Integration Of Microfluidics Research Articles

Integrating Electronics and Microfluidics on Paper.

Paper microfluidics and printed electronics have developed independently, and are incompatible in many aspects. Monolithic integration of microfluidics and electronics on paper is demonstrated. This integration makes it possible to print 2D and 3D fluidic, electrofluidic, and electrical components on paper, and to fabricate devices using them.

A portable paper-based microfluidic platform for multiplexed electrochemical detection of human immunodeficiency virus and hepatitis C virus antibodies in serum.

This paper presents a portable paper-based microfluidic platform for multiplexed electrochemical detection of antibody markers of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in serum samples. To our best knowledge, this is the first paper-based electrochemical immunosensing platform, with multiplexing and telemedicine capabilities, for diagnosing HIV/HCV co-infection. The platform consists of an electrochemical microfluidic paper-based immunosensor array (E-μPIA) and a handheld multi-channel potentiostat, and is capable of performing enzyme-linked immunosorbent assays simultaneously on eight samples within 20 min (using a prepared E-μPIA). The multiplexing feature of the platform allows it to produce multiple measurement data for HIV and HCV markers from a single run, and its wireless communication module can transmit the results to a remote site for telemedicine. The unique integration of paper-based microfluidics and mobile instrumentation renders our platform portable, low-cost, user-friendly, and high-throughput.

Gel integration for microfluidic applications.

Molecular diffusive membranes or materials are important for biological applications in microfluidic systems. Hydrogels are typical materials that offer several advantages, such as free diffusion for small molecules, biocompatibility with most cells, temperature sensitivity, relatively low cost, and ease of production. With the development of microfluidic applications, hydrogels can be integrated into microfluidic systems by soft lithography, flow-solid processes or UV cure methods. Due to their special properties, hydrogels are widely used as fluid control modules, biochemical reaction modules or biological application modules in different applications. Although hydrogels have been used in microfluidic systems for more than ten years, many hydrogels' properties and integrated techniques have not been carefully elaborated. Here, we systematically review the physical properties of hydrogels, general methods for gel-microfluidics integration and applications of this field. Advanced topics and the outlook of hydrogel fabrication and applications are also discussed. We hope this review can help researchers choose suitable methods for their applications using hydrogels.

Handheld Fluorescence Microscopy based Flow Analyzer.

Fluorescence microscopy has the intrinsic advantages of favourable contrast characteristics and high degree of specificity. Consequently, it has been a mainstay in modern biological inquiry and clinical diagnostics. Despite its reliable nature, fluorescence based clinical microscopy and diagnostics is a manual, labour intensive and time consuming procedure. The article outlines a cost-effective, high throughput alternative to conventional fluorescence imaging techniques. With system level integration of custom-designed microfluidics and optics, we demonstrate fluorescence microscopy based imaging flow analyzer. Using this system we have imaged more than 2900 FITC labeled fluorescent beads per minute. This demonstrates high-throughput characteristics of our flow analyzer in comparison to conventional fluorescence microscopy. The issue of motion blur at high flow rates limits the achievable throughput in image based flow analyzers. Here we address the issue by computationally deblurring the images and show that this restores the morphological features otherwise affected by motion blur. By further optimizing concentration of the sample solution and flow speeds, along with imaging multiple channels simultaneously, the system is capable of providing throughput of about 480 beads per second.

Discovery in Droplets.

In the summer of 2009, Daniel Chiu’s prescient review in Analytical Chemistry described droplet microfluidics, an emerging concept and malleable analytical tool.1 The article primarily discussed the potential of single droplets to aid in the study of immensely complex systems, such as single cells and organelles. But droplets, by way of miniaturization and parallelization, were also clearly poised to survey much broader swathes of chemical and biological space in discovery-oriented high-throughput experimentation. Picoliter-scale analysis coupled with microfluidic integration could very realistically deliver million-fold improvements in throughput, potentially revolutionizing diverse applications from protein engineering to drug lead identification. The technology needed to tackle these difficult problems was still just emerging, but 2009 featured a dramatic expansion in microfluidic componentry for generating and manipulating large quantities of droplets (e.g., incubation, picoinjection, sorting, etc.). Today they collectively form the microfluidic circuit engineer’s standard palette of parts. Microfluidic circuit component integration, once largely concerned with moving bulk fluid between reactions and separation-based analysis channels, has entered a digital renaissance. Single devices now generate, handle, and analyze sample collections that vastly eclipse the capabilities of even the most sophisticated robotic automation. This review highlights recent (primarily 2013-2015) themes in technology development that continue to build the foundation of droplet-based discovery platforms, and new challenges in droplet-scale information storage and retrieval that have coalesced around these new platforms.

