Chiung Wen Kuo

Exploring the Formation of Focal Adhesions on Patterned Surfaces Using Super‐Resolution Imaging

The formation of focal adhesions on various sizes of fibronectin patterns, ranging from 200 μm to 250 nm, was systematically investigated by total internal reflection fluorescence microscopy and super-resolution imaging. It was found that cells adhered to and spread on these micro/nanopatterns, forming focal adhesions. On a micrometer scale the shape of the focal adhesions was elongated. However, on the nanometer scale, the shape of focal adhesions became dotlike. To further explore the distribution of focal adhesion proteins formed on surfaces, a localization-based super-resolution imaging technique was employed in order to determine the position and density of vinculin proteins. A characteristic distance of 50 nm was found between vinculin molecules in the focal adhesions, which did not depend on the size of the fibronectin nanopatterns. This distance was found to be crucial for the formation of focal adhesions. In addition, the density of vinculin at the focal adhesions formed on the nanopatterns increased as the pattern size decreased. The density of the protein was found to be 425 ± 247, 584 ± 302, and 703 ± 305 proteins μm(-2) on the 600, 400, and 250 nm fibronectin patterns respectively. Whereas 226 ± 77 proteins μm(-2) was measured for the matured focal adhesions on homogeneous fibronectin coated substrates. The increase in vinculin density implies that an increase in mechanical load was applied to the focal adhesions formed on the smaller nanopatterns.

Investigation of size–dependent cell adhesion on nanostructured interfaces

BackgroundCells explore the surfaces of materials through membrane-bound receptors, such as the integrins, and use them to interact with extracellular matrix molecules adsorbed on the substrate surfaces, resulting in the formation of focal adhesions. With recent advances in nanotechnology, biosensors and bioelectronics are being fabricated with ever decreasing feature sizes. The performances of these devices depend on how cells interact with nanostructures on the device surfaces. However, the behavior of cells on nanostructures is not yet fully understood. Here we present a systematic study of cell-nanostructure interaction using polymeric nanopillars with various diameters.ResultsWe first checked the viability of cells grown on nanopillars with diameters ranging from 200xa0nm to 700xa0nm. It was observed that when cells were cultured on the nanopillars, the apoptosis rate slightly increased as the size of the nanopillar decreased. We then calculated the average size of the focal adhesions and the cell-spreading area for focal adhesions using confocal microscopy. The size of focal adhesions formed on the nanopillars was found to decrease as the size of the nanopillars decreased, resembling the formations of nascent focal complexes. However, when the size of nanopillars decreased to 200xa0nm, the size of the focal adhesions increased. Further study revealed that cells interacted very strongly with the nanopillars with a diameter of 200xa0nm and exerted sufficient forces to bend the nanopillars together, resulting in the formation of larger focal adhesions.ConclusionsWe have developed a simple approach to systematically study cell-substrate interactions on physically well-defined substrates using size-tunable polymeric nanopillars. From this study, we conclude that cells can survive on nanostructures with a slight increase in apoptosis rate and that cells interact very strongly with smaller nanostructures. In contrast to previous observations on flat substrates that cells interacted weakly with softer substrates, we observed strong cell-substrate interactions on the softer nanopillars with smaller diameters. Our results indicate that in addition to substrate rigidity, nanostructure dimensions are additional important physical parameters that can be used to regulate behaviour of cells.

Revealing the spatial distribution of the site enhancement for the surface enhanced Raman scattering on the regular nanoparticle arrays

The spatial distribution of the site enhancement for the surface-enhanced Raman scattering (SERS) on the regular nanoparticle arrays has been investigated by the confocal Raman microscopy. It was found that the spatial distribution of the Raman signals on the well-ordered nanoparticle arrays was very inhomogeneous and concentrated on the defects of the nanoparticle arrays. The SERS signals were also observed to depend on the thickness of silver film and the defect density. It has been demonstrated that the number of SERS active sites can be increased ten folds by trimming the size of nanoparticles using oxygen plasma.

