A microfluidic bioreactor for increased active retrovirus output

Retroviruses are one of the most commonly used vectors in ongoing gene therapy clinical trials. To evaluate and advance virus production on the microscale platform, we have created a novel microfluidic bioreactor for continuous retrovirus production. We investigated the growth kinetics of a retroviral packaging cell line in microfluidic bioreactors for several compartment sizes, and packaging cells perfused in the microdevices showed similar growth kinetics to those cultured in conventional static conditions. To evaluate the efficiency of retrovirus production, virus titers from the microdevices were compared to those obtained from static tissue culture. When retrovirus production and collection were maintained at 37 degrees C, virus production levels were comparable for the microdevices and static tissue culture conditions. However, immediate cold storage downstream of the packaging cells in the microdevices resulted in 1.4- to 3.7-fold greater active virus production levels with the microdevices compared to the conventional static conditions over a 5 day period. Lastly, the use of microfluidics for virus production provides a continuous supply of virus supernatant for immediate infection of target cells or for preservation and storage. Such devices will be valuable for the optimization of production and evaluation of retroviruses and other viral vectors for gene therapy applications.     

Cell detection and counting through cell lysate impedance spectroscopy in microfluidic devices

Cell-based microfluidic devices have attracted interest for a wide range of applications. While optical cell counting and flow cytometry-type devices have been reported extensively, sensitive and efficient non-optical methods to detect and quantify cells attached over large surface areas within microdevices are generally lacking. We describe an electrical method for counting cells based on the measurement of changes in conductivity of the surrounding medium due to ions released from surface-immobilized cells within a microfluidic channel. Immobilized cells are lysed using a low conductivity, hypotonic media and the resulting change in impedance is measured using surface patterned electrodes to detect and quantify the number of cells. We found that the bulk solution conductance increases linearly with the number of isolated cells contributing to solution ion concentration. The method of cell lysate impedance spectroscopy is sensitive enough to detect 20 cells microL(-1), and offers a simple and efficient method for detecting and enumerating cells within microfluidic devices for many applications including measurement of CD4 cell counts in HIV patients in resource-limited settings. To our knowledge, this is the most sensitive approach using non-optical setups to enumerate immobilized cells. The microfluidic device, capable of isolating specific cell types from a complex bio-fluidic and quantifying cell number, can serve as a single use cartridge for a hand-held instrument to provide simple, fast and affordable cell counting in point-of-care settings.     

微流控芯片系统在单细胞研究中的应用

微流控芯片具有网络式通道结构,扩展了在细胞和亚细胞水平进行生命科学研究的能力,为单细胞研究提供了一个新的平台.在微流控芯片通道中,人们利用气压、液压和电压,或利用介电电泳、光学陷阱、行波介电电泳以及磁场等技术,可以操纵细胞通过或驻留在通道内的任意位置,从而使单细胞计数、筛选以及胞内组分分析等操作大大简化.本文对微流控芯片系统在血液流变学、单细胞操纵与计数以及单细胞胞内组分分析中的应用进行了综述,介绍了用于单细胞研究的多种微芯片系统,讨论了芯片上进行单细胞操纵的各种方法     

Emerging trends in miniaturized and microfluidic electrochemical sensing platforms

Electrochemical sensing has established a strong presence in diverse areas. The conventional electrochemical sensing approach consumes large sample volumes and reagents and requires bulky potentiostat, macro-electrodes, and other equipment. The synergistic integration of electrochemical sensing systems with miniaturized or microfluidic electrochemical devices and microelectrodes in a single platform provides rapid analysis with a disposable, reusable, and cost-effective platform for multiplexed point-of-care detections. Such microdevices have created scope for using several materials as electrodes and sensing platforms by using appropriate fabrication techniques. One of the most recent advancements in miniaturized devices includes the integration of automation and Internet of Things to realize fully automated and robust electrochemical microdevices. The review summarizes the emerging trends in fabrication methods of miniaturized and microfluidic devices, their multiple applications in real-time, integration of Internet of Things, automation, identifying research gaps with strategies for bridging these gaps, future outlook, and recent approaches to intelligent electrochemical sensing.     

Infrared-optical properties of vapour-deposited metal films

Spectroscopic infrared ellipsometry was applied to determine the optical constants of thin metal layers deposited on dielectric substrates such as glass or CaF(2). The layers were produced by evaporating gold or silver in a vacuum, and the coverage, that is the deposited mass per area, was chosen in the range 80-1200 mg m(-2) for gold, which refers to thicknesses in the lower nanometer range; in the case of the specifically lighter silver, about half the coverage was applied. At low coverage a metal island structure is obtained, which gives rise to surface-enhanced infrared absorption (SEIRA). Depending on the coverage, the deposited films exhibit either dielectric or metallic optical properties. Atomic force microscopy and conductivity measurements complement the spectroscopic observation.     

