Cross-Species Applications of Peptide Substrate Reporters to Quantitative Measurements of Kinase Activity.

Peptide substrate reporters are short chains of amino acids designed to act as substrates for enzymes of interest. Combined with capillary electrophoresis and laser-induced fluorescence detection (CE-LIF), they are powerful molecular tools for quantitative measurements of enzyme activity even at the level of single cells. Although most peptide substrate reporters have been optimized for human or murine cells in health-related applications, their performance in nonmammalian organisms remains largely unexplored.

Emerging Areas in Undergraduate Analytical Chemistry Education: Microfluidics, Microcontrollers, and Chemometrics.

Analytical chemistry is a fast-paced field with frequent introduction of new techniques via research labs; however, incorporation of new techniques into academic curricula lags their adoption in research and industry. This review describes the recent educational literature on microfluidics, microcontrollers, and chemometrics in the undergraduate analytical chemistry curriculum. Each section highlights opportunities for nonexpert faculty to get started with these techniques and more advanced implementations suitable for experienced practitioners.

Microfluidic single-cell measurements of oxidative stress as a function of cell cycle position.

Single-cell measurements routinely demonstrate high levels of variation between cells, but fewer studies provide insight into the analytical and biological sources of this variation. This is particularly true of chemical cytometry, in which individual cells are lysed and their contents separated, compared to more established single-cell measurements of the genome and transcriptome. To characterize population-level variation and its sources, we analyzed oxidative stress levels in 1278 individual Dictyostelium discoideum cells as a function of exogenous stress level and cell cycle position.

Supported bilayer membranes for reducing cell adhesion in microfluidic devices.

The high surface area-to-volume ratio of microfluidic channels makes them susceptible to fouling and clogging when used for biological analyses, including cell-based assays. We evaluated the role of electrostatic and van der Waals interactions in cell adhesion in PDMS microchannels coated with supported lipid bilayers and identified conditions that resulted in minimal cell adhesion. For low ionic strength buffer, optimum results were obtained for a zwitterionic coating of pure egg phosphatidylcholine; for a rich growth medium, the best results were obtained for zwitterionic bilayers or those with slight negative or moderate positive charge from the incorporation of 5-10 mol% egg phosphatidylglycerol or 30 mol% ethylphosphocholine.

Effect of Loading Method on a Peptide Substrate Reporter in Intact Cells.

Studies of live cells often require loading of exogenous molecules through the cell membrane; however, effects of loading method on experimental results are poorly understood. Therefore, in this work, we compared three methods for loading a fluorescently labeled peptide into cells of the model organism Dictyostelium discoideum. We optimized loading by pinocytosis, electroporation, and myristoylation to maximize cell viability and characterized loading efficiency, localization, and uniformity.

Microfluidic single-cell analysis of oxidative stress in Dictyostelium discoideum.

Microfluidic chemical cytometry is a powerful technique for examining chemical contents of individual cells, but applications have focused on cells from multicellular organisms, especially mammals. We demonstrate the first use of microfluidic chemical cytometry to examine a unicellular organism, the social amoeba Dictyostelium discoideum. We used the reactive oxygen species indicator dichlorodihydrofluorescein diacetate to report on oxidative stress and controlled for variations in indicator loading and retention using carboxyfluorescein diacetate as an internal standard.

Interspecies comparison of peptide substrate reporter metabolism using compartment-based modeling.

Peptide substrate reporters are fluorescently labeled peptides that can be acted upon by one or more enzymes of interest. Peptide substrates are readily synthesized and more easily separated than full-length protein substrates; however, they are often more rapidly degraded by peptidases. As a result, peptide reporters must be made resistant to proteolysis in order to study enzymes in intact cells and lysates.

Microfluidic Chemical Cytometry for Enzyme Assays of Single Cells.

Cellular heterogeneity occurs, and should be probed, at multiple levels of cellular structure and physiology from the genome to enzyme activity. In particular, single-cell measures of protein levels are complemented by single-cell measurements of the activity of these proteins. Microfluidic assays of enzyme activity at the single-cell level combine moderate to high throughput with low dead volumes and the potential for automation.

Response of single leukemic cells to peptidase inhibitor therapy across time and dose using a microfluidic device.

Single-cell methodologies are revealing cellular heterogeneity in numerous biological processes and pathologies. For example, cancer cells are characterized by substantial heterogeneity in basal signaling and in response to perturbations, such as drug treatment. In this work, we examined the response of 678 individual U937 (human acute myeloid leukemia) cells to an aminopeptidase-inhibiting chemotherapeutic drug (Tosedostat) over the course of 95 days.

