**3. Synthesis of nanomaterials by controlling process parameter**

It is well known that nanoparticles have widespread applications in various field such as solar cells, photo-thermal cancer treatment, controlled drug delivery, catalysis etc., because of its tunable optical, electrical and catalytic properties which strictly depends on its structure and morphology of the particle. Therefore, nowadays, researchers have devoted substantial effort to have proper control on the shape and size of nanoparticles by simply controlling the fundamental physical and chemical parameters. One of the main advantages of In-Liquid Plasma method for nanofabrication is that it provides flexibility in controlling both the physical (plasma) as well as the chemical (solution) parameters. However, in the chemicalbased synthesis, only solution parameters and in the gas phase plasma synthesis of nanomaterial, only plasma parameters can be controlled. Process parameters have a very significant role in the morphology and composition of nanomaterials.

## **3.1 Variation of physical parameters**

In the plasma – liquid interface, due to the interaction of energetic electrons with the liquid medium, various reactive species are formed, which play a significant role as the reducing agent in nanomaterial synthesis. Interestingly, these plasmagenerated reducing agents are directly related to the discharge voltage and current applied to generate plasma. Therefore, the proper understanding of the role of discharge voltage and current on nanomaterial synthesis is very essential. Saito et al. investigated the size of Au nanoparticles by varying the discharge voltage [18]. For the applied voltage of 1600 and 3200 V, after 1 min of plasma discharge, dendrite shape nanoparticles of size 150 nm and after 5 min of discharge 50 nm nanoparticles were observed. After 20 mins of discharge, for 1600 V applied voltage, a slight change of the size of the nanoparticles was observed. However, for 3200 V applied voltage, the particle size decreased significantly with some anisotropic shapes such as triangular, pentagonal and hexagonal. After 45 mins of discharge, the size of the nanoparticles reduced up to 20 nm. During the experiment, they also observed that the pH of the solution (HAuCl4) decreased with the increase of discharge time. The change in pH explains the formation small nanoparticles. At low pH, gold nanoparticles dissolve and the reduction rate of gold ion decreases, which leads to a reduction in nanoparticle size and formation of exotic or anisotropic shapes. Ashkarran et al. reported the effect of discharge current on the size of Zirconium oxide (ZrO2) nanoparticles [16]. For 10 A and 20 A arc current, the average size (diameter) of the spherical particles were 21 nm and 42 nm respectively, i.e. size of the particles increased with the increase in discharge current. As the smaller particles have a larger specific surface area, therefore, they observed

*In-Liquid Plasma: A Novel Tool for Nanofabrication DOI: http://dx.doi.org/10.5772/intechopen.98858*

higher photocatalytic activity of the nanoparticles synthesised at lower discharge current. However, Ziashahabi et al. reported contradictory results, where they observed a decrease in the size of the Zn/ZnO nanocomposites with the increase of discharge current [25]. By maintaining the discharge current at 20, 50, 100 and 150 A, the diameter of the particles was 60, 40, 35 and 26 nm respectively. They also observed that the shape of the nanoparticles changed from spherical to bead-like at higher discharge current. For 50, 100 and 150 A discharge current, the length of bead-like aggregates was 114, 117 and 120 nm respectively. Therefore, with the increase in discharge current, the size of nanoparticles decreased in diameter and increased in length. Jin et al. observed the effect of discharge voltage on the shape and size of Ag nanoparticles [45]. To investigate the phenomenon, they fixed the other physical and chemical parameters such as pulse frequency, pulse width, discharge duration, electrode gap and solution concentration at 20 kHz, 2 μs, 600 s, 1 mm and 0.5 mM respectively. When the discharge voltage was in the range of 800–900 V, the particles formed were mostly aggregated and had some dendritic structures. Further increase in discharge voltage to 1000 V, only dendritic structure was observed. This change in morphology of the Ag nanoparticles at higher voltage has been explained by considering the orbit – limiting charging model [46]. The model relates the particle charge with the surface potential as:

$$Q = C\Phi\_i \tag{22}$$

Where, *Q*, *C* and *Φs* represents the charge, capacitance of the particle in plasma and the surface potential of the particle respectively. As the quantity of electrons is directly proportional to the particle charge. Therefore, when the discharge voltage increases, the generation of energetic electrons also increases, generating more charged particles. A considerable number of electrons surrounds the surface of these charged particles. Hence, further reduction of the Ag ion will take place quite quickly on the already nucleated negatively charged particle, which helps in forming a dendrite structure. They also investigated the effect of nanoparticle morphology by controlling the discharge duration. They also studied the impact on the morphology of the nanoparticles by varying the discharge duration and keeping the applied voltage at 1000 V. At 120 s of discharge duration, nearly spherical Ag nanoparticles of size 12.7 � 4.4 nm in diameter were observed. When the duration was increased to 500 s, aggregated nanoparticles of a dendritic shape having branch of size 61.8 � 21.8 nm was observed. However, further increase in discharge duration to 600 s showed an abrupt increase in the size of the dendritic structure to 153.8 � 54.6 nm. Moreover, during the experiment, they observed the rise of solution temperature from 333 to 368 K. From this observation of temperature rise, they explained the change of morphology of the nanoparticles by considering the Brownian motion of the particles. The below equation is used to explain the relationship between the temperature and the Brownian motion of the nanoparticles:

