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Efficient selection and enumeration of low-abundance biological cells are highly important in a variety of applications. For example, the clinical utility of circulating tumor cells (CTCs) in peripheral blood is recognized as a viable biomarker for the management of various cancers, in which the clinically relevant number of CTCs per 7.5 ml of blood is two to five. Although there are several methods for isolating rare cells from a variety of heterogeneous samples, such as immunomagnetic-assisted cell sorting and fluorescence-activated cell sorting, they are fraught with challenges. Microsystem-based technologies are providing new opportunities for selecting and isolating rare cells from complex, heterogeneous samples. Such approaches involve reductions in target-cell loss, process automation, and minimization of contamination issues. In this review, we introduce different application areas requiring rare cell analysis, conventional techniques for their selection, and finally microsystem approaches for low-abundance-cell isolation and enumeration.

We performed a series of multi-locus PCRs followed by the rapid and efficient microchip electrophoretic sorting of Alu products with LIF detection. Five polymorphic human-specific Alu insertions (RC5, A1, PV92, TPA and ACE) were used for inference of human ethnicity and two monomorphic Alu insertions for sex typing, one fixed on the X chromosome (AluSTXa) and the other on the Y chromosome (AluSTYa). These markers were used to generate unique DNA profiles for five different DNA samples. The PCR-based assays used primers that flank the insertion point to determine genotypes based on the presence or absence of the Alu element. A1, RC5, PV92, TPA and ACE were used for ethnicity determinations and have two alleles, each indicating the presence (+) or absence (-) of the Alu element on the paired chromosomes, which results in three genotypes (+/+, +/- or -/-). RC5 and A1 did not show ethnic heterogeneity resulting in a homozygous (-/-) genotype, which correctly inferred that DNA samples originating from a Caucasian male and an Asian male were not of African ancestry. The results from the five Alu markers indicated that these Alu loci could assist in identifying the individual's ethnicity using microchip electrophoresis in under 15 min of separation time. Using microchip electrophoresis and mixed genotype ratios, male DNA-to-female DNA of 1:9, corresponding to a ratio of Y-to-X chromosomes of 1:19, was also detected for both AluSTXa and AluSTYa to provide gender identification without requiring separation of female from male cells prior to the assay.

The recent outbreaks of a lethal E. coli strain in Germany have aroused renewed interest in developing rapid, specific and accurate systems for detecting and characterizing bacterial pathogens in suspected contaminated food and/or water supplies. To address this need, we have designed, fabricated and tested an integrated modular-based microfluidic system and the accompanying assay for the strain-specific identification of bacterial pathogens. The system can carry out the entire molecular processing pipeline in a single disposable fluidic cartridge and detect single nucleotide variations in selected genes to allow for the identification of the bacterial species, even its strain with high specificity. The unique aspect of this fluidic cartridge is its modular format with task-specific modules interconnected to a fluidic motherboard to permit the selection of the target material. In addition, to minimize the amount of finishing steps for assembling the fluidic cartridge, many of the functional components were produced during the polymer molding step used to create the fluidic network. The operation of the cartridge was provided by electronic, mechanical, optical and hydraulic controls located off-chip and packaged into a small footprint instrument (1 ft3). The fluidic cartridge was capable of performing cell enrichment, cell lysis, solid-phase extraction (SPE) of genomic DNA, continuous flow (CF) PCR, CF ligase detection reaction (LDR) and universal DNA array readout. The cartridge was comprised of modules situated on a fluidic motherboard; the motherboard was made from polycarbonate, PC, and used for cell lysis, SPE, CF PCR and CF LDR. The modules were task-specific units and performed universal zip-code array readout or affinity enrichment of the target cells with both made from poly(methylmethacrylate), PMMA. Two genes, uidA and sipB/C, were used to discriminate between E. coli and Salmonella, and evaluated as a model system. Results showed that the fluidic system could successfully identify bacteria in

With the advent of next-generation sequencing (NGS) systems and the associated high throughput they afford, the input to these machines requires manageable lengths of fragments (∼1000 bp) produced from chromosomal DNAs. Therefore, it is critical to develop devices that can shear DNA in a controlled fashion. We report a polymer-based microfluidic device that establishes an efficient and inexpensive platform with performance comparable to a commercially available bench-top system.

