1 Introduction

In recent times, biological synthesis of nanomaterials has emerged as an attractive alternative to traditional synthesis methods, which are inexpensive, free from toxic materials, cytotoxicity, carcinogenicity and potential hazards in environmental toxicity (Ai et al., 2011). So far, the presence of toxic agents on the synthesized nanomaterials has prevented its use and application in clinical fields. However, these tragic factors can be potentially controlled through biologically mediated fabrication which is currently a widespread interest in developing a clean, reliable, compatible, benign, and environment-friendly nanomaterials (Ahmad et al., 2003; Jain et al., 2011; Lewis et al., 2006; Ruoslahti, 2000; Mohanpuria et al., 2008; Philip, 2010). Biosynthesis of nanomaterials involve using an eco-friendly green chemistry based approach that employs unicellular and multicellular organisms such as actinomycetes (Ahmad et al., 2003; Sastry et al., 2003), bacteria (Joerger et al., 2001; Husseiny et al., 2007; Nair and Pradeep, 2002), fungus (Dameron et al., 1989;Kuber and Souza, 2006; Mukherjee et al., 2001; Kowshik et al., 2003), plants (Kumar et al., 2011; Njagi et al., 2011; Akhtar et al., 2013), viruses (Courchesne et al., 2014; Khan et al., 2013; Lee et al., 2002; Kale et al., 2013), and yeast (Ahmed et al., 2005). Moreover, biological entities like macromolecules of peptides, lipids, and enzymes can also be utilized for the biosynthesis of nanomaterials (Klem et al., 2005; Pratibha, 2009; Kaumeel et al., 2016; Gholami shabani et al., 2015). An interesting phenomenon dealing with biological entities is their ability to act as a template in the synthesis, assembly and organization of nanometre-scale materials in the fabrication of a well-defined micro and macro scale structures (Kulkarni and Muddapur, 2014; Monaliben et al., 2015). Interestingly, studies illustrate that viruses are found to be a superior group which acts as a biotemplate for constrained material synthesis at the nanoscale to microscale (Khan et al., 2013). In recent times, viral nanoparticles (VNPs) are increasingly used as templates or scaffolds for the design of novel hybrid nanomaterials to nano-smart devices by materials science researchers (Steinmetz and Manchester, 2011). A wide variety of viral nanoparticles (VNPs) both in mammalian and plant origin have been extensively studied and implemented in biomedical technology (Young et al., 2008; Bancroft et al., 1968; Fischlechner and Donath, 2007; Lewis et al., 2006). A group of plant viruses was utilized as nanoplatforms for their unique structural integrity, easy manipulation and significantly less infective to human being (Douglas and Young, 1998; Steinmetz and Manchester, 2011). In particular, because of their amazing structural diversity, capsid has been exploited as a biotemplate for material synthesis (Lee et al., 2009; Nam et al., 2006; Mao et al., 2004; Merzlyak et al., 2006). The capsid or protein coat that falls into the category of nanoscale and their dimensions typically ranges between 20 and 500 of nanometers (10⁻⁹ m). Significantly, the materials they template also fall into this size range in nanometer scale which has to be found the one can alleviate the cumbersome methodology in nanomaterials synthesis such as size and shape controlled synthesis (Alexandridis, 2011; Wang et al., 2002). Virus capsids are assembled from repeating protein subunits to form highly symmetrical architectures with precise three-dimensional (3D) structure are remarkably stable and in uniform size and shape (Sirotkin et al., 2014; Bothner et al., 1998). The repeating patterns of amino acid side chains displayed by each protein subunit can function to direct the ordered nucleation of inorganic for the display of novel hybrid molecules on either the interior or exterior surfaces (Flenniken et al., 2009; Klem et al., 2005). Synthesizing nanomaterials via virus entities acting as biological nanofactories offers a clean, nontoxic and environment-friendly method of synthesizing nanomaterials with a wide range of sizes, shapes, compositions, and physicochemical properties (Blum et al., 2004; Sirotkin et al., 2014). In this regard, plant virus nanoplatforms lately gathered much attention in the synthesis of metal virus hybrid materials which has many favourable properties like conductance, anisotropy, sensitivity and heterogeneity which can be exploited in a wide range of applications (Steinmetz and Manchester, 2011). For Instance, research revealed that Platinum-metalized TMV particles serve as a building block to fabricate a bio-memory device (Xiao et al., 2013). Living cells can offer ground-breaking solutions to some hard problems faced by the semiconductor industry. Controlled chemical reactions and molecular flows in cells are the ultimate miniaturization of electronics to the atomic and molecular scale (Susmit ang Meghna, 2015). The field of bio-semiconductor is perched for exponential growth. The ongoing miniaturization of bio-semiconductor devices is leading to new opportunities in biomedical research and commercial medical applications. Integration of semiconducting nanomaterials with biological materials has been extensively investigated in various fields. Likewise, the filamentous bacteriophage M13 has been extensively studied as a template for mineralization and has been utilized for semiconductor. Nam et al. (2006), have shown the potential of such an approach in the development of lithium ion battery electrodes using highly ordered M13-templated gold-cobalt oxide nanowires. TMV particles has been employed as a robust functional template for the fabrication of a TMV/zinc oxide field effect transistor (FET) (Sanctis et al., 2015). Likely, there are few examples of electronic devices being directly fabricated from biomaterials owing to a lack of charge transport through them and still, there is much more demand for inorganic–organic hybrid molecules with different and defined geometries for the ideal bio-electronic devices. Therefore, in this investigation, we have exploited a plant pathogenic virus Squash leaf curl China virus (SLCCNV) which belongs to Begomovirus genus and the family of Geminiviridae (Singh et al., 2008; Briddon et al., 1996). Begomoviruses are popular known viruses among plant pathologist and almost unfamiliar to the materials science researchers (Scholthof et al., 2011; Varma and Malathi, 2003). The Squash leaf curl China virus (SLCCNV) capsid are non-enveloped, about 38 nm in length and 22 nm in diameter, twinned (geminate) incomplete T = 1 icosahedral symmetry capsid that contains 22 pentameric capsomers made of 110 capsid proteins (CP). With this SLCCNV capsid structure, we have been extending its use as a nanofactory or biotemplate for fabricating noble metallic nanoparticles (Au & Ag) without any prior genetic or chemical modification of the exterior surface of virus capsid.

