Escherichia coli Adhesion and Biofilm Formation on Polymeric Nanostructured Surfaces.

Biofilm formation is a multistep process that requires initial contact between a bacterial cell and a surface substrate. Recent work has shown that nanoscale topologies impact bacterial cell viability; however, less is understood about how nanoscale surface properties impact other aspects of bacterial behavior. In this study, we examine the adhesive, viability, morphology, and colonization behavior of the bacterium Escherichia coli on 21 plasma-etched polymeric surfaces. Although we predicted that specific nanoscale surface structures of the surface would control specific aspects of bacterial behavior, we observed no correlation between any bacterial response or surface structures/properties. Instead, it appears that the surface composition of the polymer plays the most significant role in controlling and determining a bacterial response to a substrate, although changes to a polymeric surface via plasma etching alter initial bacteria colonization and morphology.

Transcriptional Response of Candida albicans to Nanostructured Surfaces Provides Insight into Cellular Rupture and Antifungal Drug Sensitization.

The rise in resistance levels against antifungal drugs has necessitated the development of strategies to combat fungal infections. Nanoscale antimicrobial surfaces, found on the cuticles of insects, have recently emerged as intriguing alternative antifungal strategies that function passively via contact and induced cell rupture. Nanostructured surfaces (NSS) offer a potentially transformative antimicrobial approach to reducing microbial biofilm formation. We examined the transcriptional response of Candida albicans, an opportunistic pathogen that is also a commensal dimorphic fungus, to the NSS found in the wings of Neotibicen spp. cicada and found characteristic changes in the expression of C. albicans genes associated with metabolism, biofilm formation, ergosterol biosynthesis, and DNA damage response after 2 h of exposure to the NSS. Further validation revealed that these transcriptional changes, particularly in the ergosterol biosynthesis pathway, sensitize C. albicans to major classes of antifungal drugs. These findings provide insights into NSS as antimicrobial surfaces and as a means of controlling biofilm formation.

Digital data storage on DNA tape using CRISPR base editors.

While the archival digital memory industry approaches its physical limits, the demand is significantly increasing, therefore alternatives emerge. Recent efforts have demonstrated DNA's enormous potential as a digital storage medium with superior information durability, capacity, and energy consumption. However, the majority of the proposed systems require on-demand de-novo DNA synthesis techniques that produce a large amount of toxic waste and therefore are not industrially scalable and environmentally friendly. Inspired by the architecture of semiconductor memory devices and recent developments in gene editing, we created a molecular digital data storage system called "DNA Mutational Overwriting Storage" (DMOS) that stores information by leveraging combinatorial, addressable, orthogonal, and independent in vitro CRISPR base-editing reactions to write data on a blank pool of greenly synthesized DNA tapes. As a proof of concept, this work illustrates writing and accurately reading of both a bitmap representation of our school's logo and the title of this study on the DNA tapes.

Digital data storage on DNA tape using CRISPR base editors.

While the archival digital memory industry approaches its physical limits, the demand is significantly increasing, therefore alternatives emerge. Recent efforts have demonstrated DNA's enormous potential as a digital storage medium with superior information durability, capacity, and energy consumption. However, the majority of the proposed systems require on-demand de-novo DNA synthesis techniques that produce a large amount of toxic waste and therefore are not industrially scalable and environmentally friendly. Inspired by the architecture of semiconductor memory devices and recent developments in gene editing, we created a molecular digital data storage system called "DNA Mutational Overwriting Storage" (DMOS) that stores information by leveraging combinatorial, addressable, orthogonal, and independent in vitro CRISPR base-editing reactions to write data on a blank pool of greenly synthesized DNA tapes. As a proof of concept, we wrote both a bitmap representation of our school's logo and the title of this study on the DNA tapes, and accurately recovered the stored data.

Dark-Field Microscopic Study of Cellular Uptake of Carbon Nanodots: Nuclear Penetrability.

