3.1 Synthesis of Ultra-thin AuNWs
AuNWs were synthesised based on the method described in the literature [53]. The synthesis reaction involved mixing 100 µl oleylamine (OA) and 150 µl triisopropylsilane (TIPS) with about 3–5 mg gold (III) chloride solution, (HAuCl4 99.9% trace metals basis, 30 wt.% in dilute HCl,) in 2.5 ml final volume of hexane. 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%. The relative concentration of the AuNWs obtained was > 50 µg/ml. A thin layer of AuNWs arrays were formed through drop coating of the AuNW solutions on silicon SEM discs (Fig. 1(A)).
3.2 Characterisation of Synthesised Ultra-Thin AuNWs and Commercial AuNWs
Samples of As-synthesised AuNWs obtained were characterised for their physicochemical properties by standard techniques. Figure 1(B) show morphology, distribution and purity of synthesised AuNWs verified by FESEM analysis with EDX. 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, we 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. In this case, the AuNWs tended to form a stack of parallel bundles which then self-assembled into 2-D network structures over macroscopic distances through spontaneous directional aggregation that occurs during solvent evaporation. The directional aggregation is typically formed via oriented attachment in which AuNWs are permitted to fuse together as the chemical potential between each chain is different [54]. Therefore, the smoothing extension process to interconnect the nanoparticles of the nanowires takes place through diffusion. The use of TIPS in the synthetic reaction at room temperature controls the acceleration of the process. AuNWs exhibit higher stability in polar solvent which is favourable for the subsequent immobilisation of biomolecules.
The primary objective of this work is to produce ultra-thin AuNWs with high aspect ratio. Also analyse quantitative and qualitative properties of the as-synthesised AuNWs by EDX analysis. Figure 1(C) shows the nanoscale characterization of the chemical composition of obtained nanowire samples. For as-synthesised AuNWs, the amount of Au is at the level of 98% with the remaining concentration of Si (substrate). The presence of high purity Au resulted from homogeneous nucleation of metallic Au as spherical clusters and hence determined the growth of 2-D nanowires. Demonstrating that in the presence of TIPS, Au seeds have a rapid kinetics in the formation of nanowires. EDX point measurements were carried out for an accurate estimation of Au amount presence in each sample.
Figure 1
(A) As-synthesised AuNWs reaction formation. The reaction product has a dark violet colour solution after 24 h. (B) FESEM and (C) EDX analyses of AuNWs. EDX spectrum recorded from the area of AuNWs presence.
During the synthesis reaction, the potential of Au + reduction to Au is higher in the presence of TIPS. The primary limiting factor for constant electron transfer rate would be the TIPS reagent. [55]. During the synthesis reaction, in the absence of heat, a colour change occurs from a light orange to red followed by dark violet during the period of reaction as shown in Fig. 1(C). For a 24 h reaction, the resultant colloid colour is dark violet which indicates extended filament-like structures. 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’ [56].
Representative FESEM images of commercial AuNWs suspended in CTAB aqueous solution are shown in Fig. 2. It can be seen that the nanowires are less than 35 µm in length (Fig. 2(A)) and build up a cross network which aggregated randomly (Fig. 2 (B and C)). The diameter of the nanowires is not uniform and has a size ranging from ~ 50 nm to ~ 200 nm. The network formed did not show any self-assembling properties.. Commercial AuNWs are randomly connected to each other. The cloudy region encircling AuNWs is due to the capping action of CTAB as the solvent evaporates leaving the residue behind. The CTAB monolayer micelle helps to stabilise Au nanowires. This stabilization effect, while making them soluble, can be an inhibitor of subsequent self-assembling process.
Bottom-up synthesis of AuNWs which is easy and highly effective method for rapid synthesis of single-crystal ultrathin (below 4 nm ) AuNWs. This method do not require any mechanical stirring, it is performed at room temperature. This method can also be applicable for the synthesis of other metal nanowires as long as the chemical combination is suitable and correct. The as-synthesised ultrathin AuNWs have a tendency to self-assemble into 2-D nanowire networks and closely packed forming parallel structures when viewed under the SEM. The nanowire networks could serve as a well-defined surface-enhanced SERS-active system. The ultrathin nanowires assembly could allow trapping of molecules for molecular recognition and signal amplification.
