The fabrication of the immunosensor is clearly shown in Fig. 1, which includes modification of GO, methylene blue, primary antibody (Ab1) modification, BSA blocking, WSSV antigen (Ag) and HRP labelled secondary antibody (Ab2 or Ab2-HRP) for virus detection. Initial experiments were focused on the optimization of the electrochemical immunosensor parameters. Figure 2(A) is a ten continuous CV response of GCE/GO@MB, prepared by immersing GO modified GCE, GCE/GO in 5 mg MB dissolved 500 μL ethanol solution for 5 min, showed a well-defined redox anodic and cathodic peak at an equilibrium potential (E 1/2 = E pa + E pc /2, where E pa and E pc are anodic and cathodic peak currents) value found to be −0.3 ± 0.005 V vs. Ag/AgCl in pH 7 PBS. The observed peak potential difference, ΔE p = 160 mV suggesting a quasi-reversible behaviour of MB immobilised electrode system. When the electrode was gently washed with distilled water and CV was performed again, there was no alteration in peak current or peak potential noticed. Calculated relative standard deviation value for anodic peak current is 2.1%. Based on the experimental results and literature data23, it can be postulated that MB dye is adsorbed strongly on the underlying GO surface and showed the redox activity. It is likely that π-π interaction between the aromatic portion of MB and sp2 carbon of GO is responsible factor for the stability of the modified electrode. This point onwards the modified electrode is designated as GCE/GO@MB.

Figure 2 CV responses of (A) GCE/GO@MB, (B) GCE/GO@MB, (C) GCE/GO@MB-Ab1 (D) GCE/GO@MB-Ab1-Ag, (E) GCE/GO@MB-Ab1-BSA-Ab2-HRP, (F)GCE/GO@MB/ Ab2-HRP, (G) GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP without (Blank) and with 500 μM of H 2 O 2 in pH 7 PBS at v = 10 mVs−1. (H) A plot of i pc of H 2 O 2 vs different modified electrodes. Full size image

Suitability of the GCE/GO@MB for electrochemical immunosensor was tested by performing hydrogen peroxide interaction in pH7 PBS as in Fig. 2(B). The GCE/GO@MB system failed to show any alteration in the peak current and potential without and with 500 μM H 2 O 2 indicating absence of any electro-catalytic activity of the underlying electrode. It is a clear advantage of using this surface-confined redox system further for H 2 O 2 biosensor application. As control experiments, individual and combinations of antigen (WSSV-vp28), Ab1 and Ab2 (i.e, Ab2-HRP) systems modified on GCE/GO@MB prepared as per the procedure given in Fig. 1, were subjected to H 2 O 2 electrochemical reduction reaction. As can be seen in the Fig. 2(C,D and E), Ab1, Ag-Ab1 and Ab1-Ab2 modified GCE/GO@MB systems showed either nil or very feeble redox peaks due to unavailability of HRP (Ab1) and improper binding of antibody systems (Ab1-Ab2) on the working electrode surface respectively. In further, Ab2 modified electrode, GCE/GO@MB-Ab2 was tested for the activity Fig. 2(F). Interestingly, a well-defined electro-catalytic reduction response for H 2 O 2 at a reduction peak potential, −0.35 V vs Ag/AgCl, where the MB redox peak exist, was noticed in Fig. 2(G). Effect of scan rate on the CV response of the modified electrodes, GCE/GO@MB and GCE/GO@MB-Ab2 showed a systematic increase in both anodic and cathodic peak currents (Fig. 3(A and B)). A plot of base-line corrected peak currents, i pa and i pc against scan rate (v) for both of the modified electrodes showed a linear line starting from origin indicating surface-confined electron-transfer mechanism of the systems (Fig. 3(C)). This observation highlights elegant immobilization and effective electron-transfer shuttling of Ag and Ab2-HRP protein with MB.