用激光诱导荧光方法研究Ca+NF3, CHF3反应的初始产物能态分布

本文使用LIF方法在气-束装置上研究了Ca+NF_3——CaF+F_2N 和Ca+CHF_3——CaF+CHF_2二个反应的初始产物能态分布。通过测得的CaF分子的LIF激发光谱和计算机模拟计算, 发现二个反应的CaF振动分布属于类Boltzmann型, 并取得其内能分配的信息。通过光谱模拟和信息论计算, 还获知NF_2和CHF_2自由基的内能态是激发的。根据分析, 本文认为这二个反应是具有后期释能特征的直接碰撞反应。     

Microfluidic exploration of the phase diagram of a surfactant/water binary system

We investigate the behavior of a binary surfactant solution (AOT/water) as it is progressively concentrated in microfluidic evaporators. We observe in time a succession of phase transitions from a dilute solution up to a dense state, which eventually grows and invades the microchannels. Analyzing these observations, we show that, with a few experiments and a limited amount of material, our microdevices permit a semiquantitative screening of the equilibrium phase diagram as well as a few kinetic observations.     

Fabrication of thermoplastics chips through lamination based techniques

In this work, we propose a novel strategy for the fabrication of flexible thermoplastic microdevices entirely based on lamination processes. The same low-cost laminator apparatus can be used from master fabrication to microchannel sealing. This process is appropriate for rapid prototyping at laboratory scale, but it can also be easily upscaled to industrial manufacturing. For demonstration, we used here Cycloolefin Copolymer (COC), a thermoplastic polymer that is extensively used for microfluidic applications. COC is a thermoplastic polymer with good chemical resistance to common chemicals used in microfluidics such as acids, bases and most polar solvents. Its optical quality and mechanical resistance make this material suitable for a large range of applications in chemistry or biology. As an example, the electrokinetic separation of pollutants is proposed in the present study.     

Monolithically integrated dye-doped PDMS long-pass filters for disposable on-chip fluorescence detection

We report the fabrication of high quality monolithically integrated optical long-pass filters, for use in disposable diagnostic microchips. The filters were prepared by incorporating dye molecules directly into the microfluidic chip substrate, thereby providing a fully integrated solution that removes the usual need for discrete optical filters. In brief, lysochrome dyes were added to a poly(dimethylsiloxane) (PDMS) monomer prior to moulding of the microchip from a structured SU-8 master. Optimum results were obtained using 1 mm layers of PDMS doped with 1200 microg mL(-1) Sudan II, which resulted in less than 0.01% transmittance below 500 nm (OD 4), >80% above 570 nm, and negligible autofluorescence. These spectral characteristics compare favourably with commercially available Schott-glass long-pass filters, indicating that high quality optical filters can be straightforwardly integrated into the form of PDMS microfluidic chips. The filters were found to be robust in use, showing only slight degradation after extended illumination and negligible dye leaching after prolonged exposure to aqueous solutions. The provision of low cost high quality integrated filters represents a key step towards the development of high-sensitivity disposable microfluidic devices for point-of-care diagnostics.     

Polarised infrared microspectroscopy of edge-oriented graphene oxide papers

We have performed FTIR transmission microspectroscopy on graphene oxide papers oriented with the nominal lattice planes parallel to the infrared optical axis. By polarising the IR light for samples of this geometry, spectral contributions of oriented oxide species are isolated from broad convoluted bands. Analysing the data alongside previous works, including experiments where samples were perturbed by reduction, dehydration and deuteration, we tabulate the most likely assignments for the observed spectral bands.     

Microfluidic chip capillary electrophoresis coupled with electrochemiluminescence for enantioseparation of racemic drugs using central composite design optimization

Optimization based on central composite design (CCD) for enantioseparation of anisodamine (AN), atenolol (AT), and metoprolol (ME) in human urine was developed using a microfluidic chip‐CE device. Coupling the flexible and wide working range of microfluidic chip‐CE device to CCD for chiral separation of AN, AT, and ME in human urine, a total of 15 experiments is needed for the optimization procedure as compared to 75 experiments using the normal one variable at a time optimization. The optimum conditions obtained are found to be more robust as shown by the curvature effects of the interaction factors. The developed microfluidic chip‐CE‐ECL system with adjustable dilution ratios has been validated by satisfactory recoveries (89.5–99% for six enanotiomers) in urine sample analysis. The working range (0.3–600 μM), repeatability (3.1–4.9% RSD for peak height and 4.0–5.2% RSD for peak area), and detection limit (0.3–0.6 μM) of the method developed are found to meet the requirements for bedside monitoring of AN, AT, and ME in patients under critical conditions. In summary, the hyphenation of CCD with the microfluidic chip‐CE device is shown to offer a rapid means for optimizing the working conditions on simultaneous separation of three racemic drugs using the microfluidic chip‐CE device developed.     

