We present the use of electrokinetic concentration to realize a continuous signal amplification scheme that increases the
sensitivity of various homogeneous mobility shift assays. By simultaneously concentrating and separating reacted and unreacted
species (with different mobilities) in this device, we can perform sensitive, quantitative and ratiometric measurement of
target biomarkers. Using this platform, we improved the sensitivity of aptamer affinity probe capillary electrophoresis to
achieve pM detection limit of IgE and HIV-RT in simple buffer and serum sample. As another application, we can perform
multiplexed detect of kinase activity from lysate concentrations corresponding to lysing a single cell.
We introduce an integrated microfluidic device consisting of a biomolecule concentrator and a microdroplet generator, which enhances the limited sensitivity of low-abundance enzyme assays by concentrating biomolecules before encapsulating them into droplet microreactors. We used this platform to detect ultralow levels of matrix metalloproteinases (MMPs) from diluted cellular supernatant and showed that it significantly (~10-fold) reduced the time required to complete the assay and the sample volume used.
In this work we investigate concentration-enhanced enzyme activity assays in nanofluidic biomolecule concentrator chips which can be used to detect and study very low abundance enzymes from cell lysates and other low volume, low concentration samples. A mathematical model is developed for a mode of operation of the assay (J. H. Lee, B. D. Cosgrove, D. A. Lauffenburger and J. Han, J. Am. Chem. Soc., 2009, 131, 10340-10341) in which enzyme and substrate are concentrated together into a plug on chip which results in a non-linear enhancement of the reaction rate. Two reaction phases, an initial quadratic enzyme-limited phase and a later, linear substrate-limited phase, are predicted and then verified with experiments. It is determined that, in most practical situations, the reaction eventually enters a substrate-limited phase, therefore mitigating the concern for non-specific reactions of biosensor substrates with off-target enzymes in such assays. We also use this mode to demonstrate a multiplexed concentration-enhanced enzyme activity assay. We then propose and demonstrate a new device and mode of operation, in which only the enzyme is concentrated and then mixed with a fixed amount of substrate in an adjacent picolitre-scale reaction chamber. This mode results in a linear enhancement of the reaction rate and can be used to perform mechanistic studies on low abundance enzymes after concentrating them into a plug on chip.
In this work, we describe a microfluidic device for the encapsulation of electrokinetically concentrated biomolecules in microdroplets and microparticles. We demonstrate the encapsulation of molecules and nanoparticles such as quantum dots into water-in-oil droplets and porous gel particles with on-the-fly adjustable concentrations by combining electrokinetic trapping and two-phase droplet generation in a single chip. Once in the droplets, the molecules and nanoparticles can then undergo chemical reactions or be delivered anywhere on the chip while maintaining programmed concentrations without dispersion loss over long time and distance scales.
We present two fast and generic methods for the fabrication of polymeric microfluidic systems using electron beam lithography: one that employs spatially varying electron-beam energy to expose to different depths a negative electron-beam resist, and another that employs a spatially varying electron-beam dose to differentially expose a bi-layer resist structure. Using these methods, we demonstrate the fabrication of various microfluidic unit structures such as microchannels of a range of geometries and also other more complex structures such as a synthetic gel and a chaotic mixer. These are made without using any separate bonding or sacrificial layer patterning and etching steps. The schemes are inherently simple and scalable, afford high resolution without compromising on speed and allow post CMOS fabrication of microfluidics. We expect them to prove very useful for the rapid prototyping of complete integrated micro/nanofluidic systems with sense and control electronics fabricated by upstream processes.
We present a novel flow-type single cell electroporation (SCE) system for low voltage electroporation of biological cells. We have used a microfabricated silicon sense-porate aperture to detect and identify a cell by its impedance and then apply the optimum electric field on it. Incorporation of a fluorescent dye into mouse embryo fibroblast NIH-3T3 cells has been demonstrated with a cell survival rate or viability much higher than conventional macro-electroporators. This paper reports the principle, design and implementation of this system and the experimental results obtained using it.
Sarkar A, Mitra B, Shastry A, Wadia S, Mulherkar R, Lal R. A single cell electroporation system, in 12th International Workshop on the Physics of Semiconductor Devices (IWPSD-2003). Chennai, India ; 2003.