A multidimensional metabolomics workflow to image biodistribution and evaluate pharmacodynamics in adult zebrafish.

An integrated evaluation of the tissue distribution and pharmacodynamic properties of a therapeutic is essential for successful translation to the clinic. To date, however, cost-effective methods to measure these parameters at the systems level in model organisms are lacking. Here, we introduce a multidimensional workflow to evaluate drug activity that combines mass spectrometry-based imaging, absolute drug quantitation across different biological matrices, in vivo isotope tracing and global metabolome analysis in the adult zebrafish. As a proof of concept, we quantitatively determined the whole-body distribution of the anti-rheumatic agent hydroxychloroquine sulfate (HCQ) and measured the systemic metabolic impacts of drug treatment. We found that HCQ distributed to most organs in the adult zebrafish 24 h after addition of the drug to water, with the highest accumulation of both the drug and its metabolites being in the liver, intestine and kidney. Interestingly, HCQ treatment induced organ-specific alterations in metabolism. In the brain, for example, HCQ uniquely elevated pyruvate carboxylase activity to support increased synthesis of the neuronal metabolite, N-acetylaspartate. Taken together, this work validates a multidimensional metabolomics platform for evaluating the mode of action of a drug and its potential off-target effects in the adult zebrafish. This article has an associated First Person interview with the first author of the paper.

Isotope tracing in adult zebrafish reveals alanine cycling between melanoma and liver.

The cell-intrinsic nature of tumor metabolism has become increasingly well characterized. The impact that tumors have on systemic metabolism, however, has received less attention. Here, we used adult zebrafish harboring BRAFV600E-driven melanoma to study the effect of cancer on distant tissues. By applying metabolomics and isotope tracing, we found that melanoma consume ~15 times more glucose than other tissues measured. Despite this burden, circulating glucose levels were maintained in disease animals by a tumor-liver alanine cycle. Excretion of glucose-derived alanine from tumors provided a source of carbon for hepatic gluconeogenesis and allowed tumors to remove excess nitrogen from branched-chain amino acid catabolism, which we found to be activated in zebrafish and human melanoma. Pharmacological inhibition of the tumor-liver alanine cycle in zebrafish reduced tumor burden. Our findings underscore the significance of metabolic crosstalk between tumors and distant tissues and establish the adult zebrafish as an attractive model to study such processes.

Translational Opportunities for Microfluidic Technologies to Enable Precision Epigenomics.

Personalizing health care by taking genetic, environmental, and lifestyle factors into account is central to modern medicine. The crucial and pervasive roles epigenetic factors play in shaping gene-environment interactions are now well recognized. However, identifying robust epigenetic biomarkers and translating them to clinical tests has been difficult due in part to limitations of available platforms to detect epigenetic features genome-wide (epigenomic assays). This Feature introduces several important prospects for precision epigenomics, highlights capabilities and limitations of current laboratory technologies, and emphasizes opportunities for microfluidic tools to facilitate translation of epigenetic analyses to the clinic, with a particular focus on methods to profile gene-associated histone modifications and their impacts on chromatin structure and gene expression.

Droplet CAR-Wash: continuous picoliter-scale immunocapture and washing.

To address current limitations in adapting solid phase sample capture and washing techniques to continuously flowing droplet microfluidics, we have developed the "Coalesce-Attract-Resegment Wash" (CAR-Wash) approach. This module provides efficient, high-throughput magnetic washing by electrocoalescing magnetic bead-laden input droplets with a washing buffer flow and magnetophoretically transporting beads through the buffer into a secondary droplet formation streamline. In this work, we first characterized the technology in terms of throughput, sample retention, and flow-based exclusion of waste volume, demonstrating >500 Hz droplet processing with >98% bead retention and >100-fold dilution in final droplets. Next, we showed that the technique can be adapted to alternative commercially available magnetic beads with lower magnetite content per particle. Then, we demonstrated the CAR-Wash module's effectiveness in washing away a small molecule competitive inhibitor to restore the activity of magnetic bead-immobilized ß-galactosidase. Finally, we applied the system to immunomagnetically enrich a green fluorescent protein-histone H2B fusion protein from cell lysate while washing away mCherry and other lysate components. We believe this approach will bridge the gap between powerful biochemical and bioanalytical techniques and current droplet microfluidic capabilities, and we envision future application in droplet-based immunoassays, solid phase extraction, and other complex, multi-step operations.

