A Microfluidic Method for the Selection of Undifferentiated Human Embryonic Stem Cells and in Situ Analysis.

Conventional cell-sorting methods such as fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) can suffer from certain shortcomings such as lengthy sample preparation time, cell modification through antibody labeling, and cell damage due to exposure to high shear forces or to attachment of superparamagnetic Microbeads. In light of these drawbacks, we have recently developed a label-free, microfluidic platform that can not only select cells with minimal sample preparation but also enable analysis of cells in situ. We demonstrate the utility of our platform by successfully isolating undifferentiated human embryonic stem cells (hESCs) from a heterogeneous population based on the undifferentiated stem-cell marker SSEA-4. Importantly, we show that, in contrast to MACS or FACS, cells isolated by our method have very high viability (~90%). Overall, our platform technology could likely be applied to other cell types beyond hESCs and to a variety of heterogeneous cell populations in order to select and analyze cells of interest.

Label-free resistive-pulse cytometry.

Numerous methods have recently been developed to characterize cells for size, shape, and specific cell-surface markers. Most of these methods rely upon exogenous labeling of the cells and are better suited for large cell populations (>10,000). Here, we review a label-free method of characterizing and screening cells based on the Coulter-counter technique of particle sizing: an individual cell transiting a microchannel (or "pore") causes a downward pulse in the measured DC current across that "pore". Pulse magnitude corresponds to the cell size, pulse width to the transit time needed for the cell to pass through the pore, and pulse shape to how the cell traverses across the pore (i.e., rolling or tumbling). When the pore is functionalized with an antibody that is specific to a surface-epitope of interest, label-free screening of a specific marker is possible, as transient binding between the two results in longer time duration than when the pore is unfunctionalized or functionalized with a nonspecific antibody. While this method cannot currently compete with traditional technology in terms of throughput, there are a number of applications for which this technology is better suited than current commercial cytometry systems. Applications include the rapid and nondestructive analysis of small cell populations (<100), which is not possible with current technology, and a platform for providing true point-of-care clinical diagnostics, due to the simplicity of the device, low manufacturing costs, and ease of use.

A resistive-pulse sensor chip for multianalyte immunoassays.

MultiAnalyte immunoassays are often required to diagnose a pathologic condition. Here, we show how resistive-pulse sensing and multiple artificial pores can be integrated together on a single chip to detect different antigens rapidly and simultaneously. We use multiple pores on a single chip to detect the size change of latex colloids upon specific antigen-antibody binding on the colloid surface. As a proof-of-principle, we demonstrate our ability to detect simultaneously human G-CSF and GM-CSF antigens on a single chip. Our novel technique is a scalable technology that can lead to the sensing of at least N2 antigens simultaneously with an N N array of pores on a single chip.

Biological sensing with an on-chip resistive pulse analyzer.

Resistive pulse sensors (or Coulter counters) detect the conductance change caused by single fluid-borne particles transiting a pore. Their simplicity in design and use, along with their capability for single-molecule sensitivity, make them well-suited to the analysis of biological particles. Here, we use standard methods of micro- and nanolithography to construct resistive-pulse devices that combine microfluidics with electronic sensing. We use the devices to detect single latex colloids, single DNA molecules, and specific antibody/antigen binding. We discuss the advantages of our design, and prospects for future applications.

Capacitance cytometry: measuring biological cells one by one.

Measuring the DNA content of eukaryotic cells is a fundamental task in biology and medicine. We have observed a linear relationship between the DNA content of eukaryotic cells and the change in capacitance that is evoked by the passage of individual cells across a 1-kHz electric field. This relationship is species-independent; consequently, we have developed a microfluidic technique-"capacitance cytometry"-that can be used to quantify the DNA content of single eukaryotic cells and to analyze the cell-cycle kinetics of populations of cells. Comparisons with standard flow cytometry demonstrate the sensitivity of this new technique.