Automated microfluidic compact disc (CD) cultivation system of Caenorhabditis elegans

Microfabricated platforms for automated cultivation and behavioral observation of animals would be a valuable tool in physiological and genetic studies. We report an automated microfluidic compact disc (CD) system specialized for cultivating and monitoring Caenorhabditis elegans, proficient in automated feeding, waste removal and live-animal microscopy. The automated CD cultivation platform contains cultivation, nutrient, and waste chamber, channels connecting the chambers, and venting holes. The feeding and waste removal processes are achieved automatically using centrifugal force driven fluidics. Compared to other microfluidic technologies for moving small amounts of fluidic or suspended particles from site to site, this centrifuge-based system is well suited for functions such as flow sequencing, mixing, capillary measuring, and switching. Those functions can be implemented by exploiting centrifugal, coriolis and capillary forces combined with specific microfluidic network. Thus, the CD cultivating system has the capacity to incorporate diverse molecular, biochemical, and pharmacological assays for real-time physiological and behavioral tests of live-animals. This compact and fully automated microfabricated platform is designed to address the unique requirements of space biology due to the stringent limitations on power, size, weight, and crew intervention.close404

Characterization of DNA hybridization kinetics in a microfluidic flow channel.

In this investigation we report on the influence of volumetric flow rate, flow velocity, complementary DNA concentration, height of a microfluidic flow channel and time on DNA hybridization kinetics. A syringe pump was used to drive Cy3-labeled target DNA through a polydimethylsiloxane (PDMS) microfluidic flow channel to hybridize with immobilized DNA from the West Nile Virus. We demonstrate that a reduction of channel height, while keeping a fixed volumetric flow rate or a fixed flow velocity, enhances mass transport of target DNA to the capture probes. Compared to a passive hybridization, the DNA hybridization in the microfluidic flow channel generates higher fluorescence intensities for lower concentration of target DNA during the same fixed period of time. Within a fixed 2 min time period the fastest DNA hybridization at a 50 pM concentration of target DNA is achieved with a continuous flow of target DNA at the highest flow rate and the lowest channel height

Cell lysis on a microfluidic CD (compact disc

Cell lysis was demonstrated on a microfluidic CD (Compact Disc) platform. In this purely mechanical lysis method, spherical particles (beads) in a lysis chamber microfabricated in a CD, cause disruption of mammalian (CHO-K1), bacterial (Escherichia coli), and yeast (Saccharomyces cerevisiae) cells. Interactions between beads and cells are generated in the rimming flow established inside a partially filled annular chamber in the CD rotating around a horizontal axis. To maximize bead-cell interactions in the lysis chamber, the CD was spun forward and backwards around this axis, using high acceleration for 5 to 7 min. Investigation on inter-particle forces (friction and collision) identified the following parameters; bead density, angular velocity, acceleration rate, and solid volume fraction as having the most significant contribution to cell lysis. Cell disruption efficiency was verified either through direct microscopic viewing or measurement of the DNA concentration after cell lysing. Lysis efficiency relative to a conventional lysis protocol was approximately 65%. In the long term, this work is geared towards CD based sample-to-answer nucleic acid analysis which will include cell lysis, DNA purification, DNA amplification, and DNA hybridization detection

Centrifugal Microfluidics with Integrated Sensing Microdome Optodes for Multiion Detection

An array of four sensing microdome optodes (potassium, sodium, calcium, and chloride) was incorporated into a centrifugal microfluidics platform to obtain a multiion analysis system. The behavior of each sensing microdome was in good agreement with a theoretical model describing the response. The selectivity of each optode over common interfering ions was established and was used to identify calibrant solutions that can be employed for the simultaneous calibration of all four optodes without significant cross-interference. The microfluidic platform was designed to facilitate both three-point calibration of the optodes and triplicate analysis of a sample within a single run, which increases the accuracy of the determination. The optimized microfluidic system was used to determine simultaneously the concentration of potassium, sodium, calcium, and chloride in aquarium water (with the composition of Lake Tanganyika water) with less than 6% error. The simple process of fabrication of these microdomes and their incorporation into a centrifugal microfluidic platform should facilitate the development of portable ion-sensing analysis systems. ??? 2007 American Chemical Society.close171

