Fully integrated digital microfluidics platform for automated immunoassay; a versatile tool for rapid, specific detection of a wide range of pathogens

© 2018 Elsevier Ltd. All rights reserved. This manuscript is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International Licence http://creativecommons.org/licenses/by-nc-nd/4.0/.With the tangible threat posed by the release of chemical and biological warfare (CBW) agents, detection of airborne pathogens is a critical military and security concern. Recent air sampling techniques developed for biocollection take advantage of Electrowetting on Dielectric (EWOD) to recover material, producing highly concentrated droplet samples. Bespoke EWOD-based digital microfluidics platforms are very well suited to take full advantage of the microlitre concentrated droplet resulting from this recovery process. In this paper we present a free-standing, fully automated DMF platform for immunoassay. Using this system, we demonstrate the automated detection of four classes of CBW agent simulant biomolecules and organisms each representing credible threat agents. Taking advantage of the full magnetic separation process with antibody-bound microbeads, rapid and complete separation of specific target antigen can be achieved with minimal washing steps allowing for very rapid detection. Here, we report clear detection of four categories of antigens achieved with assay completion times of between six and ten minutes. Detection of HSA, Bacillus atrophaeus (BG spores), MS2 bacteriophage and Escherichia coli are demonstrated with estimated limit of detection of respectively 30 ng ml -1, 4 × 10 4 cfu ml -1, 10 6 pfu ml -1 and 2 × 10 7 cfu ml -1. The fully-integrated portable platform described in this paper is highly compatible with the next generation of electrowetting-coupled air samplers and thus shows strong potential toward future in-field deployable biodetection systems and could have key implication in life-changing sectors such as healthcare, environment or food security.Peer reviewe

Label-Free Metabolic Classification of Single Cells in Droplets Using the Phasor Approach to Fluorescence Lifetime Imaging Microscopy.

Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of fluorescent tags and cell lysis followed by genomic profiling to identify the cellular heterogeneity. Identifying individual cells in a noninvasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet microfluidic technology together with the phasor approach to fluorescence lifetime imaging microscopy to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells-a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based noninvasive and label-free method to distinguish individual cells based on their metabolic states, which could be used as an upstream phenotypic platform to correlate with genomic statistics. © 2018 International Society for Advancement of Cytometry

Planar microfluidics - liquid handling without walls

The miniaturization and integration of electronic circuitry has not only made the enormous increase in performance of semiconductor devices possible but also spawned a myriad of new products and applications ranging from a cellular phone to a personal computer. Similarly, the miniaturization and integration of chemical and biological processes will revolutionize life sciences. Drug design and diagnostics in the genomic era require reliable and cost effective high throughput technologies which can be integrated and allow for a massive parallelization. Microfluidics is the core technology to realize such miniaturized laboratories with feature sizes on a submillimeter scale. Here, we report on a novel microfluidic technology meeting the basic requirements for a microfluidic processor analogous to those of its electronic counterpart: Cost effective production, modular design, high speed, scalability and programmability

Capacitive sensing of droplets for microfluidic devices based on thermocapillary actuation

The design and performance of a miniaturized coplanar capacitive sensor is presented whose electrode arrays can also function as resistive microheaters for thermocapillary actuation of liquid films and droplets. Optimal compromise between large capacitive signal and high spatial resolution is obtained for electrode widths comparable to the liquid film thickness measured, in agreement with supporting numerical simulations which include mutual capacitance effects. An interdigitated, variable width design, allowing for wider central electrodes, increases the capacitive signal for liquid structures with non-uniform height profiles. The capacitive resolution and time response of the current design is approximately 0.03 pF and 10 ms, respectively, which makes possible a number of sensing functions for nanoliter droplets. These include detection of droplet position, size, composition or percentage water uptake for hygroscopic liquids. Its rapid response time allows measurements of the rate of mass loss in evaporating droplets