Figure 3 CV responses of GCE/GO@MB (A) and GCE/GO@MB-Ab2-HRP (B) at different scan rates (5–500 mV s−1) in pH7 phosphate buffer solution. Plots of (C) anodic (i pa ) and cathodic (i pc ) peak currents vs scan rate and (D) anodic (E pa ) and cathodic (E pc ) peak potentials vs log (scan rate) for the GCE/GO@MB (A1/C1) and GCE/GO@MB-Ab2-HRP (A2/C2). CV response of (E) GCE/GO@MB-Ab2-HRP in 500 μM H 2 O 2 dissolved pH 7 PBS at different scan rate (5–500 mV s−1) and (F) Plot of i pc of H 2 O 2 vs square of scan rate for the GCE/GO@MB-Ab2-HRP. Full size image

The surface concentration of MB-electro active species (Γ MB , mol cm−2) in GCE/GO@MB and GCE/GO@MB-Ab2 can be calculated from the slope of the peak currents vs. scan rate Fig. 3(C). For a reversible reaction, the peak current is given by

where n is the number of electrons transferred, F is the faraday constant (96500), A is the geometrical area of the electrode, v is the potential scan rate. From the above equation, the calculated surface concentration of MB was estimated to 9.84 × 10−10 mol cm−2 and 8.41 × 10−10 mol cm−2 for GCE/GO@MB and GCE/GO@MB-Ab2 respectively. Electro-kinetic parameter such as transfer coefficient (α) and rate constant (k s ) values for surface-confined MB were calculated based on scan rate data and Laviron24 electrokinetic model by plotting the variation of anodic and cathodic peak potentials with logarithm of scan rate Fig. 3(D). It obeys the procedure of Laviron by indicating the E pa or E pc values are proportional to logarithm of scan rate for values higher than 0.2 V s−1. The slope of the plots can provide the kinetic parameters α c and α a (cathodic and anodic transfer coefficients). The slope of the linear segments are equal to -2.303RT/αnF and 2.303RT/(1-α)nF for the cathodic and anodic peaks25 respectively, and the calculated values for α in pH 7 PBS for with and without secondary antibody are 0.53 and 0.45 respectively.

Based on the equation (2), calculated k s values for GCE/GO@MB and GCE/GO@MB-Ab2 in PBS are 1.65 ± 0.5 s−1 and 1.04 ± 0.5 s−1 respectively. A slightly lowered surface excess and k s values observed with the GO@MB-Ab2 modified electrode than that of the unmodified electrode is due presence of the electro-inactive protein species (Ab2) in the modified electrode.

Electrocatalytic H 2 O 2 reduction rate constant of GCE/GO@MB-Ab2, k chem , can be calculated with help of the expression for the catalytic current (i pc ) given by Andrieux and Saveant for a catalytic reaction in the case of slow scan rate and large k chem 26;

It has been shown that in the case of fast scan rates and low k chem the values of the “constant” in equation are lower than 0.49627. As per Fig. 1 in the published paper by Andrieux and Saveant study,26 where a working curve for the “constant” 0.496 was given as a function of

For GO@MB-Ab2 system with 500 μM H 2 O 2 . the average value of this coefficient is found to be 0.22 (calculated by referring the working curves based on the data calculated k chem , in the scan range 100 to 500 mV s−1). Thus k chem was calculated as 1.1 × 103 mole−1 dm3 s−1.

In further, number of electrons involved in the H 2 O 2 electrochemical reduction reaction (n) was theoretically calculated using Randles Sevick equation assuming quasi-reversible behaviour of the reduction system as,

where, i pc is reduction peak current, A is the electrode area (0.0707 cm−2), D is the diffusion coefficient of methylene blue (1.71 × 10−5 cm2 s−1)28 and C o is the concentration of analyte used (500 μM). By keeping n = 2 or n = 4, respective i pc values were back calculated from the eqn (5) and plotted against v1/2 as in Fig. 3(F), curve a (n = 2) and b (n = 4). In addition, the experimental i pc value also plotted in the same graph (Fig. 3F, curve c). It is obvious that the n = 2 theoretically simulated curve is fitting very well with the experimental observation ascribing involvement of n = 2 in the overall H 2 O 2 reduction reaction. Meanwhile, a pre-peak at 0.2 V vs Ag/AgCl was specifically noticed at scan rate on optimal electrode as in Fig. 3E, which is due MB at energetically different GO sites possibly on graphitic and non-graphitic (oxygen containing surface edge) sites. Indeed, MB immobilized on graphitic sites of GO shows predominant electrocatalytic feature and thus it is taken for further analysis.

Inorder to exemplify the effect of pH, GCE/GO@MB was subjected to CV at various pH solutions viz. pH 3,5,7,9 and 11 (Fig. 4). The observed results shows that the formal potential value shifted to negative direction with increase in pH (Fig. 4A). A plot between formal potential (E°/V) vs pH is found to be linear with a slope and regression coefficient values of −45 ± 3 mV pH−1. The obtained slope value is less than that the theoretical slope value of −59 mV pH−1 for equal number of proton and electron in the electrochemical reaction suggesting that Non-Nersntian behaviour with involvement of 2 H+/3e− in the electrochemical reaction mechanism.