2 Materials and methods

3 Results

Caesium sulphate gradient ultracentrifugation yields a good quantity of SLCCNV particles (0.6 mg ml⁻¹) with homogeneity which is determined by a single intensified protein band in the silver stained SDS-PAGE gel (see Fig. S2). However, a single protein band directly denotes that the virus particles retained its capsid structure against optimum denaturing temperature, which justifies them as an obligate microorganism and its prevailing capsid integrity. In making of SLCCNV particles as a biotemplate, dialysis with different pH buffer produced the SLCCNV-biotemplate suspensions to its respective pH. It has been confirmed by simple pH analysis. Then it was assumed that all the dialysis underwent SLCCNV particles were electronically active to their respective buffer pH strength along with a customized surface net charge. Fig. S3, shown the theoretically calculated (Henderson-Hassel Bach equation) net charge values of SLCCNV-biotemplate dialyzed against different pH parameters. However, the net charge on a virus protein is zero at the isoelectric point (pI), positive at pHs (6, 7, 8 & –9) below the pI, and negative at pHs (11) above the pI. The net charge on a protein at any given pH is determined by the pKa values of the ionizable groups. The virus particles in the pH of 7–10 remain stable in their shape and structure which was observed in negative staining under electron microscopy (data not shown).

Caesium sulphate gradient ultracentrifugation yields a good quantity of SLCCNV particles (0.6 mg ml⁻¹) with homogeneity which is determined by a single intensified protein band in the silver stained SDS-PAGE gel (see Fig. S2). However, a single protein band directly denotes that the virus particles retained its capsid structure against optimum denaturing temperature, which justifies them as an obligate microorganism and its prevailing capsid integrity. In making of SLCCNV particles as a biotemplate, dialysis with different pH buffer produced the SLCCNV-biotemplate suspensions to its respective pH. It has been confirmed by simple pH analysis. Then it was assumed that all the dialysis underwent SLCCNV particles were electronically active to their respective buffer pH strength along with a customized surface net charge. Fig. S3, shown the theoretically calculated (Henderson-Hassel Bach equation) net charge values of SLCCNV-biotemplate dialyzed against different pH parameters. However, the net charge on a virus protein is zero at the isoelectric point (pI), positive at pHs (6, 7, 8 & –9) below the pI, and negative at pHs (11) above the pI. The net charge on a protein at any given pH is determined by the pKa values of the ionizable groups. The virus particles in the pH of 7–10 remain stable in their shape and structure which was observed in negative staining under electron microscopy (data not shown).