Microfluidic Integration of a Cloth-Based Hybridization Array System (CHAS) for Rapid, Colorimetric Detection of Enterohemorrhagic Escherichia coli (EHEC) Using an Articulated, Centrifugal Platform.

We describe the translation of a cloth-based hybridization array system (CHAS), a colorimetric DNA detection method that is used by food inspection laboratories for colony screening of pathogenic agents, onto a microfluidic chip format. We also introduce an articulated centrifugal platform with a novel fluid manipulation concept based on changes in the orientation of the chip with respect to the centrifugal force field to time the passage of multiple components required for the process. The platform features two movable and motorized carriers that can be reoriented on demand between 0 and 360° during stage rotation. Articulation of the chip can be used to trigger on-the-fly fluid dispensing through independently addressable siphon structures or to relocate solutions against the centrifugal force field, making them newly accessible for downstream transfer. With the microfluidic CHAS, we achieved significant reduction in the size of the cloth substrate as well as the volume of reagents and wash solutions. Both the chip design and the operational protocol were optimized to perform the entire process in a reliable, fully automated fashion. A demonstration with PCR-amplified genomic DNA confirms on-chip detection and identification of Escherichia coli O157:H7 from colony isolates in a colorimetric multiplex assay using rfbO157, fliCH7, vt1, and vt2 genes.

Recent advances in optical fiber devices for microfluidics integration.

This paper examines the recent emergence of miniaturized optical fiber based sensing and actuating devices that have been successfully integrated into fluidic microchannels that are part of microfluidic and lab-on-chip systems. Fluidic microsystems possess the advantages of reduced sample volumes, faster and more sensitive biological assays, multi-sample and parallel analysis, and are seen as the de facto bioanalytical platform of the future. This paper considers the cases where the optical fiber is not merely used as a simple light guide delivering light across a microchannel, but where the fiber itself is engineered to create a new sensor or tool for use within the environment of the fluidic microchannel.

Spheroid and Tissue Assembly via Click Chemistry in Microfluidic Flow.

Proper cell-cell contact and communication are essential for the correct development and survival of higher order organisms. In order to study complex cell interactions that occur in vivo, model systems that are able to recapitulate 3D cell-cell interactions in vitro are key to advancing new biotechnologies, therapeutics, and tissue engineering applications. Herein, we show a new strategy to rapidly and efficiently generate complex multiple cell line containing spheroids and tissues in microfluidic flow without the use of scaffolds, molecular biology, or metabolic biosynthesis. The method relies on the integration of microfluidics, liposome fusion, bio-orthogonal chemistry, and cell surface engineering to rapidly click coculture cell assemblies in flow. We demonstrate this strategy by assembling various combinations of cell types with an interfacial cell to cell click chemistry in microfluidic flow to generate a range of spheroid types and oriented tissue multilayers.

A human liver microphysiology platform for investigating physiology, drug safety, and disease models.

