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.

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.

Nanostructured Oxides Containing Ga: Materials with Unique Properties for Aqueous-Based Applications.

Oxides containing Ga have been studied by various research communities due to their stability under harsh conditions as well as conductivity and luminescence properties. Nanostructured forms of such oxides can be fabricated by a variety of methods. Advances in synthesis approaches have focused on control over size and shape that can permit adaptation in applied interfaces related to medicine, energy, and the environment. Chemical functionalization can enhance the stability of nanostructured oxides containing Ga in aqueous solutions. In this prospective, we summarize progress in making these materials as well as functionalizing them in water solutions. The prospective also identifies future opportunities with these materials in applied and fundamental materials chemistry research.

Modification of the Surface Properties of Al x Ga1-x N Substrates with Gradient Aluminum Composition Using Wet Chemical Treatments.

The surface properties of biomolecular gradients are widely known to be important for controlling cell dynamics, but there is a lack of platforms for studying them in vitro using inorganic materials. The changes in various surface properties of an Al x Ga1-x N film (0.173 ≤ x ≤ 0.220) with gradient aluminum content were quantified to demonstrate the ability to modify interfacial characteristics. Four wet chemical treatments were used to modify the surface of the film: (i) oxide passivation by hydrogen peroxide, (ii) two-step functionalization with a carboxylic acid following hydrogen peroxide pretreatment, (iii) phosphoric acid etch, and (iv) in situ functionalization with a phosphonic acid in phosphoric acid. The characterization confirmed changes in the topography, nanostructures, and hydrophobicity after chemical treatment. Additionally, X-ray photoelectron spectroscopy was used to confirm that the chemical composition of the surfaces, in particular, Ga2O3 and Al2O3 content, was dependent on both the chemical treatment and the Al content of the gradient. Spectroscopic evaluation showed red shifts in strain-sensitive Raman peaks as the Al content gradually increased, but the same peaks blue-shifted after chemical treatment. Kelvin probe force microscopy measurements demonstrated that one can modify the surface charge using the chemical treatments. There were no predictable or controllable surface charge trends because of the spontaneous oxide-based nanostructured formations of the bulk material that varied based on treatment and were defect-dependent. The reported methodology and characterization can be utilized in future interfacial studies that rely on water-based wet chemical functionalization of inorganic materials.

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.

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.

Ga Ion-Enhanced and Particle Shape-Dependent Generation of Reactive Oxygen Species in X-ray-Irradiated Composites.

The reported results test the effects of the collective behavior hypothesized to contribute to the production of more reactive oxygen species (ROS) in vitro and result in an enhanced radiosensitization. The role of particle shape in composites with gallium oxyhydroxide (GaOOH) particles and Matrigel is studied. Particles of two different shapes are embedded into the gel to understand only the materials effect on the generation of ROS rather than cell penetrating variations. The paper reports materials characterization by scanning electron microscopy and X-ray diffraction. The stability of the particles within the composite is assessed by quantification of leached metal using inductively coupled plasma mass spectrometry. The amount of ROS in each construct under variable radiation conditions is quantified in the presence and absence of PC12 cells seeded on top of the composites. The viability of cells is also recorded under different in vitro conditions. The collective materials characterization and the results from the bioassays are used to explain the role of anisotropy on the radiosensitization of nanostructures containing Ga. The presence of Ga ions in composites can have a radiosensitizing effect, and the amount of the available Ga3+ determines the magnitude of the radiosensitization. The shape of the particles determines the stability in aqueous solutions and release of Ga3+ that triggers ROS production. The concentration and shape of Ga-containing materials can be combined to generate an additive effect by increasing the amount of available free metal ions in solution. The studies with GaOOH containing composites enable one to explore the role of key parameters that lead to an increased efficiency of radiation treatments.

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.

Persistent Photoconductivity, Nanoscale Topography, and Chemical Functionalization Can Collectively Influence the Behavior of PC12 Cells on Wide Bandgap Semiconductor Surfaces.