3.1

3.1 Fabrication of SLCCNV-metallic-hybrid noble nanomaterials

The pH (6–11) altered and activated viral template was effectively reduced both precursor ions (Au & Ag) and form SLCCNV-metallic-hybrid noble nanomaterials. It was determined after the visible colour change of both gold and silver reaction solution kept in the direct sunlight about 5–6 min. The reaction carried out at least for five replications and then determined. Notably, among other reaction solution, only one reaction solution ends up with bright colours, that was observed in the pH 10 virus template. Certainly, it was a direct indication of nanoparticles synthesis with the perfect geometries. Remaining reaction solution were varied in their colour exhibition from purple to pink. The pH 10 virus template fabricated nanomaterials have got their standard UV–visible spectrum peak at 540 nm and 420 nm for both gold and silver respectively (see Fig. 1 A & B). The colour indication corroborates to the surface plasmon resonance (SPR). This signifies that “when the dimensions of a metal is reduced to the nanoscale, the optical properties are dominated by an oscillation of conduction of electrons in resonance with incident electromagnetic radiation”. Every colloidal metal nanoparticles have its own SPR and colours. Moreover, we have observed different colour (Gray to Red for Gold & Pale yellow to brown for silver) formation of reaction solution from pH 6 to 11 which may indicate the gradual synthesis of nanomaterials in the solution (Pratibha, 2009). These colour changes can be ascribed to the size and shape of NPs in the suspensions. The size variation of smaller to larger particles in the solution would apparently shows in the colour intensity (SPR) to light to dark. The absorbance peak (λmax) of both nanomaterials had shown gradual variations which directly attribute to the growth of the nanoparticles over pH altered SLCCNV template (see Fig. 1A & and B). In another experiment, optimized concentrations of both precursor and viral biotemplate produced the ideal hybrid virus nanomaterials (Au & Ag) that were primarily investigated by UV–Vis absorbance peak (λmax) at 420 nm and 520 nm for silver and gold respectively. As outlined in Fig. 2(A, B, C & –D), the UV–Vis spectral data clearly shown that the concentration of both precursors (Au & Ag) and virus biotemplate systematically played very well for the fabrication of hybrid nanomaterials. However, when precursor concentrations (Au & Ag) was high and virus template concentration was low (3:1), the corresponding absorbance peak (λmax) was shifted to broad in the wavelength indicates that both gold and silver nanoparticles are bigger in size. Similarly, while the virus template concentration was high, whereas the precursor concentration was low (3:1), the UV absorbance peak (λmax) were shifted to sharp at the corresponding wavelength of both gold (∼520 nm) and silver (∼420 nm) which indicated that fabricated metal nanomaterials was in smaller size. At the same Fig. 2(A, B, C & –D), the maximum absorbance peak (λmax) at 520 nm (gold) and 420 nm(silver) was observed for the combination of equal concentrations (1:1) of both virus templates and precursor. It apparently shows that, only equal concentrations (1:1) have yield defined nanomaterials with homogenous sized particles which was assumed by the previous study of characteristic absorption spectrum for both gold and silver nanomaterials (Wolfgang et al., 2007; Alvin et al., 2015).

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Fig. 1

UV/Vis absorbance spectrum of pH-activated-SLCCNV-biotemplate mediated fabrication of nanomaterials with gold (A) and silver (B) precursors. A & B (Inset) shows the absorbance spectrum of one time DDH₂O diluted pH 10-virus hybrid nanomaterial.

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Fig. 2

UV/Vis absorbance spectrum of concentration dependent fabrication of virus hybrid nanomaterial – (A–D); Different ratio of virus template and (Au & Ag) metal ion precursor. Graphs shows the two reaction conditions like standard concentration of virus template with the various concentration of (Ag &Au) precursors (B and C) and secondly, standard concentration of precursors with the various concentration of virus template (A and D).

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3.2

3.2 Characteristic features of SLCCNV-metallic-hybrid nanomaterials (Au & Ag)

3.2.1

3.2.1 Thermostability analysis by UV–Vis spectra

In this study, pH 10 virus template fabricated metal nanomaterials were subjected to thermostability analysis in a colloidal condition at various temperatures. Fig. S4, shows the UV–Vis absorption spectra of hybrid nanomaterials (Au & Ag) which forecasting that particles were responded differently to the temperature variation. However, at 20 °C, 35 °C, 40 °C and 45 °C there was an apparent shift of the absorption maximum (λmax) at 500 nm-–650 nm which regards the progressive particle aggregation only in virus-gold hybrid nanomaterials (see Fig. S4, A). Temperature above 45 °C, the hybrid AuNPs had shown characteristic absorption peak at 520 nm which indicated the stability of the materials even up to 55 °C (see Fig. S4, A). Whereas, in the case of hybrid AgNPs, it showed noticeable aggregation only at 40 °C and 45 °C as the gradual shift of longer wavelength in absorption spectra at 400 nm-500 nm (see Fig. S4, B). Incubated below 40 °C and above 45 °C shows excellent stability in hybrid AgNPs which reached its characteristic absorption maximum at 420 nm. Significantly, temperature at 55 °C, the hybrid AgNPs shows superior thermostability than hybrid AuNPs (see Fig. S4, A & B).

In this study, pH 10 virus template fabricated metal nanomaterials were subjected to thermostability analysis in a colloidal condition at various temperatures. Fig. S4, shows the UV–Vis absorption spectra of hybrid nanomaterials (Au & Ag) which forecasting that particles were responded differently to the temperature variation. However, at 20 °C, 35 °C, 40 °C and 45 °C there was an apparent shift of the absorption maximum (λmax) at 500 nm-–650 nm which regards the progressive particle aggregation only in virus-gold hybrid nanomaterials (see Fig. S4, A). Temperature above 45 °C, the hybrid AuNPs had shown characteristic absorption peak at 520 nm which indicated the stability of the materials even up to 55 °C (see Fig. S4, A). Whereas, in the case of hybrid AgNPs, it showed noticeable aggregation only at 40 °C and 45 °C as the gradual shift of longer wavelength in absorption spectra at 400 nm-500 nm (see Fig. S4, B). Incubated below 40 °C and above 45 °C shows excellent stability in hybrid AgNPs which reached its characteristic absorption maximum at 420 nm. Significantly, temperature at 55 °C, the hybrid AgNPs shows superior thermostability than hybrid AuNPs (see Fig. S4, A & B).

