Genetic Transformation of Plasmid DNA into Escherichia coli Using High Frequency Electromagnetic Energy.

We present a novel technique of genetic transformation of bacterial cells mediated by high frequency electromagnetic energy (HF EME). Plasmid DNA, pGLO (5.4 kb), was successfully transformed into Escherichia coli JM109 cells after exposure to 18 GHz irradiation at a power density between 5.6 and 30 kW m-2 for 180 s at temperatures ranging from 30 to 40 °C. Transformed bacteria were identified by the expression of green fluorescent protein (GFP) using confocal scanning microscopy (CLSM) and flow cytometry (FC). Approximately 90.7% of HF EME treated viable E. coli cells exhibited uptake of the pGLO plasmid. The interaction of plasmid DNA with bacteria leading to transformation was confirmed by using cryogenic transmission electron microscopy (cryo-TEM). HF EME-induced plasmid DNA transformation was shown to be unique, highly efficient, and cost-effective. HF EME-induced genetic transformation is performed under physiologically friendly conditions in contrast to existing techniques that generate higher temperatures, leading to altered cellular integrity. This technique allows safe delivery of genetic material into bacterial cells, thus providing excellent prospects for applications in microbiome therapeutics and synthetic biology.

The multi-faceted mechano-bactericidal mechanism of nanostructured surfaces.

The mechano-bactericidal activity of nanostructured surfaces has become the focus of intensive research toward the development of a new generation of antibacterial surfaces, particularly in the current era of emerging antibiotic resistance. This work demonstrates the effects of an incremental increase of nanopillar height on nanostructure-induced bacterial cell death. We propose that the mechanical lysis of bacterial cells can be influenced by the degree of elasticity and clustering of highly ordered silicon nanopillar arrays. Herein, silicon nanopillar arrays with diameter 35 nm, periodicity 90 nm and increasing heights of 220, 360, and 420 nm were fabricated using deep UV immersion lithography. Nanoarrays of 360-nm-height pillars exhibited the highest degree of bactericidal activity toward both Gram stain-negative Pseudomonas aeruginosa and Gram stain-positive Staphylococcus aureus bacteria, inducing 95 ± 5% and 83 ± 12% cell death, respectively. At heights of 360 nm, increased nanopillar elasticity contributes to the onset of pillar deformation in response to bacterial adhesion to the surface. Theoretical analyses of pillar elasticity confirm that deflection, deformation force, and mechanical energies are more significant for the substrata possessing more flexible pillars. Increased storage and release of mechanical energy may explain the enhanced bactericidal action of these nanopillar arrays toward bacterial cells contacting the surface; however, with further increase of nanopillar height (420 nm), the forces (and tensions) can be partially compensated by irreversible interpillar adhesion that reduces their bactericidal effect. These findings can be used to inform the design of next-generation mechano-responsive surfaces with tuneable bactericidal characteristics for antimicrobial surface technologies.

Programmed Death of Injured Pseudomonas aeruginosa on Mechano-Bactericidal Surfaces.

Mechano-bactericidal surfaces deliver lethal effects to contacting bacteria. Until now, cell death has been attributed to the mechanical stress imparted to the bacterial cell envelope by the surface nanostructures; however, the process of bacterial death encountering nanostructured surfaces has not been fully illuminated. Here, we perform an in-depth investigation of the mechano-bactericidal action of black silicon (bSi) surfaces toward Gram-negative bacteria Pseudomonas aeruginosa. We discover that the mechanical injury is not sufficient to kill the bacteria immediately due to the survival of the inner plasma membrane. Instead, such sublethal mechanical injury leads to apoptosis-like death (ALD) in affected bacteria. In addition, when the mechanical stress is removed, the self-accumulated reactive oxygen species (ROS) incur poststress ALD in damaged cells in a nonstressed environment, revealing that the mechano-bactericidal actions have sustained physiological effects on the bacterium. This work creates a new facet and can introduce many new regulation tools to this field.

Biomimetic Nanopillar Silicon Surfaces Rupture Fungal Spores.

