Stable chemical bonding of porous membranes and poly(dimethylsiloxane) devices for long-term cell culture

We have investigated the bonding stability of various silane treatments for the integration of track-etched membranes with poly(dimethylsiloxane) (PDMS) microfluidic devices. We compare various treatments using trialkoxysilanes or dipodal silanes to determine the effect of the organofunctional group, cross-link density, reaction solvent, and catalyst on the bond stability. We find that devices made using existing silane methods delaminated after one day when immersed in cell culture medium at 37 °C. In contrast, the dipodal silane, bis[3-(trimethoxysilyl)propyl]amine, is shown to yield stable and functional integration of membranes with PDMS that is suitable for long-term cell culture. To demonstrate application of the technique, we fabricated an open-surface device in which cells cultured on a track-etched membrane can be stimulated at their basal side via embedded microfluidic channels. C2C12 mouse myoblasts were differentiated into myotubes over the course of two weeks on these devices to demonstrate biocompatibility. Finally, devices were imaged during the basal-side delivery of a fluorescent stain to validate the membrane operation and long-term stability of the bonding technique.     

Coaxial flow focusing in poly(dimethylsiloxane) microfluidic devices

We have developed a coaxial flow focusing geometry that can be fabricated using soft lithography in poly(dimethylsiloxane) (PDMS). Like coaxial flow focusing in glass capillary microfluidics, our geometry can form double emulsions in channels with uniform wettability and of a size much smaller than the channel dimensions. However, In contrast to glass capillary coaxial flow focusing, our geometry can be fabricated using lithographic techniques, allowing it to be integrated as the drop making unit in parallel drop maker arrays. Our geometry enables scalable formation of emulsions down 7 μm in diameter, in large channels that are robust against fouling and clogging.     

Simple surface modification of poly(dimethylsiloxane) for DNA hybridization

Here, we present a simple chemical modification of poly(dimethylsiloxane) (PDMS) by curing a mixture of 2 wt% undecylenic acid (UDA) in PDMS prepolymer on a gold-coated glass slide. This gold slide had been previously pretreated with a self-assembled hydrophilic monolayer of 3-mercaptopropionic acid (MPA). During curing of the UDA∕PDMS prepolymer, the hydrophilic UDA carboxyl moieties diffuses toward the hydrophilic MPA carboxyl moieties on the gold surface. This diffusion of the UDA within the PDMS prepolymer to the surface is a direct result of surface energy minimization. Once completely cured, the PDMS is peeled off the gold substrate, thereby exposing the interfacial carboxyl groups. These groups are then available for subsequent attachment of 5(')-amino terminated DNA oligonucleotides via amide linkages. Our results show that the covalently tethered oligonucleotides can successfully capture fluorescein-labeled complementary oligonucleotides via hybridization, which are visualized using fluorescence microscopy.     

Scaffold fabrication in a perfusion culture microchamber array chip by O(2) plasma bonding of poly(dimethylsiloxane) protected by a physical mask

Extracellular matrix (ECM) proteins are required for cell culture. In this paper, we report the use of O(2) plasma bonding to fabricate a perfusion culture microchamber array chip with identical-size ECM spots in the isolated microchambers. The chip was fabricated by assembly of two poly(dimethylsiloxane) (PDMS) layers, a microfluidic network layer, and an ECM array layer, which were aligned and then bonded by O(2) plasma oxidation with protection of the ECM microarray with a physical mask made from PDMS. We successfully cultivated Chinese hamster ovary K1 cells in the microchambers with fibronectin. In the fibronectin microchambers, the cells adhered and extended after 12 h of static culture and then grew over the course of 1 d of perfusion culture.     

Parallel combinatorial chemical synthesis using single-layer poly(dimethylsiloxane) microfluidic devices

Improving methods for high-throughput combinatorial chemistry has emerged as a major area of research because of the importance of rapidly synthesizing large numbers of chemical compounds for drug discovery and other applications. In this investigation, a novel microfluidic chip for performing parallel combinatorial chemical synthesis was developed. Unlike past microfluidic systems designed for parallel combinatorial chemistry, the chip is a single-layer device made of poly(dimethylsiloxane) that is extremely easy and inexpensive to fabricate. Using the chip, a 2×2 combinatorial series of amide-formation reactions was performed. The results of this combinatorial synthesis indicate that the new device is an effective platform for running parallel organic syntheses at significantly higher throughput than with past methodologies. Additionally, a design algorithm for scaling up the 2×2 combinatorial synthesis chip to address more complex cases was developed.     

