On-chip polymerase chain reaction microdevice employing a magnetic droplet-manipulation system

An on-chip polymerase chain reaction (PCR) device employing a magnetic beads-droplet-handling system was developed. Actuation with a magnet offers a simple system for droplet manipulation that allows separation and fusion of droplets containing magnetic beads by handling with a magnet. The device consists of a reaction chamber channel and two magnet-handling channels for the manipulation of micro-droplets containing magnetic beads. Micro-droplets were placed inside a reaction chamber filled with oil and manipulated with a magnet. When a droplet containing NaOH and magnetic beads was manipulated towards a droplet containing phenol red, a color change was observed after fusion. Sample preparation was performed by fusion of droplets containing a forward primer, reverse primer, template DNA and PCR mixture, using a droplet containing magnetic beads. PCR amplification or RT-PCR was also successfully performed, with efficiency comparable to manual methods that use this device by placing it on a thermal cycler for amplification. With a magnetic beads-manipulation step, purification of amplified DNA was also accomplished by using magnetic beads as the carrier. The amplified DNA was captured on streptavidin conjugated magnetic beads using a biotinylated primer, purified by washing and digested for separation of the target DNA.     

Driving and sorting of the fluorescent droplets on digital microfluidic platform

In this paper, we present a digital microfluidic droplet sorting platform to achieve automated droplet sorting based on fluorescent detection. We design and fabricate a kind of digital microfluidic chip for manipulating nano-liter-sized liquid droplets, and the chip is integrated with a fluorescence-initiated feedback system for real-time sorting control. The driving and sorting characteristics of fluorescent droplets encapsulating fluorescent-labeled particles are studied on this platform. The droplets dispensed from on-chip reservoir electrode are transported to a fluorescence detection site and sorted according to their fluorescence signals. The fluorescent droplets and non-fluorescent droplets are successfully separated and the number of fluorescent particles inside each droplet is quantified by its fluorescent intensity. We realize droplet sorting at 20 Hz and obtain a linear relationship between the fluorescent particle concentrations and the fluorescence signals. This work is easily adapted for sorting out fluorescent-labeled microparticles, cells and bacteria and thus has the potential of quantifying catalytic or regulatory bio-activities.     

Rapid label-free DNA analysis in picoliter microfluidic droplets using FRET probes

We report a novel microfluidic system that is capable of rapidly detecting DNA and its mutants in microfluidic droplets, in addition to elucidating the dynamic hybridization process. This microfluidic picoliter droplet analysis system is able to overcome the limitations of conventional analytical techniques that utilize immobilized sensing probes on a substrate. Molecular beacon (MB), a fluorescence resonance energy transfer (FRET) molecule, was used as the DNA sensing probe in picoliter droplets. The MB-DNA duplex formation process was analyzed by the change in FRET signal, which was acquired by the time-resolved method: converting distance traveled to hybridization time. This technique demonstrates the ability to detect presence of target nucleic acids within few seconds, multiplex DNA samples in microdroplet, and distinguish single nucleotide polymorphisms. It is promising for analyzing biomolecules or reactions, such as mRNA, cells, enzymatic activity, and protein folding whose analysis requires rapid mixing and small volume.     

One-step fabrication of titania hollow spheres by controlled interfacial reaction in a droplet-based microfluidic system

We present a facile ethanol-in-oil droplet-based microfluidic approach for one-step fabrication of titania hollow spheres through controlled interfacial reaction. The method combines microfluidic generation of uniform ethanol-in-oil droplets and subsequent in situ controlled interfacial reaction within the microfluidic channel. Ethanol-based droplets are suspended in an oil continuous phase containing titanium tertabutoxide. The small amount of water in the droplet phase diffuses to the interface leading to hydrolysis and condensation, and titania solidifies around the droplet forming titania microcapsules. The vigorous reaction between titanium tetrabutoxide and water is controlled by analyzing a mass transfer model, and then by selecting suitable continuous and dispersed phases. Highly viscous paraffin oil in combination with a low-viscosity ethanol-based droplet phase facilitates the successful formation of titania at the interface rather than in the continuous phase. This research provides a new approach for the controlled fabrication of titania microcapsules having uniform particle size and unique folded and crumpled structure.     

