Mucin (MUC5AC) expression by lung epithelial cells cultured in a microfluidic gradient device

We have developed a microfluidic gradient device for controlling mucin gene expression of NCI-H292 epithelial cells derived from lung tissues. We hypothesized that gradient profiles would control mucin gene expression of lung epithelial cells. However, it was not possible to generate various stable gradient profiles using conventional culture methods. To address this limitation, we used a microfluidic gradient device to create various gradient profiles (i.e. non-linear, linear, and flat) in a temporal and spatial manner. NCI-H292 lung epithelial cells were exposed to concentration gradients of epidermal growth factor in a microfluidic gradient device with continuous medium perfusion. We demonstrated an effect of gradient profiles on mucin expression of lung epithelial cells cultured in the microfluidic gradient device. It was revealed that NCI-H292 lung epithelial cells exposed to the flat gradient profile of the epidermal growth factor exhibited high expression of mucin as compared with cells exposed to non-linear and linear gradient profiles. Therefore, this microfluidic gradient device could be a potentially useful tool for regulating the mucin expression of lung epithelial cells exposed to chemokine gradient profiles.     

Rapid spatial and temporal controlled signal delivery over large cell culture areas

Controlled chemical delivery in microfluidic cell culture devices often relies on slowly evolving diffusive gradients, as the spatial and temporal control provided by fluid flow results in significant cell-perturbation. In this paper we introduce a microfluidic device architecture that allows for rapid spatial and temporal soluble signal delivery over large cell culture areas without fluid flow over the cells. In these devices the cell culture well is divided from a microfluidic channel located directly underneath the chamber by a nanoporous membrane. This configuration requires chemical signals in the microchannel to only diffuse through the thin membrane into large cell culture area, rather than diffuse in from the sides. The spatial chemical pattern within the microfluidic channel was rapidly transferred to the cell culture area with good fidelity through diffusion. The cellular temporal response to a step-function signal showed that dye reached the cell culture surface within 45 s, and achieved a static concentration in under 6 min. Chemical pulses of less than one minute were possible by temporally alternating the signal within the microfluidic channel, enabling rapid flow-free chemical microenvironment control for large cell culture areas.     

Concentration landscape generators for shear free dynamic chemical stimulation

In this paper we first introduce a novel fabrication process, which allows for easy integration of thin track-etched nanoporous membranes, within 2D or 3D microchannel networks. In these networks, soluble chemical compounds can diffuse out of the channels through well-defined and spatially organized microfabricated porous openings. Interestingly, multiple micron-scale porous areas can be integrated in the same device and each of these areas can be connected to a different microfluidic channel and reservoir. We then present and characterize several membrane-based microdevices and their use for the generation of stable diffusible concentration gradients and complex dynamic chemical landscapes under shear free conditions. We also demonstrate how a simple flow-focusing geometry can be used to generate "on-demand" concentration profiles. In turn, these devices should provide an ideal experimental framework for high throughput cell-based assays: long term high-resolution video microscopy experiments can be performed, under multiple spatially and temporally controlled chemical conditions, with simple protocols and in a cell-friendly environment.     

Microfluidic system for measuring neutrophil migratory responses to fast switches of chemical gradients

Experimental systems that provide temporal and spatial control of chemical gradients are required for probing into the complex mechanisms of eukaryotic cell chemotaxis. However, no current technique can simultaneously generate stable chemical gradients and allow fast gradient changes. We developed a microfluidic system with microstructured membranes for exposing neutrophils to fast and precise changes between stable, linear gradients of the known chemoattractant Interleukin-8 (IL-8). We observed that rapidly lowering the average concentration of IL-8 within a gradient, while preserving the direction of the gradient, resulted in temporary neutrophil depolarization. Fast reversal of the gradient direction while increasing or decreasing the average concentration also resulted in temporary depolarization. Neutrophils adapted and maintained their directional motility, only when the average gradient concentration was increased and the direction of the gradient preserved. Based on these observations we propose a two-component temporal sensing mechanism that uses variations of chemokine concentration averaged over the entire cell surface and localized at the leading edge, respectively, and directs neutrophil responses to changes in their chemical microenvironment.     

