Jason S. Kuo

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. vivax-infected RBCs were passed through a 2-microm microfluidic channel. In contrast to P. falciparum-infected RBCs, mature P. vivax-infected RBCs readily became deformed through 2-microm constrictions. After this extreme deformation, 67% of P. vivax-infected RBCs recovered a normal appearance; however, 15% of uninfected RBCs were destroyed. Results suggest mechanisms for both avoidance of splenic clearance and anemia in vivax malaria.

Protein Quantification at the Single Vesicle Level Reveals That a Subset of Synaptic Vesicle Proteins Are Trafficked with High Precision

Protein sorting represents a potential point of regulation in neurotransmission because it dictates the protein composition of synaptic vesicles, the organelle that mediates transmitter release. Although the average number of most vesicle proteins has been estimated using bulk biochemical approaches (Takamori et al., 2006), no information exists on the intervesicle variability of protein number, and thus on the precision with which proteins are sorted to vesicles. To address this, we adapted a single molecule quantification approach (Mutch et al., 2007) and used it to quantify both the average number and variance of seven integral membrane proteins in brain synaptic vesicles. We report that four vesicle proteins, SV2, the proton ATPase, Vglut1, and synaptotagmin 1, showed little intervesicle variation in number, indicating they are sorted to vesicles with high precision. In contrast, the apparent number of VAMP2/synaptobrevin 2, synaptophysin, and synaptogyrin demonstrated significant intervesicle variability. These findings place constraints on models of protein function at the synapse and raise the possibility that changes in vesicle protein expression affect vesicle composition and functioning.

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% (n=9) with a zero false positive rate (n=8), 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.

Electro-generation of single femtoliter- and picoliter-volume aqueous droplets in microfluidic systems

This letter presents a new method to generate individual femtoliter- to picoliter-volume aqueous droplets in oil using single voltage pulses. With high-speed imaging, we characterized the displacement of the water-oil interface as a function of the time and amplitude of the voltage pulse. At high voltages where jetting and droplet formation occurred, we observed the ratio of droplet-to-jet diameter to be ∼1.84, which suggested Rayleigh instability as the primary mechanism responsible for droplet breakup. Droplets with volumes ranging from 14 fl to 8 pl were produced using this method.

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. On the basis of the fluorescence signals and labeling schemes, the count of CTCs was automatically reported. Due to the high flow rate, 1 mL of whole blood can be analyzed in less than 30 min. We applied this method in analyzing CTCs from 90 stage IV breast cancer patient samples and performed a side-by-side comparison with the results of the CellSearch assay, which is the only method approved by the U.S. Food and Drug Administration at present for enumeration of CTCs. This method has a recovery rate for cultured breast cancer cells of 94% (n = 9), with an average of 1.2 counts/mL of background level of detected CTCs from healthy donors. It detected CTCs from breast cancer patients ranging from 15 to 3375 counts/7.5 mL. Using this method, we also demonstrate the ability to enumerate CTCs from breast cancer patients that were positive for Her2 or CD44(+)/CD24(-), which is a putative cancer stem cell marker. This automated method can enumerate CTCs from peripheral blood with high throughput and sensitivity. It could potentially benefit the clinical diagnosis and prognosis of cancer.

Dynamic formation of ring-shaped patterns of colloidal particles in microfluidic systems

This letter reports the formation of patterns of micrometer-sized beads within the steady-state recirculation flow of a microvortex generated in a microfluidic system. The mechanism by which these patterns form relies on a delicate balance between the centrifugal and displacement forces experienced by the recirculating particles with a lift force exerted on the particles near the solid boundary of the microcavity. Our observation was made possible by the small dimensions of the microchannels we used and by the presence of steep velocity gradients unique to microfluidic devices.

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. We also highlight several in vitro biological studies in the areas of organ engineering, cancer, and blood coagulation that have benefited from accurate control of mass transfer.

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. One-dimensional confinement of cells offered by the capillary made cell enumeration by visual counting simple and rapid, and cell ejection from the capillary was near 100% when the appropriate surface coating and cell solution was used. The residence time of cells in the capillary, however, could affect the percentage of cells that was ejected from the capillary. To characterize the performance of this method, we enumerated the ejected cell using both visual counting under a microscope and automated detection using a chip-based flow cytometer.

The Generation, Characterization and Application of Microvortices in Microfluidic Systems (Keynote Paper)

This review describes the formation of microvortices in microfluidic systems, and discusses our experimental measurements that illustrate the velocity profiles inside such microvortices. Because of the micrometer dimensions of these vortices and the presence of high rotational velocities, we have observed a number of unique phenomena. One example is the dynamic formation of ring patterns of particles within the microvortex. The mechanism by which these patterns form relies on a balance between the centrifugal and displacement forces experienced by the re-circulating particles with a lift force exerted on the particles near the solid boundary of the microcavity. We also demonstrate the ability to orient and rotate precisely micro and nanometer -sized particles, individual DNA molecules, and single cells. Because of the high linear velocity (m/s) of fluid flow in constricted microchannels and to the small radii (< 10μm) of the microvortices, we have measured the presence of ultrahigh radial accelerations (v2 /r) in such microvortices, which can reach 107 m/s2 or 106 times the gravitational acceleration (g).Copyright