An automated high-throughput counting method for screening circulating tumor cells in peripheral blood.

Enumeration of circulating tumor cells (CTCs) has proved valuable for early detection and prognosis in cancer treatment. This paper describes an automated high-throughput counting method for CTCs based on microfluidics and line-confocal microscopy. Peripheral blood was directly labeled with multiple antibodies, each conjugated with a different fluorophore, pneumatically pumped through a microfluidic channel, and interrogated by a line-confocal microscope.

Sensitive and high-throughput isolation of rare cells from peripheral blood with ensemble-decision aliquot ranking.

This paper describes an approach called ensemble decision aliquot ranking (eDAR) for isolating rare cells from peripheral blood. eDAR has a recovery of over 93% with a zero false positive rate , and provides direct easy access to individual isolated live cells for downstream single-cell manipulation and analysis. We anticipate eDAR will enable new studies of various types of rare cells that circulate in blood.

Disposable microfluidic substrates: transitioning from the research laboratory into the clinic.

As more microfluidic applications emerge for clinical diagnostics, the choice of substrate and production method must be considered for eventual regulatory approval. In this review, we survey recent developments in disposable microfluidic substrates and their fabrication methods. We note regulatory approval for disposable microfluidic substrates will be more forthcoming if the substrates are developed with the United States Pharmacopeia's biocompatibility compliance guidelines in mind.

Controlling mass transport in microfluidic devices.

Microfluidic platforms offer exquisite capabilities in controlling mass transport for biological studies. In this review, we focus on recent developments in manipulating chemical concentrations at the microscale. Some techniques prevent or accelerate mixing, whereas others shape the concentration gradients of chemical and biological molecules.

Deformability considerations in filtration of biological cells.

Biological cells are highly sensitive to variation in local pressure because cellular membranes are not rigid. Unlike microbeads, cells deform under pressure or even lyse. In isolating or enriching cells by mechanical filtration, pressure-induced lysis is exacerbated when high local fluidic velocity is present or when a filter reaches its intended capacity.

Microfabricating high-aspect-ratio structures in polyurethane-methacrylate (PUMA) disposable microfluidic devices.

We recently reported a new UV-curable polyurethane-methacrylate (PUMA) resin that has excellent qualities as a disposable microfluidic substrate for clinical diagnostic applications. This article discusses strategies to improve the production yield of PUMA chips that contain dense and high-aspect-ratio features, which presents unique challenges in demolding and bonding steps. These fabrication improvements were deployed to produce a microfiltration device that contained closely spaced and high-aspect-ratio columns, suitable for retaining and concentrating cells or beads from a highly diluted suspension.

A new USP Class VI-compliant substrate for manufacturing disposable microfluidic devices.

As microfluidic systems transition from research tools to disposable clinical-diagnostic devices, new substrate materials are needed to meet both the regulatory requirement as well as the economics of disposable devices. This paper introduces a UV-curable polyurethane-methacrylate (PUMA) substrate that has been qualified for medical use and meets all of the challenges of manufacturing microfluidic devices. PUMA is optically transparent, biocompatible, and exhibits high electroosmotic mobility without surface modification.

Method for the accurate preparation of cell-spiking standards.

Preparation of calibration standards for cell enumeration is critical in characterizing the performance of any method or apparatus intended for recovering rare cells. Diluting a cell suspension serially is prone to statistical sampling errors as the cell suspension becomes more dilute, whereas transferring and injecting cells individually into a diluent with a micromanipulator is time-consuming. We developed a simple and robust method using a surface-modified glass capillary to siphon and eject cells.

High deformability of Plasmodium vivax-infected red blood cells under microfluidic conditions.

Maturation of Plasmodium falciparum decreases the deformability of infected red blood cells (RBCs), increasing their clearance as they attempt to pass through endothelial slits of the splenic sinus. Previous studies of Plasmodium vivax-infected RBCs led to opposite conclusions with respect to cellular deformability. To resolve this controversy, P.

Controlled shrinkage and re-expansion of a single aqueous droplet inside an optical vortex trap.

