Reusable, polyethylene glycol-structured microfluidic channel for particle immunoassays

A microfluidic channel made entirely out of polyethylene glycol (PEG), not PEG coating to silicon or polydimethylsiloxane (PDMS) surface, was fabricated and tested for its reusability in particle immunoassays and passive protein fouling, at relatively high target concentrations (1 mg ml^-1). The PEG devices were reusable up to ten times while the oxygen-plasma-treated polydimethyl siloxane (PDMS) device could be reused up to four times and plain PDMS were not reusable. Liquid was delivered spontaneously via capillary action and complicated bonding procedure was not necessary. The contact angle analysis revealed that the water contact angle on microchannel surface should be lower than ~60°, which are comparable to those on dried protein films, to be reusable for particle immunoassays and passive protein fouling.     

Applying microfluidics to electrophysiology

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities. Specifically, the motivation for the microfluidic brain slice device is discussed including how the device docks to standard perfusion chambers and the technique of passive pumping which is used to deliver boluses of neuromodulators to the brain slice. By simplifying the device design, we are able to achieve a practical solution to the current unmet electrophysiology need of applying multiple neuromodulators across multiple regions of the brain slice. This is achieved by substituting the standard coverglass substrate of the perfusion chamber with a thin microfluidic device bonded to the coverglass substrate. This was then attached to the perfusion chamber and small holes connect the open-well of the perfusion chamber to the microfluidic channels buried within the microfluidic substrate. These microfluidic channels are interfaced with ports drilled into the edge of the perfusion chamber to access and deliver stimulants. This project represents how the field of microfluidics is transitioning away from proof-of concept device demonstrations and into practical solutions for unmet experimental and clinical needs.     

A fast cell loading and high-throughput microfluidic system for long-term cell culture in zero-flow environments

We present a simple technique for cell loading, culturing, and phenotypic study in a multi-chamber microfluidic device made of polydimethylsiloxane (PDMS). This technique is based on the use of degassing induced aspiration of PDMS which allows loading cells into micro-cavities within 1 min. A large number of triangle cavities are patterned aside main flow channels with narrow connections so that cells can be loaded by aspirating into each cavity. In our device, high throughput and long-term monitoring can be done with minimum shear force of the flow. As a demonstration, we show a controlled loading at single cell level and the phenotypic variation of gene expression of the yeast strain w303 as a function of copper ion concentration of the medium.     

Validation of long-term primary neuronal cultures and network activity through the integration of reversibly bonded microbioreactors and MEA substrates

In vitro recording of neuronal electrical activity is a widely used technique to understand brain functions and to study the effect of drugs on the central nervous system. The integration of microfluidic devices with microelectrode arrays (MEAs) enables the recording of networks activity in a controlled microenvironment. In this work, an integrated microfluidic system for neuronal cultures was developed, reversibly coupling a PDMS microfluidic device with a commercial flat MEA through magnetic forces. Neurons from mouse embryos were cultured in a 100 μm channel and their activity was followed up to 18 days in vitro. The maturation of the networks and their morphological and functional characteristics were comparable with those of networks cultured in macro-environments and described in literature. In this work, we successfully demonstrated the ability of long-term culturing of primary neuronal cells in a reversible bonded microfluidic device (based on magnetism) that will be fundamental for neuropharmacological studies.     

Microfluidic chips controlled with elastomeric microvalve arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.     

Development of low-cost microfluidic systems for lab-on-a-chip biosensor applications

