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A mass manufacturable thermoplastic based microfluidic droplet generator on Cyclic Olefin Copolymer (COC)
Abstract
The rapid progress of droplet microfluidics and its wide range of applications have created a high demand for the mass fabrication of low-cost, high throughput droplet generator chips aiding both biomedical research and commercial usage. Existing polymer or glass based droplet generators have failed to successfully meet this demand which generates the need for the development of an alternate prototyping technique.This work reports the design, fabrication and characterization of a mass manufacturable thermoplastic based microfluidic droplet generator on Cyclic Olefin Copolymer (COC). COC chips with feature size as low as 20 μm have been efficiently fabricated using injection molding technology leading to a high production of inexpensive droplet generators. The novelty of this work lies in reoptimising surface treatment and solvent bonding methods to produce closed COC microchannels with sufficiently hydrophobic (contact angle of 120º) surfaces. These COC based droplet generators were shown to generate stable monodisperse droplets at a rate of 1300 droplets/second in the dripping regime. These new mass manufacturable, disposable and cheap COC droplet generators can be custom designed to cater to the rapidly increasing biomedical and clinical applications of droplet microfluidics.
... One example of the replication method is microinjection molding which involves mold design and fabrication, and molten COC injection against the fixed mold. This technique has been used for various microfluidic applications, for instance, Kim et al. [8] fabricated a 3D serpentine passive micromixer by injection molding of two COC substrates which were then aligned and bonded, and Ghosh et al. [9], fabricated a COC flow focusing microfluidic channel to generate water in oil droplets. Another form of the replication method is hot embossing, which also involves mold design and fabrication, however, in this method COC substrates are only heated to a temperature between their glass transition temperature and melting point and then pressed with the mold to create the microfluidic cavity [10]. ...
... For example, Jena et al. [19] bonded a 1 mm thick COC microchannel with a 1 mm thick COC substrate at a temperature of 125 • C and a 500 N load. On the other hand, solvent bonding relies on the solubility of COC in certain nonpolar organic solvents to create molecular entanglement of polymer chains across the interface, such solvents include cyclohexane [9,[20][21][22], decalin [23], toluene [24] and a mixture of cyclohexane and hexadecane [25]. Keller et al. [22] demonstrated solvent bonding by gently pressing a COC microchannel into a filter paper soaked with 35% cyclohexane/acetone solution followed by conformal contact with another COC substrate. ...
... Numerical simulations show that micro-droplets with different sizes and shapes can be prepared by changing the microchannel geometry and flow parameters. Ghosh et al. successfully achieved low-cost mass production of microfluidic droplet chips on thermoplastic polymers and stably generated droplets in flow-focusing microchannels (Ghosh et al. 2019). After re-optimizing the surface treatment of the COC chip, it can achieve the same effect as the microdroplets generated in the PDMS chip. ...
Gas-liquid and liquid-liquid two-phase flow are widely used in chemical engineering, biomedical engineering and other fields such as separation, reaction, and mass transfer in microfluidic systems. Studying the formation methods of droplets and bubbles in microfluidics is of great significance to the application of microchemical technology. In this review, according to the methods of droplets and bubbles formation, the research progress and development trend of droplets and bubbles formation in microfluidics in recent years are reviewed. Formation methods are divided into passive methods and active methods according to whether external energy is required. Passive methods include T-junction, flow-focusing, co-flowing and step emulsification. Active methods include surface acoustic waves, DC/AC electric fields, magnetic fields, and thermal fields. Finally, this review points out the future direction of research on liquid droplets and bubbles. This review sheds new light on monodisperses, highly controllable droplets and bubbles formation and its applications.
... However, the TOPAS E140 COC grade is a semicrystalline elastomer typically having a glass transition temperature (T g ) between − 10 • C and 15 • C and a crystallinity degree (X c ) from 5 to 40% due to its high PE content [12]. Although amorphous TOPAS grades have been used for several microfluidic applications, such as, Microvalves [13], Droplet generators [14] or Organic semiconductors and electrodes [15,16], little literature of the use of TOPAS E140 COC for microfluidics based application has been reported. Although being semicrystalline, the high transparency presented by this material in the visible and near UV regions, over 85%, makes it attractive for optical analysis methods [9]. ...
Cyclic Olefin Copolymer (COC) based on norbornene and ethylene is known to be one of the most promising materials for the fabrication of low-cost miniaturized Point-of-care (PoC) devices. Although semicrystalline materials are chemically more resistant and less brittle than amorphous ones, little literature has been reported on the unique semicrystalline COC (c-COC) grade. In this work, a thorough analysis of the crystallization behaviour of a semicrystalline COC (c-COC) is reported for the first time to better understand the relationship between the crystallization and injection moulding processing parameters of the material for the mass manufacturing of PoC devices. The complete thermomechanical characterization carried out by dynamic mechanical analysis (DMA) and differential scanning calorimetry (DSC) for the analysis of the crystallization behaviour suggested that even though a bimodal melting was detected by calorimetry, a unique crystalline structure was detected by X-ray scattering patterns confirming that the crystalline structure generated during the injection moulding of PoC parts is always the same regardless of processing conditions. In non-isothermal crystallization kinetics, the Ozawa model failed describing the crystallization behaviour of the c- COC whereas the combined Avrami-Ozawa and Avrami models successfully described the crystallization behaviour showing an excellent fitting to experimental values and suggesting a three-dimensional growth of nucleus for non-isothermal crystallization.
... Ghosh et al. exposed the thermoplastic chips to the mixture of CF 4 and O 2 for plasma treatment to have a hydrophobic surface. 13 However, this treatment is expensive and difficult to scale up. Meanwhile, treating the thin surface layer without changing the bulk properties of the thermoplastics is also a challenge for plasma treatment. ...