Although, the thinner AuNWs the better to increase the nanosensor sensitivity, commercial AuNWs are ideal for the MC deposition. Therefore, more studies need to be undertaken to elucidate the synthesis of thinner and stable AuNWs with both high defect characteristic and intrinsic quality to allow better operation and function at a higher temperature.
Correspondingly, the proof-of-concept on the cytotoxicity of the nanosensors-based nanowires requires long acquisition times. Some nanowires synthesis protocols, such as chemical reduction method can introduce toxic materials from the capping agent used to stop the reaction. Also, the metal itself can produce toxicity, being unfavourable for in-vivo applications. For this reason, we examined the potential cytotoxicity of both types of AuNWs.
3.3 Cytotoxicity Evaluation of As-Synthesised and Commercial AuNWs
To study the potential cytotoxicity of synthesised and commercial AuNWs, in this work we used a commercial bacteria Escherichia Coli (E.coli) DH5-Alpha strain (DH5α), an engineered non-pathogenic bacterial strain of used extensively as a lab Cytotoxicity microbial model system.
In the work we used several test, first use the disk diffusion method (Fig. 3). This method is performed to detect cytotoxicity by inducing a gradient of concentration around a disk loaded with AuNWs. The gradient of AuNWs halts the growth of bacteria, and creates an inhibition area around the disk. The ratio of the area (measure in mm) is directly proportional to the sample toxicity (White marks). Non-toxic samples will not form any inhibition area (red circles) (Fig. 3(A)).
For further validation of toxicity detection of our nanostructures, we performed CFU counting experiments on commercial and as-synthesized AuNWs. Pure Hexane, H2O and ionic gold at 3 different concentrations were use as negative controls (Fig. 3B). Cell-Viability assessed by CFU remained at 100% on control samples. For 10 µg/ml Au ion, the CFU counting measured a viability of 72%. At 30 µg/ml Au ion, approximates value were almost similar to the other one giving rise to 69% for CFU counting. Cells incubated with 100 µg/ml of commercial AuNWs and both 2 µg/ml and < 300 µg/ml of as-synthesised AuNWs, respectively, cell viability were 0% for all in CFU counting. Pure hexane also showed a cell viability of 0%. Assessment at 96 hours was likely to be optimal for AuNWs strain. Indeed, the effect of AuNWs and high dosage of Au-based ion seemed strong antibacterial agent in this assay. This corroborated the result that AuNWs work markedly better, meaning presents less bacteria toxicity than Au ions as happened to other metallic materials [57, 58].
Our results suggest, that nano wire-structured Au do not induce any antibacterial activity or toxicity effect on bacterial cells. Therefore, the toxicity effect observed in our experiments using commercial AuNWs could be induced by the presence of the commercial capping agent, in this case CTAB. CTAB or Cetrimonium bromide, is a quaternary ammonium surfactant. It is one of the components of the topical antiseptic cetrimide. The cetrimonium cation is indeed an effective antiseptic agent against bacteria and fungi. To clarify the inhibitory antibacterial activity observed is induced by the CTAB layer, commercial AuNWs were purified from CTAB capping layer and re-dispersed in 2 mM sodium bicarbonate (NaHCO3). Cells grown in LB media were then observed AuNWs in CTAB and NaHCO3 with an approximate volume of 250 µl for both. Water served as the standard control. The cell-nanowire-SB solutions were approximately 5 µg/ml, incubated for 90 minutes and was serially diluted to 105 times of the initial concentration with the set to dispense 100 µl onto plates. CFU showed about 130% and 4% of capture efficiency for AuNWs in NaHCO3 and CTAB solutions Fig. 3(C). The low CFU in the CTAB solution were found to be threefold lower than in NaHCO3, indicating that the toxicological effect of CTAB is quite high which significantly displayed susceptible inhibit growth of E.coli cells. NaHCO3 had lowest inhibitory effect.
3.3 Assembly of AuNWs on Cantilever
Integrating AuNWs onto a cantilever is crucial for the exploration of a multifunctional MC biosensor. To functionalise the cantilever, AuNWs were attached to an aluminum coated AFM cantilever surface. The surface of the cantilever was covered with AuNWs solution, and the nanowires on the surface repel each other and assemble. In this case, for the functionalization process we used commercial AuNWs and the evaporation process described in material and methods. The resulting attachment between the surface of the cantilever and AuNWs is shown in Fig. 4. This assembly is adequate for MC biosensor functionalization viability. Here, we demonstrated a direct depositing of AuNWs and its binding tendency onto the cantilever.