Microfluidic chemical cytometry of peptide degradation in single drug-treated acute myeloid leukemia cells.

Microfluidic systems show great promise for single-cell analysis; however, as these technologies mature, their utility must be validated by studies of biologically relevant processes. An important biomedical application of these systems is characterization of tumor cell heterogeneity. In this work, we used a robust microfluidic platform to explore the heterogeneity of enzyme activity in single cells treated with a chemotherapeutic drug.

Sample transport and electrokinetic injection in a microchip device for chemical cytometry.

Sample transport and electrokinetic injection bias are well characterized in capillary electrophoresis and simple microchips, but a thorough understanding of sample transport on devices combining electroosmosis, electrophoresis, and pressure-driven flow is lacking. In this work, we evaluate the effects of electric fields from 0 to 300  V/cm, electrophoretic mobilities from 10(-4) to 10(-6)  cm(2)/Vs, and pressure-driven fluid velocities from 50 to 250  μm/s on sample injection in a microfluidic chemical cytometry device. By studying a continuous sample stream, we find that increasing electric field strength and electrophoretic mobility result in improved injection and that COMSOL simulations accurately predict sample transport.

Measuring enzyme activity in single cells.

Seemingly identical cells can differ in their biochemical state, function and fate, and this variability plays an increasingly recognized role in organism-level outcomes. Cellular heterogeneity arises in part from variation in enzyme activity, which results from interplay between biological noise and multiple cellular processes. As a result, single-cell assays of enzyme activity, particularly those that measure product formation directly, are crucial.

Microchannel-nanopore device for bacterial chemotaxis assays.

Motile bacteria bias the random walk of their motion in response to chemical gradients by the process termed chemotaxis, which allows cells to accumulate in favorable environments and disperse from less favorable ones. In this work, we describe a simple microchannel-nanopore device that establishes a stable chemical gradient for chemotaxis assays in ≤1 min. Chemoattractant is dispensed by diffusion through 10 nm diameter pores at the intersection of two microchannels.

Effect of conical nanopore diameter on ion current rectification.

Asymmetric nanoscale conduits, such as conical track-etch pores, rectify ion current due to surface charge effects. To date, most data concerning this phenomenon have been obtained for small nanopores with diameters comparable to the electrical double layer thickness. Here, we systematically evaluate rectification for nanopores in poly(ethylene terephthalate) membranes with tip diameters of 10, 35, 85, and 380 nm.

Nanofluidics in lab-on-a-chip devices.

As the field of nanofluidics matures, fundamental discoveries are being applied to lab-on-a-chip analyses. The unique behavior of matter at the nanoscale is adding new functionality to devices that integrate nanopores or nanochannels. (To listen to a podcast about this feature, please go to the Analytical Chemistry website at pubs.

Surface-charge induced ion depletion and sample stacking near single nanopores in microfluidic devices.

We report integrated nanopore/microchannel devices in which single nanopores are isolated between two microfluidic channels. The devices were formed by sandwiching track-etched conical nanopores in a poly(ethylene terephthalate) membrane between two poly(dimethylsiloxane) microchannels. Integration of the nanopores into microfluidic devices improves mass transport to the nanopore and allows easy coupling of applied potentials.

Integrated nanopore/microchannel devices for ac electrokinetic trapping of particles.

We report integrated nanopore/microfluidic devices in which the unique combination of low pore density, conical nanopore membranes with microfluidic channels created addressable, localized high-field regions for electrophoretic and dielectrophoretic trapping of particles. A poly(ethylene terephthalate) track-etched membrane containing conical pores approximately 130 nm in diameter at the tip and approximately 1 microm in diameter at the base was used as an interconnect between two perpendicular poly(dimethylsiloxane) microfluidic channels. Integration of the nanopore membrane with microfluidic channels allowed for easy coupling of the electrical potentials and for directed transport of the analyte particles, 200 nm and 1 microm polystyrene microspheres and Caulobacter crescentus bacteria, to the trapping region.

Attoliter-scale dispensing in nanofluidic channels.

As fabrication techniques improve, functional fluidic devices with nanometer scale dimensions are rapidly being developed for chemical analysis. Here, we present fluid dispensing in nanochannels with injection volumes ranging from 42 aL to 4.1 fL.