Cell migration and polarity on microfabricated gradients of extracellular matrix proteins

This paper explores the effects of the surface density and concentration profiles of extra cellular matrix proteins on the migration of rat intestinal IEC-6 cells. Microfluidic devices were used to create linear, immobilized gradients of laminin. This study investigated both the impact of the steepness and local concentrations on the directedness of cell migration. The bulk concentrations of proteins in the feed streams in the mixing device determined the gradient profile and the local concentration of laminin in the device. Two sets of gradients were used to explore cell migration directedness: (i) gradients with similar change in local concentration, i.e., the same gradient steepness, and (ii) different gradients with similar local concentrations. Cells migrated up the gradients, independent of the steepness of the gradients used in this study. At the same local laminin concentration, the migration rate was independent of the gradient steepness. However, cell directedness decreased significantly at high laminin densities.     

Staying alive: new perspectives on cell immobilization for biosensing purposes

Intact living cells, because of their simplicity of use and their ability to provide highly valuable functional information, are well suited to biosensing applications. Cells can be genetically engineered by introduction of reporter proteins, modified to achieve analyte selectivity for their sensing capabilities, and connected to a transducer to obtain whole-cell biosensors. These bioanalytical features are increasingly attracting attention in the pharmaceutical, environmental, medical, and industrial fields. Whole-cell biosensors based on different recognition elements and transduction mechanisms have been also incorporated into portable devices and, with recent advances in micro and nanofabrication and microfluidics technology, miniaturized to achieve single-cell level analysis. Cell immobilization, widely used in, for example, microbial biofermentors or bioremediation systems, is now emerging as an appealing way of integrating whole-cell biosensors into devices, to maintain long-term cell viability, to increase the reproducibility of the cell’s response, and to avoid the spread of genetically modified cells into the environment, the latter being very important when devices are used for analysis in the field. A plethora of materials and functionalized surfaces have been proposed for immobilization of microbial or mammalian cells, each one having peculiar advantages and limitations. This critical review highlights and discusses recent trends, together with selected bioanalytical applications of immobilized viable cells. In particular the review focuses on some aspects that seem to hold great promise for future applications of immobilized cells, spanning from microbial biosensors to microbial biofilms, cell microarrays, and single-cell analysis.     

浸润性梯度表面研究与应用

浸润性梯度表面是指表面浸润性随着表面位置的变化而连续变化的一种特殊梯度表面.在过去的20年间,由于浸润性梯度表面在智能涂料、微流体器件和液体自输送等方面具有广阔的应用前景,因此人们研发并制备了各种类型的浸润性梯度表面,总体而言,可分为三类:(1)化学组成类浸润性梯度表面;(2)表面形貌类浸润性梯度表面;(3)化学组成-表面形貌复合型浸润性梯度表面.重点介绍了这三大类浸润性梯度表面的分类与区别,以及近些年来这些新型浸润性梯度表面的主要制备方法,如扩散法、浸泡法、刻蚀与打印、机械拉伸法、半月板沉积法和温度梯度法等,并归纳总结了这些方法的各自优缺点.在目前的材料科学领域,虽然关于浸润性梯度的研究还属婴儿期.可是利用梯度表面研究生物蛋白或细胞吸附,液体自输送等,已经发展成为一门成熟的学科.最后综述了近期关于浸润性梯度表面在研究生物蛋白或细胞吸附,液体自输送等方面的应用进展,并展望了该课题的未来发展.     

Complex polymer brush gradients based on nanolithography and surface-initiated polymerization

Confined surface gradients consisting of polymer brushes have great potential in various applications such as microfluidic devices, sensors, and biophysical research. Among the available fabrication approaches, nanolithographies combined with self-assembled monolayers and surface-initiated polymerization have became powerful tools to engineer confined gradients or predefined complex gradients on the nanometre size. In this tutorial review, we mainly highlight the research progress of the fabrication of confined polymer brush gradients by using electron beam, laser, and probe-based nanolithographies and the physical base for these approaches. The application of these polymer brush gradients in biomedical research is also addressed.     

