Detection of chikungunya virus DNA using two-dimensional MoS2 nanosheets based disposable biosensor

Development of platforms for a reliable, rapid, sensitive and selective detection of chikungunya virus (CHIGV) is the need of the hour in developing countries. To the best of our knowledge, there are no reports available for the electrochemical detection of CHIGVDNA. Therefore, we aim at developing a biosensor based on molybdenum disulphide nanosheets (MoS2 NSs) for the point-of-care diagnosis of CHIGV. Briefly, MoS2 NSs were synthesized by chemical route and characterized using scanning electron microscopy, transmission electron microscopy, UV-Vis spectroscopy, Raman spectroscopy and X-Ray Diffraction. MoS2 NSs were then subjected to physical adsorption onto the screen printed gold electrodes (SPGEs) and then employed for the detection of CHIGV DNA using electrochemical voltammetric techniques. Herein, the role of MoS2 NSs is to provide biocompatibility to the biological recognition element on the surface of the screen printed electrodes. The detection strategy employed herein is the ability of methylene blue to interact differentially with the guanine bases of the single and double-stranded DNA which leads to change in the magnitude of the voltammetric signal. The proposed genosensor exhibited a wide linear range of 0.1 nM to 100 µM towards the chikungunya virus DNA.

low cost, disposability and design flexibility as compared to traditional electrode materials [13][14][15] . Hence, SPEs serve as a transition away from the traditional cumbersome beaker-type electrochemical cells and bulky electrodes 16 .
Previously several DNA biosensors have been reported involving labeling of PCR products with enzymes 17 , redox active components 18 or nanoparticles 19,20 to enhance the electrochemical signal. Nanomaterials have been used as carrier beacons for indirect; however, vigorous and precise means for detecting target molecules. Two-dimensional (2D) molybdenum disulphide (MoS 2 ) nanomaterials belonging to transition-metal dichalcogenides have been gaining much attention these days 21 . This is because MoS 2 has emerged as a material with exceptional biocompatibility, good electrochemical catalysts activity, easy modification 22 , high specific surface area and large junction area of the electrode/electrolyte 23 and sensitive surface states (high surface-to-volume ratio) 24 . Each Mo is coordinated to six S atoms; by stacking covalently bound S-Mo-S via weak van der Waals interactions 25 , thereby enhancing the planar electric transportation properties 26,27 . MoS 2 nanosheets (MoS 2 NSs) are capable enough to adsorb single-stranded DNA by the van der Waals force between nucleobases and the basal plane of MoS 2 NSs 24,25 . These advantages along with the existence of suitable bandgap in comparison to graphene and graphene oxides which have small or no band gaps makes MoS 2 NSs highly suitable for sensors that can detect DNA, proteins, metal ions, and other compounds.
In the present report, an electrochemical DNA biosensor has been prepared for the detection of DNA of chikungunya virus electrochemically. Screen printed disposable gold electrodes coated with molybdenum disulphide nanosheets have been used as the platform for immobilization of the probe DNA and employed for the detection of the target DNA.

Results and Discussion
Assay design and principle. A schematic representation showing the main steps in our assay for detection of target CHIG DNA and the principle behind the detection is represented in Fig. 1. Figure 1(a) shows the various components of SPGE. The working electrode (WE) and counter electrode (CE) were made of gold and the reference electrode (RE) was made of silver. The main steps involved in the fabrication of SPGEs were presented in Fig. 1(b). The gold on the WE of SPGE shows strong affinity for the sulfur group in MoS 2 ( Fig. 1(c)). This confirms a strong immobilization of MoS 2 over SPGE. Probe DNA of CHIGV was immobilized over MoS 2 coated SPGE ( Fig. 1(b)). The MoS 2 are capable enough to adsorb single-stranded DNA by the van der Waals force between nucleobases 24,25 and the basal plane of MoS 2 NSs ( Fig. 1(d)). Thereby ensuring efficient immobilization. Further, the target DNA was added ( Fig. 1(b)) along with MB and the hybridization was allowed to occur for 60 sec. The role of MB has been presented schematically in Fig. 1(e). The interaction of MB with the free guanine bases of the single-stranded DNA leads to enhanced electrochemical response. This is due to its ability to attract to guanine via Vander Waals interaction. Upon hybridization, the MB gets intercalated between the bulky double helix of the double-stranded DNA. Thus, a decreased response was observed as shown in Fig. 1(e).
