Flow‐based analysis using microfluidics–chemiluminescence systems

This review will discuss various approaches and techniques in which analysis using microfluidics–chemiluminescence systems (MF–CL) has been reported. A variety of applications is examined, including environmental, pharmaceutical, biological, food and herbal analysis. Reported uses of CL reagents, sample introduction techniques, sample pretreatment methods, CL signal enhancement and detection systems are discussed. A hydrodynamic pumping system is predominately used for these applications. However, several reports are available in which electro‐osmotic (EO) pumping has been implemented. Various sample pretreatment methods have been used, including liquid–liquid extraction, solid‐phase extraction and molecularly imprinted polymers. A wide range of innovative techniques has been reported for CL signal enhancement. Most of these techniques are based on enhancement of the mixing process in the microfluidics channels, which leads to enhancement of the CL signal. However, other techniques are also reported, such as mirror reaction, liquid core waveguide, on‐line pre‐derivatization and the use of an opaque white chip with a thin transparent seal. Photodetectors are the most commonly used detectors; however, other detection systems have also been used, including integrated electrochemiluminescence (ECL) and organic photodiodes (OPDs). Copyright © 2012 John Wiley & Sons, Ltd.     

System Integration - A Major Step toward Lab on a Chip

Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications.     

Magnetic targeting of mechanosensors in bone cells for tissue engineering applications

Mechanical signalling plays a pivotal role in maintaining bone cell function and remodelling of the skeleton. Our previous work has highlighted the potential role of mechano-induction in tissue engineering applications. In particular, we have highlighted the potential for using magnetic particle techniques for tissue engineering applications. Previous studies have shown that manipulation of integrin attached magnetic particles leads to changes in intracellular calcium signalling within osteoblasts. However, due to the phenomenon of particle internalisation, previous studies have typically focused on short-term stimulation experiments performed within 1-2 h of particle attachment. For tissue engineering applications, bone tissue growth occurs over a period of 3-5 weeks. To date, no study has investigated the cellular responses elicited from osteoblasts over time following stimulation with internalised magnetic particles. Here, we demonstrate the long-term biocompatibility of 4.5 microm RGD-coated particles with osteoblasts up to 21 days in culture, and detail a time course of responses elicited from osteoblasts following mechanical stimulation with integrin attached magnetic particles (<2h post attachment) and internalised particles (>48h post attachment). Mechanical manipulation of both integrin attached and internalised particles were found to induce intracellular calcium signalling. It is concluded that magnetic particles offer a tool for applying controlled mechanical forces to osteoblasts, and can be used to stimulate intracellular calcium signalling over prolonged periods of time. Magnetic particle technology presents a potentially valuable tool for tissue engineering which permits the delivery of highly localised mechano-inductive forces directly to cells.     

Expanding Imaging Capabilities for Microfluidics: Applicability of Darkfield Internal Reflection Illumination (DIRI) to Observations in Microfluidics

Microfluidics is used increasingly for engineering and biomedical applications due to recent advances in microfabrication technologies. Visualization of bubbles, tracer particles, and cells in a microfluidic device is important for designing a device and analyzing results. However, with conventional methods, it is difficult to observe the channel geometry and such particles simultaneously. To overcome this limitation, we developed a Darkfield Internal Reflection Illumination (DIRI) system that improved the drawbacks of a conventional darkfield illuminator. This study was performed to investigate its utility in the field of microfluidics. The results showed that the developed system could clearly visualize both microbubbles and the channel wall by utilizing brightfield and DIRI illumination simultaneously. The methodology is useful not only for static phenomena, such as clogging, but also for dynamic phenomena, such as the detection of bubbles flowing in a channel. The system was also applied to simultaneous fluorescence and DIRI imaging. Fluorescent tracer beads and channel walls were observed clearly, which may be an advantage for future microparticle image velocimetry (μPIV) analysis, especially near a wall. Two types of cell stained with different colors, and the channel wall, can be recognized using the combined confocal and DIRI system. Whole-slide imaging was also conducted successfully using this system. The tiling function significantly expands the observing area of microfluidics. The developed system will be useful for a wide variety of engineering and biomedical applications for the growing field of microfluidics.     

Perspective: Microfluidic applications in microbiology

The application of microfluidics technology to microbiology research is an excellent platform for the analysis of microorganisms and their nucleic acids. This technology combines engineering, physics, chemistry, biology and computing to control the devices. In this perspective we discuss how microfluidics can be applied to microbiological research and used in diagnostic applications. We also summarize advantages and limitations of this technology, as well as highlight some recent microbiological applications.     

Dielectrophoretic platforms for bio-microfluidic systems

Dielectrophoresis, the induced motion of polarisable particles in a nonuniform electric field, has been proven as a versatile mechanism to transport, accumulate, separate and characterise micro/nano scale bioparticles in microfluidic systems. The integration of DEP systems into the microfluidics enables the inexpensive, fast, highly sensitive, highly selective and label-free detection and analysis of target bioparticles. This review provides an in-depth overview of state-of-the-art dielectrophoretic (DEP) platforms integrated into microfluidics aimed towards different biomedical applications. It classifies the current DEP systems in terms of different microelectrode configurations and operating strategies devised to generate and employ DEP forces in such processes, and compares the features of each approach. Finally, it suggests the future trends and potential applications of DEP systems in single cell analysis, stem cell research, establishing novel devices, and realising fully DEP-activated lab-on-a-chip systems.     

