Hybrid Microfluidic Device Research Articles

A polydimethylsiloxane-polycarbonate hybrid microfluidic device capable of generating perpendicular chemical and oxygen gradients for cell culture studies.

This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) hybrid microfluidic device capable of performing cell culture under combinations of chemical and oxygen gradients. The microfluidic device is constructed of two PDMS layers with microfluidic channel patterns separated by a thin PDMS membrane. The top layer contains an embedded PC film and a serpentine channel for a spatially confined oxygen scavenging chemical reaction to generate an oxygen gradient in the bottom layer for cell culture. Using the chemical reaction method, the device can be operated with a small amount of chemicals, without bulky gas cylinders and sophisticated flow control schemes. Furthermore, it can be directly used in conventional incubators with syringe pumps to simplify the system setup. The bottom layer contains arrangements of serpentine channels for chemical gradient generation and a cell culture chamber in the downstream. The generated chemical and oxygen gradients are experimentally characterized using a fluorescein solution and an oxygen-sensitive fluorescent dye, respectively. For demonstration, a 48 hour cell-based drug test and a cell migration assay using human lung adenocarcinoma epithelial cells (A549) are conducted under various combinations of the chemical and oxygen gradients in the experiments. The drug testing results show an increase in A549 cell apoptosis due to the hypoxia-activated cytotoxicity of tirapazamine (TPZ) and also suggest great cell compatibility and gradient controllability of the device. In addition, the A549 cell migration assay results demonstrate an aerotactic behavior of the A549 cells and suggest that the oxygen gradient plays an essential role in guiding cell migration. The migration results, under combinations of chemokine and oxygen gradients, cannot be simply superposed with single gradient results. The device is promising to advance the control of in vitro microenvironments, to better study cellular responses under various physiological conditions for biomedical applications.

Investigation and improvement of reversible microfluidic devices based on glass–PDMS–glass sandwich configuration

Reversibly assembled microfluidic devices are dismountable and reusable, which is useful for a number of applications such as micro- and nano-device fabrication, surface functionalization, complex cell patterning, and other biological analysis by means of spatial–temporal pattern. However, reversible microfluidic devices fabricated with current standard procedures can only be used for low-pressure applications. Assembling technology based on glass–PDMS–glass sandwich configuration provides an alternative sealing method for reversible microfluidic devices, which can drastically increase the sealing strength of reversibly adhered devices. The improvement mechanism of sealing properties of microfluidic devices based on the sandwich technique has not been fully characterized, hindering further improvement and broad use of this technique. Here, we characterize, for the first time, the effect of various parameters on the sealing strength of reversible PDMS/glass hybrid microfluidic devices, including contact area, PDMS thickness, assembling mode, and external force. To further improve the reversible sealing of glass–PDMS–glass microfluidic devices, we propose a new scheme which exploits mechanical clamping elements to reinforce the sealing strength of glass–PDMS–glass sandwich structures. Using our scheme, the glass–PDMS–glass microchips can survive a pressure up to 400 kPa, which is comparable to the irreversibly bonded PDMS microdevices. We believe that this bonding method may find use in lab-on-a-chip devices, particularly in active high-pressure-driven microfluidic devices.

SU-8- and PDMS-based hybrid fabrication technology for combination of permanently bonded flexible and rigid features on a single device

In this article, a novel hybrid fabrication technology is presented that uses both a flexible polymer (polydimethylsiloxane-–PDMS) and a rigid polymer (SU-8). A covalent bond between the flexible and rigid polymer layers is achieved using an oxygen plasma treatment during a layer-by-layer direct spin-on process. Precise alignment of the features in each layer and a highly repeatable method are achieved by this new process. As a proof-of-concept, we successfully fabricated PDMS-based flexible microfluidic devices with SU-8-based rigid world-to-chip/chip-to-world interconnects. The bond strength between the PDMS and SU-8 layers is measured by three methods: (1) Instron® microtester to pull apart the layers; (2) voice coil actuator to test the bond between interconnects and the substrate; and (3) microfluidic pressurization test to evaluate the bond strength along the channels. The bond strength between the flexible PDMS layer and the rigid SU-8 features is very strong; the bond between these two polymers does not fail during these evaluations although the integrity of the PDMS layer itself fails during the microtester evaluation. Additionally, the layer-by-layer direct spin-on process resulted in a repeatable process and precise alignment of the features in each layer, which are necessary in order to achieve consistent performance from the fabricated devices. The rigid SU-8 interconnects fabricated onto a flexible PDMS device serve as a world-to-chip/chip-to-world interconnects for the direct connection with Tygon® tubing. Three different designs of hybrid (PDMS and SU-8 based) microfluidic devices are designed, fabricated and tested. Each variation differed in the microchannel design in order to demonstrate the versatility of the process to make devices on multiple scales and patterns. These hybrid microfluidic devices are capable of functioning without leakage up to pressures of 85.85 ±3.56 kPa. Although microfluidic channels with interconnects are shown as a proof-of-concept, the fabrication process demonstrated herein could be utilized to develop a number of more sophisticated microfluidic and biomedical devices.