A hydrophobic surface modification followed by solvent vapor-assisted thermal bonding was developed for the fabrication of cyclic olefin copolymer (COC) microfluidic chips. The modifier species 1H,1H,2H,2H-perfluorooctyl trichlorosilane (FOTS) was used to achieve the entrapment functionalization on the COC surface, and a hydrophobic surface was developed through the formation of a Si-O-Si crosslink network. The COC surface coated with 40 vol % cyclohexane, 59 vol % acetone, and 1 vol % FOTS by ultrasonic spray 10 and 20 times maintained its hydrophobicity with the water contact angle increasing from ∼86 to ∼115° after storage for 3 weeks. The solvent vapor-assisted thermal bonding was optimized to achieve high bond strength and good channel integrity. The results revealed that the COC chips exposed to 60 vol % cyclohexane and 40 vol % acetone for 120 s have the highest bond strength, with a burst pressure of ∼17 bar, which is sufficient for microfluidics applications such as droplet generation. After bonding, the channel maintained its integrity without any channel collapse. The hydrophobicity was also maintained, proved by the water contact angle of ∼115° on the bonded film, as well as the curved shape of water flow in the chip channel by capillary test. The combined hydrophobic treatment and solvent bonding process show significant benefits for scale-up production compared to conventional hydrophilic treatment for bonding and hydrophobic treatment using surface grafting or chemical vapor deposition since it does not require nasty chemistry, long-term treatment, vacuum chamber, and can be integrated into production line easily. Such a process can also be extended to permanent hydrophilic treatment combined with the bonding process and will lay a foundation for low-cost mass production of plastic microfluidic cartridges.
... Among different droplet generator designs, T-junction (Glawdel et al. 2012;Loizou et al. 2013;Zeng et al. 2015;Charlot et al. 2015;Fan et al. 2019), flow-focusing Ghosh et al. 2019;Wu et al. 2018;Bai et al. 2017;Li 2008;Gallah et al. 2017), and co-flow (Zhu et al. 2016;Shams Khorrami and Rezai 2018;Wu et al. 2017;Lian et al. 2019) are the three most common. The T-junction design has been widely used due to its simplicity. ...
This paper presents a T-junction microdroplet generator equipped with a pneumatic actuation for controlling the droplet size. The multi-layer device is compatible with rapid manufacturing using a desktop-based laser cutter to fabricate the fluidic and pneumatic layers. A T-junction fluidic layer sits at the bottom and the control layer consists of a pneumatic chamber, as well as inlet and outlet channels are placed at the top. Our results with pneumatic control showed that the generated droplet size is inversely proportional to the pressure inside the pneumatic chamber. Pneumatic pressure of up to 0.4 MPa showed a 38% reduction in droplet size compared to without control. The droplet size can additionally be regulated by controlling the relative flow rates of the continuous and dispersed fluid. Pneumatic actuation is advantageous, as it does not require additional fluid usage and the flow rate does not need to be ramped up to decrease the droplet size. The device presented in this paper with pneumatic control and fabrication ease provides an attractive method for microdroplet manipulation.
... The vent connected to the substrate path was rendered hydrophobic by adding a drop of hydrophobic coating (A10, Joninn, Denmark) in the circular pad. Finally, the chip was sealed using solvent bonding 58,59 . COC films of 110 μm thickness (8007S-04, Topas, USA) were used for the bonding purpose. ...
There has been a considerable development in microfluidic based immunodiagnostics over the past few years which has greatly favored the growth of novel point-of-care-testing (POCT). However, the realization of an inexpensive, low-power POCT needs cheap and disposable microfluidic devices that can perform autonomously with minimum user intervention. This work, for the first time, reports the development of a new microchannel capillary flow assay (MCFA) platform that can perform chemiluminescence based ELISA with lyophilized chemiluminescent reagents. This new MCFA platform exploits the ultra-high sensitivity of chemiluminescent detection while eliminating the shortcomings associated with liquid reagent handling, control of assay sequence and user intervention. The functionally designed microchannels along with adequate hydrophilicity produce a sequential flow of assay reagents and autonomously performs the ultra-high sensitive chemiluminescence based ELISA for the detection of malaria biomarker such as PfHRP2. The MCFA platform with no external flow control and simple chemiluminescence detection can easily communicate with smartphone via USB-OTG port using a custom-designed optical detector. The use of the smartphone for display, data transfer, storage and analysis, as well as the source of power allows the development of a smartphone based POCT analyzer for disease diagnostics. This paper reports a limit of detection (LOD) of 8 ng/mL by the smartphone analyzer which is sensitive enough to detect active malarial infection. The MCFA platform developed with the smartphone analyzer can be easily customized for different biomarkers, so a hand-held POCT for various infectious diseases can be envisaged with full networking capability at low cost.
... In this work, an active microfluidic tracker using infrared sensor unit (ISU) has been developed and fully characterized for precisely tracking reagents flowing through polymer microchannels. This new microfluidic monitoring method can be used for the development of a functional microfluidic tracker for monitoring and characterizing droplets in microfluidic droplet generators [5], cells in cell counters, as well as liquid columns in immunoassay LOCs. The simple, low-cost ISU based microfluidic tracker can be easily integrated with any lab-ona-chips, cell-on-a-chips or POCTs which require precision control of moving cells or fluid columns. ...
A new microfluidic flow monitoring method has been developed using infrared sensors, and then fully implemented and characterized over an immunoassay lab-on-a-chip (LOC) which needs precise monitoring and control for a sequential flow of reagents. This new microfluidic monitoring method can be applied for the development of a functional microfluidic tracker for counting and characterizing droplets in a microfluidic droplet generator and digital PCR, cells in a cell counter, or reagent columns in an immunoassay LOC.