Detection of residual rifampicin in urine via fluorescence quenching of gold nanoclusters on paper

BackgroundRifampicin or rifampin (R) is a common drug used to treat inactive meningitis, cholestatic pruritus and tuberculosis (TB), and it is generally prescribed for long-term administration under regulated dosages. Constant monitoring of rifampicin is important for controlling the side effects and preventing overdose caused by chronic medication. In this study, we present an easy to use, effective and less costly method for detecting residual rifampicin in urine samples using protein (bovine serum albumin, BSA)-stabilized gold nanoclusters (BSA-Au NCs) adsorbed on a paper substrate in which the concentration of rifampicin in urine can be detected via fluorescence quenching. The intensity of the colorimetric assay performed on the paper-based platforms can be easily captured using a digital camera and subsequently analyzed.ResultsThe decreased fluorescence intensity of BSA-Au NCs in the presence of rifampicin allows for the sensitive detection of rifampicin in a range from 0.5 to 823xa0µg/mL. The detection limit for rifampicin was measured as 70xa0ng/mL. The BSA-Au NCs were immobilized on a wax-printed paper-based platform and used to conduct real-time monitoring of rifampicin in urine.ConclusionWe have developed a robust, cost-effective, and portable point-of-care medical diagnostic platform for the detection of rifampicin in urine based on the ability of rifampicin to quench the fluorescence of immobilized BSA-Au NCs on wax-printed papers. The paper-based assay can be further used for the detection of other specific analytes via surface modification of the BSA in BSA-Au NCs and offers a useful tool for monitoring other diseases.

Investigation of the growth of focal adhesions using protein nanoarrays fabricated by nanocontact printing using size tunable polymeric nanopillars

Here we describe a simple approach to create various sizes of protein nanoarrays for the investigation of cell adhesion. Using a combination of nanosphere lithography, oxygen plasma treatment, deep etching and nanomolding processes, well-ordered polymeric nanopillar arrays have been fabricated with diameters in the range of 50-600 nm. These nanopillar arrays were used as stamps for nanocontact printing to create fibronectin nanoarrays, which were used to study the size dependent formation of focal adhesion. It was found that cells can adhere and spread on fibronectin nanoarrays with a fibronectin pattern as small as 50 nm. It was also found that the average size of focal adhesion decreased as the size of the fibronectin pattern was reduced.

Addressable Cell Microarrays via Switchable Superhydrophobic Surfaces

Here we describe an approach to fabricate addressable cell microarrays, which are based on the patterned switchable superhydrophobic surfaces. The switchable superhydrophobic surfaces were prepared by roughening the surfaces of fluoropolymers on the electrodes. Upon the application of 150 V to the underneath electrodes, the water contact angle on the roughened fluoropolymer surfaces could be decreased from 163° to less than 10° allowing the deposition of fibronectin, which could guide the growth of the cells. Our result indicated that it was possible to control the spatial distribution of two different cells on the cell microarrays.

Poly(3,4-ethylenedioxythiophene)-Based Nanofiber Mats as an Organic Bioelectronic Platform for Programming Multiple Capture/Release Cycles of Circulating Tumor Cells