The mechano-bactericidal action of nanostructured surfaces is well-documented; however, synthetic nanostructured surfaces have not yet been explored for their antifungal properties toward filamentous fungal species. In this study, we developed a biomimetic nanostructured surface inspired by dragonfly wings. A high-aspect-ratio nanopillar topography was created on silicon (nano-Si) surfaces using inductively coupled plasma reactive ion etching (ICP RIE). To mimic the superhydrophobic nature of insect wings, the nano-Si was further functionalised with trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFTS). The viability of Aspergillus brasiliensis spores, in contact with either hydrophobic or hydrophilic nano-Si surfaces, was determined using a combination of standard microbiological assays, confocal laser scanning microscopy (CLSM), and focused ion beam scanning electron microscopy (FIB-SEM). Results indicated the breakdown of the fungal spore membrane upon contact with the hydrophilic nano-Si surfaces. By contrast, hydrophobised nano-Si surfaces prevented the initial attachment of the fungal conidia. Hydrophilic nano-Si surfaces exhibited both antifungal and fungicidal properties toward attached A. brasisiensis spores via a 4-fold reduction of attached spores and approximately 9-fold reduction of viable conidia from initial solution after 24 h compared to their planar Si counterparts. Thus, we reveal, for the first time, the physical rupturing of attaching fungal spores by biomimetic hydrophilic nanostructured surfaces.

Fluorescence Colour Control in Perylene-Labeled Polymer Chains Trapped by Nanotextured Silicon.

The ability to modulate, tune, and control fluorescence colour has attracted much attention in photonics-related research fields. Thus far, it has been impossible to achieve fluorescence colour control (FCC) for material with a fixed structure, size, surrounding medium, and concentration. Here, we propose a novel approach to FCC using optical tweezers. We demonstrate an optical trapping technique using nanotextured Si (black-Si) that can efficiently trap polymer chains. By increasing the laser intensity, the local concentration of perylene-labelled water-soluble polymer chains increased inside the trapping potential. Accordingly, the excimer fluorescence of perylene increased while the monomer fluorescence decreased, evidenced by a fluorescence colour change from blue to orange. Using nanostructure-assisted optical tweezing, we demonstrate control of the relative intensity ratio of fluorescence of the two fluorophores, thus showing remote and reversible FCC of the polymer assembly.

Tilted black-Si: ∼0.45 form-birefringence from sub-wavelength needles.

The self-organised conical needles produced by plasma etching of silicon (Si), known as black silicon (b-Si), create a form-birefringent surface texture when etching of Si orientated at angles of θi < 50 - 70° (angle between the Si surface and vertical plasma E-field). The height of the needles in the form-birefringent region following 15 min etching was d ∼ 200 nm and had a 100 µm width of the optical retardance/birefringence, characterised using polariscopy. The height of the b-Si needles corresponds closely to the skin-depth of Si ∼λ/4 for the visible spectral range. Reflection-type polariscope with a voltage-controlled liquid-crystal retarder is proposed to directly measure the retardance Δn × d/λ ≈ 0.15 of the region with tilted b-Si needles. The quantified form birefringence of Δn = -0.45 over λ = 400 - 700 nm spectral window was obtained. Such high values of Δn at visible wavelengths can only be observed in the most birefringence calcite or barium borate as well as in liquid crystals. The replication of b-Si into Ni-shim with high fidelity was also demonstrated and can be used for imprinting of the b-Si nanopattern into other materials.

Influence of nanoscale topology on bactericidal efficiency of black silicon surfaces.

The nanostructuring of materials to create bactericidal and antibiofouling surfaces presents an exciting alternative to common methods of preventing bacterial adhesion. The fabrication of synthetic bactericidal surfaces has been inspired by the anti-wetting and anti-biofouling properties of insect wings, and other topologies found in nature. Black silicon is one such synthetic surfaces which has established bactericidal properties. In this study we show that time-dependent plasma etching of silicon wafers using 15, 30, and 45 min etching intervals, is able to produce different surface geometries with linearly increasing heights of approximately 280, 430, and 610 nm, respectively. After incubation on these surfaces with Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa bacterial cells it was established that smaller, more densely packed pillars exhibited the greatest bactericidal activity with 85% and 89% inactivation of bacterial cells, respectively. The decrease in the pillar heights, pillar cap diameter and inter-pillar spacing corresponded to a subsequent decrease in the number of attached cells for both bacterial species.

