Current techniques in high-speed cell sorting are limited by the inherent coupling among three competing parameters of performance: throughput, purity, and rare cell recovery. Microfluidics provides an alternate strategy to decouple these parameters through the use of arrayed devices that operate in parallel. To efficiently isolate rare cells from complex mixtures, an electrokinetic sorting methodology was developed that exploits dielectrophoresis (DEP) in microfluidic channels. In this approach, the dielectrophoretic amplitude response of rare target cells is modulated by labeling cells with particles that differ in polarization response. Cell mixtures were interrogated in the DEP-activated cell sorter in a continuous-flow manner, wherein the electric fields were engineered to achieve efficient separation between the dielectrophoretically labeled and unlabeled cells. To demonstrate the efficiency of marker-specific cell separation, DEP-activated cell sorting (DACS) was applied for affinity-based enrichment of rare bacteria expressing a specific surface marker from an excess of nontarget bacteria that do not express this marker. Rare target cells were enriched by >200-fold in a single round of sorting at a single-channel throughput of 10,000 cells per second. DACS offers the potential for automated, surface marker-specific cell sorting in a disposable format that is capable of simultaneously achieving high throughput, purity, and rare cell recovery.cell sorting ͉ microfluidics C ell sorters are capable of separating a heterogeneous suspension of particles into purified fractions and thus have become an indispensable tool in biology and medicine. Emerging applications of cell sorting technology span a broad spectrum of pharmaceutical and biomedical fields that range from cancer diagnostics to cell-based therapies (1-3). The most widely used methodologies for cell separation are magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). MACS is a selection technique that is capable of capturing a large number of target cells in parallel (4); however, the purity and recovery in MACS typically have large variances (5). In contrast, FACS relies upon serially screening each cell, yielding high performance in cell recovery and purity (6). However, because of the serial nature of its operation, FACS allows for a comparatively low throughput, typically in the range between 10 4 and 10 5 cells per second (7). Regardless of the mechanism, the performance of cell separation is typically characterized by three metrics. ''Throughput'' gauges how many cell characterization and sorting operations can be executed per unit of time, ''purity'' is the fraction of the target cells in the collection vessel, and ''recovery'' is the fraction of the input target cells successfully sorted into the collection vessel. Demands placed on cell sorting technologies continue to increase, because cell sorting applications are expanding and biological questions are becoming more complex (7). For example, rare cell sort...

Aptamers are nucleic acid molecules that have been selected in vitro to bind to their molecular targets with high affinity and specificity. Typically, the systematic evolution of ligands by exponential enrichment (SELEX) process is used for the isolation of specific, high-affinity aptamers. SELEX, however, is an iterative process requiring multiple rounds of selection and amplification that demand significant time and labor. Here, we describe an aptamer discovery system that is rapid, highly efficient, automatable, and applicable to a wide range of targets, based on the integration of magnetic bead-based SELEX process with microfluidics technology. Our microfluidic SELEX (M-SELEX) method exploits a number of unique phenomena that occur at the microscale and implements a design that enables it to manipulate small numbers of beads precisely and isolate high-affinity aptamers rapidly. As a model to demonstrate the efficiency of the M-SELEX process, we describe here the isolation of DNA aptamers that tightly bind to the light chain of recombinant Botulinum neurotoxin type A (with low-nanomolar dissociation constant) after a single round of selection.microchannel ͉ recombinant Botulinum neurotoxin type A ͉ systematic evolution of ligands by exponential enrichment

Aptamers are nucleic acid-based reagents that bind to target molecules with high affinity and specificity. However, methods for generating aptamers from random combinatorial libraries (e.g., SELEX) are often labor-intensive and time-consuming. Recent studies suggest that microfluidic SELEX (M-SELEX) technology can accelerate aptamer isolation by enabling highly stringent selection conditions through the use of very small amounts of target molecules. We present here an alternative M-SELEX method, which employs a disposable microfluidic chip to rapidly generate aptamers with high affinity and specificity. The Micro-Magnetic Separation (MMS) chip integrates microfabricated ferromagnetic structures to reproducibly generate large magnetic field gradients within its microchannel that efficiently trap magnetic bead-bound aptamers. Operation of the MMS device is facile, robust and demonstrates high recovery of the beads (99.5%), such that picomolar amounts of target molecule can be used. Importantly, the device demonstrates exceptional separation efficiency in removing weakly-bound and unbound ssDNA to rapidly enrich target-specific aptamers. As a model, we demonstrate here the generation of DNA aptamers against streptavidin in three rounds of positive selection. We further enhanced the specificity of the selected aptamers via a round of negative selection in the same device against bovine serum albumin (BSA). The resulting aptamers displayed dissociation constants ranging from 25 to 65 nM for streptavidin but negligible affinity for BSA. Since a wide spectrum of molecular targets can be readily conjugated on magnetic beads, MMS-based SELEX should provide a general platform for rapid generation of specific aptamers.

We describe a single-step, single-component, fluorescence-based method of detecting single nucleotide polymorphisms at room temperature without exogenous reagents. The sensor is comprised of a single, self-complementary DNA strand forming a triple-stem structure, which undergoes a large conformational change only upon binding to perfectly-matched targets, resulting in a significant increase in fluorescence.