3.1 Synthesis of Ultra-Thin AuNWs
AuNWs were synthesised based on the method described in the literature . In the absence of heat, the synthesis reaction involved mixing of 100µl OA and 150 µl TIPS with about 3 to 5 mg of gold (III) chloride precursor solution, (HAuCl4 99.9% trace metals basis, 30 wt.% in dilute HCl) in 2.5 ml final volume of hexane without stirring. OA acts as a stabiliser and a template for 1-D growth with TIPS role as a highly reactive reducing agent. The observation of the reaction through the change in colour from light orange to red, followed by dark violet during the period of reaction left out at room temperature is demonstrated in Figure 1. The AuNWs products were centrifuged, washed with ethanol at least 3 times and finally re-dispersed in hexane for further characterisations. Nanowires produced, according to the published method, have diameters of about 2 nm, lengths of a few µm and self-assembled on deposition into organised structures. In this work, the resulting AuNWs were 3 nm in diameter with a standard deviation of 0.8 %. During the synthesis reaction, the potential of Au+ reduction to Au is higher in the presence of OA [61-64]. The primary limiting factor for constant electron transfer rate would be the OA reagent. The relative concentration of the AuNWs obtained was > 50µg/ml. This method can also be applicable for the synthesis of other metal nanowires as long as the chemical combination is suitable and correct.
3.2 Morphological Properties of Synthesised Ultra-Thin AuNWs and Commercial AuNWs
The as-synthesised AuNWs samples obtained were characterised for their morphological properties by standard techniques. The morphology, distribution and purity of the as-synthesised AuNWs verified by FESEM are shown in Figure 2A. For a 24 h reaction, the resultant colloid colour is dark violet which indicates extended filament-like structures. The AuNWs tend to self-assemble into 1-D networks and forming closely packed parallel structures. The use of negatively charged TIPS in the synthetic reaction at room temperature controls the acceleration of the process [65, 66]. The size distribution of parallel AuNWs bundles was difficult to measure. Higher magnification of the assembled ultra-thin AuNWs was unachievable due to their highly sensitive towards the electron beam which resulted in melted nanowires within a few seconds of exposure. However, it is expected that the ultra-thin AuNWs possess diameters of approximately smaller than 3 nm with an aspect ratio (length-to-width ratio) above 1000 nm as the method was re-created from the existing work of  and proved in the published figure with the asterisk mark. In this case, the AuNWs tended to form a stack of parallel bundles (yellow rectangular) which then self-assembled into 1-D network structures over macroscopic distances through a spontaneous directional aggregation that occurs during solvent evaporation. The directional aggregation is typically formed via oriented attachment in which AuNWs are permitted to fuse as the chemical potential between each chain is different. Therefore, the smoothing extension process to interconnect the nanoparticles of the nanowires takes place through diffusion. The AuNWs exhibit higher stability in a polar solvent which is favourable for the subsequent immobilisation of biomolecules. The ultra-thin self-assembled networks of individual crystalline AuNWs with a gap distance of ~2 nm can be of use to trap the analyte molecules and automatically locate them within the gap of closely packed parallel AuNWs making them as suitable substrate candidates for SERS studies due to the presence of closely packed ‘hotspot’.
The primary objective of this work is to produce ultra-thin AuNWs with high aspect ratio and analyse the elements of samples with the quantitative and qualitative analyses properties. Figure 2B shows the element composition of obtained AuNWs samples using EDX. The EDX point measurements were carried out for an accurate estimation of Au amount present in each sample. For as-synthesised AuNWs, the amount of Au is at the level of 98% as the major element in the sample with the remaining amount is Si (substrate). The presence of high purity Au has resulted from homogeneous nucleation of metallic Au as spherical clusters and hence determined the growth of 1-D nanowires. This condition proves that in the presence of TIPS, Au seeds have rapid kinetics in the formation of nanowires. Figure 2: A. The representative FESEM micrograph of the as-synthesised AuNWs. The enlarged figure (yellow rectangular) reveals the parallel self-assembly arrangement of close-packed AuNWs bundles. The small parallel bundle highlighted in yellow circle showed that the bundle of the nanowires has a diameter size of ~200 nm. Noted that the individual AuNWs in each bundle would possess the arrangement and the individual AuNWs size that are almost similar to the original method reported by Feng et al., (Figure with asterisk mark is reproduced from ref.  with permission from The Royal Society of Chemistry). B. The EDX spectrum analysis of the as-synthesised AuNWs recorded for the selected area of AuNWs (red rectangle) taken from image A.