Characterization of the synthesized MoS 2 nanosheets (MoS 2 NSs). SEM images clearly showed that highly dense, laminar nanosheets with curved edges. The nanosheets are folded at the edges giving them a petal like shape ( Fig. 2(a,b)). Transmission electron microscopy is an excellent tool to characterize two dimensional transition metal dichalcogenides (TMDs). The MoS 2 monolayer is composed of three atom layers: Mo layer sandwiched between two sulphur layers. The three layers are stacked via weak vander Waal interaction. TEM  Fig. 2(c,d)) clearly showed that thin layer structures of MoS 2 nanosheets. The interplanar spacing is found to be 0.64 nm which is in agreement with the earlier reports 28 . Figure 3(a) shows the UV-Vis absorption spectra of MoS 2 nanosheets. Two characteristic absorption peaks (A and B) were observed at 676 nm (1.83 eV) and 66 613 nm (2.02 eV). These exciton peaks correspond to A and B direct electronic transition of MoS 2 nanosheets, originated from the energy split of valence band and spin orbit coupling 29 . Figure 3(b) shows the Raman spectrum of as synthesized MoS 2 nanosheets. A typical two pronounced peaks were observed at 382 cm −1 and 407 cm −1 . The Raman peak at 382 cm −1 (E 1 2g ) is associated with the in-plane MoS 2 phonon mode and 407 cm −1 (A 1g ) is due to the out of plane MoS 2 phonon mode. The difference between these characteristic peaks is 25 cm −1 implying that nanosheets consist of 5 or more MoS 2 layers is stacked together. These two characteristic peaks indicate that synthesized MoS 2 nanosheets possess 2H-MoS 2 structure 30 . The crystal structure of the MoS 2 nanosheets was investigated through X-ray diffraction as shown in the Fig. 3 Fig. 4(a,b) respectively. The concentration of MoS 2 was varied from 0.5 mg/mL to 2 mg/mL and the electrochemical response was observed. The highest response occurred at 1 mg/mL ( Fig. 4(a)). Above 1 mg/mL, the response was stable so this was chosen as the concentration of MoS 2 for the future experiments. Figure 4(b) shows that the highest peak current was observed when PDNA concentration was 50 µM. At 100 µM PDNA, the signal drastically reduced. This was due to the increased thickness of the organic layer at the surface of SPGE which decreased the electron transfer rates. Consequently, 50 µM was chosen as the most favorable concentration of PDNA.

Electrochemical analysis at various stages of the SPGEs. The electrochemical behaviors of various
stages of SPGEs obtained after modification with MoS 2 NSs, PDNA or TDNA were analyzed using CV in 0.1 M phosphate buffer saline (pH 7.8) and 1 µM MB in the potential range from −0.6 to +0.4 V at a scan rate of 100 mVs −1 (Fig. 4(c)). All modified SPGEs exhibited a pair of well-defined redox peaks due to the oxidation and reduction of methylene blue at SPGE. As can be seen in Fig. 4(c), bare SPGE shows the maximum electrochemical response of 3.2 × 10 −5 A (Ia) and −3.5 × 10 −5 A (Ic) due to the presence of gold which shows good metallic conductivity. MoS 2 NSs shows decreased current response of 2.9 × 10 −5 A (Ia) and −2.8 × 10 −5 A (Ic) due to the semi-conducting nature of MoS 2 in comparison to the conducting gold (bare SPGE). In spite of the decreased electrochemical response, the MoS 2 nanosheets are preferred; due to their ability to adsorb ssDNA by the van The explanation for this behavior is explained by Raveendran et al. 31 . As per their report, the shift in the potential over the surface of SPGEs lies on the negatively charged nature of DNA and the transfer of electrons during the hybridization event. MoS 2 NSs transfer the electrons from the MB and vice versa during cyclic voltammetry to produce the characteristic voltammogram at the specified voltage. Modification of MoS 2 NSs by the probe DNA modifies this transfer of electrons reducing the potential at which this is occurring, making it easier for MB to reduce. During the hybridization of the CHIGV target DNA with the complementary strand of the probe DNA immobilized on the surface there is a restructuration of the molecules and a higher demand for electrons, which leads to reduction of MB with less available electrons for the molecule, hence increasing the reduction potential.
