Disposable microfluidic devices: fabrication, function, and application

This review article describes recent developments in microfluidics, with special emphasis on disposable plastic devices. Included is an overview of the common methods used in the fabrication of polymer microfluidic systems, including replica and injection molding, embossing, and laser ablation. Also described are the different methods by which on-chip operations--such as the pumping and valving of fluid flow, the mixing of different reagents, and the separation and detection of different chemical species--have been implemented in a microfluidic format. Finally, a few select biotechnological applications of microfluidics are presented to illustrate both the utility of this technology and its potential for development in the future.     

Monoliths for microfluidic devices in proteomics

We report here on the preparation of monolithic capillary columns in view to their integration in a microsystem for on-chip sample preparation before their on-line analysis by electrospray and mass spectrometry (ESI-MS). These monolithic columns are based on polymer materials and consist of reverse phases for peptide separation and/or desalting. They were prepared using lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA) as well as a suitable porogenic mixture composed of cyclohexanol and ethylene glycol. The resulting stationary phases present thus a C12-functionality. The LMA-based columns were first prepared in a capillary format using capillary tubing of 75 microm i.d. and tested in nanoLC-MS experiments for the separation of a commercial Cytochrome C digest composed of 12 peptidic fragments whose isoelectric point values and hydrophobic character cover a wide range. The LMA-based columns were capable of separating the peptidic fragments and their performances were seen to be similar as those of standard commercial columns dedicated to proteomic purposes with calculated separation efficiencies up to 145 x 10(3) plates/m. Monolithic LMA-based phases were then successfully polymerized in microchannels fabricated using the negative photoresist SU-8. After the polymerization, the systems were seen to withstand the pressures applied during the nanoLC-MS separation tests that were carried out in the same conditions as for the monolithic capillary columns. The pressure drop during these tests of the in-microchannel monoliths was as high as 50 bar; however, the separation was not as good as for a capillary format which could be accounted for by the monolith dimensions.     

PCR microfluidic devices for DNA amplification

The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems (MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Among many miniaturized analytical devices, the polymerase chain reaction (PCR) microchip/microdevices are studied extensively, and thus great progress has been made on aspects of on-chip micromachining (fabrication, bonding and sealing), choice of substrate materials, surface chemistry and architecture of reaction vessel, handling of necessary sample fluid, controlling of three or two-step temperature thermocycling, detection of amplified nucleic acid products, integration with other analytical functional units such as sample preparation, capillary electrophoresis (CE), DNA microarray hybridization, etc. However, little has been done on the review of above-mentioned facets of the PCR microchips/microdevices including the two formats of flow-through and stationary chamber in spite of several earlier reviews [Zorbas, H. Miniature continuous-flow polymerase chain reaction: a breakthrough? Angew Chem Int Ed 1999; 38 (8):1055–1058; Krishnan, M., Namasivayam, V., Lin, R., Pal, R., Burns, M.A. Microfabricated reaction and separation systems. Curr Opin Biotechnol 2001; 12:92–98; Schneegaβ, I., Köhler, J.M. Flow-through polymerase chain reactions in chip themocyclers. Rev Mol Biotechnol 2001; 82:101–121; deMello, A.J. DNA amplification: does ‘small’ really mean ‘efficient’? Lab Chip 2001; 1: 24N–29N; Mariella, Jr. R. MEMS for bio-assays. Biomed Microdevices 2002; 4 (2):77–87; deMello AJ. Microfluidics: DNA amplification moves on. Nature 2003; 422:28–29; Kricka, L.J., Wilding, P. Microchip PCR. Anal BioAnal Chem 2003; 377:820–825]. In this review, we survey the advances of the above aspects among the PCR microfluidic devices in detail. Finally, we also illuminate the potential and practical applications of PCR microfluidics to some fields such as microbial detection and disease diagnosis, based on the DNA/RNA templates used in PCR microfluidics. It is noted, especially, that this review is to help a novice in the field of on-chip PCR amplification to more easily find the original papers, because this review covers almost all of the papers related to on-chip PCR microfluidics.     

