Analytical Chemistry Research at Primarily Undergraduate Institutions: Training Tomorrow’s Investigators [post-print]

Primarily undergraduate institutions (PUIs) are a unique training ground for young scientists who work closely with faculty on their research. Researchers at PUIs face a number of challenges, including balancing research and teaching responsibilities, obtaining sufficient funding for projects, and training students in their first research experiences. Faculty at PUIs deploy a variety of strategies to address these challenges, and the resulting research provides important benefits to faculty, students, the analytical chemistry community, and society. This perspective discusses these challenges and benefits in detail. Additionally, several vignettes describe how specific faculty members have established and maintained productive research programs at PUIs, and select publications since 2009 by PUI researchers are highlighted

Electrokinetic Transport, Trapping, and Sensing in Integrated Micro- and Nanofluidic Devices

Thesis (Ph.D.) - Indiana University, Chemistry, 2009Microfluidics is rapidly becoming a mature field, and improved fabrication methods now routinely produce sub-micrometer features. As device dimensions shrink, physical phenomena that are negligible at larger length scales become more important, and by integrating nanofluidic elements with microchannels, new analytical techniques can be developed based on the unique behavior of matter at the nanoscale. This work addresses the fabrication, operation, and application of in-plane nanochannels and out-of-plane nanopores in lab-on-a-chip devices. In planar nanofluidic devices, we demonstrate a method to produce micro- and nanoscale features simultaneously with a single UV exposure step and evaluate flow control and sample dispensing with nanofluidic cross structures. Modification of the pinched injection method makes it applicable to variable-volume, attoliter-scale injections, including the smallest volume electrokinetically-controlled injections to date. As an alternative approach, track-etch nanopore membranes are explored as out-of-plane nanofluidic components. The random distribution of pores in these membranes is overcome by lithographic and microchannel-based methods to isolate and address specific pores. Microfluidic isolation improves mass transport to the pore(s), provides easy coupling of electrical potentials, and facilitates additional sample processing steps up- and downstream. These integrated microchannel-nanopore devices are used for diffusion-based dispensing, electrokinetic trapping, and resistive pulse sensing. In a high pore density device, diffusion-based dispensing establishes a stable chemical gradient for bacterial chemotaxis assays. For lower pore density devices, the nanopores are the most resistive components in the fluidic circuit, and application of an electric potential produces localized regions of high electric field strength and field gradient. These high field regions are applied to electrokinetic trapping of particles and cells in multiple-pore devices and to single particle detection by resistive pulse sensing in devices with a single isolated pore. To better understand factors influencing ion current in single nanoscale conduits, we systematically examine ion current rectification as a function of pore diameter, ionic strength, and pH to improve understanding of ion current through nanopores and to characterize preferred operating parameters for sensing applications. These results are applied to detection of virus capsids, and future work is proposed to investigate capsid assembly

Microfluidic single-cell analysis of oxidative stress in Dictyostelium discoideum [post-print]

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. After optimizing indicator concentration, we investigated the effect of peroxide treatment through single-cell measurements of 353 individual cells. The peak area ratio of dichlorofluorescein to carboxyfluorescein increased from 1.69 ± 0.89 for untreated cells to 5.19 ± 2.72 for cells treated with 40 mM hydrogen peroxide. Interestingly, the variance of the data also increased with oxidative stress. While preliminary, these results are consistent with the hypothesis that heterogeneous stress responses in unicellular organisms may be adaptive

Identifying interphase vs mitotic cell cycle phases using oxidative stress and a proximity-based null model

