyls in complex-matrix food samples, Anal. Chim. Acta 937 (2016) 96-105. [7] G. Bianco, G. Novario, D. Bochicchio, G. Anzilotta, T.R.I. Cataldi, Polychlorinated biphenyls in contaminated soil samples evaluated by GC–ECD with dual-column and GC– HRMS, Chemosphere 73 (2008) 104-112. [8] A.A. Ensafi, E. Heydari-Bafrooei, M. Dinari, S. Mallakpour, Improved immobilization of DNA to graphite surfaces, using amino acid modified clays, J. Mater. Chem. B 2 (2014) 30223028. [9] A.A. Ensafi, H.R. Jamei, E. Heydari-Bafrooei, B. Rezaei, Electrochemical study of quinone redox cycling: a novel application of DNA-based biosensors for monitoring biochemical reactions, Bioelectrochemistry 111, (2016) 15-22. [10] E. Heydari-Bafrooei, S. Askari, Ultrasensitive aptasensing of lysozyme by exploiting the synergistic effect of gold nanoparticle-modified reduced graphene oxide and MWCNTs in a chitosan matrix, Microchim. Acta 184 (2017) 3405-3413. [11] A.A. Ensafi, P. Nasr-Esfahani, E. Heydari-Bafrooei, B. Rezaei, Redox targeting of DNA anchored to MWCNTs and TiO2 nanoparticles dispersed in poly dialyldimethylammonium chloride and chitosan, Colloids Surf., B 121, (2014) 99-105. [12] P.F. Rostamabadi, E. Heydari-Bafrooei, Impedimetric Aptasensing of the Breast Cancer Biomarker HER2 Us-ing A Glassy Carbon Electrode Modified with Gold Nanoparticles in A Composite Consisting of Electro-chemically Reduced Graphene Oxide and Single-Walled Carbon Nanotubes. Microchim. Acta 186 (2019) 495-504. [13] A.T. Lawal, Progress in utilisation of graphene for electrochemical biosensors, Biosens. Bioelectron. 106 (2018) 149-178.

17

[14] C.I.L. Justino, A.R. Gomes, A.C. Freitas, A.C. Duarte, T.A.P. Rocha-Santos, Graphene based sensors and biosensors, TrAC, Trends Anal. Chem. 91 (2017) 53-66. [15] L. Wang, Y. Zhang, A. Wu, G. Wei, Designed graphene-peptide nanocomposites for biosensor applications: A review, Anal. Chim. Acta 985 (2017) 24-40. [16] E. Heydari-Bafrooei, A.A. Ensafi, Typically used carbon-based nanomaterials in the fabrication of biosensors, in: A.A. Ensafi (Ed.), Electrochemical biosensors, Elsevier, Amsterdam, 2019, pp. 77-94. [17] N. Mohammadian, F. Faridbod, ALS genosensing using DNA-hybridization electrochemical biosensor based on label-free immobilization of ssDNA on Sm2O3 NPsrGO/PANI composite, Sens. Actuators B 275 (2018) 432-438. [18] W. Wang, H. Tang, Y. Wu, Y. Zhang, Z. Li, Highly electrocatalytic biosensor based on Hemin@AuNPs/reduced

graphene

oxide/chitosan

nanohybrids

for

non-enzymatic

ultrasensitive detection of hydrogen peroxide in living cells, Biosens. Bioelectron. 132 (2019) 217-223 [19] M.B. Hossain, M.M. Rana, L.F. Abdulrazak, S. Mitra, M. Rahman, Graphene-MoS2 with TiO2SiO2 layers based surface plasmon resonance biosensor: Numerical development for formalin detection, Biochem. Biophys. Rep. 18 (2019) 100639. [20] S. Aksimsek, H. Jussila, Z. Sun, Graphene–MoS2–metal hybrid structures for plasmonic biosensors, Opt. Commun. 428 (2018) 233-239. [21] Y. Han, R. Zhang, C. Dong, F. Cheng, Y. Guo, Sensitive electrochemical sensor for nitrite ions based on rose-like AuNPs/MoS2/graphene composite, Biosens. Bioelectron. 142 (2019) 111529. [22] D. Lin, Z. Su, G. Wei, Three-dimensional porous reduced graphene oxide decorated with MoS2 quantum dots for electrochemical determination of hydrogen peroxide, Mater. Today Chem. 7 (2018) 76-83.