Carbon nanodots are fascinating candidates for the field of biomedicine, in applications such as bioimaging and drug delivery. However, the nuclear penetrability and process are rarely studied and lack understanding, which limits their applications for drug carriers, single-molecule detection and live cell imaging. In this study, we attempt to examine the uptake of CNDs in cells with a focus on the potential nuclear penetrability using enhanced dark-field microscopy (EDFM) associated with hyperspectral imaging (HSI) to quantitatively determine the light scattering signals of CNDs in the cells. The effects of both CND incubation time and concentration are investigated, and plausible nuclear penetration involving the nuclear pore complex (NPC) is discussed. The experimental results and an analytical model demonstrate that the CNDs' uptake proceeds by a concentration-dependent three-stage behavior and saturates at a CND incubation concentration larger than 750 µg/mL, with a half-saturated concentration of 479 µg/mL. These findings would potentially help the development of CNDs' utilization in drug carriers, live cell imaging and other biomedical applications.

Tuning Microbial Activity via Programmatic Alteration of Cell/Substrate Interfaces.

A wide portfolio of advanced programmable materials and structures has been developed for biological applications in the last two decades. Particularly, due to their unique properties, semiconducting materials have been utilized in areas of biocomputing, implantable electronics, and healthcare. As a new concept of such programmable material design, biointerfaces based on inorganic semiconducting materials as substrates introduce unconventional paths for bioinformatics and biosensing. In particular, understanding how the properties of a substrate can alter microbial biofilm behavior enables researchers to better characterize and thus create programmable biointerfaces with necessary characteristics on demand. Herein, the current status of advanced microorganism-inorganic biointerfaces is summarized along with types of responses that can be observed in such hybrid systems. This work identifies promising inorganic material types along with target microorganisms that will be critical for future research on programmable biointerfacial structures.

Cell Rupture and Morphogenesis Control of the Dimorphic Yeast Candida albicans by Nanostructured Surfaces.

Nanostructured surfaces control microbial biofilm formation by killing mechanically via surface architecture. However, the interactions between nanostructured surfaces (NSS) and cellular fungi have not been thoroughly investigated and the application of NSS as a means of controlling fungal biofilms is uncertain. Cellular yeast such as Candida albicans are structurally and biologically distinct from prokaryotic microbes and therefore are predicted to react differently to nanostructured surfaces. The dimorphic opportunistic fungal pathogen, C. albicans, is responsible for most cases of invasive candidiasis and is a serious health concern due to the rapid increase of drug resistance strains. In this paper, we show that the nanostructured surfaces from a cicada wing alter C. albicans' viability, biofilm formation, adhesion, and morphogenesis through physical contact. However, the fungal cell response to the NSS suggests that nanoscale mechanical interactions impact C. albicans differently than prokaryotic microbes. This study informs on the use of nanoscale architecture for the control of eukaryotic biofilm formation and illustrates some potential caveats with the application of NSS as an antimicrobial means.

Modulating the Stress Response of E. coli at GaN Interfaces Using Surface Charge, Surface Chemistry, and Genetic Mutations.

The surface properties of inorganic materials can be used to modulate the response of microorganisms at the interface. We used the persistent photoconductivity properties of chemically treated gallium nitride substrates to evaluate the stress response of wild-type, ΔfliC, and ΔcsgG mutant E. coli exposed to charged surfaces. Substrate surface characterization and biological assays were used to correlate the physiological response to substrate surface charge. The physiological response was evaluated by measuring the intracellular levels of reactive oxygen species (ROS) and Ca2+ cations using fluorescent probes. We evaluated the response 1, 2, and 3 h after a short exposure to the surfaces to determine generational effects of the initial exposure on the physiology of the bacteria. In general, the ROS levels 1 h after exposure were not different. However, there were differences in Ca2+ levels in E. coli 1 h after the initial exposure to charged GaN surfaces, primarily in the wild-type E. coli. The differences in Ca2+ levels depended on the substrate surface chemistry and genetic mutation that suggests the involvement of multiple factors for modulating the interactions of bacteria at interfaces.

Oxidative Stress Transcriptional Responses of Escherichia coli at GaN Interfaces.