PCR microfluidic devices for DNA amplification

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

Development of a novel disposable filter bag for separation of biomolecules with functionalized magnetic particles

The integration of disposable magnetic filters in combination with functionalized magnetic particles represents a fast and cost‐effective alternative for enzyme purification in comparison to solid/liquid separation by means of centrifugation followed by chromatographic purification. The main advantage of the particle‐based process is the solid/solid/liquid separation in one step combined with disposable equipment. Furthermore this combination provides the possibility to also process biocatalytic reactions in cell‐containing media into disposable equipment with preimmobilized enzymes onto the magnetic particles. The focus of the presented study is on the design and performance of a disposable filtration unit consisting of a plastic bag with an inlet and outlet and a stainless steel filter matrix. During magnetic separation, the magnetic particles selectively retard at the filter matrix due to the magnetic force, which counteracts the drag force. It was found that the length of a lengthwise aligned filter matrix should be longer than the magnetic pole surfaces in fluid flow direction. Hereby, a filtration capacity of 5.6 g magnetic particles was measured with a loss of below 0.5%. Introducing a two‐phase flow optimizes the cleaning of the bag after a magnetic filtration. The procedure offered a high cleaning efficiency. Herewith, the cleaned filter unit could be discarded with minimum losses of product and magnet particles.     

Microfluidic: An innovative tool for efficient cell sorting

At first mostly dedicated to molecular analysis, microfluidic systems are rapidly expanding their range of applications towards cell biology, thanks to their ability to control the mechanical, biological and fluidic environment at the scale of the cells. A number of new concepts based on microfluidics were indeed proposed in the last ten years for cell sorting. For many of these concepts, progress remains to be done regarding automation, standardization, or throughput, but it is now clear that microfluidics will have a major contribution to the field, from fundamental research to point-of-care diagnosis. We present here an overview of cells sorting in microfluidics, with an emphasis on circulating tumor cells. Sorting principles are classified in two main categories, methods based on physical properties of the cells, such as size, deformability, electric or optical properties, and methods based on biomolecular properties, notably specific surface antigens. We document potential applications, discuss the main advantages and limitations of different approaches, and tentatively outline the main remaining challenges in this fast evolving field.     

Merging microfluidics with microarray-based bioassays

Microarray technologies provide powerful tools for biomedical researchers and medicine, since arrays can be configured to monitor the presence of molecular signatures in a highly parallel fashion and can be configured to search either for nucleic acids (DNA microarrays) or proteins (antibody-based microarrays) as well as different types of cells. Microfluidics on the other hand, provides the ability to analyze small volumes (micro-, nano- or even pico-liters) of sample and minimize costly reagent consumption as well as automate sample preparation and reduce sample processing time. The marriage of microarray technologies with the emerging field of microfluidics provides a number of advantages such as, reduction in reagent cost, reductions in hybridization assay times, high-throughput sample processing, and integration and automation capabilities of the front-end sample processing steps. However, this potential marriage is also fraught with some challenges as well, such as developing low-cost manufacturing methods of the fluidic chips, providing good interfaces to the macro-world, minimizing non-specific analyte/wall interactions due to the high surface-to-volume ratio associated with microfluidics, the development of materials that accommodate the optical readout phases of the assay and complete integration of peripheral components (optical and electrical) to the microfluidic to produce autonomous systems appropriate for point-of-care testing. In this review, we provide an overview and recent advances on the coupling of DNA, protein and cell microarrays to microfluidics and discuss potential improvements required for the implementation of these technologies into biomedical and clinical applications.     

Disposable microfluidic devices: fabrication, function, and application

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

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of “lab-on-a-chip” platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique.Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives.Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.     

DNA extraction on bio-chip: history and preeminence over conventional and solid-phase extraction methods

This review covers a developmental progression on early to modern taxonomy at cellular level following the advent of electron microscopy and the advancement in deoxyribonucleic acid (DNA) extraction for expatiation of biological classification at DNA level. Here, we discuss the fundamental values of conventional chemical methods of DNA extraction using liquid/liquid extraction (LLE) followed by development of solid-phase extraction (SPE) methods, as well as recent advances in microfluidics device-based system for DNA extraction on-chip. We also discuss the importance of DNA extraction as well as the advantages over conventional chemical methods, and how Lab-on-a-Chip (LOC) system plays a crucial role for the future achievements.