18

[23] Y. Ye, J. Xie, Y. Ye, X. Cao, H. Zheng, X. Xu, Q. Zhang, A label-free electrochemical DNA biosensor based on thionine functionalized reduced graphene oxide, Carbon 129 (2018) 730-737. [24] J. Han, J. Ma, Z. Man, One-step synthesis of graphene oxide–thionine–Au nanocomposites and its application for electrochemical immunosensing, Biosens. Bioelectron. 47 (2013) 243– 247. [25] H. Lv, Y. Li, X. Zhang, X. Li, Z. Xu, L. Chen, D. Li, Y. Dong, Thionin functionalized signal amplification label derived dual-mode electrochemical immunoassay for sensitive detection of cardiac troponin I, Biosens. Bioelectron. 133 (2019) 72–78. [26] T. Wang, R. Zhu, J. Zhuo, Z. Zhu, Y. Shao, M. Li, Direct Detection of DNA below ppb Level Based on Thionin-Functionalized Layered MoS2 Electrochemical Sensors, Anal. Chem. 86 (2014) 12064−12069. [27] H.A. Alinajafi, A.A. Ensafi, B. Rezaei, Reduced graphene oxide decorated with thionine, excellent nanocomposite material for a powerful electrochemical supercapacitor, Int. J. Hydrogen Energy 43 (2018) 19102-19110. [28] E. Heydari-Bafrooei, S. Askari, Electrocatalytic activity of MWCNT supported Pd nanoparticles and MoS2 nanoflowers for hydrogen evolution from acidic media, Int. J. Hydrogen Energy 42 (2017) 2961-2969. [29] E. Heydari-Bafrooei, N.S. Shamszadeh, Synergetic effect of CoNPs and graphene as cocatalysts for enhanced electrocatalytic hydrogen evolution activity of MoS2, RSC Adv. 6 (2016) 95979–95986. [30] S. Sporring, S. Bøwadt, B. Svensmark, E. Björklund, Comprehensive comparison of classic Soxhlet extraction with Soxtec extraction, ultrasonication extraction, supercritical fluid extraction, microwave assisted extraction and accelerated solvent extraction for the determination of polychlorinated biphenyls in soil, J. Chromatogr. A 1090 (2005) 1-9.

19

[31] Y. Shabangoli, M.S. Rahmanifar, M.F. El-Kady, A. Noori, M.F. Mousavi, R.B. Kaner, Thionine Functionalized 3D Graphene Aerogel: Combining Simplicity and Efficiency in Fabrication of a Metal-Free Redox Supercapacitor, Adv. Energy Mater. 8 (2018) 1802869. [32] H. Sun, S. Ma, Y. Lia, H. Qi, Electrogenerated chemiluminescence biosensing method for the detection of DNA demethylase activity: Combining MoS2 nanocomposite with DNA super sandwich, Sens. Actuators B 244 (2017) 885-890. [33] T.A.S.L. de Sousa, T.F.D. Fernandes, M.J.S. Matos, E.N.D. Araujo, M.S.C. Mazzoni, B.R.A. Neves, F. Plentz, Thionine Self-Assembled Structures on Graphene: Formation, Organization, and Doping, Langmuir 34 (2018) 6903-6911. [34] K. Hutchinson, R.E. Hester, W.J. Albery, A.R. Hillman, Raman Spectroscopic Studies of a Thionine-modified Electrode, J. Chem. Soc., Faraday Trans. 80 (1984) 2053−2071. [35] K. Jiang, D. Nie, Q. Huang, K. Fan, Z. Tang, Y. Wu, Z. Han, Thin-layer MoS2 and thionin composite-based electrochemical sensing platform for rapid and sensitive detection of zearalenone in human biofluids, Biosens. Bioelectron. 130 (2019) 322–329. [36] X. Gan, H. Zhao, X. Quan, Two-dimensional MoS2: A promising building block for biosensors. Biosens. Bioelectron. 89 (2017) 56–71. [37] K. Yan, Y. Zhu, W. Ji, F. Chen, J. Zhang, Visible Light-Driven Membraneless Photocatalytic Fuel Cell toward Self-Powered Aptasensing of PCB77, Anal. Chem. 90 (2018) 9662−9666. [38] L. Fan, C. Zhang, H. Shi, G. Zhaob, Design of a simple and novel photoelectrochemical aptasensor for detection of 3,3′,4,4′-tetrachlorobiphenyl, Biosens. Bioelectron. 124 (2019) 8– 14. [39] S. Liang, L. Wub, H. Liu, J. Li, M. Chen, M. Zhang, Organic molecular passivation of phosphorene: An aptamer-based biosensing platform, Biosens. Bioelectron. 126 (2019) 30–35.