Simple and inexpensive fabrication of small-volume density gradients

A method is described for high speed centrifugation of small volumes of homogenates, tissue extracts, or body fluids without the use of expensive adapters. The procedure consists of introducing the samples into light polyethylene microcentrifuge tubes and then floating them in a medium contained in standard cellulose nitrate tubes. This assembly can be centrifuged at 198,000g for 90 min with no visible distortion of the sample tubes.     

Silica-immobilized enzymes for multi-step synthesis in microfluidic devices

The combinatorial synthesis of 2-aminophenoxazin-3-one (APO) in a microfluidic device is reported. Individual microfluidic chips containing metallic zinc, silica-immobilized hydroxylaminobenzene mutase and silica-immobilized soybean peroxidase are connected in series to create a chemo-enzymatic system for synthesis. Zinc catalyzes the initial reduction of nitrobenzene to hydroxylaminobenzene which undergoes a biocatalytic conversion to 2-aminophenol, followed by enzymatic polymerization to APO. Silica-immobilization of enzymes allows the rapid stabilization and integration of the biocatalyst within a microfluidic device with minimal preparation. The system proved suitable for synthesis of a complex natural product (APO) from a simple substrate (nitrobenzene) under continuous flow conditions.     

Protocol for the fabrication of enzymatically crosslinked gelatin microchannels for microfluidic cell culture

We have developed a technique for fabricating microfluidic devices from gelatin using a natural crosslinking process. By producing reusable poly(dimethyl siloxane) molds using standard photolithography, gelatin can be molded into microchannel geometries. The gelatin is crosslinked with the naturally occurring enzyme transglutaminase via a straightforward process that can produce devices suitable for cell culture. The protocol takes approximately 1 day from the start of gelatin preparation to cell seeding. Using these devices, the effects of both the extracellular matrix and soluble factors on cellular behavior and differentiation can be studied in microenvironments that more closely mimic the in vivo environment.     

In situ fabrication of a microfluidic device for immobilised metal affinity sensing

In this work a novel microfluidic device was constructed in situ containing the smallest microscopic co-polymeric immobilised metal affinity (IMA) adsorbent yet documented. This device has for the first time allowed the microlitre scale chromatographic assay of histidine-tagged proteins in a biological sample. To enable this approach, rather than using a high capacity commercial packed bed column which requires large sample volumes and would be susceptible to occlusion by cell debris, a microgram capacity co-polymeric chromatographic substrate suitable for analytical applications was fabricated within a microfluidic channel. This porous co-polymeric IMA micro-chromatographic element, only 27μl in volume, was assessed for the analytical capture of two different histidine-tagged recombinant fusion proteins. The micro-chromatographic adsorber was fabricated in situ by photo-polymerising an iminodiacetic acid (IDA) functionalised polymer matrix around a template of fused 100μm diameter NH(4)Cl particles entirely within the microfluidic channel and then etching away the salt with water to form a network of interconnected voids. The surface of the micro-chromatographic adsorber was chemically functionalised with a chelating agent and loaded with Cu(2+) ions. FTIR and NMR analysis verified the presence of the chelating agent on the adsorbent surface and its Cu(2+) ion binding capacity was determined to be 2.4μmol Cu(2+) (ml of adsorbent)(-1). Micro-scale equilibrium adsorption studies using the two different histidine-tagged proteins, LacI-His(6)-GFP and α-Synuclein-His(8)-YFP, were carried out and the protein binding capacity of the adsorbent was determined to be 0.370 and 0.802mg(g of adsorbent)(-1), respectively. The dynamic binding capacity was determined at four different flow rates and found to be comparable to the equilibrium binding capacity at low flow rates. The sensing platform was also used to adsorb LacI-His(6)-GFP protein from crude cell lysate. During adsorption, laser scanning confocal microscopy identified locations within the adsorbent where protein adsorption and desorption occurred. The findings indicate that minimal channelling, selective product capture and near quantitative elution of the captured (adsorbed) product could be achieved, supporting the application of this new device as a high-throughput process analytical tool (PAT) for the in-process monitoring of histidine-tagged proteins in manufacturing.     