New advances in electrochemical biosensors for the detection of toxins: Nanomaterials,magnetic beads and microfluidics systems. A review

The use of nanotechnology in bioanalytical devices has special advantages in the detection of toxins of interest in food safety and environmental applications. The low levels to be detected and the small size of toxins justify the increasing number of publications dealing with electrochemical biosensors, due to their high sensitivity and design versatility. The incorporation of nanomaterials in their development has been exploited to further increase their sensitivity, providing simple and fast devices, with multiplexed capabilities. This paper gives an overview of the electrochemical biosensors that have incorporated carbon and metal nanomaterials in their configurations for the detection of toxins. Biosensing systems based on magnetic beads or integrated into microfluidics systems have also been considered because of their contribution to the development of compact analytical devices. The roles of these materials, the methods used for their incorporation in the biosensor configurations as well as the advantages they provide to the analyses are summarised.     

Biomolecular gradients in cell culture systems

Biomolecule gradients have been shown to play roles in a wide range of biological processes including development, inflammation, wound healing, and cancer metastasis. Elucidation of these phenomena requires the ability to expose cells to biomolecule gradients that are quantifiable, controllable, and mimic those that are present in vivo. Here we review the major biological phenomena in which biomolecule gradients are employed, traditional in vitro gradient-generating methods developed over the past 50 years, and new microfluidic devices for generating gradients. Microfluidic gradient generators offer greater levels of precision, quantitation, and spatiotemporal gradient control than traditional methods, and may greatly enhance our understanding of many biological phenomena. For each method, we outline the salient features, capabilities, and applications.     

Generation of linear and non-linear concentration gradients along microfluidic channel by microtunnel controlled stepwise addition of sample solution

The ability to generate stable chemical gradients in microfluidics has important applications, since such gradients are useful in both chemical and biological studies. Growing evidence reveals that many cellular responses are specific to non-linear spatial gradients, hence a need to control complex concentration gradient profiles with and within microfluidics. In this paper, we present a structure-based approach to generate linear and non-linear chemical gradients, with profiles controlled by microtunnels fabricated alongside two main channels. Using single-step photolithography, microtunnels and main channels were fabricated at different heights thus having different fluidic resistance. Through these microtunnels, sample solutions were stepwise dispensed into the buffer stream to generate a chemical gradient profile. By varying the lengths of microtunnels that dictated the volume of sample solutions being dispensed, complex gradient profiles were generated. We have successfully demonstrated the formation of linear, convex and concave gradient profiles and a simple mathematical expression was established to approximate the profiles produced in our microfluidic gradient-generators.     

Luminescent Surface-Tethered Polymer Brush Materials

Surface-tethered polymers are unique molecular architectures that have been recently used in advanced sensors, electronics and biomedical applications. However, techniques for characterizing these materials in their surface-tethered form remain limited. The incorporation of luminescent functionality into these materials has enabled new characterization methods, while also unlocking new applications in optoelectronics, stenography and sensing. Micron-scale photolithography techniques have recently enabled the preparation of high-resolution patterns, as well as architectures with unique photophysical properties. Herein, we provide an overview of the techniques used to prepare luminescent polymer brush materials and their applications in stimuli-responsive sensors, cell adhesion materials, and optoelectronics. We also provide our perspective on the promising future uses of surface-tethered polymers, as well as the short-term challenges and opportunities in the field.     

Patterned two-photon photoactivation illuminates spatial reorganization in live cells

Photoactivatable fluorescent proteins offer the possibility to optically tag and track the location of molecules in their bright state with high spatial and temporal resolution. Several reports of patterned photoactivation have emerged since the development of a photoactivatable variant of the green fluorescent protein (PaGFP) and the demonstration of two-photon activation of PaGFP. To date, however, there have been few methods developed to quantify the spatial reorganization of the photoactivated population. Here we report on the use of singular value decomposition (SVD) to track the time-dependent distribution of fluorophores after photoactivation. The method was used to describe live-cell actin cytoskeleton behavior in primary murine T-cells, in which a dynamic cytoskeleton is responsible for the reorganization of membrane proteins in response to antigen peptide recognition. The method was also used to observe immortalized simian kidney (Cos-7) cells, in which the cytoskeleton is more stable. Both cell types were transfected with PaGFP fused to the F-actin binding domain of utrophin (UtrCH). Photoactivation patterns were written in the samples with a pair of galvanometric scanning mirrors in circular patterns that were analyzed by transforming the images into a time series of radial distribution profiles. The time-evolution of the profiles was well-described by the first two SVD component states. For T-cells, we find that actin filaments are highly mobile. Inward transport from the photoactivation region was observed and occurred on a 1-2 s time scale, which is consistent with retrograde cycling. For Cos-7 cells, we find that the actin is relatively stationary and does not undergo significant centripetal flow as expected for a resting fibroblast. The combination of patterned photoactivation and SVD analysis offers a unique way to measure spatial redistribution dynamics within live cells.     