20

[40] L. Wu, X. Lu, X. Fu, L. Wu, H. Liu, Gold Nanoparticles dotted Reduction Graphene Oxide Nanocomposite Based Electrochemical Aptasensor for Selective, Rapid, Sensitive and Congener-Specific PCB77 Detection, Sci. Rep. 7 (2017) 5191. [41] C. Fu, Y. Wang, G. Chen, L. Yang, S. Xu, W. Xu, Aptamer-Based Surface-Enhanced Raman Scattering-Microfluidic Sensor for Sensitive and Selective Polychlorinated Biphenyls Detection, Anal. Chem. 87 (2015) 9555−9558. [42] K. Sun, Q. Huang, G. Meng, Y. Lu, Highly Sensitive and Selective Surface-Enhanced Raman Spectroscopy Label-free Detection of 3,3′,4,4′-Tetrachlorobiphenyl Using DNA Aptamer-Modified Ag-Nanorod Arrays, ACS Appl. Mater. Interfaces 8 (2016) 5723−5728. [43] Y. Lu, Q. Huang, G. Meng, L. Wu, Z. Jingjing, Label-free selective SERS detection of PCB-77 based on DNA aptamer modified SiO2@Au core/shell nanoparticles, Analyst 139 (2014) 3083-3087. [44] R. Cheng, S. Liu, H. Shi, G. Zhao, A highly sensitive and selective aptamer-based colorimetric sensor forthe rapid detection of PCB 77, J. Hazard. Mater. 341 (2018) 373-380. [45] T. Portol, C. Sales, M. Abalos, J. Saul, E. Abad, Evaluation of the capabilities of atmospheric pressure chemical ionization source coupled to tandem mass spectrometry for the determination of dioxin-like polychlorobiphenyls in complex-matrix food samples, Anal. Chim. Acta 937 (2016) 96-105. [46] N. Ramanujam, M. Sivaselvakumar, S. Ramalingam, Fast and parallel determination of PCB 77 and PCB 180 in plasma using ultra performance liquid chromatography with diode array detection: A pharmacokinetic study in Swiss albino mouse, Biomed. Chromatogr. 31 (2017) e4000.

21

Table 1. An overview of the best previously published studies for detection of PCB77. Detection

Functional unit

Linear range (ng mL-1)

LOD (ng mL-1)

Ref.

Photoelectrochemical

g-C3N4-Au

10 -1000

4.5

37

Photoelectrochemical

CdSQDs

0.0001-0.1

0.0001

38

Target-induced conformational changes of aptamer

Electrochemical

PP-AuNP

0.0001-10

0.00003

39

Target-induced conformational changes of aptamer

Electrochemical

rGO-AuNP

0.000001-10

0.0000001

40

Strategy Change in the electron transfer after the formation of PCB77-aptamer complex on the photoanode Release of the DNA-CdSQDs probe after the formation of PCB77aptamer complex on the electrode and change in the electron transfer

Aptamer-based microfluidic sensor

SERS

Ag-nanocrown

3-30000

3

41

Target-induced conformational changes of aptamer

SERS

Ag-Nanorod Arrays

10-300

10

42

Target-induced conformational changes of aptamer

SERS

SiO2@Au core shell

300-5800

300

43

Colorimetric

AuNP

0.15-260

0.015

44

-

-

0.0025

45

-

10-3000

10

Aggregation of AuNPs due to the target-induced conformational changes of aptamer Atmospheric pressure chemical ionization source coupled to tandem

GC-MS/MS

mass spectrometry UPLC with a photodiode array detector Target-induced conformational changes of aptamer

