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

Comparing Image Quality in Phase Contrast subμ\mu X-Ray Tomography -- A Round-Robin Study

How to evaluate and compare image quality from different sub-micrometer (sub$\mu$) CT scans? A simple test phantom made of polymer microbeads is used for recording projection images as well as 13 CT scans in a number of commercial and non-commercial scanners. From the resulting CT images, signal and noise power spectra are modeled for estimating volume signal-to-noise ratios (3D SNR spectra). Using the same CT images, a time- and shape-independent transfer function (MTF) is computed for each scan, including phase contrast effects and image blur ($\mathrm{MTF_{blur}}$). The SNR spectra and MTF of the CT scans are compared to 2D SNR spectra of the projection images. In contrary to 2D SNR, volume SNR can be normalized with respect to the object's power spectrum, yielding detection effectiveness (DE) a new measure which reveals how technical differences as well as operator-choices strongly influence scan quality for a given measurement time. Using DE, both source-based and detector-based sub$\mu$ CT scanners can be studied and their scan quality can be compared. Future application of this work requires a particular scan acquisition scheme which will allow for measuring 3D signal-to-noise ratios, making the model fit for 3D noise power spectra obsolete

Development of a PDMS Based Micro Total Analysis System for Rapid Biomolecule Detection

The emerging field of micro total analysis system powered by microfluidics is expected to revolutionize miniaturization and automation for point-of-care-testing systems which require quick, efficient and reproducible results. In the present study, a PDMS based micro total analysis system has been developed for rapid, multi-purpose, impedance based detection of biomolecules. The major components of the micro total analysis system include a micropump, micromixer, magnetic separator and interdigitated electrodes for impedance detection. Three designs of pneumatically actuated PDMS based micropumps were fabricated and tested. Based on the performance test results, one of the micropumps was selected for integration. The experimental results of the micropump performance were confirmed by a 2D COMSOL simulation combined with an equivalent circuit analysis of the micropump. Three designs of pneumatically actuated PDMS based active micromixers were fabricated and tested. The micromixer testing involved determination of mixing efficiency based on the streptavidin-biotin conjugation reaction between biotin comjugated fluorescent microbeads and streptavidin conjugated paramagnetic microbeads, followed by fluorescence measurements. Based on the performance test results, one of the micromixers was selected for integration. The selected micropump and micromixer were integrated into a single microfluidic system. The testing of the magnetic separation scheme involved comparison of three permanent magnets and three electromagnets of different sizes and magnetic strengths, for capturing magnetic microbeads at various flow rates. Based on the test results, one of the permanent magnets was selected. The interdigitated electrodes were fabricated on a glass substrate with gold as the electrode material. The selected micropumps, micromixer and interdigitated electrodes were integrated to achieve a fully integrated microfluidic system. The fully integrated microfluidic system was first applied towards biotin conjugated fluorescent microbeads detection based on streptavidin-biotin conjugation reaction which is followed by impedance spectrum measurements. The lower detection limit for biotin conjugated fluorescent microbeads was experimentally determined to be 1.9 x 106 microbeads. The fully integrated microfluidic system was then applied towards immuno microbead based insulin detection. The lower detection limit for insulin was determined to be 10-5M. The total detection time was 20 min. An equivalent circuit analysis was performed to explain the impedance spectrum results

Investigation of extractable organic compounds in deep-sea hydrothermal vent fluids along the Mid-Atlantic Ridge

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here by permission of Elsevier for personal use, not for redistribution. The definitive version was published in Geochimica et Cosmochimica Acta 156 (2015): 122-144, doi:10.1016/j.gca.2015.02.022.The possibility that deep-sea hydrothermal vents may contain organic compounds produced by abiotic synthesis or by microbial communities living deep beneath the surface has led to numerous studies of the organic composition of vent fluids. Most of these studies have focused on methane and other light hydrocarbons, while the possible occurrence of more complex organic compounds in the fluids has remained largely unstudied. To address this issue, the presence of higher molecular weight organic compounds in deep-sea hydrothermal fluids was assessed at three sites along the Mid-Atlantic Ridge that span a range of temperatures (51 to >360 °C), fluid compositions, and host-rock lithologies (mafic to ultramafic). Sample were obtained at several sites within the Lucky Strike, Rainbow, and Lost City hydrothermal fields. Three methods were employed to extract organic compounds for analysis, including liquid:liquid extraction, cold trapping on the walls of a coil of titanium tubing, and pumping fluids through cartridges filled with solid phase extraction (SPE) sorbents. The only samples to consistently yield high amounts of extractable organic compounds were the warm (51-91 °C), highly alkaline fluids from Lost City, which contained elevated concentrations of C8, C10, and C12 n-alkanoic acids and, in some cases, trithiolane, hexadecanol, squalene, and cholesterol. Collectively, the C8-C12 acids can account for about 15% of the total dissolved organic carbon in the Lost City fluids. The even-carbon-number predominance of the alkanoic acids indicates a biological origin, but it is unclear whether these compounds are derived from microbial activity occurring within the hydrothermal chimney proximal to the site of fluid discharge or are transported from deeper within the system. Hydrothermal fluids from the Lucky Strike and Rainbow fields were characterized by an overall scarcity of extractable dissolved organic compounds. Trace amounts of aromatic hydrocarbons including phenanthrenes and benzothiophene were the only compounds that could be identified as indigenous components of these fluids. Although hydrocarbons and fatty acids were observed in some samples, those compounds were likely derived from particulate matter or biomass entrained during fluid collection. In addition, extracts of some fluid samples from the Rainbow field were found to contain an unresolved complex mixture (UCM) of organic compounds. This UCM shared some characteristics with organic matter extracted from bottom seawater, suggesting that the organic matter observed in these samples might represent seawater-derived compounds that had persisted, albeit with partial alteration, during circulation through the hydrothermal system. While there is considerable evidence that Rainbow and Lost City vent fluids contain methane and other light hydrocarbons produced through abiotic reduction of inorganic carbon, we found no evidence for more complex organic compounds with an abiotic origin in the same fluids.This research was supported by the NSF Ocean Sciences directorate through grants MGG-OCE 0550800 to T.M.M. and MGG-OCE 0549829 to J.S.S. and C.R.G

