Molecular rheotaxis directs DNA migration and concentration against a pressure-driven flow.

In-line preconcentration techniques are used to improve the sensitivity of microfluidic DNA analysis platforms. The most common methods are electrokinetic and require an externally applied electric field. Here we describe a microfluidic DNA preconcentration technique that does not require an external field. Instead, pressure-driven flow from a fluid-filled microcapillary into a lower ionic strength DNA sample reservoir induces spontaneous DNA migration against the direction of flow. This migratory phenomenon that we call Molecular Rheotaxis initiates in seconds and results in a concentrated DNA bolus at the capillary orifice. We demonstrate the ease with which this concentration method can be integrated into a microfluidic total analysis system composed of in-line DNA preconcentration, size separation, and single-molecule detection. Paired experimental and numerical simulation results are used to delineate the parameters required to induce Molecular Rheotaxis, elucidate the underlying mechanism, and optimize conditions to achieve DNA concentration factors exceeding 10,000 fold.

A Simple Thermoplastic Substrate Containing Hierarchical Silica Lamellae for High-Molecular-Weight DNA Extraction.

An inexpensive, magnetic thermoplastic nanomaterial is developed utilizing a hierarchical layering of micro- and nanoscale silica lamellae to create a high-surface-area and low-shear substrate capable of capturing vast amounts of ultrahigh-molecular-weight DNA. Extraction is performed via a simple 45 min process and is capable of achieving binding capacities up to 1 000 000 times greater than silica microparticles.

Fabrication of rigid microstructures with thiol-ene-based soft lithography for continuous-flow cell lysis.

In this work, we introduce a method for the soft-lithography-based fabrication of rigid microstructures and a new, simple bonding technique for use as a continuous-flow cell lysis device. While on-chip cell lysis techniques have been reported previously, these techniques generally require a long on-chip residence time, and thus cannot be performed in a rapid, continuous-flow manner. Microstructured microfluidic devices can perform mechanical lysis of cells, enabling continuous-flow lysis; however, rigid silicon-based devices require complex and expensive fabrication of each device, while polydimethylsiloxane (PMDS), the most common material used for soft lithography fabrication, is not rigid and expands under the pressures required, resulting in poor lysis performance. Here, we demonstrate the fabrication of microfluidic microstructures from off-stoichiometry thiol-ene (OSTE) polymer using soft-lithography replica molding combined with a post-assembly cure for easy bonding. With finite element simulations, we show that the rigid microstructures generate an energy dissipation rate of nearly 10(7), which is sufficient for continuous-flow cell lysis. Correspondingly, with the OSTE device we achieve lysis of highly deformable MDA-MB-231 breast cancer cells at a rate of 85%, while a comparable PDMS device leads to a lysis rate of only 40%.

High-throughput particle separation and concentration using spiral inertial filtration.

A spiral inertial filtration (SIFT) device that is capable of high-throughput (1 ml/min), high-purity particle separation while concentrating recovered target particles by more than an order of magnitude is reported. This device is able to remove large fractions of sample fluid from a microchannel without disruption of concentrated particle streams by taking advantage of particle focusing in inertial spiral microfluidics, which is achieved by balancing inertial lift forces and Dean drag forces. To enable the calculation of channel geometries in the SIFT microsystem for specific concentration factors, an equivalent circuit model was developed and experimentally validated. Large particle concentration factors were then achieved by maintaining either the average fluid velocity or the Dean number throughout the entire length of the channel during the incremental removal of sample fluid. The SIFT device was able to separate MCF7 cells spiked into whole blood from the non-target white blood cells (WBC) with a recovery of nearly 100% while removing 93% of the sample volume, which resulted in a concentration enhancement of the MCF7 cancer cells by a factor of 14.

Enrichment of tumor-initiating breast cancer cells within a mammosphere-culture microdevice.

We report for the first time a microdevice that enables the selective enrichment, culture, and identification of tumor-initiating cells on native polydimethylsiloxane (PDMS). For nearly a decade, researchers have identified tumor-initiating breast cancer cells within heterogeneous populations of breast cancer cells by utilizing low-attachment serum-free culture conditions, which lead to the formation of spheroidal colonies (mammospheres) that are enriched for tumor-initiating cells. However, the utility of this assay has been limited by difficulties in combining this culture-plate-based technique with other cellular and molecular analyses. Integrating the mammosphere technique into a microsystem can enable it to be combined directly with a number of functions, such as cell sorting, drug screens, and molecular assays. In this work, we demonstrate mammosphere culture within a PDMS microdevice. We first prove that a native hydrophobic PDMS surface is as effective as commercial low-attachment plates at selectively promoting the formation of mammospheres. We then experimentally assess the PDMS microdevice. Time-lapse images of mammosphere formation within the microdevice show that mammospheres form from single cells or small clusters of cells. Following formation of the mammospheres, it is desirable to evaluate the cells within the spheroids for enrichment of tumor initiating cells. To perform assays such as this (which require the loading and rinsing of reagents) without flushing the cells (which are in suspension) from the device, the culture chamber is separated from a reagent reservoir by a commercially available microporous membrane, and thus reagents are exchanged between the reservoir and the culture chamber by diffusion only. Using this capability, we verify that the mammospheres are enriched for tumor initiating cells by staining aldehyde dehydrogenase activity, a cancer stem cell marker. To the best of our knowledge, this is the first assay that enables the direct observation of tumor-initiating cells within a suspended mammosphere.