This paper describes the development and characterization of a microphysiology platform for drug safety and efficacy in liver models of disease that includes a human, 3D, microfluidic, four-cell, sequentially layered, self-assembly liver model (SQL-SAL); fluorescent protein biosensors for mechanistic readouts; as well as a microphysiology system database (MPS-Db) to manage, analyze, and model data. The goal of our approach is to create the simplest design in terms of cells, matrix materials, and microfluidic device parameters that will support a physiologically relevant liver model that is robust and reproducible for at least 28 days for stand-alone liver studies and microfluidic integration with other organs-on-chips. The current SQL-SAL uses primary human hepatocytes along with human endothelial (EA.hy926), immune (U937) and stellate (LX-2) cells in physiological ratios and is viable for at least 28 days under continuous flow. Approximately, 20% of primary hepatocytes and/or stellate cells contain fluorescent protein biosensors (called sentinel cells) to measure apoptosis, reactive oxygen species (ROS) and/or cell location by high content analysis (HCA). In addition, drugs, drug metabolites, albumin, urea and lactate dehydrogenase (LDH) are monitored in the efflux media. Exposure to 180 μM troglitazone or 210 μM nimesulide produced acute toxicity within 2-4 days, whereas 28 μM troglitazone produced a gradual and much delayed toxic response over 21 days, concordant with known mechanisms of toxicity, while 600 µM caffeine had no effect. Immune-mediated toxicity was demonstrated with trovafloxacin with lipopolysaccharide (LPS), but not levofloxacin with LPS. The SQL-SAL exhibited early fibrotic activation in response to 30 nM methotrexate, indicated by increased stellate cell migration, expression of alpha-smooth muscle actin and collagen, type 1, alpha 2. Data collected from the invitro model can be integrated into a database with access to related chemical, bioactivity, preclinical and clinical information uploaded from external databases for constructing predictive models.

Electrical/ Electrochemical Impedance Measurement for Biological Applications

As a principle of transduction, the impedance technique has been applied in the field of biological study ranging from microbiology to cancer cell/tissue studies. This paper summarizes the applications of electrical/electrochemical impedance spectroscopy that have been practiced in our research in the past several years, mainly including two distinct fields--the detection of foodborne bacterial pathogens and the study of oral cancer cells. Foodborne diseases caused by foodborne pathogens have been a serious threat to public health for decades and remain one of the major concerns of our society. The development of rapid, sensitive, and specific methods is central to implementing effective practice to ensure food safety and security. We have been using impedance technique as a means to detect and/or quantify foodborne pathogenic bacteria. This paper will particular introduce the recent significant development in this field, including the use of microfabricated microelectrodes, microfluidic chip-based devices, nanoparticles and integration with other techniques such as dielectrophoresis. Based on the fundamental impedance microbiology, which is a technique based on the measurement of changes in electrical impedance of a culture medium or a reaction solution resulting from the bacterial growth, we have added some new aspects to this subject that have made the impedance technique a more valuable technique for detection of foodborne bacterial pathogens. These aspects include the use of different electrode systems and the analysis of impedance components using equivalent circuits for better improvement on the detection systems. The advances in microfabrication technologies have launched the use of microfabricated microarray electrodes in impedance detection and the miniaturization of impedance microbiology into a chip format, which has shown great promising in rapid detection of bacterial growth. Further, the integration of impedance technique with biosensor technology has led to the development of impedance biosensors that is expending rapidly for bacteria detection in recent years. Impedance biosensors for bacteria detection are based on the analysis of electrical properties of bacterial cells when they are attached to or associated with the electrodes. Such impedance biosensor methods have substantially reduced the assay time down to 30 min~2 h compared with growth-based impedance methods. The dimensional compatibility of those microfabricated biosensors with the target bacterial cells has enabled them to detect the binding of bacterial cells on its surface without any amplification step. On the other hand, electrical impedance spectroscopy (EIS) enables direct sensing of cellular activities occurring on an electrode surface by measuring the induced capacitance and/or resistance changes by cells attached on the electrode. Particular, EIS is capable of distinguishing different types of cells based on the cellular activity-induced electrical signals. We have demonstrated the use of real-time impedance technique to study the cellular activities of oral cancer cells in a label-free manner, including cell proliferation, adhesion, spreading, and apoptosis induced by drug treatments. Furthermore, it is known that during transformation from non-cancer cells to caner cells, the cellular morphology, structure, and function are altered, which may includes, but are not limited to changes in the nuclei, composition of the cytoplasm, structure of the cell membrane, changes in the nuclei, composition of the cytoplasm, structure of the cell membrane, and expression levels of ion channels, etc. These changes may result in alterations in cancer cell properties that could be detected by the EIS well before these morphological features that normally used in detection can be seen or biomarkers are developed. We have explored the use of real-time impedance measurements to distinguish oral cancer cells from normal oral epithelial cells in label-free manner by studying the kinetics of cell spreading and attachment of the two cell types.