Rotating bio-reactor cell culture apparatus

A bioreactor system is described in which a tubular housing contains an internal circularly disposed set of blade members and a central tubular filter all mounted for rotation about a common horizontal axis and each having independent rotational support and rotational drive mechanisms. The housing, blade members and filter preferably are driven at a constant slow speed for placing a fluid culture medium with discrete microbeads and cell cultures in a discrete spatial suspension in the housing. Replacement fluid medium is symmetrically input and fluid medium is symmetrically output from the housing where the input and the output are part of a loop providing a constant or intermittent flow of fluid medium in a closed loop

Integration of functional materials into microfluidic devices for fluidic control and sensing

165 p.El agua es una fuente clave para el buen estado de las personas y, en la naturaleza, es una fuente nutritiva esencial responsable del crecimiento de la vegetación. Por ello, la monitorización de la calidad del agua es de gran importancia para la sociedad. En esta tesis se pretende contribuir a un futuro donde sensores altamente autónomos y eficaces son capaces de medir y compartir la información de la calidad de nuestro medio ambiente, en particular, de las diferentes matrices de agua. En este sentido, se han desarrollado diferentes módulos para contribuir con bajo coste y tecnología de rápida fabricación a la monitorización continuada de la calidad del agua. Para conseguir reducir los costes asociados a la producción de componentes convencionales, se han implementado materiales inteligentes dentro de dispositivos microfluídicos para conseguir el control fluídico y sensórico

A novel high-throughput analytical method to quantify microplastics in water by flow cytometry

Microplastics (MPs) are pervasive contaminants with unclear toxicological impacts. Current research on MP pollution relies on low-throughput methodologies, which are time-consuming and cannot directly measure MP concentration in suspensions. This study presents a qualitative and quantitative flow cytometry-based method for analysing MPs in water, offering a faster and more sustainable alternative. The method involves density separation to remove interfering particles, UV irradiation to eliminate microorganisms, and filtration to remove particles above 100 µm. The sensitivity of the method for different types of MPs, such as polystyrene (PS), polypropylene (PP), polyvinyl chloride (PVC), polyethylene terephthalate (PET), and polyamide (PA) microbeads, ranges from 2 µg/L to 1 mg/L. For these MPs, good linearity was found in matrix-matched calibration where the most concentrated standard was 5 mg/L (R2 0.9820–0.9989) although the linear range can be larger (e.g. 42 mg MP/L for PS microbeads). The repeatability and reproducibility of the method for the model PS MP were <17.0% and 8.5%, respectively. The sample treatment method consisting of density separation and UV pretreatment, when carried out independently, led to 95.0% and 93.4% recoveries. The overall trueness of the optimized method for various sizes and compositions of microbeads is about 97%, according to validation supported by microscopy analysis. This method can substitute the traditional quantitative analytical approach based on counting microbeads with microscopy

Parallel manipulation of individual magnetic microbeads for lab-on-a-chip applications

Many scientists and engineers are turning to lab-on-a-chip systems for cheaper and high throughput analysis of chemical reactions and biomolecular interactions. In this work, we developed several lab-on-a-chip modules based on novel manipulations of individual microbeads inside microchannels. The first manipulation method employs arrays of soft ferromagnetic patterns fabricated inside a microfluidic channel and subjected to an external rotating magnetic field. We demonstrated that the system can be used to assemble individual beads (1-3µm) from a flow of suspended beads into a regular array on the chip, hence improving the integrated electrochemical detection of biomolecules bound to the bead surface. In addition, the microbeads can follow the external magnet rotating at very high speeds and simultaneously orbit around individual soft magnets on the chip. We employed this manipulation mode for efficient sample mixing in continuous microflow. Furthermore, we discovered a simple but effective way of transporting the microbeads on-chip in the rotating field. Selective transport of microbeads with different size was also realized, providing a platform for effective sample separation on a chip. The second manipulation method integrates magnetic and dielectrophoretic manipulations of the same microbeads. The device combines tapered conducting wires and fingered electrodes to generate desirable magnetic and electric fields, respectively. By externally programming the magnetic attraction and dielectrophoretic repulsion forces, out-of-plane oscillation of the microbeads across the channel height was realized. Furthermore, we demonstrated the tweezing of microbeads in liquid with high spatial resolutions by fine-tuning the net force from magnetic attraction and dielectrophoretic repulsion of the beads. The high-resolution control of the out-of-plane motion of the microbeads has led to the invention of massively parallel biomolecular tweezers.Ph.D.Committee Chair: Hesketh, Peter; Committee Member: Allen, Mark; Committee Member: Degertekin, Levent; Committee Member: Lu, Hang; Committee Member: Yoda, Minam