Figure 4 (A) Effect of pH on CV of GCE/GO@MB at v = 50 mV s−1 and inset plot is E° vs pH, (B) Electrochemical impedance responses obtained with the bare GO (a), GO@MB (b), GO@MB-Ab1 (c), GO@MB-Ab1-BSA-Ag (d), GO@MB-Ab1-BSA-Ag-Ab2 (e) in 0.1 M KCl solution containing 5 mM each of [Fe(CN) 6 ]3− and [Fe(CN) 6 ]4− redox probe at an applied potential 0.3 V vs Ag/AgCl. The concentration of WSSV antigen is 1 μg μL−1. The impedance spectra were recorded in a range, 0.1 Hz–100 KHz. (C) Comparative FTIR/KBr responses of MB (a), GO (b) and GO@MB (c). Full size image

Feasibility of the GCE/GO@MB system was tested by performing electrochemical immunosensing of WSSV via electrochemical ELISA in presence of 500 μM H 2 O 2 . For the preparation of the electrochemical immunosensor, following order was followed, Ab1 → → BSA → Ag → Ab2-HRP as in the Fig. 1. CV response of the sandwich electrochemical ELISA system without and with 500 μM H 2 O 2 in pH7 PBS at a scan rate 10 mV s−1 was shown in Fig. 2(G). A clean H 2 O 2 reduction signal was observed with the electrochemical immuno-system modified electrode. Three repeated experiments of electrochemical sensing of WSSV-vp28 yielded a RSD 4.7%. This observation authenticates the facile immunochemical reaction on the GCE/GO@MB surface for further electrochemical quantification.

Part of the modified electrodes have been characterized using electrochemcial impedance spectroscopy (EIS) and FTIR techniques. EIS were employed to study the interfacial properties of the electrode after each step of modification. Figure 4B shows the impedance response in a step by step modification of the immunochemical sensor at an applied potential, 0.3 V vs Ag/AgCl with 5 mM each of [Fe(CN) 6 ]3− and [Fe(CN) 6 ]4− in 0.1 M KCl solution (as per Fig. 1). A semicircle coupled linear line like responses were uniformly observed due to charge transfer reaction (R CT ) and analyte diffusion processes respectively with all the test systems. The respective R CT values were calculated using Randles equivalent circuit, in which, a solution resistance (R s ) and double layer capacitance (C dl ) are arranged in parallel with R CT and Warburg resistance (Z W ) (Fig. 4B inset)29. Calculated R CT values of modified electrodes prepared by stepwise procedure are; GO@MB = 103 ohm, GO = 188 ohm, GO@MB-AB = 229 ohm, GO@MB- Ab1-Ag = 272 ohm, GO@MB- Ab1-Ag-Ab2 = 284 ohm. It is obvious that the R CT values increased regularly in the stepwise procedure. This observation might be due to dielectric and insulating features of the antibody and antigen that have been modified on the electrode surface. Although these results demonstrate feasibility of development EIS based electrochemical immunosensor for WSSV, with respect to selectivity and reliability bio-electrocatalytic sensor based electrochemical immunoassay system is superior than that of the impedimetric sensor.

To further characterize the immobilisation of methylene blue on graphene oxide, comparative FTIR spectroscopy was carried out with GO@MB, GO and MB as in Fig. 4C. A vibration signal due to C = C (1732 cm−1, 1728 cm−1, 1737 cm−1) and > C = O (1569 cm−1 and 1602 cm−1) were qualitatively noticed with all the samples (No > C = O signal with MB); but with a shift in the wavenumbers. In addition, specific vibrational band at 3412 cm−1 corresponds to the N-H stretching of MB was also noticed with GO@MB (3431 cm−1) ascribing MB immobilization and its GO interaction features. The morphological structure of GO and GO@MB was investigated using TEM Fig. 5(A and B). A transparent sheet like structure can be seen clearly with the GO as that of previously reported literature22. For MB modified GO case, large number of black spots of average size ~10 nm on the GO sheets was observed. This observation can be taken as a proof for the immobilization of MB on GO.