     

A method for selective isolation and aggregation of epicuticular wax nanotubes in Picea pungens

A method for selectively isolating and aggregating intact nanotubes from leaf surfaces, in sample quantities useable for their characterisation in further experimental investigations, is presented here. It uses liquid sucrose, as a saturated solution, with its wetting behaviour modified by the addition of controlled amounts of ethanol, as a temporary entrapment and release medium, for removing nanotube material from the leaf surface, here of Picea pungens (Engelmann). This harvesting technique works by the slow, gentle transition of the medium from liquid to solid, capturing the nanotubes, and then back to liquid again, releasing them, to form an aggregate sample, with little compromise to the structural integrity of individual nanotubes. Scanning electron microscopy (SEM) images are presented rigorously illustrating the technique and its effectiveness. Comparison with other recent methods reveals its advantages, and potential applications are explored.     

Array-based pharmacogenomics of molecular-targeted therapies in oncology

The advent of microarrays over the past decade has transformed the way genome-wide studies are designed and conducted, leading to an unprecedented speed of acquisition and amount of new knowledge. Microarray data have led to the identification of molecular subclasses of solid tumors characterized by distinct oncogenic pathways, as well as the development of multigene prognostic or predictive models equivalent or superior to those of established clinical parameters. In the field of molecular-targeted therapy for cancer, in particular, the application of array-based methodologies has enabled the identification of molecular targets with 'key' roles in neoplastic transformation or tumor progression and the subsequent development of targeted agents, which are most likely to be active in the specific molecular setting. Herein, we present a summary of the main applications of whole-genome expression microarrays in the field of molecular-targeted therapies for solid tumors and we discuss their potential in the clinical setting. An emphasis is given on deciphering the molecular mechanisms of drug action, identifying novel therapeutic targets and suitable agents to target them with, and discovering molecular markers/signatures that predict response to therapy or optimal drug dose for each patient.     

Assembly of linear nano-chains from iron oxide nanospheres with asymmetric surface chemistry

Besides the multifunctionality, another equally important aspect of nanoparticles is their engineerability to control the geometrical and chemical properties during fabrication. In this work, we exploited this aspect to define asymmetric surface chemistry of an iron oxide nanosphere by controlling the topology of ligand expression on its surface resulting in a particle with two faces, one displaying only amines and the other only thiols. Specifically, amine-functionalized iron oxide nanospheres were attached on a solid support via a crosslinker containing a disulfide bridge. Liberation of the nanosphere using thiolytic cleavage created thiols on the portion of the particle's surface that interacted with the solid support. Employing a solid-phase strategy and a step-by-step addition of particles, the two unique faces on the same nanosphere served as fittings to assemble them into linear nano-chains. Assembly of chains with various lengths and aspect ratios was controlled by the size and number of the added nanospheres. The characteristics of those chains showed a high degree of uniformity indicating the exceptional control of the synthetic process. Notably, one of the unique properties of the iron oxide nano-chains was an increased magnetic relaxivity, indicating their potential use as contrast agents for magnetic resonance imaging.     

“Connecting worlds – a view on microfluidics for a wider application”

From its birth, microfluidics has been referenced as a revolutionary technology and the solution to long standing technological and sociological issues, such as detection of dilute compounds and personalized healthcare. Microfluidics has for example been envisioned as: (1) being capable of miniaturizing industrial production plants, thereby increasing their automation and operational safety at low cost; (2) being able to identify rare diseases by running bioanalytics directly on the patient’s skin; (3) allowing health diagnostics in point-of-care sites through cheap lab-on-a-chip devices. However, the current state of microfluidics, although technologically advanced, has so far failed to reach the originally promised widespread use.In this paper, some of the aspects are identified and discussed that have prevented microfluidics from reaching its full potential, especially in the chemical engineering and biotechnology fields, focusing mainly on the specialization on a single target of most microfluidic devices and offering a perspective on the alternate, multi-use, “plug and play” approach. Increasing the flexibility of microfluidic platforms, by increasing their compatibility with different substrates, reactions and operation conditions, and other microfluidic systems is indeed of surmount importance and current academic and industrial approaches to modular microfluidics are presented. Furthermore, two views on the commercialization of plug-and-play microfluidics systems, leading towards improved acceptance and more widespread use, are introduced. A brief review of the main materials and fabrication strategies used in these fields, is also presented. Finally, a step-wise guide towards the development of microfluidic systems is introduced with special focus on the integration of sensors in microfluidics. The proposed guidelines are then applied for the development of two different example platforms, and to three examples taken from literature.With this work, we aim to provide an interesting perspective on the field of microfluidics when applied to chemical engineering and biotechnology studies, as well as to contribute with potential solutions to some of its current challenges.     

Particle deposition and sensory drive

The mutualism between chemical cues emitted into the air and variations in how primates respond to them using olfaction has demonstrated aspects of species‐specific adaptations. Building on this mutualism we can look at particle deposition as another means to understanding how various environments may have elicited biological changes that enable efficient communication. Research on particle movement and deposition within the nasal cavity is largely based on questions about health as it relates to drug delivery systems and overall olfactory function in modern humans. With increased access to 3D models and the use of computational fluid dynamic analysis, researchers have been able to simulate site‐specific deposition, to determine what particles are making it through the nasal cavity to the main olfactory epithelium, which ultimately leads to processing in the olfactory bulb. Here we discuss particle deposition research, sensory drive and their potential applications to evolutionary anthropology.