In this work, we develop low-cost microfluidic systems based on polydimethylsiloxane (PDMS) for lab-on-a-chip applications. PDMS microfluidic structures have been fabricated by micromolding, PDMS casting, and plasma bonding processes. The micromolding technique is used to fabricate PDMS slabs with micro-sized grooves, and the complete microchannel is formed by bonding PDMS slab with glass or PDMS substrate. The molding procedure using SU-8 photoresist patterning on silicon wafer, PDMS microchannel fabrication, and PDMS surface treatment using oxygen plasma and TiO₂ coating, are discussed. The various parameters for oxygen plasma treatment including RF power and treatment time are studied in order to obtain conditions for good bonding with the glass substrate. The best condition for plasma treatment is found to be the low RF power (8 W) with 5 min treatment time. In addition, TiO₂ coating with oxygen plasma treatment has been applied to make PDMS surface more hydrophilic to improve aqueous solution compatilbility. The microfluidic channels for various applications, including sample injection cross channel, micropump channel, T and Y sample mixers, PCR thermocyling chamber and channel, capillary electrophoresis flow channel, and conductimetric systems have been fabricated. Finally, a typical application of the PDMS chip in a flow injection conductimetric system for sodium chloride detection has been demonstrated.     

Constraining the connectivity of neuronal networks cultured on microelectrode arrays with microfluidic techniques: a step towards neuron-based functional chips

In vitro culture of small neuronal networks with pre-defined topological features is particularly desirable when the electrical activity of such assemblies can be monitored for long periods of time. Indeed, it is hoped that such networks, with pre-determined connectivity, will provide unique insights into the structure/function relationship of biological neural networks and their properties of self-organization. However, the experimental techniques that have been developed so far for that purpose have either failed to provide very long-term pattern definition and retention, or they have not shown potential for integration into more complex microfluidic devices. To address this problem, three-dimensional microfluidic systems in poly(dimethylsiloxane) (PDMS) were fabricated and used in conjunction with both custom-made and commercially available planar microelectrode arrays (pMEAs). Various types of primary neuronal cell cultures were established inside these systems. Extracellular electrical signals were successfully recorded from all types of cells placed inside the patterns, and this bioelectrical activity was present for several weeks. The advantage of this approach is that it can be further integrated with microfluidic devices and pMEAs to yield, for example, complex neuron-based biosensors or chips for pharmacological screening.     

A thin, flexible multielectrode grid for high-density surface EMG.

Although the value of high-density surface electromyography (sEMG) has already been proven in fundamental research and for specific diagnostic questions, there is as yet no broad clinical application. This is partly due to limitations of construction principles and application techniques of conventional electrode array systems. We developed a thin, highly flexible, two-dimensional multielectrode sEMG grid, which is manufactured by using flexprint techniques. The material used as electrode carrier (Polyimid, 50 microm thick) allows grids to be cut out in any required shape or size. One universal grid version can therefore be used for many applications, thereby reducing costs. The reusable electrode grid is attached to the skin by using specially prepared double-sided adhesive tape, which allows the selective application of conductive cream only directly below the detection surfaces. To explore the practical possibilities, this technique was applied in single motor unit analysis of the facial musculature. The high mechanical flexibility allowed the electrode grid to follow the skin surface even in areas with very uneven contours, resulting in good electrical connections in the whole recording area. The silverchloride surfaces of the electrodes and their low electrode-to-skin impedances guaranteed high baseline stability and a low signal noise level. The electrode-to-skin attachment proved to withstand saliva and great tensile forces due to mimic contractions. The inexpensive, universally adaptable and minimally obstructive sensor allows the principal advantages of high-density sEMG to be extended to all skeletal muscles accessible from the skin surface and may lay the foundation for more broad clinical application of this noninvasive, two-dimensional sEMG technique.     

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic microfluidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.     

Chemiluminescence determination of moxifloxacin in pharmaceutical and biological samples based on its enhancing effect of the luminol–ferricyanide system using a microfluidic chip

A sensitive determination of a synthetic fluoroquinolone antibacterial agent, moxifloxacin (MOX), by an enhanced chemiluminescence (CL) method using a microfluidic chip is described. The microfluidic chip was fabricated by a soft‐lithographic procedure using polydimethyl siloxane (PDMS). The fabricated PDMS microfluidic chip had three‐inlet microchannels for introducing the sample, chemiluminescent reagent and oxidant, and a 500 µm wide, 250 µm deep and 82 mm long microchannel. An enhanced CL system, luminol–ferricyanide, was adopted to analyze the MOX concentration in a sample solution. CL light was emitted continuously after mixing luminol and ferricyanide in the presence of MOX on the PDMS microfluidic chip. The amount of MOX in the luminol–ferricyanide system influenced the intensity of the CL light. The linear range of MOX concentration was 0.14–55.0 ng/mL with a correlation coefficient of 0.9992. The limit of detection (LOD) and limit of quantification (LOQ) were 0.06 and 0.2 ng/mL respectively. The presented method afforded good reproducibility, with a relative standard deviation (RSD) of 1.05% for 10 ng/mL of MOX, and has been successfully applied for the determination of MOX in pharmaceutical and biological samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Introduction of customized inserts for streamlined assembly and optimization of BioBrick synthetic genetic circuits