3.2.2

3.2.2 Electron microscopy and DLS analysis

With the aid of electron microscopy, the topology of fabricated virus-metal-hybrid nanomaterials was closely studied (Cao et al., 2011). Very interesting features were found in HRTEM images taken at 100 nm and 50 nm scale. However, Figs. 3 and 4A and B, electron image shown the number of gold and silver nanoparticles uniformly decorated around the viral capsid may resemble the islands of nanoparticle clusters. Similarly, electron images taken at 20 nm scale have evidently shown how majestically a single virus particle decorated with nanoparticles through surface mineralization over their entire capsid surface (see Figs. 3 and 4C). FESEM observation at 100 nm magnification also correlated with the similar morphological features seen in HRTEM images for both hybrid nanomaterials (see Figs. 3 and 4F). The FESEM image is a distinct evidence of surface mineralization capabilities of SLCCNV. From obtained electron images, we could have roughly determined the sizes of the both Au & Ag nanoparticles decorated over the virus capsid is 5–12 nm and 5–20 nm respectively. Though, the accurate size of the nanoparticles distributed over the virus capsid was determined through processing the HRTEM images of ImageJ software (video tutorials also available on YouTube). As shown Fig. 5A and B (inset), histogram represents the size distribution of nanoparticles around the virus capsid summarized by analyzing the corresponding TEM images of gold and silver nanoparticles. It was determined that gold nanoparticles have size ranges between 6 and 14 nm in diameter and silver nanoparticles contain particle size ranges between 10 and 15 nm. Also, a higher number of standard sized particles (∼14 nm) were also detected in the histogram (see Fig. 5A and B).

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Fig. 3

All images corresponding to virus fabricated hybrid gold nanomaterials (A)–(C) HRTEM images taken in various nanoscale bars such as 100 nm, 50 nm and 20 nm shows the virus particles decorated with a densely packed array of gold nanoparticles (D) EDX spectra representing the molecular species, (E) SAED diffraction pattern (F) FESEM image (G) DLS spectrum of hybrid AuNPs. Inset showing pure virus particles with similar graphical representation.

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Fig. 4

All images corresponding to virus fabricated hybrid silver nanomaterials (A)–(C) HRTEM images taken in various nanoscale bars such as 100 nm, 50 nm and 20 nm shows virus particles decorated with a densely packed array of silver nanoparticles (D) EDX spectra representing the molecular species, (E) SAED diffraction pattern, (F) FESEM image and (G) DLS spectrum of hybrid AgNPs.

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Fig. 5

Histogram showing the particle size distribution corresponding to the HRTEM images of (A) Virus hybrid gold nanomaterial and (B) Virus hybrid gold nanomaterial.

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Next Figs. 3 and 4G, the DLS spectroscopic data, we were detected two species in the colloidal medium likely to be individual virus-metallic-hybrid nanomaterial and nanoparticles detached from the virus template. However, a hydrodynamic size of the two species in the medium was varied between gold and silver. Apart from the nanoparticles size already mentioned, the size of the individual both gold and silver virus-metallic-hybrid nanomaterials were determined between 50 and 300 nm and 300–1100 nm respectively. Indeed, the virus particle size has exponentially increased by offering nucleation sites for metal precursor in which nucleation cropped up over the virus, possibly increases the size upon the precursor concentration in the medium. In Figs. 3 and 4D displays an EDX (Energy dispersive X-ray spectroscopy) elemental analysis map which shows the spectrum containing abundant gold and silver elements along with C, N environment confirmed the presence of proteinaceous groups gives higher counts in the map. Especially, there was no other contamination detected in the EDX spectrum, which demonstrates the purity of virus-metallic-hybrid nanomaterials.

The diffraction pattern obtained through SAED had shown peculiar features for hybrid AgNPs. As seen in Fig. 4E, the SAED pattern shows the crystalline orientation in bright spots that resembles the icosahedral (T = 1) symmetry of Squash leaf curl China virus capsid. In the same Fig. 4E, it is visible that the small spots of a polycrystalline pattern of the silver nanoparticle arise from the core virus crystallite. In Fig. 4E, the SAED pattern corresponding to the hybrid AuNPs had shown the polycrystalline nature (arranged in regular three-dimensional lattices) of the virus and its associated gold nanoparticles. In particular, nucleation sites present in the virus capsid transformed their amorphous phase to crystalline phase through the nanofabrication process by the nucleated noble nanomaterials.