Systematic characterization of degas-driven flow for poly(dimethylsiloxane) microfluidic devices

Degas-driven flow is a novel phenomenon used to propel fluids in poly(dimethylsiloxane) (PDMS)-based microfluidic devices without requiring any external power. This method takes advantage of the inherently high porosity and air solubility of PDMS by removing air molecules from the bulk PDMS before initiating the flow. The dynamics of degas-driven flow are dependent on the channel and device geometries and are highly sensitive to temporal parameters. These dependencies have not been fully characterized, hindering broad use of degas-driven flow as a microfluidic pumping mechanism. Here, we characterize, for the first time, the effect of various parameters on the dynamics of degas-driven flow, including channel geometry, PDMS thickness, PDMS exposure area, vacuum degassing time, and idle time at atmospheric pressure before loading. We investigate the effect of these parameters on flow velocity as well as channel fill time for the degas-driven flow process. Using our devices, we achieved reproducible flow with a standard deviation of less than 8% for flow velocity, as well as maximum flow rates of up to 3 nL∕s and mean flow rates of approximately 1-1.5 nL∕s. Parameters such as channel surface area and PDMS chip exposure area were found to have negligible impact on degas-driven flow dynamics, whereas channel cross-sectional area, degas time, PDMS thickness, and idle time were found to have a larger impact. In addition, we develop a physical model that can predict mean flow velocities within 6% of experimental values and can be used as a tool for future design of PDMS-based microfluidic devices that utilize degas-driven flow.     

Microfluidic rectifier based on poly(dimethylsiloxane) membrane and its application to a micropump

A microfluidic rectifier incorporating an obstructed microchannel and a PDMS membrane is proposed. During forward flow, the membrane deflects in the upward direction; thereby allowing the fluid to pass over the obstacle. Conversely, during reverse flow, the membrane seals against the obstacle, thereby closing the channel and preventing flow. It is shown that the proposed device can operate over a wide pressure range by increasing or decreasing the membrane thickness as required. A microfluidic pump is realized by integrating the rectifier with a simple stepper motor mechanism. The experimental results show that the pump can achieve a vertical left height of more than 2 m. Moreover, it is shown that a maximum flow rate of 6.3 ml/min can be obtained given a membrane thickness of 200 μm and a motor velocity of 80 rpm. In other words, the proposed microfluidic rectifier not only provides an effective means of preventing reverse flow but also permits the realization of a highly efficient microfluidic pump.     

A novel surface modification technique for forming porous polymer monoliths in poly(dimethylsiloxane)

A new method of surface modification is described for enabling the in situ formation of homogenous porous polymer monoliths (PPMs) within poly(dimethylsiloxane) (PDMS) microfluidic channels that uses 365 nm UV illumination for polymerization. Porous polymer monolith formation in PDMS can be challenging because PDMS readily absorbs the monomers and solvents, changing the final monolith morphology, and because PDMS absorbs oxygen, which inhibits free-radical polymerization. The new approach is based on sequentially absorbing a non-hydrogen-abstracting photoinitiator and the monomers methyl methacrylate and ethylene diacrylate within the walls of the microchannel, and then polymerizing the surface treatment polymer within the PDMS, entangled with it but not covalently bound. Four different monolith compositions were tested, all of which yielded monoliths that were securely anchored and could withstand pressures exceeding the bonding strength of PDMS (40 psi) without dislodging. One was a recipe that was optimized to give a larger average pore size, required for low back pressure. This monolith was used to concentrate and subsequently mechanical lyse B lymphocytes.     

Polyelectrolyte multilayer surface functionalization of poly(dimethylsiloxane) (PDMS) for reduction of yeast cell adhesion in microfluidic devices

Polyelectrolyte multilayers (PEMs) based on the combinations poly(diallyldimethylammonium chloride)∕poly(acrylic acid) (PDADMAC∕PAA) and poly(allylamine hydrochloride)∕PAA (PAH∕PAA) were adsorbed on poly(dimethylsiloxane) (PDMS) and tested for nonspecific surface attachment of hydrophobic yeast cells using a parallel plate flow chamber. A custom-made graft copolymer containing poly(ethylene glycol) (PEG) side chains (PAA-g-PEG) was additionally adsorbed on the PEMs as a terminal layer. A suitable PEM modification effectively decreased the adhesion strength of Saccharomyces cerevisiae DSM 2155 to the channel walls. However, a further decrease in initial cell attachment and adhesion strength was observed after adsorption of PAA-g-PEG copolymer onto PEMs from aqueous solution. The results demonstrate that a facile layer-by-layer surface functionalization from aqueous solutions can be successfully applied to reduce cell adhesion strength of S. cerevisiae by at least two orders of magnitude compared to bare PDMS. Therefore, this method is potentially suitable to promote planktonic growth inside capped PDMS-based microfluidic devices if the PEM deposition is completed by a dynamic flow-through process.     