Iris segmentation

The quality of eye image data become degraded particularly when the image is taken in the non-cooperative acquisition environment such as under visible wavelength illumination. Consequently, this environmental condition may lead to noisy eye images, incorrect localization of limbic and pupillary boundaries and eventually degrade the performance of iris recognition system. Hence, this study has compared several segmentation methods to address the abovementioned issues. The results show that Circular Hough transform method is the best segmentation method with the best overall accuracy, error rate and decidability index that more tolerant to ‘noise’ such as reflection

Fluid Patterning in a Cavity Array for High-Throughput Screening and Biotechnological Applications

Biomedical EngineeringOver the past few decades, fundamental biological understanding has advanced significantly with the help of experimental biotechnologies. Among them, synthetic biology as a rising research field has shown its capability for building new microorganisms from the scratch. In conjunction with synthetic biology, directed evolution techniques seem to be highly useful for industrial purposes such as the over-production of chemical products such as biofuels, which are expected to resolve global energy problems. However, it still requires a high-throughput screening technique and/or compartmentalized environments for cell sorting. In this thesis, two microfluidic technologies are described. First, a novel microdroplet trapping technology is developed that utilizes the difference of specific gravity between two immiscible fluids to offer simple and easy manipulation of microdroplets for time-traceable single microorganism analysis. Second, a high-throughput screening technology is developed by patterning fluid, in which individual Escherichia coli cells can be immobilized and cultured in a cavity array format. It is noted that the cavities were coated with parylene and bonded with another parylene layer to secure chemical compatibility. In addition, it was successfully demonstrated that the two technologies hold a high potential to enable not only high-throughput screening but also many biological experiments such as detection of cell-excreted products, long-term cell incubation, cell to cell communication, and detection of target molecules via a whole cell biosensor.ope

Electrowetting: from basics to applications

Electrowetting has become one of the most widely used tools for manipulating tiny amounts of liquids on surfaces. Applications range from 'lab-on-a-chip' devices to adjustable lenses and new kinds of electronic displays. In the present article, we review the recent progress in this rapidly growing field including both fundamental and applied aspects. We compare the various approaches used to derive the basic electrowetting equation, which has been shown to be very reliable as long as the applied voltage is not too high. We discuss in detail the origin of the electrostatic forces that induce both contact angle reduction and the motion of entire droplets. We examine the limitations of the electrowetting equation and present a variety of recent extensions to the theory that account for distortions of the liquid surface due to local electric fields, for the finite penetration depth of electric fields into the liquid, as well as for finite conductivity effects in the presence of AC voltage. The most prominent failure of the electrowetting equation, namely the saturation of the contact angle at high voltage, is discussed in a separate section. Recent work in this direction indicates that a variety of distinct physical effects¿rather than a unique one¿are responsible for the saturation phenomenon, depending on experimental details. In the presence of suitable electrode patterns or topographic structures on the substrate surface, variations of the contact angle can give rise not only to continuous changes of the droplet shape, but also to discontinuous morphological transitions between distinct liquid morphologies. The dynamics of electrowetting are discussed briefly. Finally, we give an overview of recent work aimed at commercial applications, in particular in the fields of adjustable lenses, display technology, fibre optics, and biotechnology-related microfluidic devices

Confinement of surface waves at the air-water interface to control aerosol size and dispersity

The precise control over the size and dispersity of droplets, produced within aerosols, is of great interest across many manufacturing, food, cosmetic, and medical industries. Amongst these applications, the delivery of new classes of high value drugs to the lungs has recently attracted significant attention from pharmaceutical companies. This is commonly achieved through the mechanical excitation of surface waves at the air liquid interface of a parent liquid volume. Previous studies have established a correlation between the wavelength on the surface of liquid and the final aerosol size. In this work, we show that the droplet size distribution of aerosols can be controlled by constraining the liquid inside micron-sized cavities and coupling surface acoustic waves into different volumes of liquid inside micro-grids. In particular, we show that by reducing the characteristic physical confinement size (i.e., either the initial liquid volume or the cavities’ diameters), higher harmonics of capillary waves are revealed with a consequent reduction of both aerosol mean size and dispersity. In doing so, we provide a new method for the generation and fine control of aerosols’ sizes distribution