Cyclic olefin copolymers (COC) are amorphous, transparent thermoplastics composed of cyclic olefin monomers (norbornene) and linear olefins (ethene). They are increasingly utilized as fabrication materials for microsystems and microfluidic devices. This is owing to their promising features of low water absorption, high electrical insulation, long‐term stability of surface treatments, and resistance to a broad variety of acids and solvents. Many manufacturing processes for COC‐based devices have been developed in recent decades. These methodologies are categorized as replication methods or fast prototyping as common in fabrication of thermoplastic microfluidic devices. This paper gives a full discussion of the features of COCs, the various production processes, and the numerous selected applications in microfluidic platforms reported in the literature. The review explores COC's composition and fundamental features, as well as fabrication processes and applications in a variety of fields, investigates the material's potential advantages and uses, and attempts to create a comprehensive list of COC's possible benefits. Due to their unique features and simplicity of fabrication, COCs are projected to advance the future of microfluidics, microsystems, and optofluidics. This article is protected by copyright. All rights reserved
Lab-on-chip (LOC) is recognised as one of the most affordable solutions for integrating electronics and fluidics devices. In this field, bonding plays a vital role because it provides the means for attaching multiple components onto a substrate, transforming them into a microfluidic circuit. Bonding is an integral step, especially when designing a device that is free from leakage and eventual clogging. A comprehensive review of the latest irreversible bonding technologies is discussed in this paper, in which the focus is on the layered microfluidic systems with large sensor arrays. This review covers microfluidic devices fabricated from a rigid-type glass-fibre-printed circuit board and a thermoplastic flexible printed circuit with 186 references whose development date back three decades ago. The bonding techniques are organised into the following four groups: (a) adhesive bonding, (b) thermal and solvent bonding, (c) surface modification and dry bonding and (d) photoresist groups. Other techniques are available beyond these groupings, but they can be classified into the nearest group to facilitate the discussion. This paper will benefit researchers and practitioners aiming to develop polymer-based LOC devices.
Personalizing health care by taking genetic, environmental, and lifestyle factors into account is central to modern medicine. The crucial and pervasive roles epigenetic factors play in shaping gene−environment interactions are now well recognized. However, identifying robust epigenetic biomarkers and translating them to clinical tests has been difficult due in part to limitations of available platforms to detect epigenetic features genome-wide (epigenomic assays). This Feature introduces several important prospects for precision epigenomics, highlights capabilities and limitations of current laboratory technologies, and emphasizes opportunities for microfluidic tools to facilitate translation of epigenetic analyses to the clinic, with a particular focus on methods to profile gene-associated histone modifications and their impacts on chromatin structure and gene expression.
This article presents a simple method to pattern the surface energy of a substrate using standard microfabrication techniques. Microfluidic devices with hydrophobic polymer substrate patterned with hydrophilic graphene oxide (GO) for the separation of liquid-liquid two-phase systems are reported. The microdevice consists of a Cyclic Olefin Copolymer (COC) substrate bonded to a Polydimethylsiloxane (PDMS) element that includes a microchannel. The patterned GO film is characterized using scanning electron, atomic force, and metallurgical microscopies. The contact angle of water on COC surface is measured to be more than 120° and is approximately 10° on the GO-patterned COC surface. Different contact angles could be achieved by tuning the GO with oxygen containing functional groups as well as using different concentrations of GO dispersions. The device is used to separate dye-water droplets dispersed in silicone oil by steering the water droplet toward a designated outlet and achieve separation. Using GO as a patterned thin film with tunable hydrophilicity allows for on-chip continuous full separation of water droplets at different flow rates.
Microfluidics is a growing field of study because these devices offer novel and versatile approaches for addressing a range of scientific problems. Microfluidics have been used in separations, cell analysis, and microreactors, to illustrate a few applications. Microfluidics have been defined in terms of microliter volumes or micrometer dimensions of channels. For this review, we utilize the latter definition, where microfluidic channels range from 1-1000 µm in width or height. Microfluidic devices are fabricated in a host of ways, with a large variety of materials. Indeed, device material can affect flow, absorptivity, biocompatibility, and function of microfluidic components.
Using polymer materials to fabricate microfluidic devices provides simple, cost effective, and disposal advantages for both lab-on-a-chip (LOC) devices and micro total analysis systems (μTAS). Polydimethylsiloxane (PDMS) elastomer and thermoplastics are the two major polymer materials used in microfluidics. The fabrication of PDMS and thermoplastic microfluidic device can be categorized as front-end polymer microchannel fabrication and post-end microfluidic bonding procedures, respectively. PDMS and thermoplastic materials each have unique advantages and their use is indispensable in polymer microfluidics. Therefore, the proper selection of polymer microfabrication is necessary for the successful application of microfluidics. In this paper, we give a short overview of polymer microfabrication methods for microfluidics and discuss current challenges and future opportunities for research in polymer microfluidics fabrication. We summarize standard approaches, as well as state-of-art polymer microfluidic fabrication methods. Currently, the polymer microfluidic device is at the stage of technology transition from research labs to commercial production. Thus, critical consideration is also required with respect to the commercialization aspects of fabricating polymer microfluidics. This article provides easy-to-understand illustrations and targets to assist the research community in selecting proper polymer microfabrication strategies in microfluidics.
Cyclic olefin copolymer (COC) is widely used in microfluidics due to its UV-transparency, its biocompatibility and high chemical resistance. Here we present a fast and cost-effective solvent bonding technique, which allows for the efficient bonding of protein-patterned COC structures. The bonding process is carried out at room temperature and takes less than three minutes. Enzyme activity is retained upon bonding and microstructure deformation does not occur.
