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

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

Microfluidic single-cell assays of peptide degradation were performed at varying inhibitor doses, and the resulting data were analyzed by regression modeling to reveal biological effects

Effect of Loading Method on a Peptide Substrate Reporter in Intact Cells [post-print]

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. We also determined how the loading method affected measurements of enzyme activity on the peptide substrate reporter using capillary electrophoresis. Loading method had a strong effect on the stability and phosphorylation of the peptide. The half-life of the intact peptide in cells was 19 ± 2, 53 ± 15, and 12 ± 1 min, for pinocytosis, electroporation, and myristoylation, respectively. The peptide was phosphorylated only in cells loaded by electroporation. Fluorescence microscopy suggested that the differences between methods were likely due to differences in peptide localization

New Software Application and Case Study That Simplify Teaching Complex Chemical Solubility and Equilibria

Chemical solubility and equilibria are paramount for understanding everyday systems. Recent events have led to more interest by the general public in chemical equilibria that occur in drinking water systems. This presents a great opportunity to increase student interest and engagement in the more complicated aspects of chemical equilibria. Shiny Apps allow exploration of complex equilibria without requiring that students (or instructors) get buried in the minutia. The application presented here, https://bazilio.shinyapps.io/LeadSolubilityCaseStudy/, can be used for instruction in chemistry or environmental science and only uses an internet browser such as Safari or Chrome; it is compatible with mobile browsers. Students are assigned a case study on the Flint, Michigan Water Crisis which guides them through lead sulfate, chloride, phosphate, and hydroxide equilibria, using the app to explore their intuitions about this complex chemical system