Electroless Deposition of Noble Metals on Rod-Shape Plant Viruses in Various Aqueous Metal Precursor Solutions

The challenge of synthesizing noble metal nanostructures sustainably has encouraged researchers to explore biological routes for nanostructure production, such as biotemplating. Plant viruses with rod-shape morphology, such as tobacco mosaic virus (TMV) and barley stripe mosaic virus (BSMV), offer promising biotemplates to produce metal nanorods. TMV and BSMV can be incubated in aqueous metal precursor solutions to mineralize metals on the coat proteins (CPs) of the viruses. Previous studies have primarily examined palladium (Pd) mineralization on TMV and BSMV using Na2PdCl4 as the Pd precursor. There is limited scientific literature on the effect of using alternative Pd precursor solutions besides Na2PdCl4 such as K2PdCl4 and PdCl2 to mineralize Pd on TMV and BSMV. Past attempts at mineralizing other noble metals such as platinum (Pt) and gold (Au) required an initial layer of Pd to be deposited on the TMV and BSMV biotemplates. In this study, we aimed to expand the understanding of using alternative Pd precursor solutions to mineralize Pd on TMV and BSMV. Additionally, the deposition of Pt and Au onto TMV and BSMV without the need for an initial Pd mineralization layer was achieved using alternative Pt and Au precursors, including K2PtCl4 and AuCl3, respectively. Pd, Pt, and Au were successfully deposited on TMV and BSMV by incubation in aqueous solutions of Na2PdCl4, K2PdCl4, PdCl2, K2PtCl4, and AuCl3. Kinetic studies were also conducted using ultraviolet–visible (UV–vis) spectroscopy to examine the rates at which Pd, Pt, and Au precursor ions were reduced during the mineralization process, mimicking their adsorption onto TMV and BSMV CPs. BSMV adsorbed noble metal precursor ions faster than TMV as determined by UV–vis spectroscopy. While palladium nanorods (PdNRs) offer high electrical conductivity desirable for electronic applications, Pd-coated TMV and BSMV may face limitations due to their organic cores, potentially compromising conductivity. To address this, one approach is to convert the organic core into conductive amorphous carbon through thermal annealing. In this study, in situ transmission electron microscopy was utilized to thermally anneal Pd-TMV2Cys, thereby transforming them into PdNRs with amorphous carbon cores.