Cell-free protein synthesis in microfluidic array devices

We report the development of a microfluidic array device for continuous-exchange, cell-free protein synthesis. The advantages of protein expression in the microfluidic array include (1) the potential to achieve high-throughput protein expression, matching the throughput of gene discovery; (2) more than 2 orders of magnitude reduction in reagent consumption, decreasing the cost of protein synthesis; and (3) the possibility to integrate with detection for rapid protein analysis, eliminating the need to harvest proteins. The device consists of an array of units, and each unit can be used for production of an individual protein. The unit comprises a tray chamber for in vitro protein expression and a well chamber as a nutrient reservoir. The tray is nested in the well, and they are separated by a dialysis membrane and connected through a microfluidic connection that provides a means to supply nutrients and remove the reaction byproducts. The device is demonstrated by synthesis of green fluorescent protein, chloramphenicol acetyl-transferase, and luciferase. Protein expression in the device lasts 5-10 times longer and the production yield is 13-22 times higher than in a microcentrifuge tube. In addition, we studied the effects of the operation temperature and hydrostatic flow on the protein production yield.     

Micro-bioreactors for fed-batch fermentations with integrated online monitoring and microfluidic devices

In this study an array of micro-bioreactors based on the format of 48-well microtiter plates (MTP) is presented. The process parameters pH-value and biomass are monitored online by a combination of different sensors, the biolector measurement technology and conductance measurements. A microfluidic device dispenses two fluids individually into each well for controlling the pH-value of fermentations. The micro-bioreactor consists of four wells and two reservoirs. In each well a polyimide foil with platinum electrodes for conductance measurements is integrated. The microfluidic device is fabricated using softlithographic techniques and utilizes pneumatically actuated microvalves. The device is able to dispense volumes below 5nl. Finally, fermentations of Escherichia coli are carried out in the micro-bioreactor system. During the fermentation, the pH-value is measured optically and the biomass development is monitored by the scattered light signal. Meanwhile, the pH-value is controlled by dispensing sodium hydroxide and phosphoric acid. This micro-bioreactor demonstrates the possibility of online monitored and pH-controlled fermentations in micro-scale. The pH-value in the uncontrolled culture varies within the range of 6.46-8.83 whereas the pH-value in the controlled cultures can be kept within 6.85-7.07. This results in an increase in biomass in the pH-controlled culture compared to the nearly completely inhibited pH-uncontrolled culture.     

Printable microfluidic systems using pressure sensitive adhesive material for biosensing devices

Background

In biosensors with a fluid analyte, the integration of a microfluidic system, which guides the analyte into the sensing area, is critical. Quicker and economical ways to build up microfluidic systems will make point of care diagnostics viable. Printing is a low-cost technology that is increasingly used in emerging organic and flexible electronics and biosensors. In this paper, we present printed fluidic systems on flexible substrates made with pressure sensitive adhesive materials.

Methods

Printable pressure sensitive adhesive materials have been used for making microfluidic systems. Flexible substrates have been used, and two types of adhesive materials, one thermally dried and another UV curable, have been tested. Top sealing layer was laminated directly on top of the printed microfluidic structure. Flow tests were done with deionized water.

Results

Flow tests with deionized water show that both adhesive materials are suitable for capillary flow driven fluidic devices. Flow test using water as dielectric material was also done successfully on a printed electrolyte gated organic field effect transistor with an integrated microfluidic system.

General significance

Due to its ease of process and low cost, printed microfluidic system is believed to find more applications in biosensing devices. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

Monolithic microfluidic mixing–spraying devices for time-resolved cryo-electron microscopy

The goal of time-resolved cryo-electron microscopy is to determine structural models for transient functional states of large macromolecular complexes such as ribosomes and viruses. The challenge of time-resolved cryo-electron microscopy is to rapidly mix reactants, and then, following a defined time interval, to rapidly deposit them as a thin film and freeze the sample to the vitreous state. Here we describe a methodology in which reaction components are mixed and allowed to react, and are then sprayed onto an EM grid as it is being plunged into cryogen. All steps are accomplished by a monolithic, microfabricated silicon device that incorporates a mixer, reaction channel, and pneumatic sprayer in a single chip. We have found that microdroplets produced by air atomization spread to sufficiently thin films on a millisecond time scale provided that the carbon supporting film is made suitably hydrophilic. The device incorporates two T-mixers flowing into a single channel of four butterfly-shaped mixing elements that ensure effective mixing, followed by a microfluidic reaction channel whose length can be varied to achieve the desired reaction time. The reaction channel is flanked by two ports connected to compressed humidified nitrogen gas (at 50 psi) to generate the spray. The monolithic mixer-sprayer is incorporated into a computer-controlled plunging apparatus. To test the mixing performance and the suitability of the device for preparation of biological macromolecules for cryo-EM, ribosomes and ferritin were mixed in the device and sprayed onto grids. Three-dimensional reconstructions of the ribosomes demonstrated retention of native structure, and 30S and 50S subunits were shown to be capable of reassociation into ribosomes after passage through the device.     