A low-cost, disposable card for rapid polymerase chain reaction

A low-cost, disposable card for rapid polymerase chain reaction (PCR) was developed in this work. Commercially available, adhesive-coated aluminum foils and polypropylene films were laminated with structured polycarbonate films to form microreactors in a card format. Ice valves [1] were employed to seal the reaction chambers during thermal cycling and a Peltier-based thermal cycler was configured for rapid thermal cycling and ice valve actuation. Numerical modeling was conducted to optimize the design of the PCR reactor and investigate the thermal gradient in the reaction chamber in the direction of sample thickness. The PCR reactor was experimentally characterized by using thin foil thermocouples and validated by a successful amplification of 10 copy of E. coli tuf gene in 27 min.close151

Gravity Force Transduced by the MEC-4/MEC-10 DEG/ENaC Channel Modulates DAF-16/FoxO Activity in Caenorhabditis elegans

The gravity response is an array of behavioral and physiological plasticity elicited by changes in ambient mechanical force and is an evolutionarily ancient adaptive mechanism. We show in Caenorhabditis elegans that the force of hypergravity is translated into biological signaling via a genetic pathway involving three factors: the degenerin/epithelial Na+ channel (DEG/ENaC) class of mechanosensory channels of touch receptor neurons, the neurotransmitter serotonin, and the FoxO transcription factor DAF-16 known to regulate development, energy metabolism, stress responses, and aging. After worms were exposed to hypergravity for 3 hr, their muscular and neuronal functions were preserved, but they exhibited DAF-16∷GFP nuclear accumulation in cells throughout the body and accumulated excess fat. Mutations in MEC-4/MEC-10 DEG/ENaC or its partners MEC-6, MEC-7, and MEC-9 blocked DAF-16∷GFP nuclear accumulation induced by hypergravity but did not affect DAF-16 response to other stresses. We show that exogenous serotonin and the antidepressant fluoxetine can attenuate DAF-16∷GFP nuclear accumulation in WT animals exposed to hypergravity. These results reveal a novel physiological role of the mechanosensory channel, showing that the perception of mechanical stress controls FoxO signaling pathways and that inactivation of DEG/ENaC may decouple mechanical loading and physiological responses

CD (compact disc)-based DNA hybridization and detection

A DNA hybridization and detection unit was developed for a compact disc (CD) platform. The compact disc was used as the fluidic platform for sample and reagent manipulation using centrifugal force. Chambers for reagent storage and conduits for fluidic functions were replicated from polydimethylsiloxane (PDMS) using an SU-8 master mold fabricated with a 2-level lithography process we developed specially for the microfluidic structures used in this work. For capture probes, we used self-assembled DNA oligonucleotide monolayers (SAMs) on gold pads patterned on glass slides. The PDMS flow cells were aligned with and sealed against glass slides to form the DNA hybridization detection units. Both an enzymatic-labeled fluorescence technique and a bioluminescent approach were used for hybridization detection. An analytical model was introduced to quantitatively predict the accumulation of hybridized targets. The flow-through hybridization units were tested using DNA samples (25-mers) of different concentrations down to 1 pM and passive assays (no flow), using samples of the same concentrations, were performed as controls. At low concentrations, with the same hybridization time, a significantly higher relative fluorescence intensity was observed in both enzymatic and bioluminescent flow-through assays compared to the corresponding passive hybridization assays. Besides the fast hybridization rate, the CD-based method has the potential for enabling highly automated, multiple and self-contained assays for DNA detection

Integration of microcolumns and microfluidic fractionators on multitasking centrifugal microfluidic platforms for the analysis of biomolecules

This work demonstrates the development of microfluidic compact discs (CDs) for protein purification and fractionation integrating a series of microfluidic features, such as microreservoirs, microchannels, and microfluidic fractionators. The CDs were fabricated with polydimethylsiloxane (PDMS), and each device contained multiple identical microfluidic patterns. Each pattern employed a microfluidic fractionation feature with operation that was based on the redirection of fluid into an isolation chamber as a result of an overflow. This feature offers the advantage of automated operation without the need for any external manipulation, which is independent of the size and the charge of the fractionated molecules. The performance of the microfluidic fractionator was evaluated by its integration into a protein purification microfluidic architecture. The microfluidic architecture employed a microchamber that accommodated a monolithic microcolumn, the fractionator, and an isolation chamber, which was also utilized for the optical detection of the purified protein. The monolithic microcolumn was polymerized "in situ" on the CD from a monolith precursor solution by microwave-initiated polymerization. This technique enabled the fast, efficient, and simultaneous polymerization of monoliths on disposable CD microfluidic platforms. The design of the CD employed allows the integration of various processes on a single microfluidic device, including protein purification, fractionation, isolation, and detection.close91