Figure 5 Transmission Electron Microscope images of GO (A) and MB surface confined GO (B). Full size image

Next, calibration plot for electrochemical immunosensing of WSSV was constructed by subjecting different concentration of the standard virus (vp28 protein) discreetly on the modified electrode as GCE/GO@MB-Ab1-BSA-Ag-Ab2 and subjected to CV experiment with fixed H 2 O 2 concentration (500 μM) in pH 7 PBS. Figure 6 is the typical calibration response for the virus obtained from different dilutions (10−1 to 10−10) of the stock WSSV, 1.37 × 107 copies μL−1. The sensor showed regular variation in the H 2 O 2 reduction current with respect to dilution. Note that the detection range (1.37 × 10−3–1.37 × 107 copies μL−1) and low-detection concentration (1.37 × 10−3 copies μL−1) obtained in this work are much better than the previous reported procedures like label-free affinity immunosensors (1.6 × 103–1.6 × 106 copies μL−1 and 1.6 × 101–1.6 × 106 copies μL−1) and colorimetric ELISA (1.6 × 103–1.6 × 107 copies μL−1)11,12,30.

Figure 6 CV responses of GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP, prepared with different dilution of antigen (10−1 to 10−10), in 500 μM H 2 O 2 containing pH 7 PBS at v = 10 mV s−1. Full size image

Electrochemical immunosensor specificity is major concern in the real sample analysis. Figure 7(A–D) is CV response of the GCE/GO@MB for various cross-reactivity samples based on different aquatic pathogens such as EHP, IHHNV, Vibrio parahaemolyticus and Vibrio harveyi. For the preparation of different aquatic pathogen modified GCE/GO@MB electrodes, procedure mentioned in Fig. 1 and Fig. 2G was adopted. Interestingly, these immunosensor systems failed to show any signal for H 2 O 2 reduction current, unlike to the WSSV-vp28, postulating the selectiveness of the present sensor for the WSSV. The specificity study was cross-confirmed with discreet PCR as well. Fig. 7(E) is the typical PCR analysis result of WSSV-infected positive DNA (PC, vp28) and healthy animals-negative DNA (NC) samples. The symbol “M” in the PCR photograph is the standard commercial 100 base pair DNA ladder. As can be seen, there were no target specific bands observed with the cross-reactivity samples when compared with the controls.

Figure 7 CV responses of different antigens modified GCE/GO@MB-Ab1-BSA-Ag-Ab2-HRP without and with 500 μM H 2 O 2 containing pH 7 PBS at v = 10 mV s−1; (A) Enterocytozoon Hepatopenaei, (B) Infectious Hypodermal and Haematapoietic Necrosis Virus, (C) Fish intestine and (D) Fish Muscle. (E) Agarose gel showing PCR amplification of WSSV vp28 gene. M-100 bp DNA Marker, NC-Negative Control, PC-positive control-WSSV (white Spot Syndrome Virus), 2-IHHNV (Infectious Hypodermal and Haematopoietic necrosis Virus) DNA, 3-EHP (Enterocytozoon Hepatopenaei) DNA, 4-vibrio parahaemolyticus infected Fish intestine, and 5-vibrio harveyi infected fish muscle samples. Full size image

As a proof of concept and usefulness of this protocol, selective detection of WSSV in raw gill tissues during the course of infection until it reaches moribund stage that usually take about 7 days were examined. Each sample is subjected to triplicate measurements. For convenience, 1st data result was presented in this work. The electrochemical immunosensor preparation procedure follows similar to Fig. 1. Figure 8(A) is a CV response of progression of the WSSV in the infected gill tissue from day 1 to day 7. In parallel, investigations were also carried out using PCR, western blot and conventional ELISA techniques along with positive and negative controls as in Fig. 8(B) to (D) respectively. As seen in Fig. 8(B), the PCR analyses gave specific bands for the 1–7 day samples relating to the qualitative information of the pathogen. There is no significant variation in the band intensities of PCR for the different time duration samples. Similarly, western blot Fig. 8(C) and ELISA Fig. 8(D) analyses results also gave signals only from the day 3 of the post infection. Interestingly, the electrochemical immunosensor results showed specific current signals for all the time duration samples. As can be seen in the Fig. 8(A) and (E), the current signals were increasing proportionately with increase in the post infection time. A plot of immunosensing signal vs post infection time (1–7 day) showed a linear line with slope value of 3.2 μA per day. In addition, a plot of electrochemical signal vs ELISA@ 405 nm OD was found to be linear Fig. 8(E), confirming suitability and reliability of the electrochemical sensor for further routine analysis.