$$D = kT/\mathfrak{G}\pi\eta r \tag{23}$$

Where, *D*, *k*, *T*, *η* and *r* represents the diffusion constant of particle, Boltzmann constant, temperature, viscosity and radius of particle respectively. Since, along with the discharge duration, the temperature of the solution increased. Therefore, the average kinetic energy of the nanoparticles also increases, which helps in aggregating the particles, due to continuous encounters with each other.

Sun et al. observed the variation on Ni – Cu bimetallic nanoparticle size and shape as well as the percentage of metal components with the plasma discharge duration [23]. With the increase of discharge duration, the size of the nanoparticles was observed to increase. At the initial stage of discharge, spherical bimetallic nanoparticles and after longer plasma treatment time flower-shaped nanoparticles were observed. Moreover, they observed higher copper content in the bimetallic particles. The atomic composition of the resulting bimetallic nanoparticles was 60% Cu, 30% Ni and 10% Oxygen. The observation can be explained by considering the standard potentials of copper and nickel, as shown by the following equations.

$$\text{Cu}^{2+}(aq) + 2\text{e}^- \rightarrow \text{Cu}(s)\text{E}^0 = +0.34\text{V} \tag{24}$$

$$\text{Ni}^{2+}(aq) + 2e^- \rightarrow \text{Ni}(s)\\E^0 = -0.25V \tag{25}$$

Since, copper has a higher standard potential i.e. lower ionisation tendency than nickel. Hence, copper ions are reduced faster than nickels to have more copper content in the resulting bimetallic nanoparticles. Kang et al. investigated a different phenomenon, where they observed the size and crystallinity of carbon nanospheres by varying the pulse frequency of a bipolar pulse power supply [47]. The voltage, pulse width and electrode gap were controlled at 1.3 kV, 2 μs and 1 mm respectively. Benzene was used as the precursor for carbon nanospheres. By adjusting the pulse frequency from 25 to 65 kHz, the average diameter of the carbon nanospheres was observed to be 20 to 100 nm. Moreover, during the discharge, another interesting phenomenon was observed. When the pulse frequency was adjusted from 25 to 50 kHz, amorphous carbon spheres were synthesised. On the other hand, at 65 kHz, synthesised carbon nanospheres composed continuous short-range graphite with turbostratic structure.

### **3.2 Variation of chemical parameters**

The chemical parameters such as concentration of the solution, pH and use of surfactant plays a significant role in the morphological and chemical compositions of the nanomaterials. In a recent work on the synthesis of Au/CuO micro/ nanocomposites, we have reported the shape transformation of CuO particles by simply varying the concentration of the gold precursor (HAuCl4) solution [28]. In the experiment, simultaneously, both the electrode (Cu) and the liquid solution act as the source of materials for the formation of Au/CuO micro/nanocomposites. At low concentration (0.1 mM HAuCl4) of the solution, the shape of CuO was found to be spindle. When the concentration was increased to 0.5 mM, along with the spindle shape, several rod-like structures of CuO were also observed. However, at higher concentration (1 mM), the spindle shape of CuO completely transformed to sheet – like structure. The shape transformation of CuO is believed to be due to the presence of a large number of foreign metal (Au3+) and halide (Cl�) ions at a higher concentration of gold precursor solution. The pH of the gold solution could also be responsible for the shape transformation process. Moreover, with the increase of solution concentration from 0.1 to 1 mM, the size of the Au nanoparticles was found to increase from 7.73 � 0.11 to 37.50 � 1.50 nm. A different work reported by Saito et al. investigated the morphology of copper/copper oxide nanoparticles synthesised from the electrode material by varying the concentration of K2CO3 solution [44]. They observed the formation of CuO nanoflowers having sharp nanorods, where size increased with increasing the solution concentration. The pH value of the precursor solution also plays a pivotal role in controlling the structure of nanomaterials. In most cases, the pH value of the initial precursor solution is controlled by using different concentrations of NaOH solution. Bratescu et al. investigated the variation of pH on the size of the Au nanoparticles [41]. They used HAuCl4.3H2O as the gold precursor. To adjust the pH of the solution, different

amount of NaOH was used. At pH 3, 6 and 12 the average size of the Au nanoparticles was measured to be 10, 4 and 2 nm respectively. They explained the size variation of Au nanoparticles with pH by considering the standard redox potential of Eqs. (11) and (12), which occur at pH 3 and 12 respectively. The standard redox potential of the reduction of *AuCl*� 4 to *Au*0 at pH 3 is 0.95 eV, whereas for *Au OH* ð Þ� 4 to *Au*0 at pH 12 is 0.60 eV [48]. As greater redox potential leads to the formation of a high number of atoms, hence, at pH 3 more number of Au atoms were formed, which aggregates and generate nanoparticles with sizes �10 nm. At higher pH, less number of atoms were formed, which lead to the formation of smaller Au nanoparticles.