The similar response was observed in Fig. 4(d) which shows Nyquist plot at various stages of SPGE. The resistance charge transfer (Rct) value of bare SPGE was lowest and that of target DNA was highest. The probe DNA show increased Rct value in comparison to MoS 2 NSs. Since, the resistance is inversely proportional to the current; therefore, the Nyquist plot and CV results were in line with one another.
Electrochemical response of PDNA/MoS 2 NSs/SPGEs at various scan rates. The effect of scan rates ranging from 10 to 100 mVs −1 on the PDNA/MoS 2 NSs/SPGEs is shown in Fig. 5(a). It shows that the anodic and cathodic peak current of MB on the PDNA biosensor increased constantly from 10 to 100 mVs −1 , confirming that the electrochemical reaction process of the biosensor is mainly diffusion controlled. However, to assure the stability of the biosensor response, 100 mVs −1 was chosen as the scan rate for subsequent studies.
The oxidation (Ia) and reduction peak current (Ip) both were proportional to square root of scan rate (v 1/2 ) in Fig. 5(b) which is expressed as Ia = 3.25 × 10 −6 × −8.31 × 10 −6 , r 2 = 0.98, Ic = −1.51 × 10 −6 × +3.07 × 10 −6 , r 2 = 0.98. This makes it clear that the process of catalysis is diffusion controlled rather than surface controlled under the condition of sufficient potential 32,33 . The diffusion controlled behavior of the reaction is confirmed by a plot between Log Ia and Log v as; Log Ia = 0.98 log v (mVs −1 ) − 6.5, r 2 = 0.96 as shown in Fig. 5(c). Analytical performance of the biosensor. A close perusal of cyclic voltammograms shows that the response signal of MB decreases with the increase of TDNA concentration in the range 0.1 nM to 100 µM ( Fig. 6(a)). The peak current has decreased due to the formation of bulky hybrid complexes which hinder the interaction of MB molecules with the pure PDNA on the electrode. MB behaves as an anionic indicator with the ability to bind with the unpaired guanine bases in the DNA strands. Therefore, higher number of unpaired guanine residues results in higher interaction of MB molecules with the DNA strand and thus gives higher current response. The hybridization of the probe and target DNA lead to the reduction in the availability of the unpaired guanine because of its hydrogen bonding with cytosine in the complimentary target DNA. This results in lesser interaction of MB and thus, lower current response. Therefore, the current response decreases after hybridization of the TDNA because of lesser interaction of MB with unpaired guanine. The similar analyses have been reported earlier as well 34,35 . Figure 6(c) shows a linear variation in peak currents with the concentrations of target nucleotide. The linear fitted relation is given below: − − Ia 5 07 10 log 1 44 10 , r 097 6 5 2 The limit of detection (LOD) was calculated to be 3.4 nM in a 3 σ rule and limit of quantification (LOQ) was calculated to be 104.81 nM in a 10 σ rule. Our proposed sensor offers a wide linear range (0.1 nM to 100 µM) and sufficiently low detection limit for CHIGV detection.