Production of uniform droplets using membrane, microchannel and microfluidic emulsification devices

This review provides an overview of major microengineering emulsification techniques for production of monodispersed droplets. The main emphasis has been put on membrane emulsification using Shirasu Porous Glass and microsieve membrane, microchannel emulsification using grooved-type and straight-through microchannel plates, microfluidic junctions and flow focusing microfluidic devices. Microfabrication methods for production of planar and 3D poly(dimethylsiloxane) devices, glass capillary microfluidic devices and single-crystal silicon microchannel array devices have been described including soft lithography, glass capillary pulling and microforging, hot embossing, anisotropic wet etching and deep reactive ion etching. In addition, fabrication methods for SPG and microseive membranes have been outlined, such as spinodal decomposition, reactive ion etching and ultraviolet LIGA (Lithography, Electroplating, and Moulding) process. The most widespread application of micromachined emulsification devices is in the synthesis of monodispersed particles and vesicles, such as polymeric particles, microgels, solid lipid particles, Janus particles, and functional vesicles (liposomes, polymersomes and colloidosomes). Glass capillary microfluidic devices are very suitable for production of core/shell drops of controllable shell thickness and multiple emulsions containing a controlled number of inner droplets and/or inner droplets of two or more distinct phases. Microchannel emulsification is a very promising technique for production of monodispersed droplets with droplet throughputs of up to 100?l?h^?1.     

Continuous generation of hydrogel beads and encapsulation of biological materials using a microfluidic droplet-merging channel

In this paper, we describe a method for encapsulation of biomaterials in hydrogel beads using a microfluidic droplet-merging channel. We devised a double T-junction in a microfluidic channel for alternate injection of aqueous fluids inside a droplet unit carried within immiscible oil. With this device, hydrogel beads with diameter <100 μm are produced, and various solutions containing cells, proteins and reagents for gelation could merge with the gel droplets with high efficiency in the broad range of flow rates. Mixing of reagents and reactions inside the hydrogel beads are continuously observed in a microchannel through a microscope. By enabling serial injection of each liquid with the dispersed gel droplets after they are produced from the oil-focusing channel, the device simplifies the sample preparation process, and gel-bead fabrication can be coupled with further assay continuously in a single channel. Instantaneous reactions of enzyme inside hydrogel and in-situ formation of cell-containing beads with high viability are demonstrated in this report.     

Fiber Bragg grating sensor for two-phase flow in microchannels

A new non-intrusive measurement technique for two-phase flow in microchannels is presented. The development of an evanescent field-based optical fiber Bragg grating (FBG) sensor is described, and experiments coupled with flow visualization demonstrating the performance of this sensor are presented. Two adjacent 1-mm FBGs in etched D-shaped fiber are embedded into the surface of a PDMS microchannel. Experiments are conducted in both droplet and slug flow regimes and high-speed digital video is captured synchronously with the sensor data. The FBGs exhibit an on?Coff type response to the passage droplets which is shown to correlate precisely with the passage of the liquid phase. This correlation enables the measurement of droplet average velocity and size using only the sensor data. In addition to the use of both FBG signals for the purpose of measuring droplet speed and size, it is shown that for droplets larger than the FBG length, a single FBG can be used to estimate the convection velocity and size of fast moving droplets. This sensing method is potentially useful for monitoring two-phase flow in fuel cells and microfluidic applications such as micro-heat exchangers and lab-on-a-chip systems.     

On-chip PCR amplification of genomic and viral templates in unprocessed whole blood

Performing medical diagnosis in microfluidic devices could scale down laboratory functions and reduce the cost for accessible healthcare. The ultimate goal of such devices is to receive a sample of blood, perform genetic amplification (polymerase chain reaction—PCR) and subsequently analyse the amplified products. DNA amplification is generally performed with DNA purified from blood, thus requiring on-chip implementation of DNA extraction steps with consequent increases in the complexity and cost of chip fabrication. Here, we demonstrate the use of unprocessed whole blood as a source of template for genomic or viral targets (human platelet antigen 1 (HPA1), fibroblast growth factor receptor 2 (FGFR2) and BK virus (BKV)) amplified by PCR on a three-layer microfluidic chip that uses a flexible membrane for pumping and valving. The method depends upon the use of a modified DNA polymerase (Phusion™). The volume of the whole blood used in microchip PCR chamber is 30 nl containing less than 1 ng of genomic DNA. For BKV on-chip whole blood PCR, about 3000 copies of BKV DNA were present in the chamber. The DNA detection method, laser-induced fluorescence, used in this article so far is not quantitative but rather qualitative providing a yes/no answer. The ability to perform clinical testing using whole blood, thereby eliminating the need for DNA extraction or sample preparation prior to PCR, will facilitate the development of microfluidic devices for inexpensive and faster clinical diagnostics.     