Variability in G-protein-coupled signaling studied with microfluidic devices

Different cells, even those that are genetically identical, can respond differently to identical stimuli, but the precise source of this variability remains obscure. To study this problem, we built a microfluidic experimental system which can track responses of individual cells across multiple stimulations. We used this system to determine that amplitude variation in G-protein-activated calcium release in RAW264.7 macrophages is generally extrinsic, i.e., they arise from long-lived variations between cells and not from stochastic activation of signaling components. In the case of responses linked to P2Y family purine receptors, we estimate that approximately one-third of the observed variability in calcium release is receptor-specific. We further demonstrate that the signaling apparatus downstream of P2Y6 receptor activation is moderately saturable. These observations will be useful in constructing and constraining single-cell models of G protein-coupled calcium dynamics.     

Uniform aligned bioconjugation of biomolecule motifs for integration within microfabricated microfluidic devices

Full details and a step-by-step guide suitable for printing proteins aligned to micron-sized sensors and subsequent integration and alignment of microfluidic structures are presented. The precise alignment and grafting of micron-sized biomolecule patterns with an underlying substrate at predefined locations is achieved using a novel semi-automated microcontact printer. Through integration of optical alignment methods in the x, y, and z directions, uniform contact of micron-sized stamps is achieved. Feature compression of the stamp is avoided by fine control of the stamp during contact. This printing method has been developed in combination with robust, compatible bioconjugate chemistry for patterning of a dextran-functionalized silicon oxide substrate with a NeutrAvidin-"inked" stamp and subsequent incubation with a biotin-functionalized protein. The bioconjugate chemistry is such that uniform coverage of the protein (without denaturation) over the printed motif is obtained and reproduction of the initial mask shape and dimensions is achieved. Later integration with a microfluidic structure aligned with the printed motif on the substrate is also described.     

三种细胞共培养微流控芯片的设计和制作

设计一种具有“微坝”和“微缝”结构的微流控芯片,能够物理隔离不同细胞,而且培养基中小分子营养物质可以自由流通。实验结果表明在芯片上可以共培养人肺腺癌细胞(A549)、人胚肺成纤维细胞(HLF-1)和人内皮细胞(HUVECs)三种细胞,在72 h培养后三种细胞生长状态良好,具有细胞图形化的特点和功能,为下一步开展多种细胞相互作用等相关研究提供重要的技术平台。     

Design and fabrication of field-deployable microbial biosensing devices

Microbial biosensing devices contain whole-cell or cell-free biosensors as core elements. They offer advantages such as sensitive detection, a wide dynamic range, and cost-effective manufacturing.

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Helical spring template fabrication of cell‐laden microfluidic hydrogels for tissue engineering

Cell‐laden microfluidic hydrogels find great potential applications in microfluidics, tissue engineering, and drug delivery, due to their ability to control mass transport and cell microenvironment. A variety of methods have been developed to fabricate hydrogels with microfluidic channels, such as molding, bioprinting, and photopatterning. However, the relatively simple structure available and the specific equipment required limit their broad applications in tissue engineering. Here, we developed a simple method to fabricate microfluidic hydrogels with helical microchannels based on a helical spring template. Results from both experimental investigation and numerical modeling revealed a significant enhancement on the perfusion ability and cell viability of helical microfluidic hydrogels compared to those with straight microchannels. The feasibility of such a helical spring template method was also demonstrated for microfluidic hydrogels with complex three‐dimensional channel networks such as branched helical microchannels. The method presented here could potentially facilitate the development of vascular tissue engineering and cell microenvironment engineering. Biotechnol. Bioeng. 2013; 110: 980–989. © 2012 Wiley Periodicals, Inc.