Although most researchers do not use any stabilising agents, a few reported the addition of surfactant to the initial solution. Surfactants mainly act as a capping agent to control the size and shape of the nanoparticles by preventing the aggregation of particles. Kim et al. reported the use of Polyvinylpyrrolidone (PVP) as the stabiliser to observe the size variation of Au nanoparticles at different concentrations [49]. For the experiment, they dissolved 0.1 mM gold precursor HAuCl4.3H2O in ethylene glycol. After the plasma discharge, Au nanoparticles with various shapes such as triangular, square and nearly spherical, having sizes 20.85 � 2.78 nm in diameter and a few nanorods with 10 nm in diameter and 40–45 nm in length, were observed. After that, they mixed potassium chloride (KCl) to generate high plasma density, as potassium ions have higher oxidation potential than hydrogen. For 0.05 and 0.1 M KCl, most of the nanoparticles were observed to be nearly spherical of diameters 17.1 � 0.48 and 16.38 � 0.48 nm respectively. Therefore, the use of KCl had a significant role in the shape of the nanoparticles. When 0.01 mM PVP was added to a mixture of 0.05 M KCl and 0.1 mM HAuCl4.3H2O solution, the size of the Au nanoparticles was reduced to 12.32 � 0.87 nm. Moreover, with the increase of the concentration of PVP, the size of the Au nanoparticles decreased and they have a high tendency to become spherical. It indicates that the use of PVP effectively protects the surface of the Au nanoparticles by limiting the crystal growth and results in spherical nanoparticles. Lee et al. reported the effect of a cationic and anionic surfactant on the size of nickel (Ni) nanoparticles [50]. For the cationic and anionic surfactant, Cetyltrimethyl ammonium bromide (CTAB) and Sodium dodecyl sulfate (SDS) respectively were used. Nickel chloride hexahydrate (NiCl2.6H2O) solution was used as the precursor for Ni nanoparticles. When SDS was added to the solution, formation of spherical nanoparticles at all molar ratios of SDS/NiCl2 were observed. On the other hand, when CTAB was added up to 20% of the molar ratio of CTAB/NiCl2, smaller spherical nanoparticles than the no surfactant case was observed. When the molar ratio was increased to 30% or greater, the formation of large polygonal or whisker-shaped particles was observed. From this experiment, it has been established that cationic surfactant play a significant role in tuning the size and shape of the nanoparticles. Use of surfactant also influenced the composition of transition metal nanoparticles, as they have very high probability to form oxides in liquid environment. For the synthesis of copper (Cu) nanoparticles, CTAB or other surfactants have to be used otherwise formation of spindle-like Cu2O/CuO structures are frequently observed [51, 52]. Change of composition of the nanoparticles from metal (Sn) to metal oxide (SnO2) with discharge time even after the use of surfactant has also been reported by Lee et al. [21]. They explained the observation by investigating the pH of the solution. With the increase in discharge time for 50 mins, solution pH decreased i.e., H2O2 and HNO3 were formed in the solution. HNO3 may react with Sn to form SnO2 nanoparticles as shown by the equation:

$$\text{Sn} + 4\text{HNO}\_3 \rightarrow \text{SnO}\_2 + 4\text{NO}\_2(\text{g}) + 2\text{H}\_2\text{O} \tag{26}$$

As HNO3 was consumed during SnO2 synthesis, the pH of the solution again increased for the discharge duration of 50 to 60 mins.


*4.1. Choice of Peptide Systems*

We chose a set of five peptides for this study: the sequence from the *p53* binding epitope, two high affinity inhibitors (*pdiq* [39] and *ATSP*-7041) [25] and two control peptides, based on the poly-Ala sequence (*Ala1* and *Ala2*; see Table 1). Of the two control sequences, *Ala1* sequence conserves the set of hydrophobic residues that allow binding, and *Ala2* does not. *ATSP*-7041 is a stapled peptide using three non-standard amino acids, where one of the three anchoring residues (Leucine) is substituted by a non-canonical amino acid.

For *p53* and *pdiq*, we used crystal structures of the peptides binding to MDM2 (PDB codes 1ycr [30] and 3jzs [39]. For *ATSP*-7041, we used the structure bound to MDMX (PDB code 4n5t [25]) and superposed the active site onto MDM2 to have the reference structure of the peptide on the active site of MDM2. For the two control peptides bases on poly-ALA, there is no native structure. We compare it to the *p53*-MDM2 conformation for those two peptides. Parameters for the *ATSP*-7041 peptide are derived from the general amber force field (GAFF) [40], deriving charges based on the AM1 model [41].