On the other hand, the representative FESEM images of commercial AuNWs suspended in CTAB aqueous solution are shown in Figure 3. It has been observed that the network formed does not assemble into ordered nanostructures (Figure 3A) as seen in the as-synthesised AuNWs sample beforehand. Instead, the commercial AuNWs are randomly self-gating forming junction connection. Some of the AuNWs showed thicker diameter with reduced lengths as demonstrated in Figures 3B and 3C. The AuNWs can be as long as 171 m (Figure 3D).
One can see that the diameters of the nanowires were averagely thin and approximately ~540 nm as indicated in Figure 3E. Interestingly, the specified by the supplier is ~30 nm. It is common to have different values of diameter and length from the specified information properties provided by the supplier as they are in a discrete condition and slight changes in the physical handling, as well as the time storage, would affect the properties of the sample. The procedure in preparing the FESEM analysis would contribute to the alteration on the morphologies and diameter size of the AuNWs. Apparently, Au, in its nature, is quite malleable [67-70]. Hence, the possibility for the AuNWs to break during sonication bath is higher [71, 72]. Although the ultrasonic bath is used for homogenising the nanowires dispersion without major damage to the nanostructures, the excess energy presence, especially towards malleable nanomaterials would lead to significant particle-particle fusion. Additionally, it is reported that the smaller nanowires with large surface-to-volume ratio would have a high surface recombination rate [73, 74] that further increase the collision frequency. Thus, it facilitates the aggregation, which also correlates to the changes of their stability property. CTAB is used to minimise the aggregation, but it would not be able to halt the entire aggregation of the AuNWs. In this case, CTAB is considered as an effective stabiliser which proved by the average diameter, distribution provided in the histogram (Figure 3E). The distribution is dominantly conquered by the 540 nm based on the highest per cent frequency distribution (68%), while the larger nanowires ( ~1.25 to 7.14 μm) with the per cent frequency is only 3% measured by the Gaussian distribution. This indicates that the 540 nm of majorly contributes to the overall properties of the sample.
The average length of nanowires, typically remained the same caused by the similar diffusion of intense high energy ions in the surrounding. The polydispersity distribution of the commercial AuNWs is shown in Figure 3F. The narrow length distribution range (up to ~ 30 m) indicates that the commercial AuNWs were stable and monodispersed. The wide distribution range is due to the more considerable length variation, which is up to ~ 171 m.
It shall be noted that the cloudy region encircling AuNWs is due to the strongly coupled capping action of CTAB upon the evaporation of the solvent, leaving the residue behind. Since cationic CTAB surfactant consists of long carbon atomic chain [75-78], it functions as space-filling of secondary material that fills up the gaps between the AuNWs and maintains the individual distribution of AuNWs. The FESEM micrographs distinctly manifest dissimilarity of CTAB grain with AuNWs, exhibiting large-sized flake grains (marked with the blue arrow in Figure 3). As stated in few reports [79-81], the tertiary ammonium ion cationic headgroup in the presence of bromide anion as a counterpart in nanowire solution results in higher binding affinity and leads to more stable bilayer on the nanowire surface. The addition of CTAB micelle helps to stabilise AuNWs when the equilibrium condition is achieved through the occupation some of the surface areas caused by the increased in the aggregation number of CTAB micelle. This stabilisation effect, while making them soluble, can be an inhibitor of subsequent self-assembling process.