Desktop aligner for fabrication of multilayer microfluidic devices.

Multilayer assembly is a commonly used technique to construct multilayer polydimethylsiloxane (PDMS)-based microfluidic devices with complex 3D architecture and connectivity for large-scale microfluidic integration. Accurate alignment of structure features on different PDMS layers before their permanent bonding is critical in determining the yield and quality of assembled multilayer microfluidic devices. Herein, we report a custom-built desktop aligner capable of both local and global alignments of PDMS layers covering a broad size range. Two digital microscopes were incorporated into the aligner design to allow accurate global alignment of PDMS structures up to 4 in. in diameter. Both local and global alignment accuracies of the desktop aligner were determined to be about 20 μm cm(-1). To demonstrate its utility for fabrication of integrated multilayer PDMS microfluidic devices, we applied the desktop aligner to achieve accurate alignment of different functional PDMS layers in multilayer microfluidics including an organs-on-chips device as well as a microfluidic device integrated with vertical passages connecting channels located in different PDMS layers. Owing to its convenient operation, high accuracy, low cost, light weight, and portability, the desktop aligner is useful for microfluidic researchers to achieve rapid and accurate alignment for generating multilayer PDMS microfluidic devices.

Controlled sealing of nanopores using an easily fabricated silicon platform combining in situ optical and electrical monitoring

Nanopores are of great interest for biosensing applications. However, using such sensing platforms requires long and costly fabrication processes and the implementation of simultaneous electrical recording and optical imaging is not straightforward. Here, we present a new approach for silicon nanopore fabrication and integration avoiding backside etching of the substrate and allowing direct microfluidics integration for electrical recording and optical imaging. We demonstrate an electrical and optical live in situ detection of the electrophoretic trapping of polystyrene microspheres on silicon nanopores. This trapping procedure could also be used to partially close already made nanopores, in order to increase their sensitivity to small analytes.

Automated cell viability assessment using a microfluidics based portable imaging flow analyzer.

In this work, we report a system-level integration of portable microscopy and microfluidics for the realization of optofluidic imaging flow analyzer with a throughput of 450 cells/s. With the use of a cellphone augmented with off-the-shelf optical components and custom designed microfluidics, we demonstrate a portable optofluidic imaging flow analyzer. A multiple microfluidic channel geometry was employed to demonstrate the enhancement of throughput in the context of low frame-rate imaging systems. Using the cell-phone based digital imaging flow analyzer, we have imaged yeast cells present in a suspension. By digitally processing the recorded videos of the flow stream on the cellphone, we demonstrated an automated cell viability assessment of the yeast cell population. In addition, we also demonstrate the suitability of the system for blood cell counting.

Analysis of fast protein phosphorylation kinetics in single cells on a microfluidic chip.

In the present study, we developed a microfluidic large-scale integration (mLSI) platform for the temporal and chemical control of cell cultures to study fast kinetics of protein phosphorylation. For in situ protein analysis the mLSI chip integrates the Proximity Ligation Assay (PLA). To investigate cell-signaling events with a time resolution of a few seconds we first engineered and optimized the fluidic layout of the chip with 128 individual addressable cell culture chambers. The functionality of the cell culture operations and PLA is demonstrated by the determination of the minimum cell sample size for obtaining robust quantitative PLA signals at the single-cell level. We show that at least 350 cells per assay condition are required to statistically evaluate single cell PLA data. In the following we used the PLA chip with over 500 hundred cells per condition to record sequential phosphorylation reactions of the canonical protein kinase within the Akt pathway, which is activated in various human cancer types. This was achieved by stimulating mouse fibroblast cell cultures with either the platelet-derived growth factor (PDGF) or insulin-like growth factor (IGF-1). Fluidic cell stimulation pulses of 5 seconds were followed by precisely time shifted cell fixation pulses to obtain a temporal resolution of 10 seconds. PLA was then performed on all fixed arrays of cell cultures to extract the characteristic phosphorylation times at the single cell level for either the PDGF, or IGF-1 receptor and the Akt and GSK3β kinases. Characteristic phosphorylation times for the receptors were between 13 and 35 seconds, whereas for downstream kinases between 25 and 200 seconds. Thus we could reveal a molecular order of the phosphorylation reactions during the signal transduction through the Akt pathway. In dependence of the stimulus we found a temporal difference for the characteristic phosphorylation time of 20 and 150 seconds for the Ser-473 and Thr-308 residues on the Akt kinase, respectively. Temporal alteration of sequential phosphorylation reactions on Akt has been proposed as molecular mechanism to differentiate between stimuli and biophysically determined in the present study.