Background  

BioBrick standard biological parts are designed to make biological systems easier to engineer (e.g. assemble, manipulate, and modify). There are over 5,000 parts available in the Registry of Standard Biological Parts that can be easily assembled into genetic circuits using a standard assembly technique. The standardization of the assembly technique has allowed for wide distribution to a large number of users -- the parts are reusable and interchangeable during the assembly process. The standard assembly process, however, has some limitations. In particular it does not allow for modification of already assembled biological circuits, addition of protein tags to pre-existing BioBrick parts, or addition of non-BioBrick parts to assemblies.     

Microfluidic PDMS (polydimethylsiloxane) bioreactor for large-scale culture of hepatocytes

Microfluidics could provide suitable environments for cell culture because of the larger surface-to-volume ratio and fluidic behavior similar to the environments in vivo. Such microfluidic environments are now used to investigate cell-to-cell interactions and behaviors in vitro, emulating situations observed in vivo, for example, microscale blood vessels modeled by microfluidic channels. These emulated situations cannot be realized by conventional technologies. In our previous works, microfluidic channels composed of two PDMS (poly(dimethylsiloxane)) layers were successfully used for Hep G2 cell culture. To achieve physiologically meaningful functions in vitro, a culture with a larger number of cells and higher density must be performed. This will require bioreactors with larger surface areas for cell attachment and sufficient amounts of oxygen and nutrition supply. For those purposes, we fabricated a bioreactor by stacking 10 PDMS layers together, i.e., four cell culture chambers, and a chamber dedicated to the oxygen supply inserted in the middle of the 10-stacked layers. The oxygen supply chamber is separated from the microfluidic channels for the culture medium perfusion by thin 300-microm PDMS walls. The high gas permeability of PDMS allows oxygen supply to the microfluidic channels through the thin walls. On the basis of the measurement of glucose consumption and albumin production, it is shown that cellular activity exhibits a gradual increase and saturation throughout the culture. We clearly observed that in the case of the microfluidic bioreactor for large-scale cultures, the oxygen chamber is indispensable to achieve longer and healthy cultures. In the present bioreactor, the cell density was found to be about 3-4 x 10(7) cells/cm(3), which is in the same order of magnitude as the conventional macroscale bioreactors. Consequently, by stacking single culture chambers and oxygen chambers in between, we could have a scalable method to realize the microfluidic bioreactor for large-scale cultures.     

Mechanosensitivity and compositional dynamics of cell–matrix adhesions

Cells perceive information about the biochemical and biophysical properties of their tissue microenvironment through integrin‐mediated cell–matrix adhesions, which connect the cytoskeleton with the extracellular matrix and thereby allow cohesion and long‐range mechanical connections within tissues. The formation of cell–matrix adhesions and integrin signalling involves the dynamic recruitment and assembly of an inventory of proteins, collectively termed the ‘adhesome’, at the adhesive site. The recruitment of some adhesome proteins, most notably the Lin11‐, Isl1‐ and Mec3‐domain‐containing proteins, depends on mechanical tension generated by myosin II‐mediated contractile forces exerted on cell–matrix adhesions. When exposed to force, mechanosensitive adhesome proteins can change their conformation or expose cryptic‐binding sites leading to the recruitment of proteins, rearrangement of the cytoskeleton, reinforcement of the adhesive site and signal transduction. Biophysical methods and proteomics revealed force ranges within the adhesome and cytoskeleton, and also force‐dependent changes in adhesome composition. In this review, we provide an overview of the compositional dynamics of cell–matrix adhesions, discuss the most prevalent functional domains in adhesome proteins and review literature and concepts about mechanosensing mechanisms that operate at the adhesion site.     