3.2.3

3.2.3 Surface Enhanced Raman spectroscopy

SERS allows us to predict the single molecule attachments on virus particles utilized for nanofabrication. In this analysis, three different vibrational excitation spectra corresponding to the reference SLCCNV and two virus-metallic-hybrid nanomaterials was recorded. Fig. 6 A shows a characteristic SER spectrum (A) taken for SLCCNV, vibration peaks are due to Amide I band 80% C⚌O stretch, near 1650 cm⁻¹, Amide II band 60% N—H bend and 40% C—N stretch, near 1550 cm⁻¹, Amide III band 40% C—N stretch, 30% N—H bend, near 1300 cm⁻¹. Near 702 cm⁻¹denoted for tryptophan amino acid and near 1000 cm⁻¹ intense band for phenylalanine amino acid (Xu et al., 2014). Fig. 6B & and C shows typical SER spectra of hybrid nanomaterials, clearly demonstrated that the silver and gold nanoparticles exhibit its originations from the virus proteins. However, comparing the spectrum of SLCCNV, there is quite sensitive relevance observed in the Raman intensity peak, which demonstrates the possibility deprotonation of amino acid (of SLCCNV) between 1000 cm⁻¹ to and 1550 cm⁻¹ by the formation of both nanoparticles. On the Spectra of Fig. 6B & and C, the vibrational energy is little higher in gold nanoparticles than silver nanoparticles. Apparently, new bands detected in spectrum B with longer wavelength 1550 cm⁻¹ to –2400 cm⁻¹corresponds to the gold atom (Seongmin and Xiao, 2013). Whereas in the case of silver nanoparticles, there is no enough vibrational peak detected at 1350 cm⁻¹ stretch which is a strong characteristic resonance peak for silver atom (Watanabe et al., 2005). It is also important to note that all the Raman scattering frequency were obtained in low intensity due to the availability of virus protein and associated nanoparticles (Kevin and Juan, 2011). This similar phenomena was already reported by Segtnan et al. (2009). In his report, the incident laser light will produce autofluorescence at biomaterials, which is usually much more intense than the Raman scattered light. This fluorescence can make the Raman signals difficult or even impossible to measure.

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Fig. 6

SERS spectra from pure SLCCNV protein, SLCCNV fabricated gold nanomaterial and SLCCNV fabricated silver nanomaterial. Characteristic Raman shifts are indicated by the wave number.

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3.3

3.3 Biocompatibility of SLCCNV-metallic-hybrid nanomaterial

Since the MTT assay is a colorimetric assay, the reduction of MTT in viable cells treated with reference SLCCNV and two SLCCNV fabricated hybrid nanomaterials were measured at 595 nm, the data shown in Fig. 7. The cytotoxicity profile of a plant virus shows good biocompatibility features at all the eleven (10–110 μl) concentrations (multiple of 600 μg/μl × 11). Whereas SLCCNV fabricated hybrid nanomaterials treated cells shows reduced viability (70%) up to the concentrations of 90 μl. Further, the cell viability outreached the cytotoxicity cut off range of the concentrations exceeds at <90 μl for both hybrid nanomaterials. In comparison with silver nanomaterials, gold has shown ample better biocompatible features up to 50 μl (>90%).

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Fig. 7

Cytotoxic effect of SLCCNV protein, SLCCNV fabricated gold nanomaterial and SLCCNV fabricated silver nanomaterial on the A549 lung cancer cell line measured by the MTT assay. The percentage of cytotoxicity or biocompatibility (%) plotted against a function of concentration. Data are shown as SD with SE for three different experiments. The line represents the fit to the experimental data points.

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3.4

3.4 Electrical conductivity of SLCCNV-metallic-hybrid nanomaterials

Both gold and silver hybrid nanomaterials visually confirm its ability to conduct the electrical charge by the glow of the LED bulb as soon as the nanomaterials coated glass substrate contact the electrode (see Fig. S1). Surprisingly, the purified virus protein has shown EC properties resulting in the glow of the bulb. Through pico-ammeter, we have obtained a solid evidence of conductivity for all three samples by measuring the resistance of samples at a different voltage. In Fig. 8A illustrates the resistance (ohm) of samples and Fig. 8B a histogram illustrates the conductivity (mS/m) of samples which was calculated from the resistance of samples using by Ohm's law. Only virus hybrid nanomaterials showed resistance properties at the input of voltage (V) from 0.1 to 1 (see Fig. 8A). The pure virus protein shows almost Zero resistance properties at the input of various voltage which means it showed good conductive properties. In the same Fig. 8A, both virus hybrid nanomaterials similarly maintain its resistance in a steady state against the application of an external voltage. Presumably, the hybrid materials will show standard resistance properties and stability against the higher electrical field. As a result, pure virus protein exhibit very low and no resistance properties, whereas hybrid nanomaterials have shown moderate resistance properties. The determination of electrical conductivity (EC) of samples is directly reciprocal of the resistance of the samples. Therefore, there might be an increased conductivity of the samples when there is the increased voltage (V). By the result of electrical conductivity equation (Ohm's law), it was determined that pure SLCCNV protein has elevated conductivity properties (0.031 mS/m) than the other two fabricated nanomaterials. It was 0.006 mS/m (6 × 10⁻⁵ S/cm) for hybrid AgNPs and 0.009 mS/m (9 × 10⁻⁵ S/cm) for hybrid AuNPs. Since the optimal semiconductivity lies between the band gap of 10⁻⁶ to –10⁻² we have achieved a good bio semiconductor material.