Electrochemical Impedance Spectroscopy (EIS) was also done in order to confirm the hybridization of the probe DNA with the various concentrations of target DNA (0.1 nM to 80 µM). Figure 6(b) shows the nyquist plot indicating the hybridization of the target DNA to the PDNA. Upon increasing the concentration of the target DNA from 0.1 nM to 80 µM, the Rct value increased. This is because the results of EIS and CV are always contrary from one another. Thus, verifying hybridization. Figure 6 Effect of pH, temperature and hybridization time on the PDNA biosensor. Figure 7(a) depicts the 3D representation of the cyclic voltammograms for the pH response of the biosensor. Since MoS 2 is generally considered to be biocompatible with DNA, the pH response of the MB solution at PDNA/MoS 2 NSs/SPGE was studied. The alkaline conditions are beneficial for MB because MB (C 16 H 18 N 3 SCl) is a heterocyclic aromatic chemical compound and under alkaline conditions, it tends to form cations, whereas OH− is adsorbed to the surface of PDNA modified to form negatively charged adsorption centers, thus promoting the adsorption of MB ions 36 . The highest current response was observed at pH 7.8 after which the response became stable. Thus, pH 7.8 was chosen for the rest of the experiments. The effect of temperature on the PDNA/MoS 2 NSs/SPGE was studied and the results are presented in Fig. 7(b)). As evident from the figure, the electrochemical response of the sensor enhanced upon increasing the temperature (though not much difference); because high temperatures are more favorable for hybridization (obvious from PCR). But in order to keep the simplicity of employment of the biosensor for general use, 35 °C was chosen as the temperature at which appropriate hybridization could take place.
The hybridization time is an important parameter in a DNA biosensor. Therefore, the hybridization time was optimized. For this, different electrodes with PDNA immobilized were prepared and TDNA was added with MB. Cyclic voltammetric measurements were recorded at various time intervals. After the analysis, 35 sec was kept as the optimum hybridization time for this biosensor.
Real samples and selectivity analysis of the biosensor. The target DNA was spiked in purchased serum sample and dropped on the surface of the PDNA/MoS 2 NSs/SPGE. The hybridization of probe DNA with target DNA in serum sample occurred and the current response observed was similar to the current response obtained when TDNA was directly added over the PDNA/MoS 2 NSs/SPGE (Fig. 8). Thus, confirming the analysis in real samples.
In order to investigate the selectivity of this biosensor, the current response obtained from PDNA/MoS 2 NSs/ SPGE and hybridized TDNA/MoS 2 NSs/SPGE was compared to the non-complimentary DNA (n DNA). A significantly different current response was observed in case of nDNA when compared with the TDNA. The non-complimentary DNA showed response nearly equal to the PDNA (Fig. 9). Thus, no hybridization occurred with the nDNA and MB is free to interact with the guanine bases available in the ssDNA. Comparison study. The sensing interface ability of present genosensor was compared with earlier reported MoS 2 based biosensors [37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52] . High sensitivity, specificity and repeatability of the sensor make it best among other biosensors (Supplementary Table 1).  Methylene blue was purchased from Thermo Fischer Scientific, India. All the chemicals were of analytical reagent grade and used without further purification. Double-distilled water was used throughout this experiment. Tris-EDTA (TE buffer) buffer of pH 8.0 was used to prepare the 100 µM stock solutions of probe DNA and target DNA. Phosphate buffer saline (PBS, 0.1 M) was prepared by mixing the stock solutions of 1 M NaH 2 PO 4 and Na 2 HPO 4 and NaCl. 1 µM methylene blue (MB) prepared in PBS was used as buffer solution in all the electrochemical measurements. DRP-220 AT (screen printed with high temperature curing ink) screen printed gold electrodes (SPGEs) with 3.4 × 1.0 × 0.05 cm dimensions were purchased from DropSens (India). The SPGEs had working electrode (diameter of 1.4 mm) and counter electrodes made of gold whereas the reference electrode and the electrical contacts were made of silver. Human serum (minus IgA/IgM/IgG) was obtained as a lyophilized powder from Sigma Aldrich (India). Electrochemical measurements like cyclic voltammetry (CV) were measured on Autolab PGSTAT 204. Morphology of the MoS 2 nanosheets was characterized by transmission electron microscope (TEM, FEI Tecnai G2, 300 KV) and scanning electron microscopy (JEOL JSM-6010LA). TEM sample preparation was done by placing a drop of MoS 2 nanosheets on carbon coated copper grid followed by drying in air and transferred to the microscope operated at an accelerating voltage of 300 KV. The structure of MoS 2 nanosheets were studied by X-Ray Diffraction. Sample was scanned in the range of 10 ° to 80 ° at a glancing angle of 1 °. The Raman spectrum was taken by using a Horiba micro-Raman confocal microscopic system (LabRAM), at room temperature in an ambient air. A spectrophotometer (Agilent Technologies, model no: Cary 100 series) was used to obtain the UV-Vis absorption spectra of the MoS 2 nanosheets, which were recorded in the wavelength range of 300-800 nm at room temperature.