Numerical and experimental study of a droplet-based PCR chip

A two-temperature continuous-flow polymerase chain reaction (PCR) polymer chip has been constructed that takes advantage of droplet technology to avoid sample contamination and adsorption at the surface. Samples contained in aqueous droplets are continuously moved by an oil carrier-fluid through various temperature zones, introducing the possibility of real-time quantitative PCR. In the present paper, we investigate many of the factors affecting droplet-based PCR chip design, including thermal mass, flow rate, and thermal resistance. The study focuses particularly on the fluid and substrate temperature distribution within the PCR chip and the droplet residence times in critical temperature zones. The simulations demonstrate that the flow rate strongly affects the temperature field within the carrier-fluid. Above a critical flow rate, the carrier-fluid fails to achieve the required temperatures for DNA amplification. In addition, the thermal resistances of the different layers in the chip are shown to have a major impact on the temperature profile in the channel.     

Controlled dispensing and mixing of pico- to nanoliter volumes using on-demand droplet-based microfluidics

We present an integrated droplet-on-demand microfluidic platform for dispensing, mixing, incubating, extracting and analyzing by mass spectrometry pico- to nanoliter-sized droplets. All of the functional components are successfully integrated for the first time into a monolithic microdevice. Droplet generation is accomplished using computer-controlled pneumatic valves. Controlled actuation of valves for different aqueous streams enables accurate dosing and rapid mixing of reagents within droplets in either the droplet generation area or in a region of widening channel cross-section. Following incubation, which takes place as droplets travel in the oil stream, the droplet contents are extracted to an aqueous channel for subsequent ionization at an integrated nanoelectrospray emitter. Using the integrated platform, rapid enzymatic digestions of a model protein were carried out in droplets and detected online by nanoelectrospray ionization mass spectrometry.     

A compact microfluidic gradient generator using passive pumping

Creating and maintaining a precise molecular gradient which is stable in space and time are essential to studies of chemotaxis. This paper describes a simple, compact, and user-friendly microfluidic device using a passive pumping method to drive liquid flow to generate a stable concentration gradient. A fluidic circuit is designed to offset the effects of the pressure imbalance between the two inlets. After loading approximately the same amount of culture media containing different concentrations of a certain chemotactic agent into the two inlet reservoirs, a linear concentration gradient will be automatically and quickly established at the downstream. Our device takes advantage of passive pumping and is compact enough to fit into a Petri dish, which is an attractive feature to biologists. Furthermore, this microfluidic gradient generator offers a platform for a facile way of long-term imaging and analysis using high-resolution microscopy.     

A droplet-based microfluidic platform for rapid immobilization of quantum dots on individual magnetic microbeads

Quantum dots (QDs) provide opportunities for the development of bioassays, biosensors, and drug delivery strategies. Decoration of the surface of QDs offers unique functions such as resistance to non-specific adsorption, selective binding to target molecules, and cellular uptake. The quality of decoration has substantial impact on the functionality of modified QDs. Single-phase microfluidic devices have been demonstrated for decorating QDs with biological molecules. The device substrate can serve as a solid-phase reaction platform, with a limitation being difficulty in the realization of reproducible decoration at high density of coverage of QDs. Magnetic beads (MBs) have been explored as an alternative form of solid-phase reaction platform for decorating QDs. As one example, controlled decoration to achieve unusually high density can be realized by first coating MBs with QDs, followed by the addition of molecules such as DNA oligonucleotides. Uniformity and high density of coatings on QDs have been obtained using MBs for solid-phase reactions in bulk solution, with the further advantage that the MBs offer simplification of procedural steps such as purification. This study explores the use of a droplet microfluidic platform to achieve solid-phase decoration of MBs with QDs, offering control of local reaction conditions beyond that available in bulk solution reactions. A microchannel network with a two-junction in-series configuration was designed and optimized to co-encapsulate one single 1 µm MB and many QDs into individual droplets. The microdroplet became the reaction vessel, and enhanced conjugation through the confined environment and fast mixing. A high density of QDs was coated onto the surface of single MB even when using a low concentration of QDs. This approach quickly produced decorated MBs, and significantly reduced QD waste, ameliorating the need to remove excess QDs. The methodology offers a degree of precision to control conjugation processes that cannot be attained in bulk synthesis methods. The proposed droplet microfluidic design can be widely adopted for nanomaterial synthesis using solid-phase assays.     