Rapid and multiplex detection of Legionella's RNA using digital microfluidics

Despite recent advances in the miniaturization and automation of biosensors, technologies for on-site monitoring of environmental water are still at an early stage of development. Prevention of outbreaks caused by pathogens such as Legionella pneumophila would be facilitated by the development of sensitive and specific bioanalytical assays that can be easily integrated in miniaturized fluidic handling systems. In this work, we report on the integration of an amplification-free assay in digital microfluidics (DMF) for the detection of Legionella bacteria based on targeting 16s rRNA. We first review the design of the developed DMF devices, which provide the capability to store up to one hundred nL-size droplets simultaneously, and discuss the challenges involved with on-chip integration of the RNA-based assay. By optimizing the various steps of the assay, including magnetic capture, hybridization duration, washing steps, and assay temperature, a limit of detection as low as 1.8 attomoles of synthetic 16s rRNA was obtained, which compares advantageously to other amplification-free detection systems. Finally, we demonstrate the specificity of the developed assay by performing multiplex detection of 16s rRNAs from a pathogenic and a non-pathogenic species of Legionella. We believe the developed DMF devices combined with the proposed detection system offers new prospects for the deployment of rapid and cost-effective technologies for on-site monitoring of pathogenic bacteria.

Fluigi

One goal of synthetic biology is to design and build genetic circuits in living cells for a range of applications. Our incomplete knowledge of the effects of metabolic load and biological “crosstalk” on the host cell make it difficult to construct multilevel genetic logic circuits in a single cell, limiting the scalability of engineered biological systems. Microfluidic technologies provide reliable and scalable construction of synthetic biological systems by allowing compartmentalization of cells encoding simple genetic circuits and the spatiotemporal control of communication among these cells. This control is achieved via valves on the microfluidics chip which restrict fluid flow when activated. We describe a Computer Aided Design (CAD) framework called “Fluigi” for optimizing the layout of genetic circuits on a microfluidic chip, generating the control sequence of the associated signaling fluid valves, and simulating the behavior of the configured biological circuits. We demonstrate the capabilities of Fluigi on a set of Boolean algebraic benchmark circuits found in both synthetic biology and electrical engineering and a set of assay-based benchmark circuits. The integration of microfluidics and synthetic biology has the capability to increase the scale of engineered biological systems for applications in DNA assembly, biosensors, and screening assays for novel orthogonal genetic parts.

Transcription factor sensor system for parallel quantification of metabolites on-chip.

Steadily growing demands for identification and quantification of cellular metabolites in higher throughput have brought a need for new analytical technologies. Here, we developed a synthetic biological sensor system for quantifying metabolites from biological cell samples. For this, bacterial transcription factors were exploited, which bind to or dissociate from regulatory DNA elements in response to physiological changes in the cellular metabolite concentration range. Representatively, the bacterial pyruvate dehydrogenase (PdhR), trehalose (TreR), and l-arginine (ArgR) repressor proteins were functionalized to detect pyruvate, trehalose-6-phosphate (T6P), and arginine concentration in solution. For each transcription factor the mutual binding behavior between metabolite and DNA, their working range, and othogonality were determined. High-throughput, parallel processing, and automation were achieved through integration of the metabolic sensor system on a microfluidic large-scale integration (mLSI) chip platform. To demonstrate the functionality of the integrated metabolic sensor system, we measured diurnal concentration changes of pyruvate and the plant signaling molecule T6P within cell etxracts of Arabidopsis thaliana rosettes. The transcription factor sensor system is of generic nature and extendable on the microfluidic chip.

