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A Lab-On-A-Chip Device For Embryo Production: Through In Vitro Oocyte Maturation To In Vitro Fertilization
Behnaz Sadeghzadeh, PhD Student, Medical University of Shahid Beheshti
oocyte maturation is the most important stage because specifies the subsequent successful fertilization, zygote formation and suitable embryo development. Application of the Lab-on-Chip system in reproductive biology provides new possibilities for the development of techniques available for the assessment of the developmental competency of mammalian oocytes/embryos. The major object of this research is improvment of oocyte maturation by use of new microfluidic device which design and constructed in present investigation and consequently improvement of embryo development.
For this purpose, dynamic culture device was generated by soft lithography method of polydimethylsiloxane (PDMS). To oocyte collection, we used adult NMRI female mice. After ovarian stimulation, total 995 immature oocytes lay in static and dynamic in vitro maturation culture media randomly for 24 hours. Then, MII stage oocytes were inseminated by sperm and all two-pronuclear (PN) zygotes followed to 4-cell stage. The percentage of matured oocytes (MII), PN and 4-cell stage embryos, in dynamic culture system were significantly higher than static system (p<0.01).
In conclusion, a portable and user-friendly micro?uidic device has been designed and constructed for immature oocyte culture in the present investigation which improved in vitro immature oocyte and embryo development considerably in comparison with traditional static culture system.
Particle extraction in plug-based microfluidics
Jan-Niklas Schönberg, Research Assistant, IMTEK
Plug-based microfluidic devices represent a fast and simple and thus attractive platform for biological applications. [1] In this field of application aqueous plugs are generated in an oil-or fluorocarbon based carrier fluid. The plugs function thus as small nanocontainers in which (bio-)chemical reactions can be performed.
An interesting aspect has been to perform biological assay reactions at the surfaces of magnetic beads and then separate the bead from the medium by magnetofection. Yet, the extraction of particles out of those plugs remains challenging. Extraction, however, becomes crucial when biological assays are performed, as all assays require washing steps, which cannot be conducted easily, when extraction is not possible. [2]
The main issue in this respect is the high interfacial energy of the plug. State of the art designs address this issue, by breaking the plug interface with a co-flowing stream. However, the compartmentalization which is one key idea of plug-based microfluidics is lost with those approaches. [2, 3]
We suggest a design, with a hydrophilic patch in a hydrophobic channel. The hydrophilic patch is easily wetted by the aqueous plug and thereby eliminates the interface, so that the particles can be magnetically attracted to the surface of the patch.
Butterfly shaped microfluidic device for investigating the invasion and chemotaxis of estrogen dependent and independent breast cancer cells
Ozlem Yesil Celiktas, Assoc.Prof.Dr., Ege University
Microfluidics-based lab-on-a-chips provide physiologically relevant settings and offer a more realistic microenvironment [1,2] thereby providing improved biological models and functionality while reducing required volumes and cost [3]. Matrigel is capable of fully supporting cell differentiation and providing a real 3D cellular microenvironment [4]. Studies indicate that purified components from rosemary such as carnosic acid display significant growth inhibitory activity on a variety of cancers, especially human breast cancers [5, 6].
The aim of this study was to demonstrate the effect of carnosic acid towards human breast cancer cells (MCF-7 and MDA-MB231) and healthy breast cells (MCF-10) along with investigation of the diffusion phenomena in butterfly-shaped microchip. IC50 values of carnosic acid for MDA-MB 231 and MCF-7 were 15 ?g/mL and 20 ?g/mL, respectively. Diffusion coefficient and cellular uptake of carnosic acid were determined experimentally by making use of a two-chambered diffusion system and 2-D diffusion equation model based on the Fick's 2nd law was generated. The results indicated that carnosic acid exhibited a 2-fold higher inhibition in MDA-MB-231 compared to the MCF-7 in 2D culture, whereas a 6-fold higher inhibition was observed in MCF-7 compared to MDA-MB-231 in the microchip, indicating the importance of mimicry in a physiologically relevant matrix.
Immobilization of beta-glucosidase in silica-based hydrogels for biotransformation of natural products in microchannel reactor
Seref Akay, Research Assistant, Ege University
Enzymes are natural catalysts used for the synthesis of useful compounds in an environmentally friendly way (1). Due to problems in their stability, cost and efficiency of reactions there are few enzymatic processes have been commercialized. Thus, there have been requirements for innovation in process engineering particularly for enzymatic reactions. Inorganic sol-gels can be used for many different applications within the area of bioengineering mainly for immobilization of biomolecules (2). During the sol-gel process, a liquid “sol” containing a precursor for the matrix formation, solvents, catalyst, and additives undergoes a gelation, which forms a porous solid structure (3). In the past few years, microreactor technology has demonstrated numerous advantages in different fields of application (4). Microreactors are generally described as miniaturized reaction systems fabricated by using methods of microtechnology and precision engineering (5). In this study, relative activity of enzyme immobilized in TEOS and EGMS derived monoliths 68% and 60% respectively. Vmax values of free and immobilized enzyme in TEOS derived monolith were determined as 0.018 U/mg and 0.009 U/mg and Km values were determined 30 mM and 79 mM, respectively. Ginseng RB1 saponin was hydrolyzed in the microchannel and conversion yield was obtained as 37.88% after 5h with 0.002 mg enzyme while it takes more than 96 h with higher amount of free enzyme.