INTRODUCTION
Noble metals such as palladium (Pd), platinum (Pt), and gold (Au) have a wide range of applications in science and technology due to their antimicrobial, 1 conductive, 2 corrosion resistive, 3 and catalytic properties. 4Nanoscale utilization of noble metals has the potential to create applications in fields like biotechnology, 5 food processing, 6 and semiconductors. 7anoscale applications of noble metals require the production of Pd, Pt, and Au nanostructures with morphologies such as nanospheres, 8 nanotubes, 9 nanowires (NWs), 10 and nanorods (NRs). 11The bottom-up chemical synthesis of 1-dimensional (1D) noble metal nanostructures, such as NWs and NRs, has captured the attention of researchers due to their application in electronics 12,13 , catalysts, 14 sensors, 15 and drug delivery. 81D metal nanostructures are conventionally synthesized by using the polyol process.However, this process is unsustainable, because it uses toxic polyols such as ethylene glycol (EG) as the reducing agent and solvent to synthesize metal nanostructures. 16,17Biotemplating is a more sustainable approach to synthesizing 1D noble metal nanostructures because it foregoes the use of hazardous reagents and requires less energy.Biotemplates, such as viruses, 18,19 virus-like particles (VLPs), 20 bacteria, 21 DNA, 22 and other biological agents, have been used for the synthesis of metal nanostructures with noteworthy properties.Rod-shape viruses are of particular interest due to their application in the production of metal NRs. 18,23Rod-shape virions such as tobacco mosaic virus (TMV) 23 and barley stripe mosaic virus (BSMV) 18 have been shown to possess the surface properties for mineralization of metals on their capsid proteins (CPs).The amino acid residues present on the CPs, such as aspartic (Asp), cysteine (Cys), threonine (Thr), and glutamic (Glu) acid, contain functional groups like hydroxyl, carboxyl, and amine groups that undergo oxidation while reducing complex metal ions to form metal atoms. 18The formed metal atoms are deposited on the CPs surface; thereby resulting in metal mineralized biotemplates. 18,23TMV and BSMV can be extracted sustainably from natural sources; therefore, using these biotemplates is an environmentally sensitive procedure for metal NR synthesis.Metal mineralization on the surfaces of TMV and BSMV is possible without the use of an external reducing agent, in an aqueous solution, and at low temperatures.Therefore, TMV and BSMV can be used for the sustainable production of noble metal NRs, such as palladium nanorods (PdNRs), 18 platinum nanorods (PtNRs), 24 and gold nanorods (AuNRs). 25TMV and BSMV with metal mineralization on their CPs have been successfully employed in a range of applications, including but not limited to electronics, 3,26,18 catalysts, 27 sensors, 28 and energy storage. 29MV is a rod-shape virus with a length of 300 nm and a diameter of 18 nm.The 2,130 CP subunits are 17.5 kDa and arranged helically with 49 subunits in three turns of the helix. 30he ribonucleic acid (RNA) of TMV is embedded around the central channel of the particle.TMV2Cys contains two additional cysteine amino acid residues added to positions 2 and 3 of the CP sequence as surface modifications.The additional cysteine residues facilitate more dense metal mineralization on the CPs of TMV2Cys. 31BSMV rod-shape particles are 110−150 nm in length and 20 nm in diameter.The 25 kDa CP subunits are helically arranged with 26 CP subunits per turn. 32TMV2Cys was explored due to improved metal mineralization promoted by the additional amino acid residues.BSMV without any additional amino acid residue modifications was also explored because BSMV adsorbs reacting metal ions using both covalent and electrostatic interactions.TMV relies solely on covalent interactions for metal ion adsorption; therefore, BSMV is a more promising biotemplate for metal mineralization than TMV. 18revious studies have comprehensively examined the effect of Pd mineralization on TMV and BSMV using kinetic and parametric studies. 18,33,34Deposition of other metals such as cobalt (Co), silver (Ag), copper (Cu), and iron (Fe) on TMV and BSMV CPs has been challenging because Co 2+ , Ag + , Cu + , and Fe 2+ ions have less positive reduction potentials compared to Pd 2+ .Therefore, Co, Ag, Cu, and Fe do not readily mineralize on TMV and BSMV similar to Pd. 33 Previous studies have achieved a higher degree of success in mineralizing Au, Co, Fe, and Ni onto TMV by employing an initial Pd coating layer.This increased effectiveness can be attributed to the catalytic reduction of metal ions facilitated by the presence of Pd on the CPs. 3,25,35This study fine-tuned the mineralization of Au and Pt to deposit Au and Pt directly onto the TMV and BSMV without an initial Pd coating.In prior studies, the mineralization of Pd onto TMV and BSMV was achieved through the incubation of the particles in solutions containing Na 2 PdCl 4 . 18,20,36This study investigated the impact of employing metal precursor solutions other than Na 2 PdCl 4 for depositing Pd onto TMV and BSMV.Transmission electron microscopy (TEM) characterization was employed to observe the mineralization of Pd, Pt, and Au on TMV and BSMV.Ultraviolet−visible (UV−vis) spectroscopy was used as an indirect measure of the progress of Pd mineralization to investigate the kinetics of adsorption and mineralization of Pd, Pt, and Au ion species onto TMV and BSMV.Our study introduces the use of alternative Pd precursors for the electroless deposition of Pd nanoparticles on TMV and BSMV, demonstrating several advantages over traditional precursors like Na 2 PdCl4 or PdCl 2 .These alternative precursors offer cost efficiency as well as higher deposition rates.Furthermore, we achieved the direct deposition of Pt and Au onto TMV and BSMV without the need for an initial Pd coating, simplifying the process and reducing the costs.Our investigation into the kinetics of metal nanoparticle mineralization provides novel insights, enhancing the versatility and applicability of TMV and BSMV as biotemplates.