3.3 Cytotoxicity Evaluation of As-Synthesised and Commercial AuNWs
The proof-of-concept on the cytotoxicity of the nanosensors-based nanowires requires long acquisition times. Some of the nanowires conventional synthesis protocols, such as the reduction of Au precursor in the presence of capping agent can introduce toxic materials from surface conditioning and chemistry [82, 83]. These including surface charge and capping stabilisation without ‘green’ physical approach profited from natural nanostructure generation [84, 85]. Additionally, the metal itself can produce toxicity and thus, being unfavourable for in-vivo applications. Taking into consideration the unique advantages of AuNWs, different capping interface, these types of AuNWs exhibits a significant effect in microbial viability. For this reason, we examined the potential cytotoxicity propensity for commercial and as-synthesised of AuNWs.
To further demonstrated the cytotoxicity tolerance in as-synthesised and commercial AuNWs samples, the commercial bacteria E.coli DH5α, an engineered non-pathogenic bacterial strain is extensively used as a lab cytotoxicity microbial model system. Two types of cytotoxicity test were carried out. The first is the disc diffusion assay. This method is performed to detect cytotoxicity by inducing a gradient of concentration (Figure 4A) around a disc (known as inhibitory effect) loaded with AuNWs. The inhibitory effect among the E.coli DH5α strains exhibited different outcomes. The ratio of the ring area (measure in mm) is directly proportional to the sample toxicity (white lines) as indicated in Figures 4B and 4C. The results show that the gradient of commercial AuNWs suspended in CTAB halts the growth of bacteria and creates an inhibition zone around the disc in both the 10 and 50 g/ml samples. Lower inhibition zone was found for 1000 g/ml Au ions. For these types of Au-based suspensions, the largest inhibition zone reached 3.9 0.2 mm, 2.9 0.3 mm and 1.2 0.1 mm for 50 g/ml commercial AuNWs-CTAB, 10 g/ml commercial AuNWs-CTAB and 1000 g/ml Au ions, respectively. Non-toxic samples with no inhibitory effect which refer to water (Figure 4D) and hexane (Figure 4E) samples. A similar observation was found for 400 g/ml Au ions and <330 g/ml as-synthesised AuNWs suspended in hexane. The six samples used to observe the average diameter of inhibition zones containing Au-based solutions is presented in Figure 4F.
The size of as-synthesised AuNWs prepared with growth solution contained AuNWs with a smaller length. Since hexane is a non-polar solvent, it has no charge. Thus, the conjugated surface of AuNWs with negatively charge of the hydrophobic OA, yield a negative surface charge of the AuNWs system [86, 87]. As a result, no electrostatic interactions due to absent of different charges in the solution between the cell surface and as –synthesised AuNWs suspended in hexane. As generally accepted, CTAB alone is highly
toxic cationic surfactant [88-90] and the conjugate systems of AuNWs-CTAB are proven to still contribute to toxicity to culture cells especially when the elevated concentrations of CTAB are present (50 g/ml commercial AuNWs-CTAB). With increasing the amount of AuNWs-CTAB solution, the cytotoxicity of the sample is increased caused by the nonspecific binding tendency of CTAB to negatively charged cell surfaces by electrostatic interactions [91, 92]. Once the interaction with the cell occurs, it forms blebs and holes on the cell and leading to cell death evident by the increase in the diameter of the ring zone. Moreover, the data also suggest that the abilities to induced cell death are also significant regardless of the aspect ratio of the AuNWs, in this case, the aspect ratio is constant for both 10 and 50 g/ml commercial AuNWs-CTAB solution. To minimise the cytotoxicity effects in AuNWs-CTAB, the interaction between the membrane cell and CTAB should be blocked by using protein types of coating on the bilayer surface of CTAB. Meanwhile, a similar trend of nonspecific binding also observed when Au ions are incubated with the cell. The nonspecific binding was evident by the increased of the ring zone when exposed to the concentration of the 1000 g/ml Au ions. This nonspecific binding did not take place with 400 g/ml Au ions might be due to the reduced content of positively charge Au ions, which in turn, decreases electrostatic interaction between the Au ions and negatively charged cell membrane. These results affirm cytotoxicity potency of commercial AuNWs -CTAB and Au ions compared to as-synthesised AuNWs suspended in hexane.