Microorganisms regulate their interactions with surfaces by altering the transcription of specific target genes in response to physicochemical surface cues. To assess the influence of surface charge and surface chemistry on the transcriptional oxidative stress response, we evaluated the expression of three genes, oxyS, katE, and sodB from the Gram-negative bacterium, Escherichia coli, after a short exposure to GaN interfaces. We observed that both surface charge and surface chemistry were the factors regulating the transcriptional response of the target genes, which indicates that reactive oxygen species (ROS) generation and the ROS response at the GaN interfaces were affected by changing surface properties. The changes in transcription did not correlate to the surface charge in all cases, indicating that there was an influence from multiple interfacial properties on the interactions. Alteration of the bacterial morphology also was a critical factor in these transcriptional responses to the surface cues. When compared to wild-type E. coli bacteria, bacteria missing either flagella or curli exhibited altered transcriptional profiles of the three oxidative stress genes when exposed to GaN materials. These results indicate that the bacterial flagella and curli modulated the oxidative stress response in different ways. The results of this work add to our understanding of the interactions of microbes at interfaces and will be useful for guiding the development of electronic biointerfaces.

Characterization of Hydrothermal Deposition of Copper Oxide Nanoleaves on Never-Dried Bacterial Cellulose.

Bacterial cellulose (BC) has attracted a great deal of interest due to its green synthesis and biocompatibility. The nanoscale dimension of BC nanofibers generates an enormous surface area that enhances interactions with water and soluble components within aqueous solution. Recent work has demonstrated that BC is a versatile platform for the formation of metal/metal oxide nanocomposites. Copper oxide (CuO) is a useful material to compare nanomaterial deposition on BC with other cellulosic materials because of copper's colorimetric reaction as it forms copper hydroxide (Cu(OH)2) and transitions to CuO. In this research, we found that never-dried BC readily deposits CuO into its matrix in a way that does not occur on cotton, dried BC, or regenerated cellulose fibers. We conclude that hydroxyl group availability does not adequately explain our results and that intrafibrillar pores in never-dried BC nanofibers play a critical role in CuO deposition.

Solid-state growth of Ag nanowires and analysis of the self-growing process on a bio-polymer chitosan film.

The growth mechanism of silver nanowires (AgNWs) in solution has been thoroughly investigated and it has been demonstrated that factors like oxidative etching and inclusion of Cl- ions in the reaction system play critical roles in the formation of AgNWs. This research is the first to report the growth mechanism of AgNWs in the solid state on a chitosan polymer film with respect to factors such as oxidative etching, Cl- ions and time. The AgNW synthetic method is a green process that involves aqueous solvents for film preparation and ambient conditions for AgNW growth. It is demonstrated that the source of the silver precursor for this solid state AgNW growth is the cuboidal AgCl nanoparticles that form during the solution preparation. Furthermore, it is shown that the 〈111〉 crystal faces of these cuboidal AgCl nanoparticles are the nucleation sites of AgNW growth. Unlike solution-based AgNW synthetic processes, the AgNWs generated by the chitosan film-based method are irregular and present lateral as well as longitudinal growth, which suggests a slightly different mechanism from the solution-based AgNW growth.

Behavior of E. coli with Variable Surface Morphology Changes on Charged Semiconductor Interfaces.

Bacterial behavior is often controlled by structural and composition elements of their cell wall. Using genetic mutant strains that change specific aspects of their surface structure, we modified bacterial behavior in response to semiconductor surfaces. We monitored the adhesion, membrane potential, and catalase activity of the Gram-negative bacterium Escherichia coli (E. coli) that were mutant for genes encoding components of their surface architecture, specifically flagella, fimbriae, curli, and components of the lipopolysaccharide membrane, while on gallium nitride (GaN) surfaces with different surface potentials. The bacteria and the semiconductor surface properties were recorded prior to the biofilm studies. The data from the materials and bioassays characterization supports the notion that alteration of the surface structure of the E. coli bacterium resulted in changes to bacterium behavior on the GaN medium. Loss of specific surface structure on the E. coli bacterium reduced its sensitivity to the semiconductor interfaces, while other mutations increase bacterial adhesion when compared to the wild-type control E. coli bacteria. These results demonstrate that bacterial behavior and responses to GaN semiconductor materials can be controlled genetically and can be utilized to tune the fate of living bacteria on GaN surfaces.