This paper describes the shrinkage and re-expansion of individual femtoliter-volume aqueous droplets that were suspended in an organic medium and held in an optical vortex trap. To elucidate the mechanism behind this phenomenon, we constructed a heat- and mass-transfer model and carried out experimental verifications of our model. From these studies, we conclude that an evaporation mechanism sufficiently describes the shrinkage of aqueous droplets held in a vortex trap, whereas a mechanism based on the supersaturation of the organic phase by water that surrounds the droplet adequately explains the re-expansion of the shrunk droplet.

Effects of ultrasmall orifices on the electrogeneration of femtoliter-volume aqueous droplets.

The ability to generate individual picoliter- and femtoliter-volume aqueous droplets on demand is useful for encapsulating and chemically manipulating discrete chemical and biological samples. This paper characterizes the effects of orifice dimensions and material choices on generating such droplets in an immiscible oil phase by using single high-voltage pulses with various amplitudes and durations. We have examined microfluidic orifices as small as 1.

Proton permeation into single vesicles occurs via a sequential two-step mechanism and is heterogeneous.

This article describes the first single-vesicle study of proton permeability across the lipid membrane of small (approximately 100 nm) uni- and multilamellar vesicles, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To follow proton permeation into the internal volume of each vesicle, we encapsulated carboxyfluorescein, a pH-sensitive dye whose fluorescence was quenched in the presence of excess protons. A microfluidic platform was used for easy exchange of high- and low-pH solutions, and fluorescence quenching of single vesicles was detected with single-molecule total internal reflection fluorescence (TIRF) microscopy.

High-power blue/UV light-emitting diodes as excitation sources for sensitive detection.

With advances in III-V nitride manufacturing processes, high-power light-emitting diode (LED) chips in the blue and UV wavelengths are now commercially available at reasonable cost and can be used as excitation sources in optical sensing. We describe the use of these high-power blue and UV LEDs for sensitive fluorescence detection, including chip-based flow cytometry, capillary electrophoresis (CE), and single-molecule imaging. By using a blue LED with a focusable power of approximately 40 mW as the excitation source for fluorescent beads, we demonstrate a simple chip-based bead sorter capable of enriching the concentration of green fluorescent beads from 63% to 95%.

Fast initiation of chemical reactions with laser-induced breakdown of a nanoscale partition.

This letter describes a new strategy for initiating a chemical reaction that is based on the laser-induced breakdown of a nanoscopic barrier, which physically separates the reactants in space. Because the breakdown of the barrier is fast ( approximately 0.3 micros) and owing to the nanometer dimension of the barrier, the reactants can be brought together and the reaction can be initiated rapidly.

Rapid prototyping of thermoset polyester microfluidic devices.

This paper presents a simple procedure for the fabrication of thermoset polyester (TPE) microfluidic systems and discusses the properties of the final devices. TPE chips are fabricated in less than 3 h by casting TPE resin directly on a lithographically patterned (SU-8) silicon master. Thorough curing of the devices is obtained through the combined use of ultraviolet light and heat, as both an ultraviolet and a thermal initiator are employed in the resin mixture.

Parametric investigation on the effect of channel topologies on electrophoretic separations.

This paper presents a systematic study that illustrates the importance of the topologies of microchannels on electrokinetically based separation. Using theoretical and numerical analyses, we designed and showed that topologies that significantly increased the surface-to-volume ratio of the channel can provide dramatic improvement in the ability of the channel both to dissipate the heat generated by Joule heating and to reduce the axial dispersion associated with the siphoning effect. The incremental benefit and tradeoff of geometric complexity was also evaluated.

Microfluidic systems: high radial acceleration in microvortices.

Microfluidic systems can conveniently be used for rapid analysis of biological samples. Here we describe a single re-circulating flow, or microvortex, that can generate a maximum fluid rotational velocity of up to 12 m s(-1) and a corresponding radial acceleration in excess of 10(6)g. Such microvortices may be exploited in centrifugal microdevices to investigate the effects of high radial acceleration on biological and chemical processes.

Selective electroless and electrolytic deposition of metal for applications in microfluidics: fabrication of a microthermocouple.

This paper describes a general strategy for the fabrication of a microthermocouple based on the spatially defined electroless deposition of metal, followed by annealing and electroplating. We present scanning electron microscopy and atomic force microscopy characterizations of the deposition and annealing process, as well as the performance of the microfabricated Ni-Ag thermocouple. The temperature-voltage curve for this Ni-Ag microthermocouple is linear over the range 0-50 degrees C with a slope of 61.