Wide bandgap semiconductors such as gallium nitride (GaN) exhibit persistent photoconductivity properties. The incorporation of this asset into the fabrication of a unique biointerface is presented. Templates with lithographically defined regions with controlled roughness are generated during the semiconductor growth process. Template surface functional groups are varied using a benchtop surface functionalization procedure. The conductivity of the template is altered by exposure to UV light and the behavior of PC12 cells is mapped under different substrate conductivity. The pattern size and roughness are combined with surface chemistry to change the adhesion of PC12 cells when the GaN is made more conductive after UV light exposure. Furthermore, during neurite outgrowth, surface chemistry and initial conductivity difference are used to facilitate the extension to smoother areas on the GaN surface. These results can be utilized for unique bioelectronics interfaces to probe and control cellular behavior.

Gallium containing composites as a tunable material to understand neuronal behavior under variable stiffness and radiation conditions.

We report a composite biomaterial containing nanostructured GaOOH and Matrigel™ that can be modulated with respect to its stiffness and radiosensitization properties. A variety of concentrations of GaOOH were added to the composite to alter the mechanical properties of the material as well as to tune the radiosensitizing properties to the composite. PC-12 cells were used to study the combined effects of different stimuli on cell behavior. NGF was given to the cells to record their morphology as well as viability. An increase in the substrate stiffness caused an increase in neurite outgrowth but a decrease in cell viability. In addition, increasing the radiation dose decreased neurite outgrowth but increased cell viability when radiosensitizing particles were present. A subtractive effect between radiosensitizing and mechanical stimuli was observed when PC-12 cells were grown on the GaOOH containing composite.

In situ and ex situ functionalization of nanostructured gallium oxy-hydroxide with a porphyrin dye.

The surface attachment of a porphyrin dye to nanocrystalline GaOOH was performed using two routes of solution-based functionalization. The first method of functionalization utilized an in situ incorporation of dissolved porphyrin salt in solution during the microwave synthesis step. Additionally, synthesized GaOOH nanorods were mixed in porphyrin solution after the microwave process to make an ex situ GaOOH/TTP-PO-3 . X-ray photoelectron spectroscopy confirmed the presence of expected surface species and provided evidence of increased surface coverage of TTP-PO3 on GaOOH in the ex situ- GaOOH/TTP-PO3 as compared to the in situ one. Size and morphology changes were investigated using SEM and, along with analysis of XRD, the in situ samples showed larger crystallite sizes. This was confirmed with PL due to the higher bandgap energy evident in the ex situ GaOOH/TTP-PO3 compared to the in situ sample. A stability study was performed using fluorescence spectroscopy which indicated no leaching of porphyrin from the in situ GaOOH/TTP-PO3 . However, porphyrin leaching was evident from the ex situ GaOOH/TTP-PO3 sample. The stability of the in situ GaOOH/TTP-PO3 makes it attractive for a number of interfacial applications. SCANNING 38:671-683, 2016. © 2016 Wiley Periodicals, Inc.

Interfaces with Tunable Mechanical and Radiosensitizing Properties.

We report the fabrication of a composite containing nanostructured GaOOH and Matrigel with tunable radiosensitizing and stiffness properties. Composite characterization was done with microscopy and rheology. The utility of the interface was tested in vitro using fibroblasts. Cell viability and reactive oxygen species assays quantified the effects of radiation dosages and GaOOH concentrations. Fibroblasts' viability decreased with increasing concentration of GaOOH and composite stiffness. During ionizing radiation experiments the presence of the scintillating GaOOH triggered a different cellular response. Reactive oxygen species data demonstrated that one can reduce the amount of radiation needed to modulate the behavior of cells on interfaces with different stiffness containing a radiosensitizing material.

Electronic Biosensors Based on III-Nitride Semiconductors.