UPLC Electrochemical

MoS2-rGO/AuNP

0.0000003-0.1

0.00000008

Abbreviation: gold nanoparticles-decorated graphitic C3N4 nanosheet (g-C3N4-Au); gold nanoparticle-dotted phosphorene (PP-AuNP); surface-enhanced Raman scattering (SERS); ultra‐performance liquid chromatography (UPLC)

22

46 This work

Table 2. Recovery of PCB77 in water and soil sample (n=4). Samples Zayandehrood River water

Tap water

Agricultural soil

PCB77 added

PCB77 found

-

undetected

20 ng L-1

21.7

60 ng L-1

RSD%

GC-MS standard method

-

undetected

108.5

4.2

21.5 ± 0.9 (ng L-1)

57.9

96.5

3.7

61.0 ± 2.7 (ng L-1)

100 ng L-1

104.9

104.9

4.4

105.5 ± 4.8 (ng L-1)

-

undetected

-

undetected

20 ng L-1

19.1

95.5

3.5

21.1 ± 0.7 (ng L-1)

60 ng L-1

59.0

98.3

5.0

60.3 ± 3.1 (ng L-1)

100 ng L-1

105.6

105.6

4.8

98.5 ± 4.4 (ng L-1)

-

undetected

-

undetected

50 ng g-1

48.8

97.6

4.0

52.2 ± 3.0 (ng g-1)

200 ng g-1

213.2

106.6

5.3

209.9 ± 9.1 (ng g-1)

500 ng g-1

491.7

98.3

5.8

512.9 ± 22.3 (ng g-1)

23

Recovery%

Figure captions

Figure 1. The sensing mechanism of MoS2-rGO based aptasensor for detection of PCB77.

Figure 2. (A) XPS spectra of S 2p region for the MoS2-rGO, Thi, and MoS2-rGO/Thi. (B) XRD patterns of rGO, MoS2 nanoflowers, MoS2-rGO, and MoS2-rGO/Thi. (C) Raman spectrum of the MoS2-rGO/Thi nanocomposite, where it is possible to detect Raman peaks associated with MoS2, rGO, and Thi. Inset is exhibited the molecular structure of Thi.

24

Figure 3. SEM image of (A) MoS2-rGO, (B) MoS2-rGO/Thi, and (C) TEM image of MoS2rGO/Thi. SEM image of (D) bare GCE (E) MoS2-rGO-modified electrode, and (F) MoS2rGO/Thi-modified electrode.

Figure 4. (A) cyclic voltammograms of (a) unmodified GCE and GCE modified with (b) MoS2-rGO, (c) MoS2-rGO/Thi, and (d) MoS2-rGO/Thi/AuNP in phosphate buffer (0.1 mol L1

, pH=7.4). (B) DP voltammograms of GCE modified with (a) MoS 2-rGO/Thi, (b) MoS2-

rGO/Thi/AuNP, aptamer-functionalized MoS2-rGO/Thi/AuNP modified electrode before (c) and after (d) interaction with PCB77 in phosphate buffer (0.1 mol L-1, pH=7.4).

25

26

Figure 5. (A) The linear calibration plot for ΔI vs. log CPCB77. The insert shows the DP voltammograms of the aptasensor for different concentrations of PCB77. The concentrations of PCB77 varied as (a) 0.0, (b) 0.0003, (c) 0.001, (d) 0.01, (e) 0.1, (f) 1, (g) 10, and (h) 100 pg mL-1. (B) Histogram for ratio of ΔI values of the proposed sensor toward 0.1 pg·mL -1 PCB77 without (ΔI') and with (ΔI") different interferents: Benzopyrene (1.0 mol L-1), p-nitrophenol (1.0 mol L-1), Atrazine (1.0 mol L-1), Biphenyl (1.0 mol L-1), Bisphenol A (1.0 mol L-1), Hydroquinone (1.0 mol L-1), PCB101 (1.0 pg·mL-1), PCB126 (1.0 pg·mL-1), PCB81 (1.0 pg·mL-1), Hg2+ (10.0 mol L-1), Fe3+ (10.0 mol L-1), Co2+ (10.0 mol L-1), Cu2+ (10.0 mol L-1), Cd2+ (10.0 mol L-1), and Zn2+ (10.0 mol L-1).

27