RESULTS AND DISCUSSION
2.1.TMV2Cys and BSMV Biotemplates.TMV2Cys and BSMV were characterized using TEM as shown in Figure 1.The widths of the biotemplates were measured and recorded to be 20 ± 0.9 and 18 ± 0.7 nm for BSMV and TMV2Cys, respectively.
2.2.Palladium Mineralization on TMV2Cys and BSMV Using Different Pd Precursors.TMV2Cys and BSMV were coated with Pd using the directions presented in the experimental details section.The images obtained from TEM characterization of Pd-coated BSMV (BSMV-Pd) and Pd- coated TMV2Cys (TMV2Cys-Pd) are displayed in Figure 2a− f.The presence of Pd mineralization on the surface of TMV2Cys incubated in Na 2 PdCl 4 solution was further confirmed using energy-dispersive X-ray spectroscopy (EDS) characterization as shown in Figure 2g.
No Pd mineralization was observed when TMV2Cys and BSMV were incubated in Na 2 PdBr 4 solution; therefore, the samples were not characterized using TEM.The average diameter of the BSMV-Pd and TMV2Cys-Pd produced using the different Pd precursor solutions were measured and are tabulated in Table 1.Based on Table 1, BSMV-Pd particles were thicker than TMV2Cys-Pd produced using each Pd precursor solution.The thickest layers of Pd deposition were observed on the CPs of BSMV-Pd and TMV2Cys-Pd that were produced by using PdCl 2 as the metal precursor.
Pd mineralization occurred exclusively on TMV2Cys and BSMV when the biotemplates were incubated in Pd precursor solutions containing chlorides, including Na 2 PdCl 4 , K 2 PdCl 4 , and PdCl 2 .This phenomenon is attributed to the adsorption of metal precursor ions onto the CPs of the biotemplates through the ligand-switching mechanism of chlorides. 18   BSMV-Pd virions were thicker than those of TMV2Cys-Pd.BSMV isoelectric point is 4.5 compared to that of TMV2Cys at 3.5; therefore, there are more positively charged functionalities in BSMV. 40,41The positively charged functionalities of BSMV permit the adsorption of metal precursor ions onto the CPs using both electrostatic and covalent interactions; therefore, resulting in the adsorption of more Pd precursor ions.TMV2Cys depends solely on covalent interactions; therefore, fewer Pd precursor ions are adsorbed during incubation. 42SMV-Pd and TMV2Cys-Pd, produced by incubating BSMV and TMV2Cys in PdCl 2 , exhibit thicker layers of Pd mineralization compared to BSMV-Pd and TMV2Cys-Pd produced using Na 2 PdCl 4 and K 2 PdCl 4 solutions.Chloride ions are released into the aqueous solution after the PdCl 4 2− ions are converted to Pd atoms by the amino acid residues of the CPs.As a result, a 0.75 mM PdCl 2 solution has a lower concentration of chloride ions compared to 0.75 mM Na 2 PdCl 4 and K 2 PdCl 4 solutions.43 The chloride ions present in the incubation solution react with the amino acid residues of TMV2Cys and BSMV CPs, leading to the chlorination of these amino acid residues.44 The higher concentration of chloride ions in the Na 2 PdCl 4 and K 2 PdCl 4 solutions results in comparatively greater number of amino acid residues of TMV2Cys and BSMV being chlorinated during incubation.Chlorinated amino acid residues are unable to participate in the ligand-switching process necessary for PdCl 4 2− ions to adsorb onto biotemplates.Based on Figures 3 and 4 Based on Figures 3 and 4, the adsorption of PdCl 4 2− ions occurred most rapidly when BSMV and TMV2Cys were incubated in a PdCl 2 solution.This can be attributed to the higher availability of amino acid residues for adsorbing PdCl 4 2− ions.According to Figures 3 and 4, the concentration of PdBr 4 2− ions remained constant during the 20 min incubation period.The PdBr 4 2− ions lack chlorides required for ligand switching necessary to adsorb the PdBr 4 2− ions onto the outer surfaces of BSMV and TMC2Cys.