Impact of multiscale surface topography characteristics on Candida albicans biofilm formation: From cell repellence to fungicidal activity.

While there has been significant research conducted on bacterial colonization on implant materials, with a focus on developing surface modifications to prevent the formation of bacterial biofilms, the study of Candida albicans biofilms on implantable materials is still in its infancy, despite its growing relevance in implant-associated infections. C. albicans fungal infections represent a significant clinical concern due to their severity and associated high fatality rate. Pathogenic yeasts account for an increasing proportion of implant-associated infections, since Candida spp. readily form biofilms on medical and dental device surfaces. In addition, these biofilms are highly antifungal-resistant, making it crucial to explore alternative solutions for the prevention of Candida implant-associated infections. One promising approach is to modify the surface properties of the implant, such as the wettability and topography of these substrata, to prevent the initial Candida attachment to the surface. This review summarizes recent research on the effects of surface wettability, roughness, and architecture on Candida spp. attachment to implantable materials. The nanofabrication of material surfaces are highlighted as a potential method for the prevention of Candida spp. attachment and biofilm formation on medical implant materials. Understanding the mechanisms by which Candida spp. attach to surfaces will allow such surfaces to be designed such that the incidence and severity of Candida infections in patients can be significantly reduced. Most importantly, this approach could also substantially reduce the need to use antifungals for the prevention and treatment of these infections, thereby playing a crucial role in minimizing the possibility contributing to instances of antimicrobial resistance. STATEMENT OF SIGNIFICANCE: In this review we provide a systematic analysis of the role that surface characteristics, such as wettability, roughness, topography and architecture, play on the extent of C. albicans cells attachment that will occur on biomaterial surfaces. We show that exploiting bioinspired surfaces could significantly contribute to the prevention of antimicrobial resistance to antifungal and chemical-based preventive measures. By reducing the attachment and growth of C. albicans cells using surface structure approaches, we can decrease the need for antifungals, which are conventionally used to treat such infections.

Piercing of the Human Parainfluenza Virus by Nanostructured Surfaces.

This paper presents a comprehensive experimental and theoretical investigation into the antiviral properties of nanostructured surfaces and explains the underlying virucidal mechanism. We used reactive ion etching to fabricate silicon (Si) surfaces featuring an array of sharp nanospikes with an approximate tip diameter of 2 nm and a height of 290 nm. The nanospike surfaces exhibited a 1.5 log reduction in infectivity of human parainfluenza virus type 3 (hPIV-3) after 6 h, a substantially enhanced efficiency, compared to that of smooth Si. Theoretical modeling of the virus-nanospike interactions determined the virucidal action of the nanostructured substrata to be associated with the ability of the sharp nanofeatures to effectively penetrate the viral envelope, resulting in the loss of viral infectivity. Our research highlights the significance of the potential application of nanostructured surfaces in combating the spread of viruses and bacteria. Notably, our study provides valuable insights into the design and optimization of antiviral surfaces with a particular emphasis on the crucial role played by sharp nanofeatures in maximizing their effectiveness.

Modulation of MG-63 Osteogenic Response on Mechano-Bactericidal Micronanostructured Titanium Surfaces.

Despite recent advances in the development of orthopedic devices, implant-related failures that occur as a result of poor osseointegration and nosocomial infection are frequent. In this study, we developed a multiscale titanium (Ti) surface topography that promotes both osteogenic and mechano-bactericidal activity using a simple two-step fabrication approach. The response of MG-63 osteoblast-like cells and antibacterial activity toward Pseudomonas aeruginosa and Staphylococcus aureus bacteria was compared for two distinct micronanoarchitectures of differing surface roughness created by acid etching, using either hydrochloric acid (HCl) or sulfuric acid (H2SO4), followed by hydrothermal treatment, henceforth referred to as either MN-HCl or MN-H2SO4. The MN-HCl surfaces were characterized by an average surface microroughness (Sa) of 0.8 ± 0.1 µm covered by blade-like nanosheets of 10 ± 2.1 nm thickness, whereas the MN-H2SO4 surfaces exhibited a greater Sa value of 5.8 ± 0.6 µm, with a network of nanosheets of 20 ± 2.6 nm thickness. Both micronanostructured surfaces promoted enhanced MG-63 attachment and differentiation; however, cell proliferation was only significantly increased on MN-HCl surfaces. In addition, the MN-HCl surface exhibited increased levels of bactericidal activity, with only 0.6% of the P. aeruginosa cells and approximately 5% S. aureus cells remaining viable after 24 h when compared to control surfaces. Thus, we propose the modulation of surface roughness and architecture on the micro- and nanoscale to achieve efficient manipulation of osteogenic cell response combined with mechanical antibacterial activity. The outcomes of this study provide significant insight into the further development of advanced multifunctional orthopedic implant surfaces.

