Microfluidic generation of polydopamine gradients on hydrophobic surfaces.

Engineered surface-bound molecular gradients are of great importance for a range of biological applications. In this paper, we fabricated a polydopamine gradient on a hydrophobic surface. A microfluidic device was used to generate a covalently conjugated gradient of polydopamine (PDA), which changed the wettabilty and the surface energy of the substrate. The gradient was subsequently used to enable the spatial deposition of adhesive proteins on the surface. When seeded with human adipose mesenchymal stem cells, the PDA-graded surface induced a gradient of cell adhesion and spreading. The PDA gradient developed in this study is a promising tool for controlling cellular behavior and may be useful in various biological applications.

Absolute quantitation of isoforms of post-translationally modified proteins in transgenic organism.

Post-translational modification isoforms of a protein are known to play versatile biological functions in diverse cellular processes. To measure the molar amount of each post-translational modification isoform (P(isf)) of a target protein present in the total protein extract using mass spectrometry, a quantitative proteomic protocol, absolute quantitation of isoforms of post-translationally modified proteins (AQUIP), was developed. A recombinant ERF110 gene overexpression transgenic Arabidopsis plant was used as the model organism for demonstration of the proof of concept. Both Ser-62-independent (14)N-coded synthetic peptide standards and (15)N-coded ERF110 protein standard isolated from the heavy nitrogen-labeled transgenic plants were employed simultaneously to determine the concentration of all isoforms (T(isf)) of ERF110 in the whole plant cell lysate, whereas a pair of Ser-62-dependent synthetic peptide standards were used to quantitate the Ser-62 phosphosite occupancy (R(aqu)). The P(isf) was finally determined by integrating the two empirically measured variables using the following equation: P(isf) = T(isf) · R(aqu). The absolute amount of Ser-62-phosphorylated isoform of ERF110 determined using AQUIP was substantiated with a stable isotope labeling in Arabidopsis-based relative and accurate quantitative proteomic approach. The biological role of the Ser-62-phosphorylated isoform was demonstrated in transgenic plants.

A simple fabricated thickness-based stiffness gradient for cell studies.

In this work, we developed a simple method to fabricate a thickness-based continuous stiffness gradient for biological studies. It was made by glass slides, polydimethylsiloxane (PDMS) pre-polymer, spacer and clips only, without any sophisticated equipment. It is easy to fabricate in any general biological and pharmaceutical laboratories. The stiffness gradient was characterized in terms of apparent Young's modulus by atomic force microscopy (AFM) and the Young's modulus along the gradient was found to be 8.5-120kPa, which is within the physiological relevant range. HeLa-C3 cells were cultured on the gradient to study their morphological behavior according to the substrate stiffness. Furthermore, the drug efficiency of etoposide, an anti-cancer drug, was studied along the substrate stiffness gradient. It was found that HeLa-C3 cells cultured on the soft region of the gradient (8.5-11kPa) are more sensitive to etoposide. We believe the proposed device could promote cell investigations and drug screenings on a substrate with comparable stiffness to the native tissue.

Poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA): a bioactive copolymer for specific targeting to folate receptor-positive cancer cells.

In this study, we present the preparation, characterization and application of a novel bioactive copolymer poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA), which has a specific interaction with folate receptor (FR)-positive cancer cells. Glass surface immobilized with PLL-g-PMOXA-c-FA was demonstrated to be adhesive to FR-positive cancer cells (HeLa, JEG-3) while nonadhesive to FR-negative ones (MCF-7, HepG2) in 3 h. The specific interaction between conjugated FA on the substrate and FRs on the cells could hardly be inhibited unless a high concentration (5 mM) of free FA was used due to the multivalent nature of it. The FA functionality ratio of the copolymer on the substrate had a significant influence on the adhesion of HeLa cells, and our experiments revealed that the affinity of the substrate to the cells declined dramatically with the decrease of functionality ratio. This was believed to be caused by the polydispersity of PMOXA tethers, as supported by GPC and ToF-SIMS data. As a proof of concept in the application of our material, we demonstrated successful recovery of HeLa cells from mixture with MCF-7 (1:100) on the copolymer-coated glass, and our results showed that both high sensitivity (95.6 ± 13.3%) and specificity (24.3 ± 8.6%) were achieved.

Recent developments in microfluidics for cell studies.

As a technique for precisely manipulating fluid at the micrometer scale, the field of microfluidics has experienced an explosive growth over the past two decades, particularly owing to the advances in device design and fabrication. With the inherent advantages associated with its scale of operation, and its flexibility in being incorporated with other microscale techniques for manipulation and detection, microfluidics has become a major enabling technology, which has introduced new paradigms in various fields involving biological cells. A microfluidic device is able to realize functions that are not easily imaginable in conventional biological analysis, such as highly parallel, sophisticated high-throughput analysis, single-cell analysis in a well-defined manner, and tissue engineering with the capability of manipulation at the single-cell level. Major advancements in microfluidic device fabrication and the growing trend of implementing microfluidics in cell studies are presented, with a focus on biological research and clinical diagnostics.

Stretchable and micropatterned membrane for osteogenic differentation of stem cells.

Stem cells have emerged as potentially useful cells for regenerative medicine applications. To fully harness this potential, it is important to develop in vitro cell culture platforms with spatially regulated mechanical, chemical, and biological cues to induce the differentiation of stem cells. In this study, a cell culture platform was constructed that used polydopamine (PDA)-coated parafilm. The modified parafilm supports cell attachment and proliferation. In addition, because of the superb plasticity and ductility of the parafilm, it can be easily micropatterned to regulate the spatial arrangements of cells, and can exert different mechanical tensions. Specifically, we constructed a PDA-coated parafilm with grooved micropatterns to induce the osteogenic differentiation of stem cells. Adipose-derived mesenchymal stem cells that were cultured on the PDA-coated parafilm exhibited significantly higher osteogenic commitment in response to mechanical and spatial cues compared to the ones without stretch. Our findings may open new opportunities for inducing osteogenesis of stem cells in vitro using the platform that combines mechanical and spatial cues.

Highly emissive and biocompatible dopamine-derived oligomers as fluorescent probes for chemical detection and targeted bioimaging.

We prepared highly emissive and biocompatible dopamine-derived oligomers, and demonstrated their applications as novel fluorescent probes for sensitive detection of Fe(3+) ions and targeted bioimaging in live cells.

Periosteum-mimetic structures made from freestanding microgrooved nanosheets.

A "sticker-like" PLGA nanosheet with microgrooved patterns is developed through a facile combination of spin coating and micropatterning techniques. The resulting microgrooved PLGA nanosheets can be physically adhered on flat or porous surfaces with excellent stability in aqueous environments and can harness the spatial arrangements of cells, which make it a promising candidate for generating biomimic periosteum for bone regenerative applications.