Detecting communities in large complex networks has found a wide range of applications in physical, biological, and social sciences by identifying mesoscopic groups based on the links between individual units. Moreover, community detection approaches have been generalized to various data analysis tasks by constructing networks whose links depend on individual units' measurements. However, identifying well separated subpopulations in data sets, e.g., multimodality, still presents challenges due to both the inherent spatial nature of the resulting networks and the generic emergence of communities in such networks and the similarity between network structures and distance-dependent null models. Here we introduce a new spatially informed null model for this task that takes into account spatial structure but does not explicitly depend on distances between measurements. We find that community detection using this null model successfully identifies subpopulations in multimodal data and accurately does not for unimodal data. We apply this new null model to the task of identifying interphase vs mitotic cell cycle phases in a group of Dictyostelium discoideum cells using measurements of oxidative stress, which have been shown to correlate strongly with cell cycle behaviors

Supported bilayer membranes for reducing cell adhesion in microfluidic devices [post-print]

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. In both solutions, the presence of 10 g L-1 glucose in the cell suspension reduced cell adhesion. Under optimum conditions, all cells were consistently removed from the channels, demonstrating the utility of these coatings for whole-cell microfluidic assays. These results provide practical information for immediate application and suggest future research areas on cell-lipid interactions

Interspecies Comparison of Peptide Substrate Reporter Metabolism using Compartment-Based Modeling [post-print]

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. This is typically achieved by optimizing the reporter sequence in a single cell type or model organism, but studies of reporter stability in a variety of organisms are needed to establish the robustness and broader utility of these molecular tools. We measured peptidase activity toward a peptide substrate reporter for protein kinase B (Akt) in E. coli, D. discoideum, and S. cerevisiae using capillary electrophoresis with laser-induced fluorescence (CE-LIF). Using compartment-based modeling, we determined individual rate constants for all potential peptidase reactions and explored how these rate constants differed between species. We found the reporter to be stable in D. discoideum (t1/2 = 82–103 min) and S. cerevisiae (t1/2 = 279–314 min), but less stable in E. coli (t1/2 = 21–44 min). These data suggest that the reporter is sufficiently stable to be used for kinase assays in eukaryotic cell types while also demonstrating the potential utility of compartment-based models in peptide substrate reporter design

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. Recent innovations have yielded a range of techniques to obtain these data, including image-, flow- and separation-based assays. Research to date has focused on easy-to-measure glycosylases and clinically-relevant kinases. Expansion of these techniques to a wider range and larger number of enzymes will answer contemporary questions in proteomics and glycomics, specifically with respect to biological noise and cellular heterogeneity

Microfluidic Chemical Cytometry of Peptide Degradation in Single Drug-Treated Acute Myeloid Leukemia Cells

Microfluidic systems show great promise for single-cell analysis, but 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. Using chemical cytometry, we measured peptide degradation in the U937 acute myeloid leukemia (AML) cell line in the presence and absence of the aminopeptidase inhibitor Tosedostat (CHR-2797). Analysis of 99 untreated cells revealed rapid and consistent degradation of the peptide reporter within 20 min of loading. Results from drug-treated cells showed inhibited, but on-going degradation of the reporter. Because the device operates at an average sustained throughput of 37 ± 7 cells/h, we were able to sample cells over the course of this time-dependent degradation. In data from 498 individual drug-treated cells, we found a linear dependence of degradation rate on amount of substrate loaded superimposed upon substantial heterogeneity in peptide processing in response to inhibitor treatment. Importantly, these data demonstrated the potential of microfluidic systems to sample biologically-relevant analytes and time-dependent processes in large numbers of single cells

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 0–300 V/cm, electrophoretic mobilities from 10−4–10−6 cm2/Vs, and pressure-driven fluid velocities from 50–250 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. The effects of pressure-driven fluid velocity on injection are complex, and relative concentration values lie on a surface defined by pressure-driven flow rates. For high mobility analytes, this surface is flat, and sample injection is robust despite fluctuations in flow rate. For lower mobility analytes, the surface becomes steeper, and injection depends strongly on pressure-driven flow. These results indicate generally that device design must account for analyte characteristics and specifically that this device is suited to high mobility analytes. We demonstrate that for a suitable pair of peptides fluctuations in injection volume are correlated; electrokinetic injection bias is minimized; and electrophoretic separation achieved