Droplet-based microfluidics enables assays to be carried out at very high throughput (up to thousands of samples per second) and enables researchers to work with very limited material, such as primary cells, patient's biopsies or expensive reagents. An additional strength of the technology is the possibility to perform large-scale genotypic or phenotypic screens at the single-cell level. Here we critically review the latest developments in antibody screening, drug discovery and highly multiplexed genomic applications such as targeted genetic workflows, single-cell RNAseq and single-cell ChIPseq. Starting with a comprehensive introduction for non-experts, we pinpoint current limitations, analyze how they might be overcome and give an outlook on exciting future applications.
We report on a novel microfluidic strategy for the continuous fabrication of monodisperse asymmetric vesicles with customized membrane composition, size, and luminal content. The microfluidic device encompasses a triangular post region and two flow-focusing regions. The major steps involved in the vesicle fabrication process include: (1) forming highly uniform water emulsions in an oil/inner-leaflet-lipid solution, (2) replacing the inner-leaflet-lipid solution with an outer-leaflet-lipid solution inside the microchannel network, (3) forming water-in-oil-in-water double emulsions, and (4) extracting excess oil/outer-leaflet-lipid solution from the double emulsions. Bilayer membrane asymmetry and unilamellarity are evaluated using a fluorescence quenching assay and a transmembrane protein insertion assay, respectively. Our approach addresses many of the deficiencies found in existing technologies for building vesicles, and yields strong membrane asymmetry. The ability to create and sustain membrane asymmetry is an important feature, as it is a characteristic of nearly all natural membranes. Over 80% of the vesicles remain stable for at least 6 weeks and the membrane asymmetry is maintained for over 30 hours. The asymmetric vesicles built using this strategy are collected off-chip and hold the potential to be used as model systems in membrane biology or as vehicles for drug delivery.
Recent advances in microfluidics to generate and control picoliter emulsions of water in oil have enabled ultra-sensitive assays for small molecules, proteins, nucleic acids, and cells. Unfortunately, the conventional fluorescence detection used to measure the outcome of these droplet-based assays has not proven suited to match the time and space multiplexing capabilities of microfluidic systems. To address this challenge, we developed an in-flow fluorescence detection platform that enables multiple streams of droplets to be monitored using only a single photodetector and no lenses. The key innovation of our technology is the amplitude modulation of the signal from fluorescent droplets using distinct micro-patterned masks for each channel. By taking advantage of the high bandwidth of electronics, our technique enables the velocity-independent recovery of weak fluorescent signals (SNR << 1) using only simple hardware, obviating the need for lasers, bulky detectors, and complex fluid control. We demonstrated a handheld-sized device that simultaneously monitors four independent channels with the capability to be scaled-up to more than sixteen, limited primarily by the droplet density.
Microfluidic emulsification yields droplets with extremely narrow size distribution, multiple emulsions with a precisely controlled number of inner droplets, and higher order emulsions ranging from double to quintuple emulsions. However, production rates are inherently small and scale-up can be achieved by massive parallelization only. Can the control that microfluidic emulsification offers for drop production nevertheless be used for the production of materials? What will it take to develop it into an industrial process? Many obstacles need to be addressed ranging from technological, and commercial issues to a fragmented and confusing conference and supplier landscape. This contribution gives a large chemical industry's perspective on formulating materials in massively parallelized microfluidic processes.
We fabricated phospholipid liposomes from double emulsion templates produced in a microfluidic 3D flow focusing PDMS droplet generator. We combined the easing of wetting criteria due to 3D flow focusing geometry with a highly effective hydrophilic surface modification by corona discharge for a spatially patterned droplet generator, allowing the reliable generation of double emulsions in water and oleic acid without added surfactants. By controlling the Capillary numbers of the immiscible flows, we defined regions for the stable generation of double emulsions which were transformed into liposomes after solvent extraction.
A microfluidic device has been used to create novel monodisperse polymeric oil-in-water emulsions of diameter 15–100 µm, stabilised by surfactants and polymers. The pulse field gradient nuclear magnetic resonance (PFG-NMR) signal attenuation function showed minima that allow a simple calculation of the droplet size and polydispersity. The presence of the minima is due to the restricted diffusion of molecules within the droplets, which can be analysed using standard solutions of the diffusion equation with a spherical boundary condition. However, even when a small population of polymer is unrestricted, the deep troughs in the attenuation function are obscured. The simultaneous water NMR attenuation function gives structural information about the continuous-phase matrix. The NMR size measurements were compared with those obtained by optical and confocal microscopy, and laser diffraction analysis.
Microfluidic technologies have emerged recently as a promising new route for the fabrication of uniform emulsions. In this paper, we review microfluidic methods for synthesizing uniform streams of droplets and bubbles, focusing on those that utilize pressure-driven flows. Three categories of microfluidic geometries are discussed, including co-flowing streams, cross-flowing streams, and flow focusing devices. In each category we summarize observations that have been reported to date in experiments and numerical simulations. We describe these results in the context of physical mechanisms for droplet breakup, and simple theoretical models that have been proposed. Applications of droplets in microfluidic devices are briefly reviewed.
This paper present a method of rapid replication of polymeric high aspect ratio microstructures (HARMs) and a method of rapid
reproduction of metallic micromold inserts for HARMs using polydimethylsiloxane (PDMS) casting and standard LIGA processes.
A high aspect ratio (HAR) metallic micromold insert, featuring a variety of test microstructures made of electroplated nickel
with 15:1 height-to-width ratio for 300 μm microstructures, was fabricated by the standard LIGA process using deep X-ray lithography
(DXRL). A 10:1 mixture of pre-polymer PDMS and a curing agent were cast onto the HAR metallic micromold insert, cured and
peeled off to create reverse images of the HAR metallic micromold insert in PDMS. In addition to the replication of polymeric
HARMs, replicated PDMS HARMS were coated with a metallic sacrificial layer and electroplated in nickel to reproduce another
metallic micromold insert. This method can be used to rapidly and massively reproduce HAR metallic micromold inserts in low
cost mass production manner without further using DXRL.
Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.
We present a tunable three-dimensional (3D) self-assembled droplet packing method to achieve high-density micro-reactor arrays for greater imaging efficiency and higher-throughput chemical and biological assays. We demonstrate the capability of this platform's high-density imaging method by performing single molecule quantification using digital polymerase chain reaction, or digital PCR, in multiple self-assembled colloid-like crystal lattice configurations. By controlling chamber height to droplet diameter ratios we predictively control three-dimensional packing configurations with varying degrees of droplet overlap to increase droplet density and imaging sensor area coverage efficiency. Fluorescence imaging of the densely packed 3D reactor arrays, up to three layers high, demonstrates high throughput quantitative analysis of single-molecule reactions. Now a greater number of microreactors can be observed and studied in a single picture frame without the need for confocal imaging, slide scanners, or complicated image processing techniques. Compared to 2D designs, tunable 3D reactor arrays yield up to a threefold increase in density and use 100% of the sensor's imaging area to enable simultaneous imaging a larger number of reactions without sacrificing digital quantification performance. This novel approach provides an important advancement for ultra-high-density reactor arrays.
Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented.
The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify approximately 100 variants comparable in activity to the parent from an initial population of approximately 10(7). After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen approximately 10(8) individual enzyme reactions in only 10 h, using < 150 microL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.
We present a high throughput microfluidic device for continuous-flow polymerase chain reaction (PCR) in water-in-oil droplets of nanoliter volumes. The circular design of this device allows droplets to pass through alternating temperature zones and complete 34 cycles of PCR in only 17 min, avoiding temperature cycling of the entire device. The temperatures for the applied two-temperature PCR protocol can be adjusted according to requirements of template and primers. These temperatures were determined with fluorescence lifetime imaging (FLIM) inside the droplets, exploiting the temperature-dependent fluorescence lifetime of rhodamine B. The successful amplification of an 85 base-pair long template from four different start concentrations was demonstrated. Analysis of the product by gel-electrophoresis, sequencing, and real-time PCR showed that the amplification is specific and the amplification factors of up to 5 x 10(6)-fold are comparable to amplification factors obtained in a benchtop PCR machine. The high efficiency allows amplification from a single molecule of DNA per droplet. This device holds promise for convenient integration with other microfluidic devices and adds a critical missing component to the laboratory-on-a-chip toolkit.
An innovative polymer lab-on-a-chip (LOC) for reverse transcription (RT)-polymerase chain reaction (PCR) has been designed, fabricated, and characterized for point-of-care testing (POCT) clinical diagnostics. In addition, a portable analyzer that consists of a non-contact infrared (IR) based temperature control system for RT-PCR process and an optical detection system for on-chip detection, has also been developed and used to monitor the RT-PCR LOC. The newly developed LOC and analyzer have been interfaced and optimized for performing RT-PCR procedures and chemiluminescence assays in sequence. As a clinical diagnostic application, human immunodeficiency virus (HIV) for the early diagnosis of acquired immune deficiency syndrome (AIDS) has been successfully detected and analyzed using the newly developed LOC and analyzer, where the primer sets for p24 and gp120 were used as the makers for HIV. The developed polymer LOC and analyzer for RT-PCR can be used for POCT for the analysis of HIV with the on-chip RT-PCR and chemiluminescence assays in shorter than one hour with minimized cross-contamination.
Plastic materials have the potential to substitute for glass substrates used in microfluidic and microTAS systems adding flexibility in materials' choices. Optical quality plastic materials with a low autofluorescence are crucial for optimal detection by fluorescence and laser induced fluorescence techniques. This paper summarizes a series of optical investigations on commercially available plastic chip materials (PMMA, COC, PC, PDMS) and chips made from those materials. Intrinsic optical constants of plastic materials-refractive index for bulk materials-determined by spectroscopic ellipsometry and transmission spectroscopy in the visible range are presented. The laser-induced autofluorescence of materials and chips was assessed at four laser wavelengths, namely, 403, 488, 532 and 633 nm. Considerable bleaching of the autofluorescence was observed under continuous laser illumination. Overall, the longer wavelength laser excitation sources yielded less autofluorescence. PDMS exhibited the least autofluorescence and was comparable to BoroFloat glass. In all cases, chips exhibited slightly higher autofluorescence than the raw plastic materials from which they had been made.
Droplet-based microfluidic systems have been shown to be compatible with many chemical and biological reagents and capable of performing a variety of "digital fluidic" operations that can be rendered programmable and reconfigurable. This platform has dimensional scaling benefits that have enabled controlled and rapid mixing of fluids in the droplet reactors, resulting in decreased reaction times. This, coupled with the precise generation and repeatability of droplet operations, has made the droplet-based microfluidic system a potent high throughput platform for biomedical research and applications. In addition to being used as microreactors ranging from the nano- to femtoliter range; droplet-based systems have also been used to directly synthesize particles and encapsulate many biological entities for biomedicine and biotechnology applications. This review will focus on the various droplet operations, as well as the numerous applications of the system. Due to advantages unique to droplet-based systems, this technology has the potential to provide novel solutions to today's biomedical engineering challenges for advanced diagnostics and therapeutics.
