3.1 Synthesis of Au microwires
Au microwires 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 Au microwires products were centrifuged, washed with ethanol at least 3 times and finally re-dispersed in hexane for further characterisations. In this work, the resulting Au microwires were more than 100 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 [40, 41]. The primary limiting factor for constant electron transfer rate would be the OA reagent. The relative concentration of the Au microwires obtained was > 50µg/ml. This method can also be applicable for the synthesis of other metal microwires as long as the chemical combination is suitable and correct.
3.2 Morphological Properties of As-Synthesised and Commercial Au Microwires
The as-synthesised Au microwires samples obtained were characterised for their morphological properties by standard techniques. The morphology, distribution and purity of the as-synthesised Au microwires 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 Au microwires 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 [42, 43]. The size distribution of parallel Au microwires bundles was difficult to measure. Higher magnification of the assembled ultra-thin Au microwires was unachievable due to their highly sensitive towards the electron beam which resulted in melted microwires within a few seconds of exposure. However, it is expected that the Au microwires possess diameters of approximately larger than 100 nm with an aspect ratio (length-to-width ratio) above 1000 nm. In this case, the Au microwires 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 Au microwires are permitted to fuse as the chemical potential between each chain is different. Therefore, the smoothing extension process to interconnect the of the microwires takes place through diffusion. The Au microwires exhibit higher stability in a polar solvent which is favourable for the subsequent immobilisation of biomolecules.
The primary objective of this work is to produce Au microwires 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 Au microwires samples using EDX. The EDX point measurements were carried out for an accurate estimation of Au amount present in each sample. For as-synthesised Au microwires, 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 microwires. This condition proves that in the presence of TIPS, Au seeds have rapid kinetics in the formation of microwires.
On the other hand, the representative FESEM images of commercial Au microwires suspended in CTAB aqueous solution are shown in Figure 3. It has been observed that the network formed does not assemble into ordered microstructures (Figure 3A) as seen in the as-synthesised Au microwires sample beforehand. Instead, the commercial Au microwires are randomly self-gating forming junction connection. Some of the Au microwires showed thicker diameter with reduced lengths as demonstrated in Figures 3B and 3C. The Au microwires can be as long as 171 m (Figure 3D).
One can see that the diameters of the microwires 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. Apparently, Au, in its nature, is quite malleable [44-47]. 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 Au microwires. 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 microwires ( ~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 microwires, typically remained the same caused by the similar diffusion of intense high energy ions in the surrounding. The polydispersity distribution of the commercial Au microwires is shown in Figure 3F. The narrow length distribution range (up to ~ 30 m) indicates that the commercial Au microwires 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 Au microwires 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 [48-50], it functions as space-filling of secondary material that fills up the gaps between the Au microwires and maintains the individual distribution of Au microwires. The FESEM micrographs distinctly manifest dissimilarity of CTAB grain with Au microwires, exhibiting large-sized flake grains (marked with the blue arrow in Figure 3). As stated in few reports [51-53], the tertiary ammonium ion cationic headgroup in the presence of bromide anion as a counterpart in microwire solution results in higher binding affinity and leads to more stable bilayer on the microwire surface. The addition of CTAB micelle helps to stabilise Au microwires 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 Au Microwires
The proof-of-concept on the cytotoxicity of the sensors-based microwires requires long acquisition times. Some of the microwires 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 [36, 54, 55]. These including surface charge and capping stabilisation without ‘green’ physical approach profited from natural microstructure generation. Additionally, the metal itself can produce toxicity and thus, being unfavourable for in-vivo applications. Taking into consideration the unique advantages of Au microwires, different capping interface, these types of Au microwires exhibits a significant effect in microbial viability. For this reason, we examined the potential cytotoxicity propensity for commercial and as-synthesised of Au microwires.
To further demonstrated the cytotoxicity tolerance in as-synthesised and commercial Au microwires 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 Au microwires. 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 Au microwires 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 Au microwires -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 Au microwires 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 Au microwires prepared with growth solution contained Au microwires with a smaller length. Since hexane is a non-polar solvent, it has no charge. Thus, the conjugated surface of Au microwires with negatively charge of the hydrophobic OA, yield a negative surface charge of the Au microwires system [34, 56]. As a result, no electrostatic interactions due to absent of different charges in the solution between the cell surface and as –synthesised Au microwires suspended in hexane. As generally accepted, CTAB alone is highly toxic cationic surfactant [57-59] and the conjugate systems of Au microwires -CTAB are proven to still contribute to toxicity to culture cells especially when the elevated concentrations of CTAB are present (50 g/ml commercial Au microwires-CTAB). With increasing the amount of Au microwires-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 [57, 60]. 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 Au microwires, in this case, the aspect ratio is constant for both 10 and 50 g/ml commercial Au microwires-CTAB solution. To minimise the cytotoxicity effects in Au microwires-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 Au microwires-CTAB and Au ions compared to as-synthesised Au microwires suspended in hexane.
For further validation of cytotoxicity detection on commercial and as-synthesised Au microwires, 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 Au microwires suspended in CTAB, <300 g/ml as-synthesised Au microwires 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 Au microwires strain. Indeed, the effect of both commercial and as-synthesised Au microwires 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 Au microwires, it is suggested that the Au microwires 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 Au microwires at those particular concentrations, another study is carried out to compare the inhibiting capability of commercial Au microwires-CTAB with Au microwires-sodium bicarbonate (NaHCO3) towards the cell growth. The focused study is purposely done on the commercial Au microwires-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 Au microwires were purified from CTAB capping layer and re-dispersed in 2 mM NaHCO3. Cells grown in LB media with Au microwires-CTAB and Au microwires-NaHCO3 with an approximate volume of 250 µl for both were observed. Water served as the standard control. The cell-microwire-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 Au microwires-CTAB and Au microwires-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 Au microwires. CTAB proved the capability to induce E.coli DH5α cell death and destroy bacteria growth. As regards to the surface modification, the Au microwires 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 Au microwires-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 microwires. The microwires 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 Au microwires surface would able to enter the cells effectively. The proper conjugated Au microwires able to recognise the proteins on the cell membrane. Thus, adsorption of the protein to the surface of the microwires mediate the direct penetration of microwires. Typically, smaller Au microwires 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 microstructures. Hence, since cytotoxicity was controlled by the physicochemical surface properties (surface charge number and dimensions) of Au microwires, it is crucial to employ riskless surface modification reagent to govern the biological effects of microwires to ensure the safety of microstructures used in medical applications.