Integrated liquid and droplet dielectrophoresis for biochemical assays

Surface microfluidic systems have emerged as an attractive alternative to conventional closed-channel microfluidic devices. In many such systems, electric fields are leveraged for the manipulation and transport of discrete nanoliter droplets on open planar surfaces. The present research work discusses dielectrophoretic liquid and droplet actuations, which provide an attractive methodology for dispensing and manipulating nanoliter and picoliter droplets on planar surfaces. We demonstrate the integration of two independent sample actuation schemes, namely liquid dielectrophoresis (L-DEP) and droplet dielectrophoresis, and furthermore validate its applicability through model biochemical assays (DNA-PicoGreen^® assay and DNA FRET assay). We also describe and present ‘tapering L-DEP’ actuation scheme, whereby we demonstrate how to simultaneously create multiple droplets of different sizes and volumes in the range of nanoliter and picoliters, from a given larger parent sample droplet.     

Optical separation of droplets on a microfluidic platform

This paper describes the optical separation of microdroplets according to their refractive indices. The behavior of the droplets was characterized in terms of the optical force and the hydrodynamic effects present upon illumination of the droplets in a direction normal to the flow direction in a rectangular microfluidic channel. The optical forces acting on the droplets and the resultant droplet trajectories were analyzed and compared with the numerically predicted values. The relationship between the drag force and optical force was examined to understand the system performance properties in the context of screening applications involving the removal of unwanted droplets. Two species of droplets were compared for their photophoretic displacements by varying the illumination intensity. Because the optical forces exerted on the droplets were functions of the refractive indices and sizes of the droplets, a variety of chemical species could be separated simultaneously.     

Non-destructive on-chip imaging flow cell-sorting system for on-chip cellomics

We have developed a non-destructive imaging flow cell-sorting system using an ultra-high-speed camera (shutter speed of 1/10,000 s) with a real-time image analysis unit and a poly(methyl methacrylate) (PMMA)-based disposable microfluidic chip for single-cell-based on-chip cellomics. It has a 3-D micropipetting device that supports fully automated sorting and collection of samples. The entire fluidic system is implemented in a disposable plastic chip, enabling biological samples to be lined up in a laminar flow using hydrodynamic focusing. Its optical system enables direct observation-based cell identification using specific image indexes and phase-contrast/fluorescence microscopy, real-time image processing. It has a non-destructive, wider dynamic range, sorting procedure using mild electrostatic force in a laminar flow; agarose gel electrodes are used to prevent electrode loss and electrolysis bubble formation. The microreservoir used for recultivating collected target cells is contamination-free. An integrated ultra-high-speed droplet polymerase chain reaction measurement module is used for DNA/mRNA analysis of the collected target cells. This system was used to separate cardiomyocyte cells from a mixture of various cells. All the operations were automated using the 3-D micropipetting device. The results demonstrate that this imaging flow cell-sorting system is practically applicable for biological research and clinical diagnosis.     

MCU based real-time temperature control system for universal microfluidic PCR chip

The analysis of genetic materials in the post-human genome project era has become an ever-expanding branch of research and thus routinely employed in majority of biochemical laboratories. Most of the diagnostic research area emphasizes on polymerase chain reaction for detecting pathogenic organisms. However, the conventional polymerase chain reaction requires expensive and sophisticated thermal cycler and is not handy owing to its large dimensions. Therefore, we fabricated a continuous-flow polymerase chain reaction chip on a PDMS based microfluidic platform to ease the hardship of the conventional system. Temperature being the most crucial factor in polymerase chain reaction, was monitored and regulated by thermostatic action using an on-line computer system. Indium tin oxide coated glass platform was used for heating as it is transparent and has good thermal conductivity under the influence of DC bias. The heating circuit used an ATMega 128 MCU to control the temperature. As a result, a precise and quick heating environment was maintained on the microfluidic chip to amplify the target DNA. We successfully amplified Lambda phage and Escherichia coli DNA on our chip to prove the practicality of the device.     