Disposable parallel poly(dimethylsiloxane) microbioreactor with integrated readout grid for germination screening of Aspergillus ochraceus

In this work a disposable, parallel microbioreactor (MBR) suitable for screening in batch or continuous mode is presented. The reactor consists of five parallel microchambers made of poly(dimethylsiloxane) bonded to a glass substrate. A grid structure is engraved on each chamber, allowing subsequent morphology imaging. Measurements are recorded over the entire cultivation period with constant parameters, namely, position and focus in the z-axis. The microdevice may be used for either parallel, uni- or multiparametric screening, and overcomes the drawback of gridless microwell plates which require expensive equipment such as an inverted microscope with an automatic stage. To validate the scalability from laboratory scale to microscale, and thus the cultivation protocol in the MBR, the germination of fungal spores (A. ochraceus) is evaluated for two different key magnitudes (pH and temperature) and compared to the results obtained from conventional laboratory scale systems (flasks and agar plates). Information on germination capacity with regard to interspecies' variability allows for optimization of industrial processes as optimal pH and temperature matched to the mesoscopic cultivation systems. The germination conditions therefore remain unaffected inside the MBR, while providing the following advantages: (i) dramatic reduction of medium consumption, (ii) submerged cultivation with constant oxygen supply, (iii) assured low cost and disposability, and (iv) possibility of a continuous cultivation mode.     

DNA capture-probe based separation of double-stranded polymerase chain reaction amplification products in poly(dimethylsiloxane) microfluidic channels

Herein, we describe the development of a novel primer system that allows for the capture of double-stranded polymerase chain reaction (PCR) amplification products onto a microfluidic channel without any preliminary purification stages. We show that specially designed PCR primers consisting of the main primer sequence and an additional “tag sequence” linked through a poly(ethylene glycol) molecule can be used to generate ds-PCR amplification products tailed with ss-oligonucleotides of two forensically relevant genes (amelogenin and human c-fms (macrophage colony-stimulating factor) proto-oncogene for the CSF-1 receptor (CSF1PO). Furthermore, with a view to enriching and eluting the ds-PCR products of amplification on a capillary electrophoretic-based microfluidic device we describe the capture of the target ds-PCR products onto poly(dimethylsiloxane) microchannels modified with ss-oligonucleotide capture probes.     

Rapid bench-top fabrication of poly(dimethylsiloxane)/polystyrene microfluidic devices incorporating high-surface-area sensing electrodes

The development of widely applicable point-of-care sensing and diagnostic devices can benefit from simple and inexpensive fabrication techniques that expedite the design, testing, and implementation of lab-on-a-chip devices. In particular, electrodes integrated within microfluidic devices enable the use of electrochemical techniques for the label-free detection of relevant analytes. This work presents a novel, simple, and cost-effective bench-top approach for the integration of high surface area three-dimensional structured electrodes fabricated on polystyrene (PS) within poly(dimethylsiloxane) (PDMS)-based microfluidics. Optimization of PS-PDMS bonding results in integrated devices that perform well under pressure and fluidic flow stress. Furthermore, the fabrication and bonding processes are shown to have no effect on sensing electrode performance. Finally, the on-chip sensing capabilities of a three-electrode electrochemical cell are demonstrated with a model redox compound, where the high surface area structured electrodes exhibit ultra-high sensitivity. We propose that the developed approach can significantly expedite and reduce the cost of fabrication of sensing devices where arrays of functionalized electrodes can be used for point-of-care analysis and diagnostics.     

