Laser-based directed release of array elements for efficient collection into targeted microwells.

A cell separation strategy capable of the systematic isolation and collection of moderate to large numbers (25-400) of single cells into a targeted microwell is demonstrated. An array of microfabricated, releasable, transparent micron-scale pedestals termed pallets and an array of microwells in poly(dimethylsiloxane) (PDMS) were mated to enable selective release and retrieval of individual cells. Cells cultured on a pallet array mounted on a custom designed stage permitted the array to be positioned independently of the microwell locations. Individual pallets containing cells were detached in a targeted fashion using a pulsed Nd:YAG laser. The location of the laser focal point was optimized to transfer individual pallets to designated microwells. In a large-scale sort (n = 401), the accuracy, defined as placing a pallet in the intended well, was 94% and the collection efficiency was 100%. Multiple pallets were observed in only 4% of the targeted wells. In cell sorting experiments, the technique provided a yield and purity of target cells identified by their fluorescence signature of 91% and 93%, respectively. Cell viability based on single-cell cloning efficiency at 72 h post collection was 77%.

Micromolded arrays for separation of adherent cells.

We present an efficient, yet inexpensive, approach for isolating viable single cells or colonies from a mixed population. This cell microarray platform possesses innovations in both the array manufacture and the manner of target cell release. Arrays of microwells with bases composed of detachable concave elements, termed microrafts, were fabricated by a dip-coating process using a polydimethylsiloxane mold as the template and the array substrate. This manufacturing approach enabled the use of materials other than photoresists to create the array elements. Thus microrafts possessing low autofluorescence could be fabricated for fluorescence-based identification of cells. Cells plated on the microarray settled and attached at the center of the wells due to the microrafts' concavity. Individual microrafts were readily dislodged by the action of a needle inserted through the compliant polymer substrate. The hard polymer material (polystyrene or epoxy resin) of which the microrafts were composed protected the cells from damage by the needle. For cell analysis and isolation, cells of interest were identified using a standard inverted microscope and microrafts carrying target cells were dislodged with the needle. The released cells/microrafts could be efficiently collected, cultured and clonally expanded. During the separation and collection procedures, the cells remained adherent and provided a measure of protection during manipulation, thus providing an extremely high single-cell cloning rate (>95%). Generation of a transfected cell line based on expression of a fluorescent protein demonstrated an important application for performing on-chip cell separations.

Microdevice to capture colon crypts for in vitro studies.

There is a need in biological research for tools designed to manipulate the environment surrounding microscopic regions of tissue. In the current work, a device for the oriented capture of an important and under-studied tissue, the colon crypt, has been designed and tested. The objective of this work is to create a BioMEMs device for biological assays of living colonic crypts. The end goal will be to subject the polarized tissue to user-controlled fluidic microenvironments in a manner that recapitulates the in vivo state. Crypt surrogates, polymeric structures of similar dimensions and shape to isolated colon crypts, were used in the initial design and testing of the device. Successful capture of crypt surrogates was accomplished on a simple device composed of an array of micron-scale capture sites that enabled individual structures to be captured with high efficiency (92+/-3%) in an ordered and properly oriented fashion. The device was then evaluated using colon crypts isolated from a murine animal model. The capture efficiency attained using the fixed biologic sample was 37+/-5% due to the increased variability of the colon crypts compared with the surrogate structures, yet 94+/-3% of the captured crypts were properly oriented. A simple approach to plug the remaining capture sites in the array was performed using inert glass beads. Blockage of unfilled capture sites is an important feature to establish a chemical gradient across the arrayed crypts. A chemical concentration gradient (Cluminal/Cbasal>10) was demonstrated across the arrayed crypts for over 8 h. Finally unfixed colon crypts were demonstrated to be effectively captured by the micromesh array and to remain viable on the capture sites at 5 h after mouse sacrifice. The present study demonstrates the feasibility and potential for rationally microengineered technologies to address the specific needs of the biologic researcher.

Electric field gradient focusing in microchannels with embedded bipolar electrode.