Rapid electrochemical impedance spectroscopy for protein detection in Lab-on-a-Chip devices
Tamas Pardy, Student, Tallinn University of Technology
Lab-on-a-Chip devices form an ever-growing segment of the IVD market, and there is a pronounced need for reliable, rapid detection methods for various biomarkers, especially label-free methods. Electrochemical impedance spectroscopy (EIS) in liquids means the determination of passive electrical properties of ingredients in a continuous or segmented flow of fluids. More specifically, it means label-free discovery, counting and characterization of particles, mostly concentrations of various ionized molecules in chemical solutions or biological particles in fluids. We present results in rapid solution impedance spectroscopy to detect protein interactions (antibody-antigen) in human serum. The experimental setup was based on screen-printed electrodes (Dropsens DRP-C220AT) and cuvettes (Brandtech 7592 00), and the serum was buffered in PBS. Two different serum-antibody mixtures were created and impedance spectra recorded over 15 minutes to determine whether the system was capable of discerning solution compositions. Significant shifts in impedance magnitude and phase were detected in the 10 Hz-100 kHz range, with a clear difference between peaks for both solutions. Although in the described experiment, the solution was static, this setup could be adapted to be part of a flow cell.
A combined cell placement and migration assay device for cancer cell anti-migration drug screening
Colin Hisey, Student, The Ohio State University
Highly migratory cancer cells often lead to recurrence and secondary tumors and are responsible for high mortality rates in cancers such as glioblastoma multiforme. Current treatments focus on resection and destruction of the primary tumor with radiation as well as chemical targeting of proliferative cells. Recently, drugs which specifically target highly migratory cells have been developed, but robust in vitro platforms for quantifying the efficacy of these drugs are still lacking. An ideal platform would provide high repeatability and predictability to automate quantification as well as mimic the migratory behavior of in vivo cells, all while minimizing reagent use. We have developed a microfluidic device capable of hydrodynamically trapping cancer cells from solution onto 10 and 15 micron polystyrene and polycaprolactone lines which encourage 1D migratory behavior based on the cells’ tendency to follow topographical cues. This device enables the use of a variety of other polymers via spin-dewetting/stamping that can be tuned to mimic the stiffness and/or surface chemistry of in vivo, fiber-like structures. Furthermore, its design will allow precise microfluidic assessment of potential drug targets to limit cancer cell migration.
Lighting up micromotors with quantum dots for smart chemical sensing
Beatriz Jurado Sanchez, Postdoctoral Researcher, University of Alcala
A new “on-the-fly” chemical optical detection strategy based on the incorporation of fluorescence CdTe quantum dots (QDs) on the surface of self-propelled tubular micromotors is presented. The preparation protocol is highly versatile, allowing for the incorporation of QDs with different emission wavelengths for the simultaneous detection of multiple analytes. The potential of such nanocrystal-functionalized microengines was evaluated for the detection of mercury under the presence of other heavy metal ions in biological samples. The motion-accelerated binding of trace Hg to the QDs selectively quenches the fluorescence emission and leads to an effective discrimination between different mercury species and other co-existing ions. The prolonged and efficient propulsion of the microengines allowed for its direct application in raw biological samples such saliva and blood. This new QDs-based microsensors can be readily as mobile sensor for “on-chip” application, avoiding thus the use of external forces to move fluids (as pumps or electrophoresis) and achieving further miniaturization.
Finite element modelling for the optimization of temperature control in Lab-on-a-Chip devices
Tamas Pardy, Student, Tallinn University of Technology
Some of the assays actively developed in a Lab-on-a-Chip format require temperature control, mostly heating to a specified range for the duration of the assay. In a highly portable system with limited power supply, heating microliters of liquids poses technical challenges, which may be costly to overcome solely by experimental means. The way computer-aided design helps in rapid prototyping, so does finite element modelling help in validating the functionality of these prototypes in silico, saving time and costs. The model we present was developed for the geometric optimization of Lab-on-a-Chip devices and tested for experimental setups emulating the targeted device format. The model is presented for multiple temperature control scenarios, i.e. heaters and device geometries.
Technical development of a biochip microfluidic immunoassay for out-of-laboratory analysis of food allergens
Niamh O'Reilly, Assay Development Scientist, Institute of Technology Tallaght
MiCRA Biodiagnostics in partnership with Allogen Biotech Limited is overseeing the technical and commercial development of electronic biochip sensors for rapid on-site detection of allergens in food manufacturing processes.
The food industry is responding to public and regulatory demands for more informative product labeling with respect to the health risks associated with allergen contaminants such as peanut and gluten proteins.
Allergen biochip technology integrates a patented microfluidics mechanism with immuno-responsive sensors that detect and quantify a target allergen. Established micro-well immunoassay techniques such as ELISA were translated onto the MiCRA biochip platform. It is intended the biochip prototypes will perform point-of-site testing at reduced cost with minimal time delay in production.
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