This paper describes the first microelectromechanical systems
(MEMS) demonstration device that adopts surface tension as the driving
force. A liquid-metal droplet can be driven in an electrolyte-filled
capillary by locally modifying the surface tension with electric
potential. We explore this so-called continuous electrowetting
phenomenon for MEMS and present crucial design and fabrication
technology that reduce the surface-tension-driving principle, inherently
powerful in microscale, into practice. The key issues that are
identified and investigated include the problem of material
compatibility, electrode polarization, and electrolysis, as well as the
micromachining process. Based on the results from the initial test
devices and the design concept for a long-range movement of the
liquid-metal droplet, we demonstrate a liquid micromotor, an electrolyte
and liquid-metal droplets rotating along a microchannel loop. Smooth and
wear-free rotation of the liquid system is shown at a speed of ~40 mm/s
(or 420 r/min along a 2-mm loop) with a driving voltage of only 2.8 V
and little power consumption (10-100 μW)
This paper presents the development of a disposable plastic biochip incorporating smart passive microfluidics with embedded on-chip power sources and integrated biosensor array for applications in clinical diagnostics and point-of-care testing. The fully integrated disposable biochip is capable of precise volume control with smart microfluidic manipulation without costly on-chip microfluidic components. The biochip has a unique power source using on-chip pressurized air reservoirs, for microfluidic manipulation, avoiding the need for complex microfluidic pumps. In addition, the disposable plastic biochip has successfully been tested for the measurements of partial oxygen concentration, glucose, and lactate level in human blood using an integrated biosensor array. This paper presents details of the smart passive microfluidic system, the on-chip power source, and the biosensor array together with a detailed discussion of the plastic micromachining techniques used for chip fabrication. A handheld analyzer capable of multiparameter detection of clinically relevant parameters has also been developed to detect the signals from the cartridge type disposable biochip. The handheld analyzer developed in this work is currently the smallest analyzer capable of multiparameter detection for point-of-care testing.
Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. VIDEO ABSTRACT.
Copyright © 2015 Elsevier Inc. All rights reserved.
This research demonstrates an integrated microfluidic titration assay to characterize the cation concentrations in working buffer to rapidly optimize the signal-to-noise ratio (SNR) of molecular beacons (MBs). The "Microfluidic Droplet Array Titration Assay" (MiDATA) integrated the functions of sample dilution, sample loading, sample mixing, fluorescence analysis, and re-confirmation functions all together in a one-step process. It allows experimentalists to arbitrarily change sample concentration and acquire SNR measurements instantaneously. MiDATA greatly reduces sample dilution time, number of samples needed, sample consumption, and the total titration time. The maximum SNR of molecular beacons is achieved by optimizing the concentrations of the monovalent and divalent cation (i.e., Mg(2+) and K(+)) of the working buffer. MiDATA platform is able to reduce the total consumed reagents to less than 50 μL, and decrease the assay time to less than 30 min. The SNR of the designated MB is increased from 20 to 126 (i.e., enhanced the signal 630 %) using the optimal concentration of MgCl2 and KCl determined by MiDATA. This novel microfluidics-based titration method is not only useful for SNR optimization of molecular beacons but it also can be a general method for a wide range of fluorescence resonance energy transfer (FRET)-based molecular probes.
The nucleation mechanisms behind crystallized products remain mysterious. In this communication, we describe experiments performed using small volumes, microdroplets, to control nucleation and thus product properties. The effect of small-volume systems on nucleation is discussed.RésuméIl apparait clairement que lʼétape de nucléation est décisive dans le contrôle des propriétés des matériaux. On sʼintéresse à son contrôle en suivant deux objectifs : le premier, très fondamental, concerne la détection du germe critique, la théorie de la nucléation et la compréhension des mécanismes ; le second, plus applicatif, concerne la maitrise de la cristallisation des matériaux et de leurs propriétés (pour lʼindustrie pharmaceutique, la biocristallographie ou la biominéralisation…). Cʼest dans ce cadre que nous avons mené des expérimentations vers le confinement, la localisation spatiale et temporelle de la nucléation, démontrant les possibilités dʼun unique événement de cristallisation.Dans ce contexte, nous avons développé une approche en deux temps, basée sur la génération de gouttes de petit volume, du nanolitre au femtolitre, en utilisant deux montages différents, mais complémentaires. Un premier outil microfluidique simple et polyvalent a été mis en place pour étudier la nucléation, en réduisant le volume de nucléation au nanolitre. Sans pour autant traiter le problème du confinement, il permet dʼaccéder à la cinétique de nucléation. Lʼinfluence directe du volume de manipulation sur la cristallisation est traitée, dans un second temps, à travers un deuxième outil expérimental que nous avons aussi développé ; utilisant un micro-injecteur couplé à des nano-déplaceurs, il permet dʼatteindre des volumes du picolitre au femtolitre et montre la possibilité dʼagir sur la nucléation par le confinement.
The results reported here confirm that environmental stress cracking phenomena in plastics depend on the size and shape of the test liquid's molecules as well as their Hansen solubility parameters (HSP) relative to those of the polymer. The behavior of other, untested liquids can be estimated from HSP correlations based on a limited number of test liquids. A simple test has been developed for the cracking tendency of immersed polymer tubes having different levels of externally applied, controlled stresses. This uses standard tapered NS Teflon (most glass is too rough) stoppers placed on a toploader analytical balance. Polymer tubes are pressed onto the stoppers until the balance shows the given desired force. The tube and stopper are then immersed in a test liquid and observed at regular time intervals for cracking. It has been assumed that the Teflon stoppers are inert in this application. The experiments described here used a COC (cyclo-olefinic copolymer) type polymer (Topas 6013, Ticona). The samples cracked in a number of solvents without external stress. Methyl isobutyl ketone gives rapid cracking of tubes of this polymer at low external stress while olive oil, for example, is somewhat slower to give cracking and requires moderate external stress and longer times.