A novel paper-based microfluidic enhanced chemiluminescence biosensor for facile, reliable and highly-sensitive gene detection of Listeria monocytogenes

This paper describes the development of a paper-based microfluidic device that detects long DNA amplicons on the basis of hybridization reactions with a covalently immobilized DNA probe and biotin-labeled signal DNA strands, and chemiluminescent (CL) reactions catalyzed by a horseradish peroxidase (HRP)-streptavidin conjugate. The paper-based microfluidic CL biosensor is developed for detection of 198-bp DNA fragments obtained by PCR amplification of the Listeria monocytogenes hlyA gene, where the CL intensity is detected by a simple, inexpensive CCD. To our knowledge, the combined use of the CCD sensing and paper-based microfluidic CL detection is first proposed here. The paper-based device includes wax-screen printed channels, and further explores origami-type folds to create an easy-to-operate valve that makes it possible to successively wash the detection zone in-between hybridization steps. Compared with other paper-based wax-screen-printing methods, the proposed method is faster and needs lower heating temperature. In our DNA biosensor, CL signals generated using a HRP-luminol-H2O2 system are heightened with p-iodine phenol (PIP) and detected with a CCD system. Under optical conditions, a linear range of 1.94×10−1pmol/L to 1.94×104pmol/L is achieved and the limit of detection is found to be 6.3×10−2pmol/L. And, this detection limit is 3–5 orders of magnitude lower than those obtained from other CCD-based microfluidic CL DNA assays. The results show that the proposed integration of paper-based microfluidics, CL method and CCD sensing offers great promises in providing a highly-sensitive, reliable and cost-effective solution for gene biosensing applications.

Multi-focus parallel detection of fluorescent molecules at picomolar concentration with photonic nanojets arrays

Fluorescence sensing and fluorescence correlation spectroscopy (FCS) are powerful methods to detect and characterize single molecules; yet, their use has been restricted by expensive and complex optical apparatus. Here, we present a simple integrated design using a self-assembled bi-dimensional array of microspheres to realize multi-focus parallel detection scheme for FCS. We simultaneously illuminate and collect the fluorescence from several tens of microspheres, which all generate their own photonic nanojet to efficiently excite the molecules and collect the fluorescence emission. Each photonic nanojet contributes to the global detection volume, reaching FCS detection volumes of several tens of femtoliters while preserving the fluorescence excitation and collection efficiencies. The microspheres photonic nanojets array enables FCS experiments at low picomolar concentrations with a drastic reduction in apparatus cost and alignment constraints, ideal for microfluidic chip integration.

Field-programmable lab-on-a-chip based on microelectrode dot array architecture.

The fundamentals of electrowetting-on-dielectric (EWOD) digital microfluidics are very strong: advantageous capability in the manipulation of fluids, small test volumes, precise dynamic control and detection, and microscale systems. These advantages are very important for future biochip developments, but the development of EWOD microfluidics has been hindered by the absence of: integrated detector technology, standard commercial components, on-chip sample preparation, standard manufacturing technology and end-to-end system integration. A field-programmable lab-on-a-chip (FPLOC) system based on microelectrode dot array (MEDA) architecture is presented in this research. The MEDA architecture proposes a standard EWOD microfluidic component called 'microelectrode cell', which can be dynamically configured into microfluidic components to perform microfluidic operations of the biochip. A proof-of-concept prototype FPLOC, containing a 30 × 30 MEDA, was developed by using generic integrated circuits computer aided design tools, and it was manufactured with standard low-voltage complementary metal-oxide-semiconductor technology, which allows smooth on-chip integration of microfluidics and microelectronics. By integrating 900 droplet detection circuits into microelectrode cells, the FPLOC has achieved large-scale integration of microfluidics and microelectronics. Compared to the full-custom and bottom-up design methods, the FPLOC provides hierarchical top-down design approach, field-programmability and dynamic manipulations of droplets for advanced microfluidic operations.