Fabrication and Characterization of Nanostructured Antibacterial Surfaces

Abstract

Bacterial antibiotic resistance is becoming wide spread due to the excessive and unregulated use of antibiotics in healthcare and agriculture. At the same time the development of new antibiotics has become slow. Adding antibiotics to surfaces result in poor long-term performance in preventing bacterial build-up. This also increases the risk of development of more drug resistant strains. Hence, approaches for realising antibacterial action through physical surface topography have become increasingly important and interesting to the community in recent years. The complex and strain dependent nature of the bacterial cell wall interactions with nanostructured surfaces leads to many challenges while the design of nanostructured antibacterial surfaces is concerned. First part of this work focuses on enhancing the antibacterial activity of the nanostructured surfaces by coating them with different chemistries. Using two different categories of coatings firstly metals (Cu and Ag) and secondly biocompatible polymer chitosan, we demonstrate efficient bactericidal activity against a range of bacteria. The second part of the work focuses on developing processing technology for demonstration of such nanostructured antibacterial surfaces for practical applications. The focus was to demonstrate a low-cost processing technology which can be easily scaled to large area. Finally, we test the bactericidal efficacy of the developed surfaces against the drug resistant strains obtained from a hospital. In nature several insects such as cicada wing, dragonfly wing, dronefly wing possess sharp nanostructures on their wing which kill bacteria by contact killing mechanism. When a bacterium sits on such surfaces, they get stretched and deformed while trying to settle on maximum anchoring points. When the threshold of stretching is reached, cell wall is compromised, and cell lysis takes place. In this work a range of surfaces with distinct surface topography and chemistry has been studied. Initially, inspired from dragonfly wing, high-aspect ratio silicon nanostructured surface (NSS) was fabricated using a single-step deep reactive ion etching (DRIE) technique. The nanostructures were found to be random in both size (300-1100 nm) and spatial distribution (300-500 nm). Post fabrication the surfaces were coated with a thin layer of copper (NSS_Cu) and silver (NSS_Ag). The bactericidal efficacy of the NSS_Cu, NSS_Ag and NSS surfaces were tested and compared against Gram-negative bacterium E. coli. NSS_Cu was found to have the highest bactericidal efficacy killing 97% of the bacteria in just 90 minutes. The results from this study suggests that the addition of a surface chemistry to the physical nanostructures enhances the bactericidal efficacy. However, copper is not stable when exposed to environment and oxidises to form CuO, Cu2O etc. To overcome this problem, we replaced copper with a stable biocompatible polymer “Chitosan (CHI)”. Unlike copper coating where sputtering tool was used, CHI can be coated on any substrate by a simple dip coating technique making the process simpler and cost-effective. CHI was coated on flat silicon (Si_CHI) and NSS surfaces (NSS_CHI). The bactericidal efficacy of the surfaces was tested against Gram-negative E. coli and Gram-positive S. aureus. NSS_CHI surface was found to be the most efficient in killing bacteria as compared to the Si_CHI and NSS surfaces. Also, the antibiofilm characteristics of these surfaces was studied. NSS_CHI surface was found to have the least amount of bacterial bio mass on its surface after a period of 5 days of bacterial incubation in Luria Broth (LB) medium for both E. coli and S. ii aureus. Also, the CHI coating was found to be very stable when exposed to PBS for 7 days showing its durability for a longer period. CHI coating was cost-effective and easy as compared to the sputtering technique. However, fabrication of NSS using DRIE still comes with a cost. To overcome this issue, we fabricated ZnO nanostructured surface using simple chemical synthesis method at near room temperature (~20O C). Neither sophisticated tool like DRIE nor clean room environment was required for this process. Sharp ZnO nanostructured surface was fabricated in an alkaline solution containing zinc nitrate hexahydrate and potassium hydroxide. The synthesis time was set to 12 hours (h). The ZnO nanostructures possess a length of 1.5-2 𝜇m, tip diameter ~20 nm, tip angle ~ 10O. Also, this technique was used to grow the ZnO nanostructures on a variety of substrates such as copper sheet, glass, polydimethylsiloxane (PDMS) showing the versatility of the fabrication technique. The antibacterial performance of the ZnO nanostructured surface was evaluated against Gram-negative E. coli. Flat silicon surface and silicon surface coated with 20 nm of ZnO thin film were taken as controls. Bacterial attachment was seen on the flat silicon and flat ZnO substrates after a 24 h of incubation. In contrary no bacterial colony was observed on the nanostructured ZnO surface showing its bacteriophobic behaviour. The simplicity and cost effectiveness of this process makes it possible for this surface to be used in practical applications. Also, large scale fabrication is possible using this technique. Despite several advances in this area, it is well understood that the micro/nano structures are mechanically fragile. This reduces their reliability and hence increases the cost of use. Moreover, several applications such as aprons, gloves, temporary mats etc. require these surfaces to be flexible. The above requirements call for the development of flexible antibacterial surfaces with mechanical reliability. In addition, the surfaces should be low-cost so that they can be periodically replaced to address the issues with reliability. To achieve this, transferring of copper hydroxide nanostructures onto a curable silicone polymer, polydimethylsiloxane (PDMS), was carried out by a two-step process: (i) copper etching to form nanostructures and (ii) transfer of the copper based nanostructures onto the PDMS surface by mechanical tearing. This PDMS surface decorated with the copper nanostructures (PDMS_Cu) is unique in displaying two functionalities; superhydrophobicity preventing bacterial adhesion and a potent bactericidal effect from the copper nanowires as copper has been regarded as a very good antimicrobial agent from centuries. This process was scaled for large area fabrication for real world applications. Absence of a micro-fabricated template makes this process significantly cheaper and easily scalable as it is not limited by the size of the template. In addition, as the cured polymer strongly holds these nanowires in place, these surfaces showed reliability against abrasion, tape peel and solid weight impact. Also, the surface was superhydrophobic after dry heat, moist heat and UV exposure. The fabricated PDMS_Cu surface was tested against drug resistant E. coli, S. aureus and K. pneumoniae. The surface exhibited excellent antibacterial behaviour against all the drug resistant bacteria. Also, the PDMS_Cu surfaces were kept at several infectious places in the hospital. The flora count on the PDMS_Cu was lesser than the control surfaces showing its superior antibacterial property. The ability of the PDMS_Cu surface to support RAW Macrophage and HeLA cells proliferation was also evaluated using confocal microscopy by staining the cells with DAPI and tubulin. Both the Macrophage and HeLa cells attachment was found to be higher on the coverslip and PDMS substrates as compared to the PDMS_Cu surface which can be attributed to the superhydrophobic property of the PDMS_Cu iii surface. MTT assay method was also used to assess the cell metabolic activity. ~50% RAW macrophage and ~71% HeLa cells were found to be viable after an incubation period of 5 h. Taken together, these data confirm that the PDMS_Cu surface was not cytotoxic to the RAW macrophage and the HeLa cells. To demonstrate its application in healthcare, heartbeat sound recording was carried out via the PDMS_Cu surface. Good quality heart beat sound was recorded showing its plausible use as a thin covering on the stethoscope diaphragm to prevent the transmission of pathogenic flora from one person to another in the hospital. Every surface studied in this work exhibits unique topography and surface chemistry and they can be used in several applications such as photovoltaic, high efficiency photo detector and sensors, water treatment, food packaging, health care etc.