Polyacrylonitrile nanofibrous mat from electrospinning: Born with potential anti-fungal functionality.

Electrospun nanofibers have been found in many applications such as air/water filtration, performance apparel, drug delivery, and scaffold for tissue engineering and started to be integrated in commercial products, which leads to their exposure to environment. Electrospun nanofibrous material is a relatively new material to microorganism in nature and little is known about the biological implication of interactions between electrospun nanofibrous mats and cellular fungal cells. Herein the interaction between electrospun polyacrylonitrile (ESPAN) nanofibrous mat and representative non-pathogenic/pathogenic cellular yeasts (Saccharomyces cerevisiae and Candida albicans) was investigated. It is demonstrated for the first time that when these cellular yeasts, species of the kingdom fungi, were exposed to ESPAN nanofibrous mat, they exhibited lower growth rate, radical change to morphology, and reduced viability without presence of any chemical antifungal agent. These responses were distinct from the cellular interactions with other forms of PAN materials (e.g. solid film or microfibrous mat). Exploration of mechanism indicated that the interaction between yeast cell and electrospun nanofibrous mat is a complex phenomenon in which both nanofibrous morphology and fiber surface composition/property play significant roles. The inherent anti-yeast and potential anti-fungal functionality of ESPAN nanofibrous mat may make an immediate impact on environmental microorganism and could also benefit the next-generation material design to control microbial growth through solely physical contact.

Nanosphere Lithography of Chitin and Chitosan with Colloidal and Self-Masking Patterning.

Complex surface topographies control, define, and determine the properties of insect cuticles. In some cases, these nanostructured materials are a direct extension of chitin-based cuticles. The cellular mechanisms that generate these elaborate chitin-based structures are unknown, and involve complicated cellular and biochemical "bottom-up" processes. We demonstrated that a synthetic "top-down" fabrication technique-nanosphere lithography-generates surfaces of chitin or chitosan that mimic the arrangement of nanostructures found on the surface of certain insect wings and eyes. Chitin and chitosan are flexible and biocompatible abundant natural polymers, and are a sustainable resource. The fabrication of nanostructured chitin and chitosan materials enables the development of new biopolymer materials. Finally, we demonstrated that another property of chitin and chitosan-the ability to self-assemble nanosilver particles-enables a novel and powerful new tool for the nanosphere lithographic method: the ability to generate a self-masking thin film. The scalability of the nanosphere lithographic technique is a major limitation; however, the silver nanoparticle self-masking enables a one-step thin-film cast or masking process, which can be used to generate nanostructured surfaces over a wide range of surfaces and areas.

Characterization of Pseudomonas aeruginosa Films on Different Inorganic Surfaces before and after UV Light Exposure.

The changes of the surface properties of Au, GaN, and SiO x after UV light irradiation were used to actively influence the process of formation of Pseudomonas aeruginosa films. The interfacial properties of the substrates were characterized by X-ray photoelectron spectroscopy and atomic force microscopy. The changes in the P. aeruginosa film properties were accessed by analyzing adhesion force maps and quantifying the intracellular Ca2+ concentration. The collected analysis indicates that the alteration of the inorganic materials' surface chemistry can lead to differences in biofilm formation and variable response from P. aeruginosa cells.

Noninvasive Stimulation of Neurotypic Cells Using Persistent Photoconductivity of Gallium Nitride.

The persistent photoconductivity (PPC) of the n-type Ga-polar GaN was used to stimulate PC12 cells noninvasively. Analysis of the III-V semiconductor material by atomic force microscopy, Kelvin probe force microscopy, photoconductivity, and X-ray photoelectron spectroscopy quantified bulk and surface charge, as well as chemical composition before and after exposure to UV light and cell culture media. The semiconductor surface was made photoconductive by illumination with UV light and experienced PPC, which was utilized to stimulate PC12 cells in vitro. Stimulation was confirmed by measuring the changes in intracellular calcium concentration. Control experiments with gallium salt verified the stimulation of neurotypic cells. Inductively coupled plasma mass spectrometry data confirmed the lack of gallium leaching and toxic effects during the stimulation.