Localization of nanospheres in pheochromocytoma-like cells following exposure to high-frequency electromagnetic fields at 18 GHz.

Exposure to high-frequency (HF) electromagnetic fields (EMFs) at 18 GHz was previously found to induce reversible cell permeabilization in eukaryotic cells; however, the fate of internalized foreign objects inside the cell remains unclear. Here, silica core-shell gold nanospheres (Au NS) of 20 ± 5 nm diameter were used to study the localization of Au NS in pheochromocytoma (PC 12) cells after exposure to HF EMFs at 18 GHz. Internalization of Au NS was confirmed using fluorescence microscopy and transmission electron microscopy. Analysis based on corresponding scanning transmission electron microscopy energy-dispersive spectroscopy revealed the presence of the Au NS free within the PC 12 cell membrane, cytoplasm, enclosed within intracellular vesicles and sequestered in vacuoles. The results obtained in this work highlight that exposure to HF EMFs could be used as an efficient technique with potential for effective delivery of drugs, genetic material, and nanomaterials into cells for the purpose of cellular manipulation or therapy.

Surface Architecture Influences the Rigidity of Candida albicans Cells.

Atomic force microscopy (AFM) was used to investigate the morphology and rigidity of the opportunistic pathogenic yeast, Candida albicans ATCC 10231, during its attachment to surfaces of three levels of nanoscale surface roughness. Non-polished titanium (npTi), polished titanium (pTi), and glass with respective average surface roughness (Sa) values of 389 nm, 14 nm, and 2 nm, kurtosis (Skur) values of 4, 16, and 4, and skewness (Sskw) values of 1, 4, and 1 were used as representative examples of each type of nanoarchitecture. Thus, npTi and glass surfaces exhibited similar Sskw and Skur values but highly disparate Sa. C. albicans cells that had attached to the pTi surfaces exhibited a twofold increase in rigidity of 364 kPa compared to those yeast cells attached to the surfaces of npTi (164 kPa) and glass (185 kPa). The increased rigidity of the C. albicans cells on pTi was accompanied by a distinct round morphology, condensed F-actin distribution, lack of cortical actin patches, and the negligible production of cell-associated polymeric substances; however, an elevated production of loose extracellular polymeric substances (EPS) was observed. The differences in the physical response of C. albicans cells attached to the three surfaces suggested that the surface nanoarchitecture (characterized by skewness and kurtosis), rather than average surface roughness, could directly influence the rigidity of the C. albicans cells. This work contributes to the next-generation design of antifungal surfaces by exploiting surface architecture to control the extent of biofilm formation undertaken by yeast pathogens and highlights the importance of performing a detailed surface roughness characterization in order to identify and discriminate between the surface characteristics that may influence the extent of cell attachment and the subsequent behavior of the attached cells.

Lethal Interactions of Atomically Precise Gold Nanoclusters and Pseudomonas aeruginosa and Staphylococcus aureus Bacterial Cells.

Ultrasmall metal nanoclusters (NCs) are employed in an array of diagnostic and therapeutic applications due to their tunable photoluminescence, high biocompatibility, polyvalent effect, ease of modification, and photothermal stability. However, gold nanoclusters' (AuNCs') intrinsically antimicrobial properties remain poorly explored and are not well understood. Here, we share an insight into the antimicrobial action of atomically precise AuNCs based on their ability to passively translocate across the bacterial membrane. Functionalized by a hydrophilic modified-bidentate sulfobetaine zwitterionic molecule (AuNC-ZwBuEt) or a more hydrophobic monodentate-thiolate, mercaptohexanoic acid (AuNC-MHA) molecule, 2 nm AuNCs were lethal to both Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacteria. The bactericidal efficiency was found to be bacterial strain-, time-, and concentration-dependent. The direct visualizations of the translocation of AuNCs and AuNC-cell and subcellular interactions were investigated using cryo-soft X-ray nano-tomography, transmission electron microscopy (TEM), and scanning TEM energy-dispersive spectroscopy analyses. AuNC-MHA were identified in the bacterial cytoplasm within 30 min, without evidence of the loss of membrane integrity. It is proposed that the bactericidal effect of AuNCs is attributed to their size, which allows for efficient energy-independent translocation across the cell membrane. The internalization of both AuNCs caused massive internal damage to the cells, including collapsed subcellular structures and altered cell morphology, leading to the eventual loss of cellular integrity.