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Fig. 8

Electrical conductivity (EC) analysis of SLCCNV protein, SLCCNV fabricated gold nanomaterial and SLCCNV fabricated silver nanomaterial, (A) Pico-ammeter measured materials resistance properties at various voltage sources (0.1–1 V), (B) Histogram represents the electrical conductivity of materials (Data are shown as standard deviation with error bar).

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Both gold and silver hybrid nanomaterials visually confirm its ability to conduct the electrical charge by the glow of the LED bulb as soon as the nanomaterials coated glass substrate contact the electrode (see Fig. S1). Surprisingly, the purified virus protein has shown EC properties resulting in the glow of the bulb. Through pico-ammeter, we have obtained a solid evidence of conductivity for all three samples by measuring the resistance of samples at a different voltage. In Fig. 8A illustrates the resistance (ohm) of samples and Fig. 8B a histogram illustrates the conductivity (mS/m) of samples which was calculated from the resistance of samples using by Ohm's law. Only virus hybrid nanomaterials showed resistance properties at the input of voltage (V) from 0.1 to 1 (see Fig. 8A). The pure virus protein shows almost Zero resistance properties at the input of various voltage which means it showed good conductive properties. In the same Fig. 8A, both virus hybrid nanomaterials similarly maintain its resistance in a steady state against the application of an external voltage. Presumably, the hybrid materials will show standard resistance properties and stability against the higher electrical field. As a result, pure virus protein exhibit very low and no resistance properties, whereas hybrid nanomaterials have shown moderate resistance properties. The determination of electrical conductivity (EC) of samples is directly reciprocal of the resistance of the samples. Therefore, there might be an increased conductivity of the samples when there is the increased voltage (V). By the result of electrical conductivity equation (Ohm's law), it was determined that pure SLCCNV protein has elevated conductivity properties (0.031 mS/m) than the other two fabricated nanomaterials. It was 0.006 mS/m (6 × 10⁻⁵ S/cm) for hybrid AgNPs and 0.009 mS/m (9 × 10⁻⁵ S/cm) for hybrid AuNPs. Since the optimal semiconductivity lies between the band gap of 10⁻⁶ to –10⁻² we have achieved a good bio semiconductor material.