Disposable roll-to-roll hot embossed electrophoresis chip for detection of antibiotic resistance gene mecA in bacteria

We present a high-throughput roll-to-roll (R2R) manufacturing process for foil-based polymethyl methacrylate (PMMA) chips of excellent optical quality. These disposable, R2R hot embossed microfluidic chips are used for the identification of the antibiotic resistance gene mecA in Staphylococcus epidermidis. R2R hot embossing is an emerging manufacturing technology for polymer microfluidic devices. It is based on continuous feeding of a thermoplastic foil through a pressurized area between a heated embossing cylinder and a blank counter cylinder. Although mass fabrication of foil-based microfluidic chips and their use for biological applications were foreseen already some years ago, no such studies have been published previously.     

Adsorption of SDS and PEG on calcium fluoride studied by sum frequency generation vibrational spectroscopy

The adsorption of sodium dodecyl sulfate (SDS) from aqueous solution onto a calcium fluoride substrate (CaF(2)), in the presence of polyethylene glycol (PEG) of different molecular weights, has been investigated using the interface specific nonlinear optical technique of sum frequency generation (SFG) vibrational spectroscopy. Spectra of adsorbed SDS (in the C-H stretching region) were recorded at the surface of a CaF(2) prism in contact with SDS solutions at concentrations up to the cmc (8 mM) of the pure surfactant and in contact with binary solutions containing SDS and PEG with molecular weights from 400 to 12 000. In contrast with SFG spectra from the same combinations of surfactant and polymer on a hydrophobic surface, there was no evidence of spectra arising from the actual polymer adsorbed on CaF(2) at any polymer molecular weight either in the absence or presence of surfactant. However, there was indirect evidence for the presence of adsorbed polymer from changes in the SDS SFG spectra in the presence of polymer compared with spectra when the polymer was absent. The SFG spectra of SDS at 0.8 mM were closely similar to each other at all polymer molecular weights and different from the spectra in the absence of the polymer. The spectral differences between the polymer present and polymer absent was much smaller when the solution concentration of surfactant was 8 mM.     

Brain slice on a chip: opportunities and challenges of applying microfluidic technology to intact tissues

Isolated brain tissue, especially brain slices, are valuable experimental tools for studying neuronal function at the network, cellular, synaptic, and single channel levels. Neuroscientists have refined the methods for preserving brain slice viability and function and converged on principles that strongly resemble the approach taken by engineers in developing microfluidic devices. With respect to brain slices, microfluidic technology may 1) overcome the traditional limitations of conventional interface and submerged slice chambers and improve oxygen/nutrient penetration into slices, 2) provide better spatiotemporal control over solution flow/drug delivery to specific slice regions, and 3) permit successful integration with modern optical and electrophysiological techniques. In this review, we highlight the unique advantages of microfluidic devices for in vitro brain slice research, describe recent advances in the integration of microfluidic devices with optical and electrophysiological instrumentation, and discuss clinical applications of microfluidic technology as applied to brain slices and other non-neuronal tissues. We hope that this review will serve as an interdisciplinary guide for both neuroscientists studying brain tissue in vitro and engineers as they further develop microfluidic chamber technology for neuroscience research.     

Electroporation of cells in microfluidic devices: a review

In recent years, several publications on microfluidic devices have focused on the process of electroporation, which results in the poration of the biological cell membrane. The devices involved are designed for cell analysis, transfection or pasteurization. The high electric field strengths needed are induced by placing the electrodes in close proximity or by creating a constriction between the electrodes, which focuses the electric field. Detection is usually achieved through fluorescent labeling or by measuring impedance. So far, most of these devices have only concerned themselves solely with the electroporation process, but integration with separation and detection processes is expected in the near future. In particular, single-cell content analysis is expected to add further value to the concept of the microfluidic chip. Furthermore, if advanced pulse schemes are employed, such microdevices can also enhance research into intracellular electroporation.     

Single cell manipulation, analytics, and label-free protein detection in microfluidic devices for systems nanobiology

Single cell analytics for proteomic analysis is considered a key method in the framework of systems nanobiology which allows a novel proteomics without being subjected to ensemble-averaging, cell-cycle, or cell-population effects. We are currently developing a single cell analytical method for protein fingerprinting combining a structured microfluidic device with latest optical laser technology for single cell manipulation (trapping and steering), free-solution electrophoretical protein separation, and (label-free) protein detection. In this paper we report on first results of this novel analytical device focusing on three main issues. First, single biological cells were trapped, injected, steered, and deposited by means of optical tweezers in a poly(dimethylsiloxane) microfluidic device and consecutively lysed with SDS at a predefined position. Second, separation and detection of fluorescent dyes, amino acids, and proteins were achieved with LIF detection in the visible (VIS) (488 nm) as well as in the deep UV (266 nm) spectral range for label-free, native protein detection. Minute concentrations of 100 fM injected fluorescein could be detected in the VIS and a first protein separation and label-free detection could be achieved in the UV spectral range. Third, first analytical experiments with single Sf9 insect cells (Spodoptera frugiperda) in a tailored microfluidic device exhibiting distinct electropherograms of a green fluorescent protein-construct proved the validity of the concept. Thus, the presented microfluidic concept allows novel and fascinating single cell experiments for systems nanobiology in the future.     