A microfluidic multi-injector for gradient generation

This paper describes a microfluidic multi-injector (MMI) that can generate temporal and spatial concentration gradients of soluble molecules. Compared to conventional glass micropipette-based methods that generate a single gradient, the MMI exploits microfluidic integration and actuation of multiple pulsatile injectors to generate arbitrary overlapping gradients that have not previously been possible. The MMI device is fabricated in poly(dimethylsiloxane) (PDMS) using multi-layer soft lithography and consists of fluidic channels and control channels with pneumatically actuated on-chip barrier valves. Repetitive actuation of on-chip valves control pulsatile release of solution that establishes microscopic chemical gradients around the orifice. The volume of solution released per actuation cycle ranged from 30 picolitres to several hundred picolitres and increased linearly with the duration of valve opening. The shape of the measured gradient profile agreed closely with the simulated diffusion profile from a point source. Steady state gradient profiles could be attained within 10 minutes, or less with an optimized pulse sequence. Overlapping gradients from 2 injectors were generated and characterized to highlight the advantages of MMI over conventional micropipette assays. The MMI platform should be useful for a wide range of basic and applied studies on chemotaxis and axon guidance.     

Endothelial cell migration on surface-density gradients of fibronectin, VEGF, or both proteins

Cell migration is essential to many physiological processes, including angiogenesis, which is critical to the success of implanted biomaterials and tissue-engineered constructs. Gradients play an important role in cell migration. Previous work on cell migration has been mostly executed either in the concentration gradients of stimuli (e.g., VEGF) in bulk or hydrogels or on the surface-density gradients of ECM proteins (e.g., fibronectin) or small ligands (e.g., RGD). Little work has been done to investigate how cell migration responds to the surface-density gradients of growth factors. No work has been done to study how the surface gradients of both adhesive proteins and growth factors influence cell migration. In this work, we studied the effect of the surface-density gradients of fibronectin (FN), VEGF, or both proteins on endothelial cell migration. Gradients with different slopes were prepared to study how the gradient slope affects cell migration. The gradients were generated by first forming a counter-propagating C15COOH/C11OH self-assembled monolayer (SAM) gradient using a surface electrochemistry approach, followed by activating the -COOH moieties and covalently immobilizing proteins onto the surface. Fourier transform infrared spectra and X-ray photoelectron spectroscopy were used to characterize the SAM and protein gradients, respectively. A free cell migration assay using bovine aortic endothelial cells was performed on various gradient surfaces or on surfaces with uniform protein density. Results showed that cells on the surface-density gradients of FN, VEGF, or both proteins moved faster along the gradient direction than on the respective uniform control surface after 24-h cell culture. It is also shown that for each protein or protein combination, the directional cell displacement was not statistically different between two gradients with different slopes. Results show that the directional cell migration was increased by about 2-fold on the VEGF gradient as compared to the FN gradient and was further increased by another 2-fold on the combined gradients of both proteins as compared to the VEGF gradient alone. This is the first work to create surface-density gradients of VEGF and the first study to generate a combined surface gradient of growth factor and ECM protein to investigate their effect on cell migration on surfaces. This work broadens our understanding of the directional movement of endothelial cells. Our findings provide useful information for directing cell migration into tissue-engineered constructs and can be potentially used for those applications where cell migration is critical, such as angiogenesis.     