Bead-based polymerase chain reaction on a microchip

We present a bead-based approach to microfluidic polymerase chain reaction (PCR), enabling fluorescent detection and sample conditioning in a single microchamber. Bead-based PCR, while not extensively investigated in microchip format, has been used in a variety of bioanalytical applications in recent years. We leverage the ability of bead-based PCR to accumulate fluorescent labels following DNA amplification to explore a novel DNA detection scheme on a microchip. The microchip uses an integrated microheater and temperature sensor for rapid control of thermal cycling temperatures, while the sample is held in a microchamber fabricated from (poly)dimethylsiloxane and coated with Parylene. The effects of key bead-based PCR parameters, including annealing temperature and concentration of microbeads in the reaction mixture, are studied to achieve optimized device sensitivity and detection time. The device is capable of detecting a synthetically prepared section of the Bordetella pertussis genome in as few as 10 temperature cycles with times as short as 15?min. We then demonstrate the use of the procedure in an integrated device; capturing, amplifying, detecting, and purifying template DNA in a single microfluidic chamber. These results show that this method is an effective method of DNA detection which is easily integrated in a microfluidic device to perform additional steps such as sample pre-conditioning.     

Thin-film electrode based droplet detection for microfluidic systems

We report on a droplet-producing microfluidic system with electrical impedance-based detection. The microfluidic devices are made of polydimethylsiloxane (PDMS) and glass with thin film electrodes connected to an impedance-monitoring circuit. Immiscible fluids containing the hydrophobic and hydrophilic phases are injected with syringe pumps and spontaneously break into water-in-oil droplet trains. When a droplet passes between a pair of electrodes in a medium having different electrical conductivity, the resulting impedance change signals the presence of the particle for closed-loop feedback during processing. The circuit produces a digital pulse for input into a computer control system. The droplet detector allows estimation of a droplet's arrival time at the microfluidic chip outlet for dispensing applications. Droplet detection is required in applications that count, sort, and direct microfluidic droplets. Because of their low cost and simplicity, microelectrode-based droplet detection techniques should find applications in digital microfluidics and in three-dimensional printing technology for rapid prototyping and biotechnology.     

Magnetic liquid marbles, their manipulation and application in optical probing

Magnetic liquid marbles, an encapsulation of liquid droplet with hydrophobic magnetic particles, show remarkable responsiveness to external magnetic force and great potential to be used as a discrete droplet microfluidic system. In this study, we presented the manipulation of a magnetic liquid marble under an external magnetic field and calculated the maximum frictional force, the magnetic force required for actuating the liquid marbles and the effective surface tension of the magnetic liquid marble, as well as the threshold volume for the transition from quasi-spherical to puddle-like shape. By taking advantage of the unique feature of being opened and closed reversibly, we have proven the encapsulated droplets can be detected optically with a reflection-mode probe. Combining the open-close and optical detection also enables to probe chemical reactions taking place within liquid marbles. These remarkable features offer a simple yet powerful alternative to conventional discrete microfluidic systems and may have wide applications in biomedical and drug discovery.     

On-Demand DNA Synthesis on Solid Surface by Four Directional Ejectors on a Chip

This paper presents a synthesis technique for any random deoxyribonucleic acid (DNA) sequences on different substrates such as glass, plastic or silicon by an array of directional droplet ejectors. Any DNA sequence can be synthesized by ejecting droplets of DNA bases by an ultrasonic transducer having lens with air-reflectors (LWARs) that requires no nozzle. The LWAR is capable of ejecting liquid droplets around 80 mum in diameter, and reduces the amount of reagents needed for the synthesis from most of conventional microarray techniques. One major advantage of the nozzleless ejector is that it can eject droplets in any direction, so that a spot can be inked by four ejectors (carrying four DNA bases) without moving the ejector. The directional ejection of the droplets removes the need for aligning the substrate with the ejector, and minimizes the automation and control circuitry. To demonstrate the DNA synthesis capability of the directional droplet ejectors, four LWAR ejectors were used to synthesize a 15-mer 5'-CGCCAAGCAGTTCGT-3' on a substrate surface. This paper describes the concept and scheme of the on-demand DNA synthesis (with MEMS ejector integrated with microfluidic components) along with experimental results of an actual DNA synthesis by four directional droplet ejectors.