This work is motivated by the recent experimental development of microfluidic flow-focusing devices that produce highly monodisperse simple or compound drops. Using finite elements with adaptive meshing in a diffuse-interface framework, we simulate the breakup of simple and compound jets in coflowing conditions, and explore the flow regimes that prevail in different parameter ranges. Moreover, we investigate the effects of viscoelasticity on interface rupture and drop pinch-off. The formation of simple drops exhibits a dripping regime at relatively low flow rates and a jetting regime at higher flow rates. In both regimes, drops form because of the combined effects of capillary instability and viscous drag. The drop size increases with the flow rate of the inner fluid and decreases with that of the outer fluid. Viscoelasticity in the drop phase increases the drop size in the dripping regime but decreases it in the jetting regime. The formation of compound drops is a delicate process that takes place in a narrow window of flow and rheological parameters. Encapsulation of the inner drop depends critically on coordination of capillary waves on the inner and outer interfaces. © 2006 American Institute of Physics.
Soft-lithography-based microfluidics and droplet break-off dynamics that can be combined to produce uniform colloidal assemblies from amonodisperse suspension was demonstrated. Experimental results showed that colloidal assemblies with a reasonable uniformity were prepared and the uniformity was enhanced as the number of the embedded particles was increased. For colloidal assemblies with a smaller number of particles, the present microfluidic device needed to be incorporated with a subsequent soft-microfluidic device to sort the assemblies by the number of the internal particles.
LIGA process includes three processes as X-ray lithography, electroforming to fabricate metalic molds and replication, and can be fabricated nano and micro parts for various devices that it is difficult to product by conventional machining methods. A key technology which gathers mass-production efficiency in the LIGA process is micro-replication technology. We choiced hot embossing and injection molding methods for replication. For a demonstration, two kinds of Ni molds, a mesh pattern within a line width of 100 m, and an aspect ratio of 1.0 and a mesh pattern within a line width of 40 m, and an aspect ratio of 2.5, were prepared. These were produced with X-ray lithography and nickel electrofoming technique. In hot embossing, an experiment of micro-replication using polymethyl methacrylate (PMMA) and polycarbonate (PC) sheets succeeded. At injection molding, it could not transfer well with PMMA and PC, but injection temperature was set up highly, and it succeeded by cycloolefin polymer. Furthermore, we measured sidewalls surface roughness of microstructures produced at each steppes of the LIGA process, and it checked that the LIGA process had processing accuracy higher than a conventional machining method.
As the less familiar cousin of quantitative PCR moves mainstream, researchers have more options to choose from.
A microfluidic device designed to generate monodispersed picoliter to femtoliter sized droplet emulsions at controlled rates is presented. This PDMS microfabricated device utilizes the geometry of the channel junctions in addition to the flow rates to control the droplet sizes. An expanding nozzle is used to control the breakup location of the droplet generation process. The droplet breakup occurs at a fixed point due to the focused velocity gradient created by the nozzle shape geometry. The system not only creates monodispersed primary droplets with sizes controlled by the applied flow rates, but also generates monodispersed submicron droplets. Droplets with radii as less than 100 nm can be produced without use of surfactants. Numerical results relating flow rates to the size of primary droplets, satellite droplets and generation rates are reported.
Double emulsions are useful templates for microcapsules and complex particles, but no method yet exists for making double emulsions with both high uniformity and high throughput. We present a parallel numbering-up design for microfluidic double emulsion devices, which combines the excellent control of microfluidics with throughput suitable for mass production. We demonstrate the design with devices incorporating up to 15 dropmaker units in a two-dimensional or three-dimensional array, producing single-core double emulsion drops at rates over 1 kg day(-1) and with diameter variation less than 6%. This design provides a route to integrating hundreds of dropmakers or more in a single chip, facilitating industrial-scale production rates of many tons per year.
This paper details the development of a digital microfluidic platform for multiplexed real-time polymerase chain reactions (PCR). Liquid samples in discrete droplet format are programmably manipulated upon an electrode array by the use of electrowetting. Rapid PCR thermocycling is performed in a closed-loop flow-through format where for each cycle the reaction droplets are cyclically transported between different temperature zones within an oil-filled cartridge. The cartridge is fabricated using low-cost printed-circuit-board technology and is intended to be a single-use disposable device. The PCR system exhibited remarkable amplification efficiency of 94.7%. To test its potential application in infectious diseases, this novel PCR system reliably detected diagnostic DNA levels of methicillin-resistant Staphylococcus aureus (MRSA), Mycoplasma pneumoniae , and Candida albicans . Amplification of genomic DNA samples was consistently repeatable across multiple PCR loops both within and between cartridges. In addition, simultaneous real-time PCR amplification of both multiple different samples and multiple different targets on a single cartridge was demonstrated. A novel method of PCR speed optimization using variable cycle times has also been proposed and proven feasible. The versatile system includes magnetic bead handling capability, which was applied to the analysis of simulated clinical samples that were prepared from whole blood using a magnetic bead capture protocol. Other salient features of this versatile digital microfluidic PCR system are also discussed, including the configurability and scalability of microfluidic operations, instrument portability, and substrate-level integration with other pre- and post-PCR processes.
Microfabricated integrated circuits revolutionized computation by vastly reducing the space, labor, and time required for calculations. Microfluidic systems hold similar promise for the large-scale automation of chemistry and biology, suggesting the possibility of numerous experiments performed rapidly and in parallel, while consuming little reagent. While it is too early to tell whether such a vision will be realized, significant progress has been achieved, and various applications of significant scientific and practical interest have been developed. Here a review of the physics of small volumes (nanoliters) of fluids is presented, as parametrized by a series of dimensionless numbers expressing the relative importance of various physical phenomena. Specifically, this review explores the Reynolds number Re, addressing inertial effects; the Péclet number Pe, which concerns convective and diffusive transport; the capillary number Ca expressing the importance of interfacial tension; the Deborah, Weissenberg, and elasticity numbers De, Wi, and El, describing elastic effects due to deformable microstructural elements like polymers; the Grashof and Rayleigh numbers Gr and Ra, describing density-driven flows; and the Knudsen number, describing the importance of noncontinuum molecular effects. Furthermore, the long-range nature of viscous flows and the small device dimensions inherent in microfluidics mean that the influence of boundaries is typically significant. A variety of strategies have been developed to manipulate fluids by exploiting boundary effects; among these are electrokinetic effects, acoustic streaming, and fluid-structure interactions. The goal is to describe the physics behind the rich variety of fluid phenomena occurring on the nanoliter scale using simple scaling arguments, with the hopes of developing an intuitive sense for this occasionally counterintuitive world.