A novel surface modification technique for forming porous polymer monoliths in poly(dimethylsiloxane).

A new method of surface modification is described for enabling the in situ formation of homogenous porous polymer monoliths (PPMs) within poly(dimethylsiloxane) (PDMS) microfluidic channels that uses 365 nm UV illumination for polymerization. Porous polymer monolith formation in PDMS can be challenging because PDMS readily absorbs the monomers and solvents, changing the final monolith morphology, and because PDMS absorbs oxygen, which inhibits free-radical polymerization. The new approach is based on sequentially absorbing a non-hydrogen-abstracting photoinitiator and the monomers methyl methacrylate and ethylene diacrylate within the walls of the microchannel, and then polymerizing the surface treatment polymer within the PDMS, entangled with it but not covalently bound. Four different monolith compositions were tested, all of which yielded monoliths that were securely anchored and could withstand pressures exceeding the bonding strength of PDMS (40 psi) without dislodging. One was a recipe that was optimized to give a larger average pore size, required for low back pressure. This monolith was used to concentrate and subsequently mechanical lyse B lymphocytes.

Influence of the semi-permeable membrane on the performance of dynamic field gradient focusing.

This paper is part of our continued effort to understand the underlying principles of dynamic field gradient focusing. In this investigation, we examined three problems associated with the use of a semi-permeable membrane. First, the influence of steric and ionic exclusion of current carrying ions through the membrane was examined. It was found that resistance to the transport of ions across the membrane resulted in a shallowing of the electric field profile and an increase in the size of the defocusing zone, which is where the slope of the electric field is reversed so that it disperses rather than concentrates solutes. These problems could be reduced by using a membrane with large pores relative to the size of the buffering ions and completely void of fixed charges. Next, a numerical simulation was used to investigate concentration polarization of protein onto the surface of the membrane. Due to the presence of a transverse electric field, species were pulled toward the membrane. If the membrane is restrictive to those species, a concentrated, polarized layer will form on the surface. The simulation showed that by decreasing the channel to a depth of 20 microm, the concentrated region next to the membrane could be reduced. Finally, it was found that changes in column volume due to loss of membrane structural integrity could be mitigated by including a porous ceramic support. The variation in peak elution times was decreased from greater than 20% to less than 3%.

Development of a membrane-less dynamic field gradient focusing device for the separation of low-molecular-weight molecules.

Dynamic field gradient focusing uses an electric field gradient generated by controlling the voltage profile of an electrode array to separate and concentrate charged analytes according to their individual electrophoretic mobilities. This study describes a new instrument in which the electrodes have been placed within the separation channel. The major challenge faced with this device is that when applied voltages to the electrodes are larger than the redox potential of water, electrolysis will occur, producing hydrogen ions (H+) plus oxygen gas on the anodes and hydroxide (OH(-)) plus hydrogen gas on the cathodes. The resulting gas bubbles and pH excursions can cause problems with system performance and reproducibility. An on-column, degassing system that can remove gas bubbles "on-the-fly" is described. In addition, the use of a high capacity, low-conductivity buffer to address the problem of the pH shift that occurs due to the production of H+ on the anodes is illustrated. Finally, the successful separation of three, low-molecular-weight dyes (amaranth, bromophenol blue and methyl red) is described.

Simultaneous separation of negatively and positively charged species in dynamic field gradient focusing using a dual polarity electric field.

Dynamic field gradient focusing (DFGF) utilizes an electric field gradient established by a computer-controlled electrode array to separate and concentrate charged analytes at unique axial positions. Traditionally, DFGF has been restricted to the analysis of negatively charged species due to limitations in the software of our voltage controller. This paper introduces a new voltage controller capable of operating under normal polarity (positive potentials applied to the electrode array) and reversed polarity (negative potentials applied to the electrode array) for the separation of negatively and positively charged analytes, respectively. The experiments conducted under normal polarity and reversed polarity illustrate the utility of the new controller to perform reproducible DFGF separations (elution times showing less than 1% run-to-run variation) over a wide pH range (3.08 to 8.5) regardless of the protein charge. A dual polarity experiment is then shown in which the separation channel has been divided into normal polarity and reversed polarity regions. This simultaneous separation of negatively charged R-phycoerythrin (R-PE) and positively charged cytochrome c (CYTC) within the same DFGF apparatus is shown.

Characterization of voltage degradation in dynamic field gradient focusing.

Dynamic field gradient focusing (DFGF) is an equilibrium gradient method that utilizes an electric field gradient to simultaneously separate and concentrate charged analytes based on their individual electrophoretic mobilities. This work describes the use of a 2-D nonlinear, numerical simulation to examine the impact of voltage loss from the electrodes to the separation channel, termed voltage degradation, and distortions in the electric field on the performance of DFGF. One of the design parameters that has a large impact on the degree of voltage degradation is the placement of the electrodes in relation to the separation channel. The simulation shows that a distance of about 3 mm from the electrodes to the separation channel gives the electric field profile with least amount of voltage degradation. The simulation was also used to describe the elution of focused protein peaks. The simulation shows that elution under constant electric field gradient gives better performance than elution through shallowing of the electric field. Qualitative agreement between the numerical simulation and experimental results is shown. The simulation also illustrates that the presence of a defocusing region at the cathodic end of the separation channel causes peak dispersion during elution. The numerical model is then used to design a system that does not suffer from a defocusing region. Peaks eluted under this design experienced no band broadening in our simulations. Preliminary experimental results using the redesigned chamber are shown.