Modification of the Glass Surface Property in PDMS-Glass Hybrid Microfluidic Devices

This paper presents a simple method to change the hydrophilic nature of the glass surface in a poly(dimethylsiloxane) (PDMS)-glass hybrid microfluidic device to hydrophobic by an extra-heating step during the fabrication process. Glass substrates bonded to a native or oxygen plasma-treated PDMS chip having microchambers (12.5 mm diameter, 110 µm height) were heated at 200°C for 3 h, and then the hydrophobicity of the glass surfaces on the substrate was evaluated by measuring the contact angle of water. By the extra-heating process, the glass surfaces became hydrophobic, and its contact angle was around 109°, which is nearly the same as native PDMS surfaces. To demonstrate the usefulness of this surface modification method, a PDMS-glass hybrid microfluidic device equipped with microcapillary vent structures for pneumatic manipulation of droplets was fabricated. The feasibility of the microcapillary vent structures on the device with the hydrophobic glass surfaces are confirmed in practical use through leakage tests of the vent structures and liquid handling for the electrophoretic separation of DNA molecules.

Controlling gas/liquid exchange using microfluidics for real-time monitoring of flagellar length in living Chlamydomonas at the single-cell level

Chlamydomonas reinhardtii is widely used for studying cilia/flagella, organelles important for human health and disease. In situ monitoring of flagellar assembly/disassembly kinetics in single living cells has been difficult with conventional methods because of time-consuming media exchange and the requirement of whole cell fixation. Here, we develop a PDMS/glass hybrid microfluidic device for real-time tracking of flagellar length in single living cells of Chlamydomonas. Media exchange is precisely controlled by sequential gas-liquid plugs and complete medium replacement occurs within seconds. Rapid medium exchange allows the capture of transient flagellar dynamics. We show that Chlamydomonas cells respond to acidic medium exchange and deflagellate. However, the two flagella may shed asynchronously. After subsequent medium exchange, cells regenerate full-length flagella. Cells are also induced to shorten their flagella after being exposed to extracellular stimuli. The long-term kinetics of flagellar regeneration and disassembly for the whole cell population on the chip are comparable to those from conventional methods; however, individual cells display non-uniform response kinetics. We also find that flagellar growth rate is dependent on flagellar length. This device provides a potential platform to continuously monitor molecular activities associated with changes in flagellar length and to capture transient molecular changes upon flagellar loss, and initiation of flagellar assembly/disassembly.

Microfluidic synthesis of atto-liter scale double emulsions toward ultrafine hollow silica spheres with hierarchical pore networks

A facile PDMS-glass hybrid microfluidic device is developed for generating uniform submicrometer-scale double emulsion droplets with unprecedented simplicity and controllability. Compared with planar flow-focusing geometries, our three-dimensional flow-focusing geometry is advantageous for stably producing femto- to atto-liter droplets without the retraction problem of the dispersed phase fluid. In addition, this microfluidic platform can withstand the use of strong organic solvents (e.g. tetrahydrofuran (THF) and toluene) as a dispersed phase without deforming PDMS devices because the dispersed phase containing organic solvents does not directly contact the PDMS wall. In particular, monodisperse double emulsions are generated spontaneously via the internal phase separation of single emulsions driven by the diffusion of a co-solvent (tetrahydrofuran) in microfluidic devices. Finally, we demonstrated that the double emulsions can be used as morphological templates of ultrafine spherical silica capsules with controlled hierarchical pore networks via the evaporation-induced self-assembly (EISA) method. During EISA, triblock copolymers (Pluronic F127) act as a surfactant barrier separating the internal droplet from the continuous oil phase, resulting in the 'inverse' morphology (i.e. hydrophobic polymer-in-water-in-oil emulsions). Depending on the precursor composition and kinetic condition, various structural and morphological features, such as mesoporous hollow silica spheres with a single central core, multi-cores, or a combination of these with robust controllability can be seen. Electron microscopy (SEM, STEM, HR-TEM), small angle X-ray scattering (SAXS), and N(2) adsorption-desorption confirm the well-controlled hierarchical pore structure of the resulting particles.