Preparation of CHIG probe and target DNA. All the oligonucleotides were synthesized by Integrated DNA Technology (IDT) as lyophilized translucent films. The sequences were listed as follows:

Synthesis of Molybdenum disulphide nanosheets (MoS 2 NSs).
MoS 2 NSs were synthesized by dissolving 3 mM of sodium molybdate dihydrate and 9 mM of thioacetamide in 50 mL of distilled water. Further 2.8 mM silicon tungstic acid was added into the reaction solution under violent stirring. The resultant solution was transferred into a 100 mL Teflon-lined stainless autoclave and was kept at 220 °C for 24 h. Then the autoclave was allowed to cool and the resulting products were filtered off, washed with 1 M NaOH, ethanol and distilled water for several times, and dried in vacuum at 50 °C for 8 h.
Bioelectrode fabrication, immobilization of PDNA and hybridization of TDNA. The bare SPGEs were cleaned by washing them subsequently with gold cleaning solution and 0.1 M phosphate buffer saline. The synthesized MoS 2 NSs (2.5 µL) were deposited physically on the surface of the working electrode (WE) of SPGEs. The drop-casted electrodes were left for drying at room temperature for 1.5 h. Since gold has a high affinity for sulfur, modification of SPGEs was made easy.
The MoS 2 NSs/SPGEs were further interacted with the CHIG probe DNA. After drying, 3 µL of the probe DNA was deposited over the MoS 2 NSs. The probe DNA immobilized MoS 2 NSs/SPGEs were left for 3 h to allow complete immobilization. The probe DNA immobilized SPGE was further used for hybridization of the target DNA. The target DNA was added along with the hybridization indicator (MB) and after appropriate time (optimized); the electrochemical analysis was done to confirm hybridization.
Optimization of pH, temperature and hybridization time. The probe DNA immobilized SPGE was optimized for hybridization at various pH (5.8 to 8) and temperature (20 to 40 °C). The pH, temperature showing best electrochemical response was finally used for hybridization with the target DNA. The hybridization time is also an important factor in a DNA biosensor and thus, the hybridization time between PDNA and TDNA was also optimized. Six electrodes having fixed concentration of probe DNA (50 µM) over MoS 2 NSs were prepared and were incubated with complimentary target DNA (80 µM) for different intervals of time (10 to 60 s).
Analysis of the stability, specificity and performance of the biosensor with real sample. The real sample analysis was done by adding known concentration of the target oligonucleotides in the purchased serum. This mixture along with PBS containing MB was dropped over probe DNA modified SPGE and sensing was done further. The stability analysis was performed by storing the probe DNA modified SPGE at 4 °C and periodically measuring the signal strength corresponding to it by CV and DPV upon addition of the target DNA. For selectivity analysis of the proposed DNA biosensor, the probe DNA modified SPGE was exposed to complementary and non-complementary target samples. The concentration of the non-complementary nucleotide sample was kept 3 orders higher than that of the complementary sample for determining the sensor selectivity. The probe modified electrodes was exposed to complementary and non-complementary target samples subsequently.

Conclusions
A reliable electrochemical CHIGV DNA detection system has been developed in the present work. MoS 2 nanosheets deposited screen printed gold electrodes proved efficient for probe DNA binding. The sensor shows good linear range from 0.1 nM to 100 µM with 3.4 nM as the limit of detection. Point of care technologies (POCTs) are the need of the hour. The features like less response time, high linearity and economic feasibility makes the present sensor beneficial to be miniaturized as POCTs.