In this investigation, we employed a novel one-step electrospinning process to fabricate poly(ethylene oxide) (PEO)/poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) core/shell nanofiber structures with improved water resistance and good electrochemical properties and characterized them using scanning electron microscopy, transmission electron microscopy, and time-of-flight secondary ion mass spectrometry imaging. We then integrated a biotinylated poly-(l-lysine-graft-ethylene glycol) (PLL-g-PEG-biotin) coating with three-dimensional (3D) PEDOT-based nanofiber devices for dynamic control over the capture/release performance of rare circulating tumor cells (CTCs) on-chip. The detailed capture/release behavior of the circulating tumor cells was studied using an organic bioelectronic platform comprising PEO/PEDOT:PSS nanofiber mats with 3 wt % (3-glycidyloxypropyl)trimethoxysilane as an additive. We have demonstrated that these nanofiber mats deposited on five-patterned indium tin oxide finger electrodes are excellent candidates for use as functional bioelectronic interfaces for the isolation, detection, sequential collection, and enrichment of rare CTCs through electrical activation of each single electrode. This combination behaved as an ideal model system displaying a high cell-capture yield for antibody-positive cells while resisting the adhesion of antibody-negative cells. Taking advantage of the electrochemical doping/dedoping characteristics of PEDOT:PSS materials, the captured rare cells could be electrically triggered release through the desorption phenomena of PLL-g-PEG-biotin on device surface. More than 90% of the targeted cancer cells were captured on the 3D PEDOT-based nanofiber microfluidic device; over 87% of captured cancer cells were subsequently released for collection; approximately 80% of spiked cancer cells could be collected in a 96-well plate. Therefore, this 3D PEDOT-based nanofiber approach appears to be an economical route for the large-scale preparation of systems for enhancing the downstream characterization of rare CTCs.

Flexible nanopillars to regulate cell adhesion and movement

Flexible polymer nanopillar substrates were used to systematically demonstrate cell alignment and migration guided by the directional formation of focal adhesions. The polymer nanopillar substrates were constructed to various height specifications to provide an extensive variation of flexibility; a rectangular arrangement created spatial confinement between adjacent nanopillars, providing less spacing in the horizontal and vertical directions. Three polymer nanopillar substrates with the diameter of 400 nm and the heights of 400, 800, and 1200 nm were fabricated. Super-resolution localization imaging and protein pair-distance analysis of vinculin proteins revealed that Chinese hamster ovary (CHO) cells formed mature focal adhesions on 1200 nm high nanopillar substrates by bending adjacent nanopillars to link dot-like adhesions. The spacing confinement of the adjacent nanopillars enhanced the orthogonal directionality of the formation tendency of the mature focal adhesions. The directional formation of the mature focal adhesions also facilitated the organization of actin filaments in the horizontal and vertical directions. Moreover, 78% of the CHO cells were aligned in these two directions, in conformity with the flexibility and nanotopographical cues of the nanopillars. Biased cell migration was observed on the 1200 nm high nanopillar substrates.

Study of oxygen tension variation within live tumor spheroids using microfluidic devices and multi-photon laser scanning microscopy

Three-dimensional cell spheroid culture using microfluidic devices provides a convenient in vitro model for studying tumour spheroid structures and internal microenvironments. Recent studies suggest that oxygen deprived zones inside solid tumors are responsible for stimulating local cytokines and endothelial vasculature proliferation during angiogenesis. In this work, we develop an integrated approach combining microfluidic devices and multi-photon laser scanning microscopy (MPLSM) to study variations in oxygen tension within live spheroids of human osteosarcoma cells. Uniform shaped, size-controlled spheroids are grown and then harvested using a polydimethylsiloxane (PDMS) based microfluidic device. Fluorescence live imaging of the harvested spheroids is performed using MPLSM and a commercially available oxygen sensitive dye, Image-iT Red, to observe the oxygen tension variation within the spheroids and those co-cultured with monolayers of human umbilical vein endothelial cells (HUVECs). Oxygen tension variations are observed within the spheroids with diameters ranging from 90 ± 10 μm to 140 ± 10 μm. The fluorescence images show that the low-oxygenated cores diminish when spheroids are co-cultured with HUVEC monolayers for 6 hours to 8 hours. In the experiments, spheroids subjected to HUVEC conditioned medium treatment and with a cell adherent substrate are also measured and analyzed to study their significance on oxygen tension within the spheroids. The results show that the oxygenation within the spheroids is improved when the spheroids are cultured under those conditions. Our work presents an efficient method to study oxygen tension variation within live tumor spheroids under the influence of endothelial cells and conditioned medium. The method can be exploited for further investigation of tumor oxygen microenvironments during angiogenesis.