DNA hybridization detection in a microfluidic channel using two fluorescently labelled nucleic acid probes

A conceptually new technique for fast DNA detection has been developed. Here, we report a fast and sensitive online fluorescence resonance energy transfer (FRET) detection technique for label-free target DNA. This method is based on changes in the FRET signal resulting from the sequence-specific hybridization between two fluorescently labelled nucleic acid probes and target DNA in a PDMS microfluidic channel. Confocal laser-induced microscopy has been used for the detection of fluorescence signal changes. In the present study, DNA hybridizations could be detected without PCR amplification because the sensitivity of confocal laser-induced fluorescence detection is very high. Two probe DNA oligomers (5'-CTGAT TAGAG AGAGAA-TAMRA-3' and 5'-TET-ATGTC TGAGC TGCAGG-3') and target DNA (3'-GACTA ATCTC TCTCT TACAG GCACT ACAGA CTCGA CGTCC-5') were introduced into the channel by a microsyringe pump, and they were efficiently mixed by passing through the alligator teeth-shaped PDMS microfluidic channel. Here, the nucleic acid probes were terminally labelled with the fluorescent dyes, tetrafluororescein (TET) and tetramethyl-6-carboxyrhodamine (TAMRA), respectively. According to our confocal fluorescence measurements, the limit of detection of the target DNA is estimated to be 1.0 x 10(-6) to 1.0 x 10(-7)M. Our result demonstrates that this analytical technique is a promising diagnostic tool that can be applied to the real-time analysis of DNA targets in the solution phase.     

An environmental impact comparison of single-use and reusable thermally controlled shipping containers

Purpose

Pharmaceutical and biological materials require thermally controlled environments when being transported between manufacturers, clinics, and hospitals. It is the purpose of this report to compare the life cycle impacts of two distinct logistical approaches to packaging commonly used in cold chain logistics and to identify the method of least environmental burden. The approaches of interest are single-use packaging utilizing containers insulated with either polyurethane or polystyrene and reusable packaging utilizing containers with vacuum-insulated panels.

Methods

This study has taken a cradle-to-grave perspective, which covers material extraction, manufacture, assembly, usage, transportation, and end-of-life realities. The functional unit of comparison is a 2-year clinical trial consisting of 30,000 individual package shipments able to maintain roughly 12 L of payload at a controlled 2–8 °C temperature range for approximately 96 h. Published life-cycle inventory data were used for process and material emissions. A population-centered averaging method was used to estimate transportation distances to and from clinical sites during container use. Environmental impacts of the study include global warming potential, eutrophication potential, acidification potential, photochemical oxidation potential, human toxicity potential, and postconsumer waste.

Results and discussion

The average single-use approach emits 1,122 tonnes of CO₂e compared with 241 tonnes with the reusable approach over the functional unit. This is roughly a 75 % difference in global warming potential between the two approaches. Similar differences exist in other impact categories with the reusable approach showing 60 % less acidification potential, 65 % less eutrophication potential, 85 % less photochemical ozone potential, 85 % less human toxicity potential, and 95 % less postconsumer waste. The cradle-to-gate emissions of the single-use container were the overwhelming cause of its high environmental burden as 30,000 units were required to satisfy the functional unit rather than 772 for the reusable approach. The reusable container was about half the mass of the average single-use container, which lowered its transportation emissions below the single-use approach despite an extra leg of travel.

Conclusions

The reusable logistical approach has shown to impose a significantly smaller environmental burden in all impact categories of interest. A sensitivity analysis has shown some leeway in the degree of the environmental advantage of the reusable approach, but it confirms the conclusion as no case proved otherwise.     