Mechano-bactericidal actions of nanostructured surfaces.

Antibiotic resistance is a global human health threat, causing routine treatments of bacterial infections to become increasingly difficult. The problem is exacerbated by biofilm formation by bacterial pathogens on the surfaces of indwelling medical and dental devices that facilitate high levels of tolerance to antibiotics. The development of new antibacterial nanostructured surfaces shows excellent prospects for application in medicine as next-generation biomaterials. The physico-mechanical interactions between these nanostructured surfaces and bacteria lead to bacterial killing or prevention of bacterial attachment and subsequent biofilm formation, and thus are promising in circumventing bacterial infections. This Review explores the impact of surface roughness on the nanoscale in preventing bacterial colonization of synthetic materials and categorizes the different mechanisms by which various surface nanopatterns exert the necessary physico-mechanical forces on the bacterial cell membrane that will ultimately result in cell death.

Towards antiviral polymer composites to combat COVID-19 transmission.

Polymer matrix composite materials have the capacity to aid the indirect transmission of viral diseases. Published research shows that respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or COVID-19), can attach to polymer substrata as a result of being contacted by airborne droplets resulting from infected people sneezing or coughing in close proximity. Polymer matrix composites are used to produce a wide range of products that are "high-touch" surfaces, such as sporting goods, laptop computers and household fittings, and these surfaces can be readily contaminated by pathogens. This article reviews published research on the retention of SARS-CoV-2 and other virus types on plastics. The factors controlling the viral retention time on plastic surfaces are examined and the implications for viral retention on polymer composite materials are discussed. Potential strategies that can be used to impart antiviral properties to polymer composite surfaces are evaluated. These strategies include modification of the surface composition with biocidal agents (e.g., antiviral polymers and nanoparticles) and surface nanotexturing. The potential application of these surface modification strategies in the creation of antiviral polymer composite surfaces is discussed, which opens up an exciting new field of research for composite materials.

Incoherent Optical Tweezers on Black Titanium.

Optical tweezers enable the manipulation of micro- and nanodielectric particles through entrapment using a tightly focused laser. Generally, optical trapping of submicron size particles requires high-intensity light in the order of MW/cm2. Here, we demonstrate a technique of stable optical trapping of submicron polymeric beads on nanostructured titanium surfaces (black-Ti) without the use of lasers. Fluorescent polystyrene beads with a diameter d = 20-500 nm were successfully trapped on black-Ti by low-intensity focused illumination of incoherent light at λ = 370 m from a Hg lamp. Light intensity was 5.5 W/cm2, corresponding to a reduced light intensity of 6 orders of magnitude. Upon switching off illumination, trapped particles were released from the illuminated area, indicating that trapping was optically driven and reversible. Such trapping behavior was not observed on nonstructured Ti surfaces or on nanostructured silicon surfaces. Thus, the Ti nanostructures were demonstrated to play a key role.

Antifungal versus antibacterial defence of insect wings.