Platinum and Gold Mineralization on TMV2Cys
and BSMV.TMV2Cys and BSMV biotemplates were incubated in solutions of K 2 PtCl 4 and AuCl 3 to mineralize Pt and Au on their CPs, respectively.The BSMV-Pt, BSMV-Au, TMV2Cys-Pt, and TMV2Cys-Au samples generated from the reactions were subjected to TEM microscopy for the acquisition of the images depicted in Figure 5.The thickness of Pt and Au mineralization on the biotemplates was measured and is provided in Table 1.Based on Table 1, the layers of Pt mineralization on BSMV-Pt and TMV2Cys-Pt were thicker compared to Pd mineralization on BSMV-Pd and TMV2Cys-Pd.The layers of Au mineralization on BSMV-Au and TMV2Cys-Au were thicker compared with Pt mineralization on BSMV-Pt and TMV2Cys-Pt.The metals that dissociate into ions with more positive reduction potentials are more easily mineralized on the CPs of the BSMV and TMV2Cys. 33hicker layers of metal coating were achieved when BSMV or TMV2Cys was incubated in K 2 PtCl 4 , compared to BSMV or TMV2Cys incubated in Pd precursors, because Pt 2+ ions have a more positive reduction potential than Pd 2+ ions.Similarly, thicker layers of metal coating were achieved when biotemplates were incubated in AuCl 3 , compared to biotemplates incubated in K 2 PtCl 4 .
BSMV-Pt and BSMV-Au exhibited thicker metal coatings compared to those of TMV2Cys-Pt and TMV2Cys-Au.This difference is attributed to BSMV having a higher isoelectric point than TMV2Cys, resulting in a greater abundance of positively charged functionalities on BSMV. 40,41The positive functionalities of BSMV facilitate the adsorption of metal precursor ions onto the CPs through a combination of electrostatic and covalent interactions, leading to the increased adsorption of metal precursor ions.TMV2Cys relies exclusively on covalent interactions, resulting in fewer Pt and Au precursor ions being adsorbed during incubation.This led . 45The faster rate of ion diffusion contributes to the faster adsorption of AuCl 4 − ions by  2.6.Thermal Annealing of Pd-Coated TMV2Cys.To achieve a thicker layer of metal mineralization suitable for thermal annealing, five cycles of Pd mineralization was conducted.The resulting TMV2Cys-Pd was characterized by using EDS prior to thermal annealing, as illustrated in Figure 8. EDS mapping was also conducted to investigate the elemental composition of the Pd-coated TMV, as displayed in Figure 8.The presence of carbon, oxygen, and nitrogen, confirmed by Figures 8a, b, and c, respectively, signifies that the biotemplate is composed of proteins.The presence of the mineralized Pd coating is also confirmed, as shown in Figure 8d.
Surface changes on the virion were observed at approximately 200 °C, where smaller particles began to coalesce into larger ones.The particle sizes continued to increase as the temperature was raised to 300 and 400 °C, as depicted in Figure 9.This phenomenon can be attributed to Ostwald ripening, wherein larger particles grow at the expense of smaller ones in pursuit of a more thermodynamically favorable state.The resulting larger particles reduce the number of grain boundaries present on the Pd coating, thereby improving conductivity.
The surface of the TMV2Cys with Pd coating appears smoothest at 200 °C, despite the formation of fewer grain boundaries at 300 and 400 °C due to the aggregation of larger particles.The smooth surface observed at 200 °C makes it the appropriate temperature for annealing Pd-coated TMV2Cys.