We review recent advances of AlGaN/GaN high-electron-mobility transistor (HEMT)-based electronic biosensors. We discuss properties and fabrication of III-nitride-based biosensors. Because of their superior biocompatibility and aqueous stability, GaN-based devices are ready to be implemented as next-generation biosensors. We review surface properties, cleaning, and passivation as well as different pathways toward functionalization, and critically analyze III-nitride-based biosensors demonstrated in the literature, including those detecting DNA, bacteria, cancer antibodies, and toxins. We also discuss the high potential of these biosensors for monitoring living cardiac, fibroblast, and nerve cells. Finally, we report on current developments of covalent chemical functionalization of III-nitride devices. Our review concludes with a short outlook on future challenges and projected implementation directions of GaN-based HEMT biosensors.

Comparison of the Stability of Functionalized GaN and GaP.

Surface functionalization via 1 H,1 H,2 H,2H-perfluoro octanephosphonic acid was done in the presence of phosphoric acid to provide a simplified surface passivation technique for gallium nitride (GaN) and gallium phosphide (GaP). In an effort to identify the leading causes of surface instabilities, hydrogen peroxide was utilized as an additional chemical modification to cap unsatisfied bonds. The stability of the surfaces was studied in an aqueous environment and subsequently characterized. A physical characterization was carried out to evaluate the surface roughness and water hydrophobicity pre and post stability testing via atomic force microscopy and water goniometry. Surface-chemistry changes and solution leaching were quantified by X-ray photoelectron spectroscopy and inductively coupled plasma mass spectrometry. The results indicate a sensitivity to hydroxyl terminated species for both GaN and GaP under aqueous environments, as the increase of the degree of leaching was more significant for hydrogen peroxide treated samples. The results support the notion that hydroxyl species act as precursors to gallium oxide formation and lead to subsequent instability in aqueous solutions.

Adsorption and adhesion of common serum proteins to nanotextured gallium nitride.

As the broader effort towards device and material miniaturization progresses in all fields, it becomes increasingly important to understand the implications of working with functional structures that approach the size scale of molecules, particularly when considering biological systems. It is well known that thin films and nanostructures feature different optical, electrical, and mechanical properties from their bulk composites; however, interactions taking place at the interface between nanomaterials and their surroundings are less understood. Here, we explore interactions between common serum proteins - serum albumin, fibrinogen, and immunoglobulin G - and a nanotextured gallium nitride surface. Atomic force microscopy with a carboxyl-terminated colloid tip is used to probe the 'activity' of proteins adsorbed onto the surface, including both the accessibility of the terminal amine to the tip as well as the potential for protein extension. By evaluating the frequency of tip-protein interactions, we can establish differences in protein behaviour on the basis of both the surface roughness as well as morphology, providing an assessment of the role of surface texture in dictating protein-surface interactions. Unidirectional surface features - either the half-unit cell steppes of as-grown GaN or those produced by mechanical polishing - appear to promote protein accessibility, with a higher frequency of protein extension events taking place on these surfaces when compared with less ordered surface features. Development of a full understanding of the factors influencing surface-biomolecule interactions can pave the way for specific surface modification to tailor the bio-material interface, offering a new path for device optimization.

Engineering the cell-semiconductor interface: a materials modification approach using II-VI and III-V semiconductor materials.

Developing functional biomedical devices based on semiconductor materials requires an understanding of interactions taking place at the material-biosystem interface. Cell behavior is dependent on the local physicochemical environment. While standard routes of material preparation involve chemical functionalization of the active surface, this review emphasizes both biocompatibility of unmodified surfaces as well as use of topographic features in manipulating cell-material interactions. Initially, the review discusses experiments involving unmodified II-VI and III-V semiconductors - a starting point for assessing cytotoxicity and biocompatibility - followed by specific surface modification, including the generation of submicron roughness or the potential effect of quantum dot structures. Finally, the discussion turns to more recent work in coupling topography and specific chemistry, enhancing the tunability of the cell-semiconductor interface. With this broadened materials approach, researchers' ability to tune the interactions between semiconductors and biological environments continues to improve, reaching new heights in device function.