The complex interplay of electrophoretic, electroosmotic, bulk convective, and diffusive mass/charge transport in a hybrid poly(dimethylsiloxane) (PDMS)/glass microchannel with embedded floating electrode is analyzed. The thin floating electrode attached locally to the wall of the straight microchannel results in a redistribution of local field strength after the application of an external electric field. Together with bulk convection based on cathodic electroosmotic flow, an extended field gradient is formed in the anodic microchannel segment. It imparts a spatially dependent electrophoretic force on charged analytes and, in combination with the bulk convection, results in an electric field gradient focusing at analyte-specific positions. Analyte concentration in the enriched zone approaches a maximum value which is independent of its concentration in the supplying reservoirs. A simple approach is shown to unify the temporal behavior of the concentration factors under general conditions.

Electrokinetics in microfluidic channels containing a floating electrode.

Electrokinetic transport within a buffer-filled microchannel incorporating a flat bipolar electrode is investigated. The key finding is that the presence of the electrode disrupts the passage of electrical current through the microchannel and thereby alters the uniformity of the local electric field. Electroosmotic flow further modulates the local field gradient. These dynamics are demonstrated experimentally by utilizing the field gradient for concentration enrichment of negatively charged tracer molecules, and a set of computer simulations is presented to interpret the underlying electrokinetics.

The influence of membrane ion-permselectivity on electrokinetic concentration enrichment in membrane-based preconcentration units.

The performance of nanoporous hydrogel microplugs with varying surface charge density is described in concentrating charged analytes electrokinetically in a microfluidic device. A neutral hydrogel plug with a mean pore size smaller than the size of charged analytes acts as a simple size-exclusion membrane. The presence of fixed charges on the backbone of a nanoporous hydrogel creates ion-permselectivity which results in charge-selective transport through the hydrogel. This leads to the development of concentration polarization (CP) in the adjoining bulk electrolyte solutions under the influence of an applied electrical field. CP strongly affects the distribution of the local electrical field strength, in particular, in the vicinity of the hydrogel plug which can significantly reduce the concentration enrichment factors compared to the neutral hydrogel. A theoretical model and simulations are presented, together with experimental data, to explain the interplay of hydrogel or membrane cation-selectivity, electrical field-induced CP, and the distribution of the local electrical field strength with respect to concentration enrichment of negatively charged analytes at the cathodic membrane-solution interface.

Transient effects on microchannel electrokinetic filtering with an ion-permselective membrane.

The electrokinetics and hydrodynamics in a hybrid microfluidic/nanofluidic pore network configuration and its effect on the concentration enrichment of charged analytes are described. A hydrogel microplug, photopolymerized in a microfluidic channel, with negative surface charge serves as a nanoporous membrane and dictates the electrokinetic behavior within the adjoining microchannel compartments. The nanoporous hydrogel with a mean pore size on the order of the electrical double layer thickness imparts ion-permselectivity (cation-selectivity) to the migration of ionic species which, under the influence of an applied electrical field, drives concentration polarization in bulk solution near the interfaces between the two microchannel compartments and the hydrogel-based nanopores. The concentration enrichment efficiency for charged analytes depends on this concentration polarization, which strongly affects the distribution of local electrical field strength. In addition, electroosmotic flow in the device plays a critical role in determining the location of the analyte enrichment zone. A theoretical model and simulations are presented to explain the interplay of concentration polarization and electroosmotic flow with respect to the observed concentration enrichment of negatively charged analytes at the cathodic hydrogel plug-microchannel solution interface.

Electrokinetic concentration enrichment within a microfluidic device using a hydrogel microplug.

A simple and efficient approach for concentration of charged molecules in microfluidic devices is described. The functional component of the system is a hydrogel microplug photopolymerized within the main channel of a microfluidic device. When an appropriately biased voltage is applied across the hydrogel, charged analyte molecules move from the source well toward the hydrogel. Transport of the analyte through the hydrogel is slow compared to its velocity in the microfluidic channel, however, and therefore it concentrates at the hydrogel/solution interface. For an uncharged hydrogel, a bias of 100 V leads to a approximately 500-fold enrichment of the DNA concentration within 150 s, while the same conditions result in an enrichment of only 50-fold for fluorescein. Somewhat lower enrichment factors are observed when a negatively charged hydrogel is used. A qualitative model is proposed to account for the observed behavior.