CONCLUSIONS
Based on the findings of this study, noble metal mineralization was observed when TMV2Cys and BSMV were incubated in aqueous solutions of Na 2 PdCl 4 , K 2 PdCl 4 , PdCl 2 , K 2 PtCl 4 , and AuCl 3 .Specifically, BSMV-Pd and TMV2Cys-Pd were produced when BSMV and TMV2Cys were incubated in Pd precursors, such as Na 2 PdCl 4 , K 2 PdCl 4 , and PdCl 2 .However, when BSMV and TMV2Cys were incubated in a Na 2 PdBr 4 solution, Pd mineralization did not occur.This is because Na 2 PdBr 4 dissociates into PdBr 4 2− ions, which contain bromide ligands.Metal ion complexes with bromide ligands have reduction potentials lower than those containing chlorides; therefore, amino acid residues are unable to reduce PdBr Future research on metal NR synthesis using TMV2Cys and BSMV should explore their applications in various novel areas.This includes their utilization as nanocatalysts with the organic core serving as the substrate for supporting noble metal particles for catalytic applications.Additionally, conducting Xray photoelectron spectroscopy, X-ray diffraction, and X-ray absorption spectroscopy will further elucidate the structural and oxidation state information on the Pd coatings, while providing deeper insights into the mineralization mechanism.Optimization of thermal annealing processes can be pursued to convert the metal-coated biotemplates into single-crystal metal NRs; thereby producing noble metal−carbon electrodes suitable for electronic applications.One potential application is the formulation of conductive inks and the manufacturing of transparent conductive patterns using thermally annealed noble metal-coated TMV and BSMV.Additionally, parametric studies are essential for the mineralization of Au and Pt on TMV2Cys and BSMV, as there is limited scientific literature on coating viral biotemplates with these noble metals compared to the more commonly studied Pd.VLPs of TMV2Cys and BSMV can be produced in bacteria, such as Escherichia coli (E.coli), with designer CPs, which could not be produced in plants because they would be noninfectious.The combination of VLPs with the methods established in this study, along with biotemplate removal through thermal annealing, represents the comprehensive series of steps required for the sustainable production of noble metal NRs using rod-shape viral biotemplates.

Figure 1 .
Figure 1.TEM images of BSMV (a) and TMV2Cys (b) without a metal coating.
Na 2 PdCl 4 , K 2 PdCl 4 , and PdCl 2 dissociates into PdCl 4 2− ions in aqueous solution.PdCl 4 2− is a decahedral ion with four chlorides connected to a central Pd atom.The chlorides of PdCl 4 2− participate in ligand exchange with the functional groups of the amino acid residues present on the CPs of TMV2Cys and BSMV; thereby, adsorbing the PdCl 4 2− ions onto the outer surface of the virions.The adsorbed PdCl 4 2− ions are converted into Pd atoms by the electron-rich functional groups such as amine and carboxyl groups present in amino

2 . 3 .
Kinetic Study of Palladium Precursor Reduction on TMV2Cys and BSMV.A kinetic study was conducted to examine the mechanism of adsorption of Pd precursor ions onto TMV2Cys and BSMV biotemplates.Pd mineralization reactions were conducted with a spectrophotometer at 55 °C.UV−vis readings were taken in 5 min intervals, and the intensity of the absorption peak at 425 nm (corresponding to the PdCl 4 2− ion precursor) was converted to PdCl 4 2− ion concentration using the calibration curve.The concentration of PdCl 4 2− ions recorded at a specific time represents the Pd precursor remaining in the reaction solution that has not yet mineralized on the CPs of TMV2Cys or BSMV.The change in the concentration of PdCl 4 2− ions with time demonstrates the rate at which PdCl 4 2− ions are adsorbed onto the viral biotemplates.The concentration of Pd precursor ions versus time for BSMV incubated in Na 2 PdCl 4 , K 2 PdCl 4 , Na 2 PdBr 4 , and PdCl 2 solutions is displayed in Figure 3, and the concentration of Pd precursor ions versus time for TMV2Cys incubated in Na 2 PdCl 4 , K 2 PdCl 4 , Na 2 PdBr 4 , and PdCl 2 solution is displayed in Figure 4.
, PdCl 4 2− ions were adsorbed rapidly by the BSMV and TMV2Cys during the first 5 min of the incubation, followed by a more gradual rate of PdCl 4 2− ion adsorption during the remaining 15 min of incubation.The initially rapid rate of PdCl 4 2− ion adsorption is due to the large number of amino acid residues available for metal mineralization on the CPs of BSMV and TMV2Cys that adsorb the PdCl 4 2− . 18As the incubation period progresses, Pd covers a greater number of sites on the CPs.Consequently, fewer amino acid residues remain available to adsorb PdCl 4 2− ions, leading to a reduction in the rate of PdCl 4 2− ion adsorption after the initial 5 min of incubation.PdCl 4 2− ions were adsorbed faster by BSMV compared to that by TMV2Cys.PdCl 4 2− ion concentration after the first 5 min of incubation is about 20% lower when BSMV is incubated in Pd precursor solutions, compared to TMV2Cys.BSMV virions use both electrostatic and covalent interactions to adsorb PdCl 4 2− ions onto the CPs of the virions, unlike TMV2Cys which depends solely on covalent interactions to adsorb PdCl 4 2− ions; therefore, BSMV adsorbs precursor ions faster compared to TMV2Cys.PdCl 2 solution contains a lower concentration of chloride ions compared to Na 2 PdCl 4 and K 2 PdCl 4 solutions; therefore, fewer amino acid residues are chlorinated when BSMV and TMV2Cys are incubated in PdCl 2 .