HYPOTHESIS: The ability exhibited by insect wings to resist microbial infestation is a unique feature developed over 400 million years of evolution in response to lifestyle and environmental pressures. The self-cleaning and antimicrobial properties of insect wings may be attributed to the unique combination of nanoscale structures found on the wing surface. EXPERIMENTS: In this study, we characterised the wetting characteristics of superhydrophobic damselfly Calopteryx haemorrhoidalis wings. We revealed the details of air entrapment at the micro- and nano scales on damselfly wing surfaces using a combination of spectroscopic and electron microscopic techniques. Cryo-focused-ion-beam scanning electron microscopy was used to directly observe fungal spores and conidia that were unable to cross the air-liquid interface. By contrast, bacterial cells were able to cross the air-water interface to be ruptured upon attachment to the nanopillar surface. The robustness of the air entrapment, and thus the wing antifungal behaviour, was demonstrated after 1-week of water immersion. A newly developed wetting model confirmed the strict Cassie-Baxter wetting regime when damselfly wings are immersed in water. FINDINGS: We provide evidence that the surface nanopillar topography serves to resist both fungal and bacterial attachment via a dual action: repulsion of fungal conidia while simultaneously killing bacterial cells upon direct contact. These findings will play an important role in guiding the fabrication of biomimetic, anti-fouling surfaces that exhibit both bactericidal and anti-fungal properties.

Lensless Three-Dimensional Quantitative Phase Imaging Using Phase Retrieval Algorithm.

Quantitative phase imaging (QPI) techniques are widely used for the label-free examining of transparent biological samples. QPI techniques can be broadly classified into interference-based and interferenceless methods. The interferometric methods which record the complex amplitude are usually bulky with many optical components and use coherent illumination. The interferenceless approaches which need only the intensity distribution and works using phase retrieval algorithms have gained attention as they require lesser resources, cost, space and can work with incoherent illumination. With rapid developments in computational optical techniques and deep learning, QPI has reached new levels of applications. In this tutorial, we discuss one of the basic optical configurations of a lensless QPI technique based on the phase-retrieval algorithm. Simulative studies on QPI of thin, thick, and greyscale phase objects with assistive pseudo-codes and computational codes in Octave is provided. Binary phase samples with positive and negative resist profiles were fabricated using lithography, and a single plane and two plane phase objects were constructed. Light diffracted from a point object is modulated by phase samples and the corresponding intensity patterns are recorded. The phase retrieval approach is applied for 2D and 3D phase reconstructions. Commented codes in Octave for image acquisition and automation using a web camera in an open source operating system are provided.

Black-Si as a Photoelectrode.

The fabrication and characterization of photoanodes based on black-Si (b-Si) are presented using a photoelectrochemical cell in NaOH solution. B-Si was fabricated by maskless dry plasma etching and was conformally coated by tens-of-nm of TiO2 using atomic layer deposition (ALD) with a top layer of CoO x cocatalyst deposited by pulsed laser deposition (PLD). Low reflectivity R < 5 % of b-Si over the entire visible and near-IR ( λ < 2   µ m) spectral range was favorable for the better absorption of light, while an increased surface area facilitated larger current densities. The photoelectrochemical performance of the heterostructured b-Si photoanode is discussed in terms of the n-n junction between b-Si and TiO2.

Nanoscale Surface Roughness Influences Candida albicans Biofilm Formation.

The microbial contamination of surfaces presents a significant challenge due to the adverse effects associated with biofilm formation, particularly on implantable devices. Here, the attachment and biofilm formation of the opportunistic human pathogen, Candida albicans ATCC 10231, were studied on surfaces with decreasing magnitudes of nanoscale roughness. The nanoscale surface roughness of nonpolished titanium, polished titanium, and glass was characterized according to average surface roughness, skewness, and kurtosis. Nonpolished titanium, polished titanium, and glass possessed average surface roughness (Sa) values of 350, 20, and 2.5 nm; skewness (Sskw) values of 1.0, 4.0, and 1.0; and (Skur) values of 3.5, 16, and 4, respectively. These unique characteristics of the surface nanoarchitecture were found to play a key role in limiting C. albicans attachment and modulating the functional phenotypic changes associated with biofilm formation. Our results suggest that surfaces with a specific combination of surface topographical parameters could prevent the attachment and biofilm formation of C. albicans. After 7 days, the density of attached C. albicans cells was recorded to be 230, 70, and 220 cells mm-2 on nonpolished titanium, polished titanium, and glass surfaces, respectively. Despite achieving a very low attachment density, C. albicanscells were only observed to produce hyphae associated with biofilm formation on nonpolished titanium surfaces, possessing the highest degree of surface roughness (Sa = 350 nm). This study provides a more comprehensive picture of the impact of surface architectures on C. albicans attachment, which is beneficial for the design of antifungal surfaces.