This paper presents the development of an easy-to-handle and disposable clinical diagnostic lab-on-a-chip using fully integrated plastic microfluidic components, which has the sampling/identifying capability to make fast and reliable measurements of metabolic parameters from human whole blood. A smart and functional lab-on-a-chip cartridge, which incorporates a full on-chip auto-calibration function for in the field applications, has been developed, and then fully characterized using a portable analyzer (3 (1/4)''x 5''x 1'') with multi-analyte detection capability. In addition, several new approaches in realizing smart and functional lab-on-a-chips on polymer have been adopted, which include the pinch valve for automatic fluidic sealing, a by-pass channel as the sampling indicator, and a robust connector design for long analyzer lifetimes. Metabolic parameters such as glucose, lactate, and partial oxygen from human whole blood have been successfully measured using the functional polymer lab-on-a-chips and the portable analyzer developed in this work.
Protein crystallization is a major bottleneck in determining tertiary protein structures from genomic sequence data. This paper describes a microfluidic system for screening hundreds of protein crystallization conditions using less than 4 nL of protein solution for each crystallization droplet. The droplets are formed by mixing protein, precipitant, and additive stock solutions in variable ratios in a flow of water-immiscible fluids inside microchannels. Each droplet represents a discrete trial testing different conditions. The system has been validated by crystallization of several water-soluble proteins.
Cells have been encapsulated inside lipid vesicles by using a new microfluidic lipid vesicle formulation technique. Lipid vesicles are formulated within minutes without using toxic lipid solvents. The encapsulation efficiency inside the vesicles is controlled by the microfluidic flows. Green fluorescent proteins (GFP), carcinoma cells, and bead encapsulated vesicles have mean diameters of 27.2 mum, 62.4 mum, and 55.9 mum, respectively. The variations of vesicle sizes are approximately 20% for the GFP and cell encapsulated vesicles and approximately 10% for the bead encapsulated vesicles.
Biochemical and genetic assays can be both miniaturized and parallelized by compartmentalization in living cells. In vitro compartmentalization (IVC) offers an alternative strategy based on partitioning reactions in water droplets dispersed to form microscopic compartments in water-in-oil emulsions. The cell-like volumes of these compartments (as low as one femtolitre), the ability to freely determine and regulate their content and the large number of compartments (>10(10) per millilitre emulsion) have provided the basis for a range of new, ultra-high-throughput, cell-free technologies. This review describes the scope and potential of IVC in areas such as in vitro evolution of proteins and RNAs, cell-free cloning and sequencing, genetics, genomics, and proteomics.
The manipulation of fluids in channels with dimensions of tens of micrometres--microfluidics--has emerged as a distinct new field. Microfluidics has the potential to influence subject areas from chemical synthesis and biological analysis to optics and information technology. But the field is still at an early stage of development. Even as the basic science and technological demonstrations develop, other problems must be addressed: choosing and focusing on initial applications, and developing strategies to complete the cycle of development, including commercialization. The solutions to these problems will require imagination and ingenuity.
Fundamental and applied research in chemistry and biology benefits
from opportunities provided by droplet-based microfluidic systems.
These systems enable the miniaturization of reactions by compartmentalizing reactions in droplets of femoliter to microliter volumes.
Compartmentalization in droplets provides rapid mixing of reagents,
control of the timing of reactions on timescales from milliseconds to
months, control of interfacial properties, and the ability to synthesize
and transport solid reagents and products. Droplet-based microfluidics can help to enhance and accelerate chemical and biochemical
screening, protein crystallization, enzymatic kinetics, and assays.
Moreover, the control provided by droplets in microfluidic devices can
lead to new scientific methods and insights.
This paper presents an overview of our recent work on the use of soft lithography and two-phase fluid flow to form arrays of droplets. The crucial issues in the formation of stable arrays of droplets and alternating droplets of two sets of aqueous solutions include the geometry of the microchannels, the capillary number, and the water fraction of the system. Glass capillaries could be coupled to the PDMS microchannels and droplets could be transferred into glass capillaries for long-term storage. The arrays of droplets have been applied to screen the conditions for protein crystallization with microbatch and vapor diffusion techniques.
In this study, we report the mass production of monodisperse emulsion droplets and particles using microfluidic large-scale integration on a chip. The production module comprises a glass microfluidic chip with planar microfabricated 16-256 droplet-formation units (DFUs) and a palm-sized stainless steel holder having several layers for supplying liquids into the inlets of the mounted chip. By using a module having 128 cross-junctions (i.e., 256 DFUs) arranged circularly on a 4 cm x 4 cm chip, we could produce droplets of photopolymerizable acrylate monomer at a throughput of 320.0 mL h(-1). The product was monodisperse, having a mean diameter of 96.4 microm, with a coefficient of variation (CV) of 1.3%. Subsequent UV polymerization off the module yielded monodisperse acrylic microspheres at a throughput of approximately 0.3 kg h(-1). Another module having 128 co-flow geometries could produce biphasic Janus droplets of black and white segments at 128.0 mL h(-1). The product had a mean diameter of 142.3 microm, with a CV of 3.3%. This co-flow module could also be applied in the mass production of homogeneous monomer droplets.