The potential utility of genome-related research in terms of evolving basic discoveries in biology has generated widespread use of DNA diagnostics and DNA forensics and driven the accelerated development of fully integrated microfluidic systems for genome processing. To produce a microsystem with favorable performance characteristics for genetic-based analyses, several key operational elements must be strategically chosen, including device substrate material, temperature control, fluidic control, and reaction product readout. As a matter of definition, a microdevice is a chip that performs a single processing step, for example microchip electrophoresis. Several microdevices can be integrated to a single wafer, or combined on a control board as separate devices to form a microsystem. A microsystem is defined as a chip composed of at least two microdevices. Among the many documented analytical microdevices, those focused on the ability to perform the polymerase chain reaction (PCR) have been reported extensively due to the importance of this processing step in most genetic-based assays. Other microdevices that have been detailed in the literature include those for solid-phase extractions, microchip electrophoresis, and devices composed of DNA microarrays used for interrogating DNA primary structure. Great progress has also been made in the areas of chip fabrication, bonding and sealing to enclose fluidic networks, evaluation of different chip substrate materials, surface chemistries, and the architecture of reaction conduits for basic processing steps such as mixing. Other important elements that have been developed to realize functional systems include miniaturized readout formats comprising optical or electrochemical transduction and interconnect technologies. These discoveries have led to the development of fully autonomous and functional integrated systems for genome processing that can supply “sample in/answer out” capabilities. In this chapter, we focus on microfluidic systems that are composed of two or more microdevices directed toward DNA analyses. Our discussions will primarily be focused on the integration of various processing steps with microcapillary electrophoresis (μCE) or microarrays. The advantages afforded by fully integrated microfluidic systems to enable challenging applications, such as single-copy DNA sequencing, single-cell gene expression analysis, pathogen detection, and forensic DNA analysis in formats that provide high throughput and point-of-analysis capabilities will be discussed as well.

A 96-well solid-phase reversible immobilization (SPRI) reactor in a micro molded, photo-activated polycarbonate (PPC) chip was designed, fabricated, and used to purify genomic DNA (gDNA) in a highly parallelized fashion. A SPRI reactor was designed to fit in a 96-well microtiter plate format with compatibility to the existing samples/reagents loading technologies. A high quality, metallic, large area mold insert (LAMI) was fabricated using a SU-8 based UV-LIGA technique. Micro molding of polycarbonate (PC) was done using hot embossing, resulting in good replication fidelity over the large surface area. The 96-well PPC-SPRI reactor, assembled by thermal fusion bonding, showed 100% efficiency for simple and fast purification of gDNA from whole cell lysates of bacterial species. The 96- well PPC-SPRI reactor will open up new avenues for low cost, disposable DNA sample purification in high throughput applications.

Highly parallelized biochemical analysis is a significant step toward achieving high throughput processing of patient samples for diagnosis and treatment monitoring. The standard microtiter plate is used to carry out multiple reactions for high throughput screening. By incorporating polymer microfluidic devices at each well in the microtiter plate format, the capability of the format could be significantly enhanced for high throughput processing of large numbers of biochemical samples in a cost-effective manner. Low cost replication of the microtiter plates is done using micro molding techniques, so microfabrication technology for making large area mold inserts (LAMIs) containing microfluidic devices at each well of a microtiter plate format is needed. A large area mold insert (LAMI) in the footprint of the standard microtiter plate was fabricated using an SU-8 based UV-LIGA technique. Excellent lithography results, with vertical sidewalls, were obtained by utilizing flycutting to minimize SU-8 film thickness variation and a UV filter for attenuating high absorbance UV wavelengths. Overplating of nickel in the SU-8 polymeric molds was used to make high quality metallic mold inserts with vertical sidewalls. Micro molding of polycarbonate (PC) was done using hot embossing, resulting in good replication fidelity over the large surface area. Thermal fusion bonding of the molded PC chips yielded good sealing results and the developed polymer microfluidic platforms showed good fluidic uniformity.Copyright © 2006 by ASME