For further validation of cytotoxicity detection on commercial and as-synthesised AuNWs, the CFU enumeration is carried out after 24 h incubation (Figure 5A). As evident from Figure 5B, the CFU enumeration for 10 g/ml Au ion measured viability of 72%. At 30 g/ml Au ion approximates value were almost similar to the other one giving rise to 69%. Cell-viability assessed by CFU remained at 100% on the control (water) sample. Compared with 10 and 30 g/ml of Au ions, the cell incubated with a higher concentration of Au ions (100 g/ml) was observed to be 0% cell viability. In the case of 2 g/ml of commercial AuNWs suspended in CTAB, <300 g/ml as-synthesised AuNWs and hexane also, 0% were viable for all in CFU enumeration. Pure hexane, water and the ionic Au at three different concentrations (10, 30 and 100 g/ml) were used as negative controls. Assessment at 96 hours was likely to be optimal for AuNWs strain. Indeed, the effect of both commercial and as-synthesised AuNWs along with a high dosage of 100 g/ml Au-based ion seemed strong antibacterial agent in this assay. Considering the high amount concentration use for as-synthesised AuNWs, it is suggested that the AuNWs work markedly better, meaning presents less bacteria toxicity than Au ions (tenfold lower in concentration) as happened to other metallic materials.
Endowing the high antibacterial potency or toxicity effect based on the viability results for both types of AuNWs at those particular concentrations, another study is carried out to compare the inhibiting capability of commercial AuNWs-CTAB with AuNWs-sodium bicarbonate (NaHCO3) towards the cell growth. The focused study is purposely done on the commercial AuNWs-CTAB since a relatively low amount of sample concentration used proved to affect the cell viability significantly. To further clarify the inhibitory antibacterial activity induced by the CTAB layer, commercial AuNWs were purified from CTAB capping layer and re-dispersed in 2 mM NaHCO3. Cells grown in LB media with AuNWs-CTAB and AuNWs NaHCO3 with an approximate volume of 250 µl for both were observed. Water served as the standard control. The cell-nanowire-LB solutions were approximately 5µg/ml, incubated for 90 minutes and was serially diluted to 105 times of the initial concentration with the set to dispense for 100 µl onto plates. As shown in Figure 5C, CFU enumeration showed 4 % and 130% of capture efficiency for AuNWs-CTAB and AuNWs-NaHCO3 solutions, respectively. The low CFU in the CTAB solution was found to be threefold lower than in NaHCO3, indicating that the toxicological effect of CTAB is relatively high, which significantly displayed susceptible inhibit growth of E.coli cells. NaHCO3 had a low inhibitory effect.
The results demonstrated that surface modification is the decisive factor governing the cytotoxicity of AuNWs. CTAB proved the capability to induce E.coli DH5α cell death and destroy bacteria growth. As regards to the surface modification, the AuNWs capped with NaHCO3 (well-known as baking soda), indicated minimal toxicity effect and had almost no effect on cell viability in an alkaline environment. The alkaline environment retarded the activity of the toxicity which inhibits the cellular uptake. When combining with NaHCO3, the acidic environment that usually leads to cell death is halts and decreasing the risk of adverse effects and toxicity.
It is believed that the primary toxicity mechanism of the AuNWs-CTAB was via the activation of intracellular cell damage, which rigidly controls the per cent of cell survival. As the cell tries protecting itself from toxicity created by the molecule, other existing plasma proteins will adsorb on the surface of nanowires. The nanowires eventually penetrate the cells after adjusting the interfacial properties of the adsorbed protein shell. However, some of the adjustments are not always a success as not all modified AuNWs surface would able to enter the cells effectively. The proper conjugated AuNWs able to recognise the proteins on the cell membrane. Thus, adsorption of the protein to the surface of the nanowires mediate the direct penetration of nanowires. Typically, smaller AuNWs are frequently deemed as more toxic due to the ability to fully penetrate at intracellular locations (e.g., nucleus) which is not able to get through by larger nanostructures. Hence, since cytotoxicity was controlled by the physicochemical surface properties (surface charge number and dimensions) of AuNWs, it is crucial to employ riskless surface modification reagent to govern the biological effects of nanowires to ensure the safety of nanostructures used in medical applications.