A microfluidic device that forms and redirects pheromone gradients to study chemotropism in yeast

Chemotropism, or directed cell growth in response to a chemical gradient, is integral to many biological processes. The mating response of the budding yeast, Saccharomyces cerevisiae, is a well studied model chemotropic system. Yeast cells of opposite mating type signal their positions by secreting soluble mating pheromones. The mutual exchange of pheromones induces the cells to grow towards one another, resulting in mating projections or "shmoos." Yeast cells exhibit a remarkable ability to orient their growth toward the nearest potential mating partner, and to reorient (i.e., bend their mating projections) in response to a change in the direction of the pheromone gradient. Although a number of microfluidic devices have been used to generate linear pheromone gradients and to measure initial orientation, none of them have the capability to change the direction of the gradient, other than to invert it. We have developed a microfluidic device that can produce stable pheromone gradients and rapidly rotate them in 90° increments, mimicking the dynamic gradients yeast are exposed to in situ, and allowing for the study of reorientation as well as initial orientation. The mean angle of orientation exhibited by gradient-stimulated yeast cells in this device was 56.9°. In control experiments, cells subjected to pheromone coming from all four directions showed no evidence of orientation. Switching the direction of the pheromone source by 90° induced 83.6% of the polarized cells to change their direction of growth. Of these, 85.2% bent their mating projections toward the second source, demonstrating the utility of this device in the study of reorientation with specifically controlled gradients.     

Normal-phase high-performance liquid chromatographic separations using ethoxynonafluorobutane as hexane alternative. I. Analytical and chiral applications.

A novel, environmentally friendly, fluorinated solvent--ethoxynonafluorobutane--has been used to replace n-hexane in normal-phase HPLC applications. Fast gradients of methanol in ethoxynonafluorobutane on a cyano column have been successfully applied to the separation of steroids, benzodiazepines, NSAIDs, tricyclic antidepressants, beta-adrenergic blocking agents and mixtures of purines and pyrimidines. Small amounts of triethylamine and trifluoroacetic acid added to such gradients significantly improved peak shape and column performance for basic and acidic solutes. Ethoxynonafluorobutane and its mixtures with methanol have also been demonstrated to have a unique selectivity in chiral HPLC applications.     

A microfluidics-based turning assay reveals complex growth cone responses to integrated gradients of substrate-bound ECM molecules and diffusible guidance cues

Neuronal growth cones contain sophisticated molecular machinery precisely regulating their migration in response to complex combinatorial gradients of diverse external cues. The details of this regulation are still largely unknown, in part due to limitations of the currently available experimental techniques. Microfluidic devices have been shown to be capable of generating complex, stable and precisely controlled chemical gradients, but their use in studying growth cone migration has been limited in part due to the effects of shear stress. Here we describe a microfluidics-based turning-assay chip designed to overcome this issue. In addition to generating precise gradients of soluble guidance cues, the chip can also fabricate complex composite gradients of diffusible and surface-bound guidance cues that mimic the conditions the growth cones realistically counter in vivo. Applying this assay to Xenopus embryonic spinal neurons, we demonstrate that the presence of a surface-bound laminin gradient can finely tune the polarity of growth cone responses (repulsion or attraction) to gradients of brain-derived neurotrophic factor (BDNF), with the guidance outcome dependent on the mean BDNF concentration. The flexibility inherent in this assay holds significant potential for refinement of our understanding of nervous system development and regeneration, and can be extended to elucidate other cellular processes involving chemotaxis of shear sensitive cells.     

Optochemical Nanosensors and Subcellular Applications in Living Cells

What may be the smallest anthropogenic devices to date, spherical sensors (wireless and fiberless) with radii as small as 10?nm have been produced. This class of optochemical PEBBLE (Probe Encapsulated By Biologically Localized Embedding) sensors covers a wide range of analytes (pH, calcium, oxygen and potassium included here) with excellent spatial, temporal and chemical resolution. Examples of such sensors for the monitoring of intracellular analytes are given. Methods, such as pico-injection, liposomal delivery and gene gun bombardment, are used to inject PEBBLE sensors into single cells. These PEBBLEs have caused minimal perturbation when delivered and operated inside single mammalian cells, such as human neuroblastoma, mouse oocytes or rat alveolar macrophage.