Figure 6 .
Figure 6.Metal precursor ion concentration versus time for BSMV incubated in a K 2 PtCl 4 and AuCl 3 solution.

Figure 7 .
Figure 7. Metal precursor ion concentration versus time for TMV2Cys incubated in K 2 PtCl 4 and AuCl 3 solution.

Figure 8 .
Figure 8. EDS mapping of TMV2Cys-Pd coated with five cycles of Pd coating.

Figure 9 .
Figure 9. TEM images of TMV2Cys-Pd with five layers of coating at different temperatures during in situ TEM.

4 2 −
ions to cause Pd mineralization.TMV2Cys and BSMV effectively adsorb only salts containing chlorides.Therefore, Na 2 PdCl 4 , K 2 PdCl 4 , and PdCl 2 are suitable candidates for depositing Pd on TMV2Cys and BSMV, as these salts dissociate into PdCl 4 2− ions.The thickest coating of Pd mineralization was produced when TMV2Cys or BSMV were incubated in PdCl 2 because fewer chloride ions were present in its solution compared to Na 2 PdCl 4 and K 2 PdCl 4 .The lower occurrence of chloride ions reduces the chlorination of amino acid residues present on TMV2Cys and BSMV CPs; therefore, more amino acid residues are available to adsorb PdCl 4 2− ions when TMV2Cys and BSMV are incubated in PdCl 2 .Thicker Pd coating was observed when BSMV was incubated in Pd precursor solutions because BSMV uses both electrostatic and covalent interactions to adsorb the precursor ions onto its CPs, unlike TMV which depends solely on covalent interactions.K 2 PtCl 4 and AuCl 3 were chosen as the precursor salts for mineralizing Pt and Au on TMV2Cys and BSMV CPs.K 2 PtCl 4 and AuCl 3 are soluble in aqueous solution and dissociate into PtCl 4 2− and AuCl 4 − ions, respectively.Both PtCl 4 2− and AuCl 4 − ions contain chlorides that participate in the ligandswitching process, which cause the ions to adsorb onto TMV2Cys and BSMV during incubation.BSMV-Pt and TMV2Cys-Pt were successfully produced by incubating BSMV and TMV2Cys in K 2 PtCl 4 .BSMV-Au and TMV2Cys-Au were successfully produced by incubating BSMV and TMV2Cys in AuCl 3 .There were thicker layers of Au mineralization on BSMV-Au and TMV2Cys-Au, compared to Pt mineralization on BSMV-Pt and TMV2Cys-Pt.BSMV-Pt and TMV2Cys-Pt had thicker layers of Pt mineralization compared to those of BSMV-Pd and TMV2Cys-Pd.Au mineralizing on BSMV or TMV2Cys CPs at a faster rate compared with Pt resulted in faster adsorption of Au relative to Pt.The more rapid adsorption rates produced thicker layers of metal mineralization.TMV2Cys-Pd was also annealed and characterized using in situ heating TEM, and it was demonstrated that the PdNRs with best surface smoothness were achieved at a temperature of around 200 °C.

Figure 10 .
Figure 10.Schematic of depositing Pd, Pt, and Au on TMV2Cys and BSMV via incubation in the corresponding metal precursor solutions.