Magnetophoretic-based microfluidic device for DNA Concentration

Abstract Nucleic acids serve as biomarkers of disease and it is highly desirable to develop approaches to extract small number of such genomic extracts from human bodily fluids. Magnetic particles-based nucleic acid extraction is widely used for concentration of small amount of samples and is followed by DNA amplification in specific assays. However, approaches to integrate such magnetic particles based capture with micro and nanofluidic based assays are still lacking. In this report, we demonstrate a magnetophoretic-based approach for target-specific DNA extraction and concentration within a microfluidic device. This device features a large chamber for reducing flow velocity and an array of μ-magnets for enhancing magnetic flux density. With this strategy, the device is able to collect up to 95 % of the magnetic particles from the fluidic flow and to concentrate these magnetic particles in a collection region. Then an enzymatic reaction is used to detach the DNA from the magnetic particles within the microfluidic device, making the DNA available for subsequent analysis. Concentrations of over 1000-fold for 90 bp dsDNA molecules is demonstrated. This strategy can bridge the gap between detection of low concentration analytes from clinical samples and a range of micro and nanofluidic sensors and devices including nanopores, nanocantilevers, and nanowires

Light-Responsive Polymeric Micellar Nanoparticles with Enhanced Formulation Stability.

Light-sensitive polymeric micelles have recently emerged as promising drug delivery systems for spatiotemporally controlled release of payload at target sites. Here, we developed diazonaphthoquinone (DNQ)-conjugated micellar nanoparticles that showed a change in polarity of the micellar core from hydrophobic to hydrophilic under UV light, releasing the encapsulated anti-cancer drug, doxetaxel (DTX). The micelles exhibited a low critical micelle concentration and high stability in the presence of bovine serum albumin (BSA) solution due to the hydrophobic and π-π stacking interactions in the micellar core. Cell studies showed enhanced cytotoxicity of DTX-loaded micellar nanoparticles upon irradiation. The enhanced stability would increase the circulation time of the micellar nanoparticles in blood, and enhance the therapeutic effectiveness for cancer therapy

Quantitative Analysis of the Membrane Affinity of Local Anesthetics Using a Model Cell Membrane.

Local anesthesia is a drug that penetrates the nerve cell membrane and binds to the voltage gate sodium channel, inhibiting the membrane potential and neurotransmission. It is mainly used in clinical uses to address the pain of surgical procedures in the local area. Local anesthetics (LAs), however, can be incorporated into the membrane, reducing the thermal stability of the membrane as well as altering membrane properties such as fluidity, permeability, and lipid packing order. The effects of LAs on the membrane are not yet fully understood, despite a number of previous studies. In particular, it is necessary to analyze which is the more dominant factor, the membrane affinity or the structural perturbation of the membrane. To analyze the effects of LAs on the cell membrane and compare the results with those from model membranes, morphological analysis and 50% inhibitory concentration (IC50) measurement of CCD-1064sk (fibroblast, human skin) membranes were carried out for lidocaine (LDC) and tetracaine (TTC), the most popular LAs in clinical use. Furthermore, the membrane affinity of the LAs was quantitatively analyzed using a colorimetric polydiacetylene assay, where the color shift represents their distribution in the membrane. Further, to confirm the membrane affinity and structural effects of the membranes, we performed an electrophysiological study using a model protein (gramicidin A, gA) and measured the channel lifetime of the model protein on the free-standing lipid bilayer according to the concentration of each LA. Our results show that when LAs interact with cell membranes, membrane affinity is a more dominant factor than steric or conformational effects of the membrane

Detection and quantification of methylation in DNA using solid-state nanopores.

Epigenetic modifications in eukaryotic genomes occur primarily in the form of 5-methylcytosine (5 mC). These modifications are heavily involved in transcriptional repression, gene regulation, development and the progression of diseases including cancer. We report a new single-molecule assay for the detection of DNA methylation using solid-state nanopores. Methylation is detected by selectively labeling methylation sites with MBD1 (MBD-1x) proteins, the complex inducing a 3 fold increase in ionic blockage current relative to unmethylated DNA. Furthermore, the discrimination of methylated and unmethylated DNA is demonstrated in the presence of only a single bound protein, thereby giving a resolution of a single methylated CpG dinucleotide. The extent of methylation of a target molecule could also be coarsely quantified using this novel approach. This nanopore-based methylation sensitive assay circumvents the need for bisulfite conversion, fluorescent labeling, and PCR and could therefore prove very useful in studying the role of epigenetics in human disease

Hydrodynamic loading and viscous damping of patterned perforations on microfabricated resonant structures

We examined the hydrodynamic loading of vertically resonating microfabricated plates immersed in liquids with different viscosities. The planar structures were patterned with focused ion beam, perforating various shapes with identical area but varying perimeters. The hydrodynamic loading of various geometries was characterized from resonant frequency and quality factor. In water, the damping increased linearly with the perimeter at 45.4 × 10 -3 Ns/m 2, until the perforation\u27s radius was 123% ± 13% of the depth of penetration of fluid\u27s oscillation. The added mass effect decreased with perforations and recovered to the level of un-perforated structures when the perforation\u27s radius became smaller than the depth of penetration. © 2012 American Institute of Physics

Direct Visualization of Single-Molecule Translocations through Synthetic Nanopores Comparable in Size to a Molecule

A nanopore is the ultimate analytical tool. It can be used to detect DNA, RNA, oligonucleotides, and proteins with submolecular sensitivity. This extreme sensitivity is derived from the electric signal associated with the occlusion that develops during the translocation of the analyte across a membrane through a pore immersed in electrolyte. A larger occluded volume results in an improvement in the signal-to-noise ratio, and so the pore geometry should be made comparable to the size of the target molecule. However, the pore geometry also affects the electric field, the charge density, the electro-osmotic flow, the capture volume, and the response time. Seeking an optimal pore geometry, we tracked the molecular motion in three dimensions with high resolution, visualizing with confocal microscopy the fluorescence associated with DNA translocating through nanopores with diameters comparable to the double helix, while simultaneously measuring the pore current. Measurements reveal single molecules translocating across the membrane through the pore commensurate with the observation of a current blockade. To explain the motion of the molecule near the pore, finite-element simulations were employed that account for diffusion, electrophoresis, and the electro-osmotic flow. According to this analysis, detection using a nanopore comparable in diameter to the double helix represents a compromise between sensitivity, capture volume, the minimum detectable concentration, and response time

Light-Responsive Polymeric Micellar Nanoparticles with Enhanced Formulation Stability

Light-sensitive polymeric micelles have recently emerged as promising drug delivery systems for spatiotemporally controlled release of payload at target sites. Here, we developed diazonaphthoquinone (DNQ)-conjugated micellar nanoparticles that showed a change in polarity of the micellar core from hydrophobic to hydrophilic under UV light, releasing the encapsulated anti-cancer drug, doxetaxel (DTX). The micelles exhibited a low critical micelle concentration and high stability in the presence of bovine serum albumin (BSA) solution due to the hydrophobic and π–π stacking interactions in the micellar core. Cell studies showed enhanced cytotoxicity of DTX-loaded micellar nanoparticles upon irradiation. The enhanced stability would increase the circulation time of the micellar nanoparticles in blood, and enhance the therapeutic effectiveness for cancer therapy