This paper describes a multiwell biochip for simultaneous parallel recording of ion current through transmembrane pores reconstituted in planar lipid bilayer arrays. Use of a thin poly(p-xylylene) (parylene) film having micrometer-sized apertures (phi=15-50 microm, t=20 microm) led to formation of highly stable bilayer lipid membranes (BLMs) for incorporation of transmembrane pores; thus, a large number of BLMs could be arrayed without any skillful technique. We optically confirmed the simultaneous formation of BLMs in a 5x5 matrix, and in our durability test, the BLM lasted more than 15 h. Simultaneous parallel recording of alamethicin and gramicidin transmembrane pores in multiple contiguous recording sites demonstrated the feasibility of high-throughput screening of transmembrane ion currents in artificial lipid bilayers.
We present a microarray system that enables simultaneous monitoring of multiple ionic currents through transmembrane alpha-hemolysin nanopores arrayed at bilayer lipid membranes. We applied the self-assembling ability of lipid molecules interfaced between an aqueous solution and organic solvent to induce bilayer membrane formation at a microfluidic device; the device consists of a hydrophobic polymer film that serves to suspend the lipid-containing solvent at micrometer-sized apertures as well as to separate the aqueous solution into two chambers. In this study, we confirmed that expeditious and reproducible bilayer formation is realized by control of the composition of the solvent, a mixture of n-decane and 1-hexanol, which permits simultaneous incorporation of the alpha-hemolysin nanopores to the membrane array. Monitoring the eight wells on the array at once, we obtained a maximum of four relevant, synchronous signals of translocating ionic current through the nanopores. The system was also able to detect translocation events of nucleic acid molecules through the pore via the profile of a blocked current, promising its potential for high-throughput applications.
Red blood cells (RBCs) are deformable and flow through vessels narrower than their own size. Their deformability is most stringently challenged when they cross micrometer-wide slits in the spleen. In several inherited or acquired RBC disorders, blockade of small vessels by stiff RBCs can trigger organ damage, but a functional spleen is expected to clear these abnormal RBCs from the circulation before they induce such complications. We analyzed flow behavior of RBCs in a microfluidic chip that replicates the mechanical constraints imposed on RBCs as they cross the human spleen. Polymer microchannels obtained by soft lithography with a hydraulic diameter of 25 lm drove flow into mechanical filtering units where RBCs flew either slowly through 5-to 2-lm-wide slits or rapidly along 10-lm-wide channels, these parallel paths mimicking the splenic microcirculation. Stiff heated RBCs accumulated in narrow slits seven times more frequently than normal RBCs infused simultaneously. Stage-dependent retention of Plasmodium falciparuminfected RBCs was also observed in these slits. We also analyzed RBCs from patients with hereditary spherocytosis and observed retention for those having the most altered mechanical properties as determined by ektacytometry. Thus, in keeping with previous observations in vivo and ex vivo, the chip successfully discriminated poorly deformable RBCs based on their distinct mechanical properties and on the intensity of the cell alteration. Applications to the exploration of the pathogenesis of malaria, hereditary spherocytosis, sickle cell disease and other RBC disorders are envisioned.
Electrical detection based on single nanopores is an efficient tool to detect biomolecules, particles and study their morphology. Nevertheless the surface of the solid-state membrane supporting the nanopore should be better controlled. Moreover, nanopore should be integrated within microfluidic architecture to facilitate control fluid exchanges. We built a reusable microfluidic system integrating a decorated membran, rendering the drain and refill of analytes and buffers easier. This process enhances strongly ionic conductance of the nanopore and its lifetime. We highlight the reliability of this device by detecting gold nanorods and spherical proteins.
We present a micro fluidic chip for parallel ion channel recording in a large array of artificial planar lipid bilayer membranes. To realize a composite structure that features an array of recording wells with free-standing microapertures for lipid bilayer reconstitution, the device was fabricated by the hybrid stereolithography technology, in which a Parylene film with pre-formed microapertures was inserted during the rapid stereolithography process. We designed and tested a hybrid chip that has 96 (12x8) addressable recording wells to demonstrate recording of ion channel current in high-throughput manner. Measurement was done by sequentially moving the recording electrode, and, as a result, the channel current of model membrane protein was detected in 44 wells out of 96. We also showed that this hybrid fabrication process was capable of integrating micropatterned electrodes suitable for automated recording. These results support the efficiency of our present architecture of the parallel ion channel recording chip toward realization of the high-throughput screening of ion channel proteins in the artificial lipid bilayer system.
In this paper, a new nonlinear control strategy is proposed for a permanent-magnet salient-pole synchronous motor. This control strategy simultaneously achieves accurate torque control and copper losses minimization without recurring to an internal current loop nor to any feedforward compensation. It takes advantage of the rotor saliency by allowing the current (i d ) to have nonzero values. This, in turn, allows us to increase the power factor of the machine and to raise the maximum admissible torque. We apply input-output linearization techniques where the inputs are the stator voltages and the outputs are the torque and a judiciously chosen new output. This new output insures a well-defined relative degree and is linked to the copper losses in such a way that, when forced to zero, it leads to maximum machine efficiency. The performance of our nonlinear controller is demonstrated by a real-time implementation using a digital signal processor (DSP) chip on a permanent-magnet salient-pole synchronous motor with sinusoidal flux distribution. The results are compared to the ones obtained with a scheme which forces the i d current to zero.
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