4 Discussion

The destructive plant pathogenic virus of Squash leaf curl China virus presents a confined environment and unique twin icosahedral protein surface topology for noble metallic nanoparticle synthesis and are amenable to nanotechnology manipulations. The metal deposition on SLCCNV has successfully produced the SLCCNV-metallic-hybrid noble nanomaterials at optimized condition related to the ionic strength of virus template and the concentration of both template and precursor ions. In the first approach, the SLCCNV capsid actively potentiated the reduction of both Au and Ag metal precursors by protonation from electronically activated solvent exposed amino acid residues resulting into the virus-metallic hybrid nanomaterials (AuNPs and AgNPs). It was found that viral reduction appeared to be selective for noble metals like gold and silver and the similar work was reported using Tobacco mosaic virus earlier by Bromely et al. (2008) and Dujardin et al. (2003). We are absolutely agree with the another report stated that the preparation of virus templates with altered surface charge by pH is certainly a significant guiding factor for nanofabrication process (Namba and Stubbs, 1986). When the pH (10) conditions were adjusted equal to the isoelectric point of virus protein, produced a highly reactive amino acid functional groups rendered a successful nanofabrication, also evidenced by previous work (Balci et al., 2006; Knez et al., 2004). Amino acids are highly reactive in its native conditions (Tan et al., 2010). Therefore, metal ion binding possibly occur via N-terminal amino and C-terminal carbonyl groups, or through side chains such as the ring nitrogen atom of histidine, or the carboxylate groups of aspartic and glutamic acid. Moreover, metal ion reduction may be facilitated by hydroxyl groups in tyrosine and carboxyl groups in asparagine and glutamine, and indole groups on tryptophan (Tan et al., 2010). All 20 amino acids have their pI (Isoelectric point) and pKa values (the acid ionization constant of a solution) determined by the pH of the medium in which the protein exists. In our case, we are not certain about what type of amino acids involved in the surface mineralization process. However, it can be determined by the pH-dependent characteristics of the virus protein and its pKa Values, where pH = pI (the number of negative and positive charge is equal). Following the same phenomenon, we have used pH 10 buffer dialyzed viral protein as a template for surface mineralization. The pH-activated virus template at their neutral charge facilitates a highly reactive nucleation sites (primary COO⁻) which could draw charged metal ions (Au⁺ and Ag⁺) to initiate the mineralization process and that point of time are called as nucleation point (Douglas and Young, 1998; Dujardin et al., 2003). After the initial nucleation, more precursor ions in the reaction solution electrostatically drive to the nucleation site to further growth of nanoparticles over the virus template (Nguyen et al., 2014). Reduction of the gold precursor at the virus template produced the colloidal suspension in wine red colour with the characteristic absorbance spectra near 520 nm and reduction of silver precursor produced a yellow coloured colloidal suspension with absorbance spectra near 420 nm. Both gold and silver virus hybrid nanomaterials were fabricated within 5–10 min of exposure to direct sunlight. At the time, the concentration factor also plays a significant role in this nanomaterial fabrication (Chandran et al., 2006; Monaliben et al., 2015) where evidenced by UV–Vis absorption spectra, DLS and nanoparticles size distribution analysis by ImageJ (see Figs. 2, 3G, 4G and 5). As a result, it produced a narrow size distribution during a metal deposition over virus templates. Where the lower concentration of precursors produced smaller particles and higher concentration produced larger ones. Similarly, low concentration of viral template offers a minimum number of nucleation sites and a higher concentration of viral template offer a higher number of nucleation sites where possibly produce the homogenous size nanoparticle by adjusting the precursor concentration. An important feature perceived in all electron microscopy images is all nanoparticles seems to be unconnected to each other with sub-nanometer scale distances (Figs. 3 and 4). Basically, the virus capsid has well defined structural symmetry with multiple protein subunits. Every subunit in the capsid is eligible to act as a biotemplate for nanoparticle synthesis. Therefore a single virus can act as a “nanofactory” to synthesis 'n' number of particles with a defined size. Figs. 3 and 4F FESEM images also reveal the shape of the nanoparticles such as gold nanoparticles are observed in truncated octahedron (sphere) shape, whereas silver nanoparticle was found to have anisotropic nanostructures like truncated octahedron and tetrahedron (Pedro et al., 2015). The EDX observations confirm the presence of virus protein and associated metal precursor in the pure form. It also suggested that there are no other elements involved in the hybrid nanomaterials fabrication (Figs. 3 and 4D). SAED diffraction pattern of both hybrid nanomaterials paves the new way to study the symmetry of Begomovirus capsid protein. However, upon analyzing the diffraction pattern, it seems that every point of symmetry on virus capsid possibly embedded by metal nanoparticles principally gave the diffraction orientation pattern where exactly mimics the icosahedral symmetry of Begomovirus (Fig. 4E). By indexing the SAED crystal diffraction pattern, it is elegant to predict the number of nucleation sites rendered by SLCCNV capsid (Figs. 3 and 4E). But it requires some comprehensive physical data analysis.

The in-vitro cytotoxicity assay (MTT) supports our fabricated nanomaterials that are robust to use for biomedical applications near future. Gold hybrid nanomaterials treated A549 lung cancer cells shown better viability and biocompatible features than silver hybrid nanomaterials treated at the same concentration (Fig. 7). We also identified statistically significant differences between virus protein and its fabricated nanomaterials. However, the purified virus protein shows no significant cytotoxicity level at higher concentrations when compared to both virus fabricated nanomaterials. We, therefore, believes that a combination of bio and inorganic and the relatively large surface area of hybrid nanomaterials were slightly elevated the toxicity level compared to virus particles. These results are consistent with Blandino et al. (2015), who demonstrated no toxicity by plant viruses to human cells. In contrast, both virus fabricated nanomaterials had shown better biocompatibility in low concentrations ranging from 10 to 90 μl (Fig. 7). The 24 h incubation period for virus fabricated nanomaterials is considered as a stringent time duration to study the viability of cells, whereas it would increase if the incubation period reduced from 24 h (Nagvi et al., 2010).

A functional LED light device was fabricated by embedding the SLCCNV-noble metal hybrids and SLCCNV protein in a glass substrate which was connected between two copper electrodes along with the battery (Fig. S1). The embedded materials including, virus protein exhibited electrical conductance switching behaviour were easily detectable by LED light. Tremendously, SLCCNV protein ignites the LED due to its proven electrical conductive capacity. We had never envisaged this result from pure SLCCNV particles that have electrical conductivity properties. However, it is possible because of the architecture of the virus capsid made of mutiple protein monomers which resembles a crystalline arrangement of molecules. There are few similar reports found on peptides that can act as biological conductors (Hauser and Zhang, 2010; Flynn et al., 2003). At the same time, conductivity measurement also provides detailed confirmation about the electrical conductivity properties of SLCCNV templated nanomaterials (Fig. 8A), which make them appear under the semi-conductive band gap since it contains both resistance and conductivity properties (Gagan et al., 2015). A semiconductor is a material which has electrical conductivity to a degree between a conductor and an insulator. E.g., Silicon, Germanium, carbon, etc. (Sze, 2002). Thus, virus templates can be used to produce non-toxic, high semi-conductive organic based nanomaterials. These results suggest that nanoscale virus metallic hybrid material thought to be an indispensable component in future electronics. The nanoscale integrated circuit also produces other fringe benefits. The nanoscale circuits consume less power than conventional circuits, and they cost less to operate. They generate less heat and, therefore, generally do not require elaborate cooling or ventilation systems. The nanoscale circuits are also capable of operating at higher speeds because it takes nanoseconds for signals to travel through them. Their virus hybrid nanomaterials semi-conductivity properties may be altered in useful ways by the controlled deposition of metal ion precursors.