A microfluidic in situ analyzer for ATP quantification in ocean environments

We have developed and tested a functionally integrated in situ analyzer, the IISA-ATP system, for microbial activity assays based on a quantitative determination of the total (particulate and dissolved) ATP in ocean environments. The IISA-ATP utilizes a PDMS-glass hybrid microfluidic device as its core functional element, which can perform cell lysis and total ATP quantification by a luciferin-luciferase bioluminescence assay in situ. Transparent heaters and a temperature sensor fabricated on a glass substrate provide temperature control. As a result of the evaluation using the microfluidic device with ATP standard solutions, the bioluminescence intensity was linearly correlated with 2 × 10(-12) to 2 × 10(-8) M of ATP. A detection limit of 1.1 × 10(-11) M was determined using the completed IISA-ATP system, which includes a miniature pumping module and a control module. As a result of the evaluation using the environmental seawater sample collected from Tokyo Bay, Japan, 2.7 × 10(-10) M of total ATP was successfully determined in the laboratory by the IISA-ATP. The system was operated at a shallow submarine hot spring area in Okinawa, Japan for an in situ trial. The result shows the system was successfully operated in situ and the total ATP was determined to be 3.4 × 10(-10) M.

Reduction of water evaporation in polymerase chain reaction microfluidic devices based on oscillating-flow

Producing polymeric or hybrid microfluidic devices operating at high temperatures with reduced or no water evaporation is a challenge for many on-chip applications including polymerase chain reaction (PCR). We study sample evaporation in polymeric and hybrid devices, realized by glass microchannels for avoiding water diffusion toward the elastomer used for chip fabrication. The method dramatically reduces water evaporation in PCR devices that are found to exhibit optimal stability and effective operation under oscillating-flow. This approach maintains the flexibility, ease of fabrication, and low cost of disposable chips, and can be extended to other high-temperature microfluidic biochemical reactors.

Rapid ultraviolet monitoring of multiple psychotropic drugs with a renewable microfluidic device

A rapid method for sensitive ultraviolet detection of multiple psychotropic drugs in human plasma was developed on a low-cost and expediently fabricated hybrid microfluidic device. The device was composed of one fused-silica capillary with a sampling fracture, a poly(methyl methacrylate) board with four reservoirs, and a printed circuit board. At the optimal separation and detection conditions, the baseline separation of three kinds of psychotropic drugs including barbiturates (phenobarbital and barbital), benzodiazepines (nitrazepam, clonazepam, chlordiazepoxide, alprazolam and diazepam) and tricyclic antidepressant drugs (amitriptyline) was achieved within 200 s with separation efficiency up to 3.80 × 10 5 plates m −1. The linear ranges for ultraviolet detection were from 2.0 to 1000.0 μg mL −1 for chlordiazepoxide and 1.0 to 1000.0 μg mL −1 for other seven drugs. Combining with solid-phase extraction, this novel protocol could successfully be used to screen naturally existing psychotropic drugs in a known human plasma sample. The minimum detectable concentration was down to 27 ng mL −1 for phenobarbital spiked in plasma. This work provided a promising way to initially screen different psychotropic drugs with high resolution, rapid separation and low-cost.

Multilayer Hybrid Microfluidics: A Digital-to-Channel Interface for Sample Processing and Separations

Microchannels can separate analytes faster with higher resolution, higher efficiency and with lower reagent consumption than typical column techniques. Unfortunately, an impediment in the path toward fully integrated microchannel-based laboratories-on-a-chip is the integration of preseparation sample processing. In contrast, the alternative format of digital microfluidics (DMF), in which discrete droplets are manipulated on an array of electrodes, is well-suited for carrying out sequential chemical reactions such as those commonly employed in proteomic sample preparation. We recently reported a new paradigm of "hybrid microfluidics," integrating DMF with microchannels for in-line sample processing and separations. Here, we build on our initial efforts, introducing a second-generation hybrid microfluidic device architecture. In the new multilayer design, droplets are manipulated by DMF in the two-plate format, an improvement that facilitates dispensing samples from reservoirs, as well as droplet splitting and storage for subsequent analysis. To demonstrate the capabilities of the new method, we implemented an on-chip serial dilution experiment, as well as multistep enzymatic digestion. Given the myriad applications requiring preprocessing and chemical separations, the hybrid digital-channel format has the potential to become a powerful new tool for micro total analysis systems.

