Facile fabrication of ordered discontinuous nanotopography on photosensitive substrates for enhanced neuronal differentiation.

Controlling the development and morphology of neurons is important for basic neuroscience research as well as for applications in nerve regeneration and neural interfaces. Various studies have shown that nanoscale topographies can promote the development of neuronal cells and the differentiation of neural stem cells; however, the fabrication of these nanotopographical features often involves expensive and sophisticated techniques. Here, we employ nanosphere lens lithography combined with UV-LED technology to create nanopatterns on an SU-8 photoresist. We develop a facile method to create a reusable polystyrene nanosphere (PS-NS) lens array by the spontaneous formation of a hexagonal close-packed array of PS-NSs at a water-air interface and its subsequent transfer to a polydimethylsiloxane carrier film without using any special equipment. We show that this simple technique can create ordered arrays of nanodots on an SU-8 film, the dimensions of which can be controlled by the size of the PS-NSs. When used as a substrate for the neuronal differentiation of pheochromocytoma (PC12) cells, the nanopatterned SU-8 films exhibit enhanced differentiation parameters with respect to conventional tissue culture plastic as compared with their flat counterparts. The method proposed here can greatly facilitate the nanopatterning of various photosensitive substrates for the development of implants for nerve regeneration and neural interfacing.

Unraveling the Morphological Evolution and Etching Kinetics of Porous Silicon Nanowires During Metal-Assisted Chemical Etching.

Many potential applications of porous silicon nanowires (SiNWs) fabricated with metal-assisted chemical etching are highly dependent on the precise control of morphology for device optimization. However, the effects of key etching parameters, such as the amount of deposited metal catalyst, HF-oxidant molar ratio (χ), and solvent concentration, on the morphology and etching kinetics of the SiNWs still have not been fully explored. Here, the changes in the nanostructure and etch rate of degenerately doped p-type silicon in a HF-H2O2-H2O etching system with electrolessly deposited silver catalyst are systematically investigated. The surface morphology is found to evolve from a microporous and cratered structure to a uniform array of SiNWs at sufficiently high χ values. The etch rates at the nanostructure base and tip are correlated with the primary etching induced by Ag and the secondary etching induced by metal ions and diffused holes, respectively. The H2O concentration also affects the χ window where SiNWs form and the etch rates, mainly by modulating the reactant dilution and diffusion rate. By controlling the secondary etching and reactant diffusion via χ and H2O concentration, respectively, the fabrication of highly doped SiNWs with independent control of porosity from length is successfully demonstrated, which can be potentially utilized to improve the performance of SiNW-based devices.

Non-Faradaic electrical impedimetric investigation of the interfacial effects of neuronal cell growth and differentiation on silicon nanowire transistors.

Silicon nanowire field-effect transistor (SiNW FET) devices have been interfaced with cells; however, their application for noninvasive, real-time monitoring of interfacial effects during cell growth and differentiation on SiNW has not been fully explored. Here, we cultured rat adrenal pheochromocytoma (PC12) cells, a type of neural progenitor cell, directly on SiNW FET devices to monitor cell adhesion during growth and morphological changes during neuronal differentiation for a period of 5-7 d. Monitoring was performed by measuring the non-Faradaic electrical impedance of the cell-SiNW FET system using a precision LCR meter. Our SiNW FET devices exhibited changes in impedance parameters during cell growth and differentiation because of the negatively charged cell membrane, seal resistance, and membrane capacitance at the cell/SiNW interface. It was observed that during both PC12 cell growth and neuronal differentiation, the impedance magnitude increased and the phase shifted to more negative values. However, impedance changes during cell growth already plateaued 3 d after seeding, while impedance changes continued until the last observation day during differentiation. Our results also indicate that the frequency shift to above 40 kHz after growth factor induction resulted from a larger coverage of cell membrane on the SiNWs due to distinctive morphological changes according to vinculin staining. Encapsulation of PC12 cells in a hydrogel scaffold resulted in a lack of trend in impedance parameters and confirmed that impedance changes were due to the cells. Moreover, cytolysis of the differentiated PC12 cells led to significant changes in impedance parameters. Equivalent electrical circuits were used to analyze the changes in impedance values during cell growth and differentiation. The technique employed in this study can provide a platform for performing investigations of growth-factor-induced progenitor cell differentiation.

Investigation of the interfacial effects of small chemical-modified TiO2 nanotubes on 3T3 fibroblast responses.

In order to gain insight into how interfacial effects influence cell responses, chemically modified anodized TiO2 nanotubes (ATNs) were used to simultaneously investigate the effects of nanoscale substrate structure and angstrom-scale chemicals on cell morphological change and cell growth. Two small chemicals were used to modify the ATNs, namely, 3-aminopropyltrimethoxysilane (APTMS) and 3-mercaptopropyltrimethoxysilane (MPTMS), resulting in APTMS-modified ATNs (APTMS-ATNs) and MPTMS-modified ATNs (MPTMS-ATNs), respectively. In our in vitro observation of NIH/3T3 fibroblasts, cells thrived on both unmodified and modified ATNs. Quantitative analyses of cell numbers exhibited that APTMS-ATNs effectively facilitated cell proliferation and directed cell orientation owing to full cell-substrate contact caused by positively charged amino groups (-NH3(+)) on the surface. In addition, scanning electron microscopy and fluorescence images showed different cell morphologies on APTMS-ATNs and MPTMS-ATNs. APTMS-ATNs resulted in flat spreading of fibroblasts, while MPTMS-ATNs resulted in fibroblasts with a three-dimensional solid shape and clear contours. The results indicate that the synergistic effects of nanotube surface topology and small chemical modification and, to a lesser extent, surface hydrophilicity, alter the interfacial interactions between cells and substrates, significantly affecting cell morphology, attachment, and growth. Using ATNs with different interfacial effects from various small chemicals, orientation of cells into various patterns can be achieved and investigation of cell fates, such as proliferation or stem cell differentiation, can be performed for future advanced medical or biological applications.