A functional LED light device was fabricated by embedding the SLCCNV-noble metal hybrids and SLCCNV protein in a glass substrate which was connected between two copper electrodes along with the battery (Fig. S1). The embedded materials including, virus protein exhibited electrical conductance switching behaviour were easily detectable by LED light. Tremendously, SLCCNV protein ignites the LED due to its proven electrical conductive capacity. We had never envisaged this result from pure SLCCNV particles that have electrical conductivity properties. However, it is possible because of the architecture of the virus capsid made of mutiple protein monomers which resembles a crystalline arrangement of molecules. There are few similar reports found on peptides that can act as biological conductors (Hauser and Zhang, 2010; Flynn et al., 2003). At the same time, conductivity measurement also provides detailed confirmation about the electrical conductivity properties of SLCCNV templated nanomaterials (Fig. 8A), which make them appear under the semi-conductive band gap since it contains both resistance and conductivity properties (Gagan et al., 2015). A semiconductor is a material which has electrical conductivity to a degree between a conductor and an insulator. E.g., Silicon, Germanium, carbon, etc. (Sze, 2002). Thus, virus templates can be used to produce non-toxic, high semi-conductive organic based nanomaterials. These results suggest that nanoscale virus metallic hybrid material thought to be an indispensable component in future electronics. The nanoscale integrated circuit also produces other fringe benefits. The nanoscale circuits consume less power than conventional circuits, and they cost less to operate. They generate less heat and, therefore, generally do not require elaborate cooling or ventilation systems. The nanoscale circuits are also capable of operating at higher speeds because it takes nanoseconds for signals to travel through them. Their virus hybrid nanomaterials semi-conductivity properties may be altered in useful ways by the controlled deposition of metal ion precursors.

5 Conclusion

It is likely that most virus capsids are more dynamic in structure, rather than static cages, whose transitions are only now being revealed (Whaley et al., 2000). The viral capsid interacts with its environment primarily at the external surface. Modification of the external surfaces with small molecules can direct the cage toward specific interactions with both biological and non-biological surfaces. The repetitive structure of the capsid exterior surface serves as a platform for multivalent presentation, providing a significant advantage over traditional materials processing (Sarikaya et al., 2003; Lewis et al., 2006; Sun et al., 1993). Few viruses displaying nucleation properties have been used as templates for material synthesis. Also, the nucleation peptides identified by phage display can be engineered into another virus and virus-like protein cage architectures for direct nucleation and spatial control over mineral particle growth (Flynn et al., 2003; Porta et al., 2003; Chatterji et al., 2002).

In this research study, we have achieved to fabricate the noble metal nanomaterials using destructive plant pathogenic virus of Squash leaf curl China virus as a biotemplate. Both fabricated virus-metallic-hybrid nanomaterials are of great importance while it has synthesised at a high degree of control. All the characteristic features analyzed by instrumental methods provide sufficient information about the virus-hybrid-metallic nanomaterials and have revealed few unique properties. Using SLCCNV biotemplates as a “nanofactory” can produce customized size variant metal nanoparticles depending on the amount of precursor and virus template. Though it can be easily separated from the template, it might be implemented in numerous applications like gene delivery, drug delivery and highly sensitive diagnostic assays where the size of the particles is crucial. The durability of hybrid nanomaterials towards higher temperature shows as a sensitive part of semi-conductor material which directly impact the conduction band (Varshni, 1967). Intriguingly, the biocompatibility study along with wild SLCCNV and its fabricated hybrid nanomaterial shows good biocompatible features for the first time ever in the history of viral nanotechnology (Steinmetz and Manchester, 2011). In the consequences of the result, we ultimately reached our objective of this research work, that is a virus hybrid nanomaterials have shown good electrical conductivity which was validated by the simple handmade device along with pico-ammeter. Hence, using SLCCNV biotemplate, it is possible to generate a conductive matrix within a few minutes at any place under the direct sunlight which is an important criterion to enable access to advances in semi-conductive material systems. Since naturally occurring viral particles rarely feature the functional groups, SLCCNV can mediate the surface mineralization with its available amino acid residues (Klem et al., 2003; Andrew et al., 2015; Blum et al., 2005; Kramer et al., 2004; Lee et al., 2002).

Therefore, from a materials science point of view, there are many interesting possibilities to be explored using this Squash leaf curl China virus combined with inorganic materials to develop new applications which is cost effective, simple and rapid. Remarkably, we discovered some novel characters in many instances on our SLCCNV fabricated hybrid metallic nanomaterials, which may contribute to the advances of virus nanotechnology without any doubt.