Sol–gel based fabrication of hybrid microfluidic devices composed of PDMS and thermoplastic substrates

Fabrication of microfluidic devices using both rigid and flexible plastic substrates offers benefits for making pressure-actuated membrane valves, mechanically active components, and low-cost but highly functional 3D microchannel networks. Here we present a simple and versatile process for bonding flexible polydimethylsiloxane (PDMS) and rigid thermoplastics like poly(methyl methacrylate) (PMMA), by utilizing the sol–gel method. The silica sol, obtained by oligomerizing tetraethoxysilane monomers, was spin-coated on a thermoplastic plate and further polymerized to form a thin silica layer (silica gel) with a thickness of 140–300nm. The silica-coated surface could be covalently and strongly bonded with an O2-plasma-activated PDMS plate, just by bringing them into contact. We applied the presented process to preparing multi-layer PDMS–PMMA microdevices having 3D crossing channels or pneumatically controlled membrane valves, and demonstrated the parallel flow distribution, mixing, and droplet generation. In addition, bonding strengths between PDMS and various thermoplastics, including PMMA, polyvinyl chloride (PVC), polycarbonate (PC), and polypropylene (PP), were examined.

High flow rate microfluidic device for blood plasma separation using a range of temperatures

A hybrid microfluidic device that uses hydrodynamic forces to separate human plasma from blood cells has been designed and fabricated and the advantageous effects of temperature and flow rates are investigated in this paper. The blood separating device includes an inlet which is reduced by approximately 20 times to a small constrictor channel, which then opens out to a larger output channel with a small lateral channel for the collection of plasma. When tested the device separated plasma from whole blood using a wide range of flow rates, between 50 microl min(-1) and 200 microl min(-1), at the higher flow rates injected by hand and at temperatures ranging from 23 degrees C to 50 degrees C, the latter resulting in an increase in the cell-free layer of up to 250%. It was also tested continuously using between 5% and 40% erythrocytes in plasma and whole blood without blocking the channels or hemolysis of the cells. The mean percentage of plasma collected after separation was 3.47% from a sample of 1 ml. The percentage of cells removed from the plasma varied depending on the flow rate used, but at 37 degrees C ranged between 95.4 +/- 1% and 97.05 +/- 05% at 100 microl min(-1) and 200 microl min(-1), respectively. The change in temperature also had an effect on the number of cells removed from the plasma which was between 93.5 +/- 0.65% and 97.01 +/- 0.3% at 26.9 degrees C and 37 degrees C, respectively, using a flow rate of 100 microl min(-1). Due to its ability to operate in a wide range of conditions, it is envisaged that this device can be used in in vitro 'lab on a chip' applications, as well as a hand-held point of care (POC) device.

Long-term high-resolution imaging and culture of C. elegans in chip-gel hybrid microfluidic device for developmental studies.

Developmental studies in multicellular model organisms such as Caernohabditis elegans rely extensively on the ability to cultivate and image animals repeatedly at the cell or subcellular level. However, standard high-resolution imaging techniques require the use of anaesthetics for immobilization, and may have undesirable side effects on development. Thus such techniques are not ideal in allowing the same animals to grow and be imaged throughout development to observe specific developmental processes. In this paper, we present a microfluidic system designed to overcome these difficulties. The system allows for long-term culture of C. elegans starting at L1 larval stage and repeated high-resolution imaging at physiological temperatures without using anaesthetics. We use a commercially available biocompatible polymer, Pluronic F127 for immobilization; this polymer is capable of a reversible thermo-sensitive sol-gel transition within approximately 2 degrees C, which is well-controlled in the microfluidic chip. The gel phase is sufficient to immobilize the animals. While animals are not imaged, they are cultured in individual chambers in media containing nutrients required for development. We show here that this method facilitates time-lapse studies of single animals at high-resolution and lends itself to live imaging experiments on developmental processes and dynamic events.

Hybrid microfluidics: A digital-to-channel interface for in-line sample processing and chemical separations

Microchannels can separate analytes faster with higher resolution, higher efficiency and with lower reagent consumption than typical column techniques. Unfortunately, an impediment in the path toward fully integrated microchannel-based labs-on-a-chip is the integration of pre-separation sample processing. Although possible in microchannels, such steps are challenging because of the difficulty in maintaining spatial control over many reagents simultaneously. In contrast, the alternative format of digital microfluidics (DMF), in which discrete droplets are manipulated on an array of electrodes, is well-suited for carrying out sequential chemical reactions. Here, we report the development of the first digital-channel hybrid microfluidic device for integrated pre-processing reactions and chemical separations. The device was demonstrated to be useful for on-chip labeling of amino acids and primary amines in cell lysate, as well as enzymatic digestion of peptide standards, followed by separation in microchannels. Given the myriad applications requiring pre-processing and chemical separations, the hybrid digital-channel format has the potential to become a powerful new tool for micro total analysis systems.