Microchip free-flow electrophoresis on glass substrate using laser-printing toner as structural material

In this work, a microfluidic free-flow electrophoresis device, obtained by thermal toner transferring on glass substrate, is presented. A microdevice can be manufactured in only 1 h. The layout of the microdevice was designed in order to improve the fluidic and electrical characteristics. The separation channel is 8 microm deep and presents an internal volume of 1.42 microL. The deleterious electrolysis effects were overcome by using a system that isolates the electrolysis products from the separation channel. The Joule heating dissipation in the separation channel was found to be very efficient up to a current density of 8.83 mA/mm(2) that corresponds to a power dissipation per unit volume of running electrolyte of 172 mW/microL. Promising results were obtained in the evaluation of the microdevices for the separation of ionic dyes. The microfluidic device can be used for a continuous sample pretreatment step for micro total analysis system.     

Microfluidic devices for culturing primary mammalian neurons at low densities

Microfluidic devices have been used to study high-density cultures of many cell types. Because cell-to-cell signaling is local, however, there exists a need to develop culture systems that sustain small numbers of neurons and enable analyses of the microenvironments. Such cultures are hard to maintain in stable form, and it is difficult to prevent cell death when using primary mammalian neurons. We demonstrate that postnatal primary hippocampal neurons from rat can be cultured at low densities within nanoliter-volume microdevices fabricated using polydimethylsiloxane (PDMS). Doing so requires an additional fabrication step, serial extractions/washes of PDMS with several solvents, which removes uncrosslinked oligomers, solvent and residues of the platinum catalyst used to cure the polymer. We found this step improves the biocompatibility of the PDMS devices significantly. Whereas neurons survive for > or = 7 days in open channel microdevices, the ability to culture neurons in closed-channel devices made of untreated, native PDMS is limited to < or = 2 days. When the closed-channel PDMS devices are extracted, biocompatibility improves allowing for reliable neuron cultures at low densities for > or = 7 days. Comparisons made to autoclaved PDMS and native, untreated PDMS reveal that the solvent-treated polymer is superior in sustaining low densities of primary neurons in culture. When neuronal affinity for local substrates is observed directly, we find that axons localize to channel corners and prefer PDMS surfaces to glass in hybrid devices. When perfusing the channels with media by gravity flow, cultured hippocampal neurons survive for > or = 11 days. Extracting PDMS improves biocompatibility of microfluidic devices and thus enables the study of differentiation of identifiable neurons and the characterization of local extracellular signals.     

微流控芯片测定单细胞内化学组分的进展

细胞是生命的基本单元。由于细胞的个体差异,传统分析群体细胞的方法难以得到单细胞的重要信息。准确可靠地测定单细胞内化学组分的含量能大大提高从正常细胞中辨别不正常细胞的能力,为进一步研究和发展生物化学、医学和临床检验等领域奠定基础。近年来,用微流控芯片进行单细胞分析已引起广泛的兴趣。微流控芯片可以集成单细胞进样、溶膜、电泳分离胞内化学组分和高灵敏度测定等一系列操作步骤,为分析单细胞内的化学组分提供了新的技术平台。本文主要综述了近年来微流控芯片测定单细胞内化学组分的进展。重点在于利用电渗流、压力结合电渗流和激光镊子等技术操控单细胞在微流控芯片上完成单细胞进样、溶膜、细胞内化学组分的电泳分离和高灵敏度测定等一系列操作步骤。对在微流控芯片上的衍生技术也做了较为详细的阐述。     

Carbon nanotube and diamond as electrochemical detectors in microchip and conventional capillary electrophoresis

As two important polymorphs of carbon, carbon nanotube (CNT) and diamond have been widely employed as electrode materials for electrochemical sensing. This review focuses on recent advances and the key strategies in the fabrication and application of electrochemical detectors in microchip and conventional capillary electrophoresis (CE) using CNT and boron-doped diamond. The subjects covered include CNT-based electrochemical detectors in microchip CE, CNT-based electrochemical detectors in conventional CE, boron-doped diamond electrochemical detectors in microchip CE, and boron-doped diamond electrochemical detectors in conventional CE. The attractive properties of CNT and boron-doped diamond make them very promising materials for the electrochemical detection in microchip and conventional CE systems and other microfluidic analysis systems.