Bioelectronics communication: encoding yeast regulatory responses using nanostructured gallium nitride thin films.

Baker's yeast, S. cerevisiae, is a model organism that is used in synthetic biology. The work demonstrates how GaN nanostructured thin films can encode physiological responses in S. cerevisiae yeast. The Ga-polar, n-type, GaN thin films are characterized via Photocurrent Measurements, Atomic Force Microscopy and Kelvin Probe Force Microscopy. UV light is used to induce persistent photoconductivity that results in charge accumulation on the surface. The morphological, chemical and electronic properties of the nanostructured films are utilized to activate the cell wall integrity pathway and alter the amount of chitin produced by the yeast. The encoded cell responses are induced by the semiconductor interfacial properties associated with nanoscale topography and the accumulation of charge on the surface that promotes the build-up of oxygen species and in turn cause a hyperoxia related change in the yeast. The thin films can also alter the membrane voltage of yeast. The observed modulation of the membrane voltage of the yeast exposed to different GaN samples supports the notion that the semiconductor material can cause cell polarization. The results thus define a strategy for bioelectronics communication where the roughness, surface chemistry and charge of the wide band gap semiconductor's thin film surface initiate the encoding of the yeast response.

Variably doped nanostructured gallium nitride surfaces can serve as biointerfaces for neurotypic PC12 cells and alter their behavior.

Neurotypic PC12 cells behavior was studied on nanostructured GaN and rationalized with respect to surface charge, doping level, and chemical functionalization. The semiconductor analysis included atomic force microscopy, Kelvin probe force microscopy, and X-ray photoelectron spectroscopy. The semiconductor surfaces were then evaluated as biointerfaces, and the in vitro cell behavior was quantified based on cell viability, reactive oxygen species production, as well as time dependent intracellular Ca concentration, [Ca2+]i, a known cell-signaling molecule. In this work, we show that persistent photoconductivity (PPC) can be used to alter the surface properties prior to chemical functionalization, the concentration of dopants can have some effect on cellular behavior, and that chemical functionalization changes the surface potential before and after exposure to UV light. Finally, we describe some competing mechanisms of PPC-induced [Ca2+]i changes, and how researchers looking to control cell behavior non-invasively can consider PPC as a useful control knob.

Solid-state synthesis of silver nanowires using biopolymer thin films.

In this paper, we describe a novel method of silver nanowire (AgNW) synthesis. Silver nanoparticles (AgNPs) were synthesized under ambient conditions by a chitosan/chitin-based method. These crystalline AgNPs then served as seeds for the solid-state formation of AgNWs within a drop-cast chitosan/chitin thin film. To the best of our knowledge, this is the first report of AgNW growth on a bio-polymer thin film. Chemical analysis demonstrated that AgNPs and AgNWs produced by this synthetic process have distinct interactions with polysaccharide polymers, and unlike AgNWs produced by other methods, the AgNWs formed in the chitin/chitosan matrix display an irregular twisted morphology. The flexible AgNW/chitosan nanocomposite material is conductive, and we incorporate this new material into a peroxide sensor to demonstrate of its potential applications in chemical sensing devices.

Alternative SiO2 Surface Direct MDCK Epithelial Behavior.

The mechanical interactions of cells are mediated through adhesive interactions. In this study, we examined the growth, cellular behavior, and adhesion of MDCK epithelial cells on three different SiO2 substrates: amorphous glass coverslips and the silicon oxide layers that grow on ⟨111⟩ and ⟨100⟩ wafers. While compositionally all three substrates are almost similar, differences in surface energy result in dramatic differences in epithelial cell morphology, cell-cell adhesion, cell-substrate adhesion, actin organization, and extracellular matrix (ECM) protein expression. We also observe striking differences in ECM protein binding to the various substrates due to the hydrogen bond interactions. Our results demonstrate that MDCK cells have a robust response to differences in substrates that is not obviated by nanotopography or surface composition and that a cell's response may manifest through subtle differences in surface energies of the materials. This work strongly suggests that other properties of a material other than composition and topology should be considered when interpreting and controlling interactions of cells with a substrate, whether it is synthetic or natural.