Separation of catecholamines and dopamine-derived DNA adduct using a microfluidic device with electrochemical detection

Dopamine (DA) can produce quinones that form protein and depurinating DNA adducts; the latter are thought to be involved in some neurodegenerative diseases (e.g. Parkinson's disease) [E.L. Cavalieri et al., Carcinogenesis 23 (2002) 1071–1077]. Methods with high resolving power are needed to detect neurotransmitters (e.g. DA) and DA-derived DNA adducts in human fluids, as their presence is difficult to determine by standard chromatography with UV absorbance detection. We propose using microfluidic devices with an electrochemical (EC) detection system to determine the presence of DA, DA-6- N7Gua adduct, l-tyrosine, dihydroxyphenylalanine ( l-DOPA), and catechol (used as an internal standard). A PDMS glass-based, hybrid microfluidic device for free solution electrophoresis with totally integrated electrodes as well as improved and simplified gated injection was fabricated and tested. The best position and optimal width for minimizing background current of the working electrode was experimentally established. Using a single power supply simplified the operation of the microfluidic device and provided control of the plug size (length), leading to increased separation quality. It is shown that excellent separation of the above analytes can be accomplished in a relatively short time; the response is linear in the range between 2 and 300 μM, and reproducible with a limit of detection (LOD) for DA-6- N7Gua adducts in the sub-femtomole range. Thus, future separation and identification of various neurotransmitters and/or their products in human fluids could be accomplished using microfluidic devices with electrochemical detection.

A SU-8/PDMS Hybrid Microfluidic Device with Integrated Optical Fibers for Online Monitoring of Lactate

A microfluidic device with integrated optical fibres was developed for online monitoring of lactate. The device consists of a SU-8 waveguide, microfluidic channels and grooves for the insertion of optic fibres. It was fabricated by one-step photolithography of SU-8 polymer resist. Different channel widths (50-300 microm) were tested in terms of detection sensitivity. A wide range of flow rates were applied to investigate the influence of flow rate on signal fluctuations. The separation between optical fibre sensor and microfluidic channel and the width of fluidic channel have been optimized to maximize the detection sensitivity. It was revealed that 250 microm of channel width is the optimum light path length for a compromise between detection sensitivity and interference of ambient light. The independence of detection signals on flow rates was demonstrated within the range of flow rate (0.5-5 ml/hr) tested. Compared with conventional lactate detection, the device is proved to have high accuracy, relatively low limit of detection (50 mg/L) and reasonably fast response time (100 sec). The fabrication of device is simple and low cost. The present work has provided some fundamental data for further system optimization to meet specific detection requirements.

Field-effect flow control in a polydimethylsiloxane-based microfluidic system

The application of the field-effect for direct control of electroosmosis in a polydimethylsiloxane (PDMS)-based microfluidic system, constructed on a silicon wafer with a 2.0 microm electrically insulating layer of silicon dioxide, is demonstrated. This microfluidic system consists of a 2.0 cm open microchannel fabricated on a PDMS slab, which can reversibly adhere to the silicon wafer to form a hybrid microfluidic device. Aside from mechanically serving as a robust bottom substrate to seal the channel and support the microfluidic system, the silicon wafer is exploited to achieve field-effect flow control by grounding the semiconductive silicon medium. When an electric field is applied through the channel, a radial electric potential gradient is created across the silicon dioxide layer that allows for direct control of the zeta potential and the resulting electroosmotic flow (EOF). By configuring this microfluidic system with two power supplies at both ends of the microchannel, the applied electric potentials can be varied for manipulating the polarity and the magnitude of the radial electric potential gradient across the silicon dioxide layer. At the same time, the longitudinal potential gradient through the microchannel, which is used to induce EOF, is held constant. The results of EOF control in this hybrid microfluidic system are presented for phosphate buffer at pH 3 and pH 5. It is also demonstrated that EOF control can be performed at higher solution pH of 6 and 7.4 by modifying the silicon wafer surface with cetyltrimethylammonium bromide (CTAB) prior to assembly of the hybrid microfluidic system. Results of EOF control from this study are compared with those reported in the literature involving the use of other microfluidic devices under comparable solution conditions.