Mechanisms underlying sawtoothed grain beetle (Oryzaephilus surinamensis [L.]) (Coleoptera: Silvanidae) infestation of consumer food packaging materials

The sawtoothed grain beetle, Oryzaephilus surinamensis (L.), is an extremely destructive pest of packaged consumer food products. The beetle is not believed to chew directly through packaging materials, but to use openings or flaws in damaged or improperly sealed packages to gain entry. We investigated the behavioral mechanisms by which the sawtoothed grain beetle infests packages with flaws. Significantly more sawtoothed grain beetles infested consumer food packages that had been punctured with 0.4 mm diameter holes, to simulate packaging flaws that preclude adults, than when packages had no flaws. In a test arena, females laid more eggs into or near the hole in a plastic packaging film, when they were able to contact the food through the hole than when they could not contact the food. First instar larvae placed either 1 mm or 1 cm away entered holes when food was present, indicating that packages could become infested if eggs were laid near holes. In the absence of food, neither adults nor larvae responded to holes. This study has shown the importance of sound packaging in preventing insect infestation.     

Enhanced Compliant Adhesive Design and Fabrication with Dual-Level Hierarchical Structure

<正> Synthetic dry adhesives inspired by the nano-and micro-scale hairs found on the feet of geckos and some spiders have beendeveloped for almost a decade. Elastomeric single level micro-scale mushroom shaped fibres are currently able to function evenbetter than natural dry adhesives on smooth surfaces under normal loading. However, the adhesion of these single level syntheticdry adhesives on rough surfaces is still not optimal because of the reduced contact surface area. In nature, contact area ismaximized by hierarchically structuring different scales of fibres capable of conforming surface roughness. In this paper, weadapt the nature's solution arid propose a novel dual-level hierarchical adhesive design using Polydimethylsiloxane (PDMS),which is tested under peel loading at different orientations. A negative macro-scale mold is manufactured by using a laser cutterto define holes in a Poly(methyl methacrylate) (PMMA) plate. After casting PDMS macro-scale fibres by using the obtainedPMMA mold, a previously prepared micro-fibre adhesive is bonded to the macro-scale fibre substrate. Once the bondingpolymer is cured, the micro-fibre adhesive is cut to form macro scale mushroom caps. Each macro-fibre of the resulting hierarchicaladhesive is able to conform to loads applied in different directions. The dual-level structure enhances the peel strengthon smooth surfaces compared to a single-level dry adhesive, but also weakens the shear strength of the adhesive for a given areain contact. The adhesive appears to be very performance sensitive to the specific size of the fibre tips, and experiments indicatethat designing hierarchical structures is not as simple as placing multiple scales of fibres on top of one another, but can requiresignificant design optimization to enhance the contact mechanics and adhesion strength.     

Brain slice stimulation using a microfluidic network and standard perfusion chamber

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.     

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and K_ATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.     

聚合物微流控芯片消毒灭菌技术研究进展

聚合物微流控芯片成本低、易加工,目前在医药、生物检测和化学合成等领域得到了普遍应用。以热塑性聚合物聚甲基丙烯酸甲酯(polymethylmethacrylate,PMMA)和热固型聚合物聚二甲基硅氧烷(polydimethy lsiloxane,PDMS)为基材的高分子聚合物材料因具有较好的生物相容性和光学透明性,已逐渐成为聚合物微流控芯片加工的主导材料,被广泛应用于生物医药类微流控芯片的制备。鉴于该类芯片应用场景的特殊性,需在使用前进行消毒灭菌处理以避免微生物干扰。目前,针对PMMA和PDMS的消毒灭菌方法包括高压蒸汽灭菌、紫外线灭菌、电子束、60Co γ射线辐射灭菌、超临界二氧化碳灭菌、乙醇消毒、环氧乙烷灭菌、过氧化氢低温等离子体灭菌、绿原酸消毒、清洗剂消毒。本文从基本原理、消毒灭菌方法、应用场景等方面,回顾和总结了相关技术在PMMA和PDMS基体微流控芯片中的实现方法,并在芯片材质、适用范围等方面分析了所适用的消毒灭菌方法,为以聚合物为基材的生物医药类微流控芯片的消毒灭菌提供有益参考。