Formation of lipid bilayer membrane in a poly(dimethylsiloxane) microchip integrated with a stacked polycarbonate membrane support and an on-site nanoinjector

This paper describes a new and facile approach for the formation of pore-spanning bilayer lipid membranes (BLMs) within a poly(dimethylsiloxane) (PDMS) microfluidic device. Commercially, readily available polycarbonate (PC) membranes are employed for the support of BLMs. PC sheets with 5 μm, 2 μm, and 0.4 μm pore diameters, respectively, are thermally bonded into a multilayer-stack, reducing the pore density of 0.4 μm-pore PC by a factor of 200. The BLMs on this support are considerably stable (a mean lifetime: 17 h). This multilayer-stack PC (MSPC) membrane is integrated into the PDMS chip by an epoxy bonding method developed to secure durable bonding under the use of organic solvents. The microchip has a special channel for guiding a micropipette in the proximity of the MSPC support. With this on-site injection technique, tens to hundreds of nanoliters of solutions can be directly dispensed to the support. Incorporating gramicidin ion channels into BLMs on the MSPC support has confirmed the formation of single BLMs, which is based on the observation from current signals of 20 pS conductance that is typical to single channel opening. Based on the bilayer capacitance (1.4 pF), about 15% of through pores across the MSPC membrane are estimated to be covered with BLMs.     

广西蕗蕨属一新种

 One new species of the genus Mecodium, M. paramnioides H. G.Zhou et W. M. Chu, is described from Guangxi, China.     

民间金融的类型与法理分析(上)

民间金融是目前我国比较突出的金融问题,也是法律上的难点问题.对于民间金融的合法性,有关专家学者和相关政府部门都提出了许多观点和意见.但在金融和法律层面上对它还缺少完整的认识,其合法与非法的认定标准还没有明确的定论.为更好地判定民间金融的合法性,在具体的民间金融的司法实践中,应从金融权的公平性,民间金融的行为目的,公众财产利益保护,社会综合金融环境,整体的金融效率、秩序与安全等五个方面入手,找到它们最佳的边际均衡点,并将之作为合法与非法金融的判断标准.     

Surface modification on polydimethylsiloxane-based microchannels with fragmented poly(l-lactic acid) nanosheets

Surface modification is a critical issue in various applications of polydimethylsiloxane (PDMS)-based microfluidic devices. Here, we describe a novel method through which PDMS-based microchannels were successfully modified with fragmented poly(l-lactic acid) (PLLA) nanosheets through a simple patchwork technique that exploited the high level of adhesiveness of PLLA nanosheets. Compared with other surface modification methods, our method required neither complicated chemical modifications nor the use of organic solvents that tend to cause PDMS swelling. The experimental results indicated that the modified PDMS exhibited excellent capacity for preventing the adhesion and activation of platelets. This simple yet efficient method can be used to fabricate the special PDMS microfluidic devices for biological, medical, and even hematological purposes.     

Robust sulfonated poly (ether ether ketone) nanochannels for high-performance osmotic energy conversion

The membrane-based reverse electrodialysis (RED) technique has a fundamental role in harvesting clean and sustainable osmotic energy existing in the salinity gradient. However, the current designs of membranes cannot cope with the high output power density and robustness. Here, we construct a sulfonated poly (ether ether ketone) (SPEEK) nanochannel membrane with numerous nanochannels for a membrane-based osmotic power generator. The parallel nanochannels with high space charges show excellent cation-selectivity, which could further be improved by adjusting the length and charge density of nanochannels. Based on numerical simulation, the system with space charge shows better conductivity and selectivity than those of a surface-charged nanochannel. The output power density of our proposed membrane-based device reaches up to 5.8 W/m² by mixing artificial seawater and river water. Additionally, the SPEEK membranes exhibit good mechanical properties, endowing the possibility of creating a high-endurance scale-up membrane-based generator system. We believe that this work provides useful insights into material design and fluid transport for the power generator in osmotic energy conversion.     

Improved single-cell culture achieved using micromolding in capillaries technology coupled with poly (HEMA)

Cell studies at the single-cell level are becoming more and more critical for understanding the complex biological processes. Here, we present an optimization study investigating the positioning of single cells using micromolding in capillaries technology coupled with the cytophobic biomaterial poly (2-hydroxyethyl methacrylate) (poly (HEMA)). As a cytophobic biomaterial, poly (HEMA) was used to inhibit cells, whereas the glass was used as the substrate to provide a cell adhesive background. The poly (HEMA) chemical barrier was obtained using micromolding in capillaries, and the microchannel networks used for capillarity were easily achieved by reversibly bonding the polydimethylsiloxane mold and the glass. Finally, discrete cell adhesion regions were presented on the glass surface. This method is facile and low cost, and the reagents are commercially available. We validated the cytophobic abilities of the poly (HEMA), optimized the channel parameters for higher quality and more stable poly (HEMA) patterns by investigating the effects of changing the aspect ratio and the width of the microchannel on the poly (HEMA) grid pattern, and improved the single-cell occupancy by optimizing the dimensions of the cell adhesion regions.