Droplet Microfluidics in Thermoplastics: Device Fabrication, Droplet Generation, and Content Manipulation using Integrated Electric and Magnetic Fields.

We have developed droplet microfluidic devices in thermoplastics and demonstrated the integration of key functional components that not only facilitate droplet generation, but also include electric field-assisted reagent injection, droplet splitting, and magnetic field-assisted bead extraction. We manufactured devices in poly(methyl methacrylate) and cyclic olefin polymer using a hot-embossing procedure employing silicon masters fabricated via photolithography and deep reactive ion etching techniques. Device characterization showed robust fabrication with uniform feature transfer and good embossing yield. Channel modification with heptadecafluoro-1,1,2,2-tetrahydrodecyltrichlorosilane increased device hydrophobicity, allowing stable generation of 330-pL aqueous droplets using T-junction configuration. Picoinjector and K-channel motifs were also both successfully integrated into the thermoplastic devices, allowing for robust control over electric field-assisted reagent injection, as well as droplet splitting with the K-channel. A magnetic field was also introduced to the K-channel geometry to allow for selective concentration of magnetic beads while decanting waste volume through droplet splitting. To show the ability to link multiple, modular features in a single thermoplastic device, we integrated droplet generation, reagent injection, and magnetic field-assisted droplet splitting on a single device, realizing a magnetic bead washing scheme to selectively exchange the fluid composition around the magnetic particles, analogous to the washing steps in many common biochemical assays. Finally, integrated devices were used to perform a proof-of-concept in-droplet ß-galactosidase enzymatic assay combining enzyme-magnetic bead containing droplet generation, resorufin-ß-D-galactopyranoside substrate injection, enzyme-substrate reaction, and enzyme-magnetic bead washing. By integrating multiple droplet operations and actuation forces we have demonstrated the potential of thermoplastic droplet microfluidic devices for complex (bio)chemical analysis, and we envision a path toward mass fabrication of droplet microfluidic devices for a range of (bio)chemical applications.

K-Channel: A Multifunctional Architecture for Dynamically Reconfigurable Sample Processing in Droplet Microfluidics.

By rapidly creating libraries of thousands of unique, miniaturized reactors, droplet microfluidics provides a powerful method for automating high-throughput chemical analysis. In order to engineer in-droplet assays, microfluidic devices must add reagents into droplets, remove fluid from droplets, and perform other necessary operations, each typically provided by a unique, specialized geometry. Unfortunately, modifying device performance or changing operations usually requires re-engineering the device among these specialized geometries, a time-consuming and costly process when optimizing in-droplet assays. To address this challenge in implementing droplet chemistry, we have developed the "K-channel," which couples a cross-channel flow to the segmented droplet flow to enable a range of operations on passing droplets. K-channels perform reagent injection (0-100% of droplet volume), fluid extraction (0-50% of droplet volume), and droplet splitting (1:1-1:5 daughter droplet ratio). Instead of modifying device dimensions or channel configuration, adjusting external conditions, such as applied pressure and electric field, selects the K-channel process and tunes its magnitude. Finally, interfacing a device-embedded magnet allows selective capture of 96% of droplet-encapsulated superparamagnetic beads during 1:1 droplet splitting events at ∼400 Hz. Addition of a second K-channel for injection (after the droplet splitting K-channel) enables integrated washing of magnetic beads within rapidly moving droplets. Ultimately, the K-channel provides an exciting opportunity to perform many useful droplet operations across a range of magnitudes without requiring architectural modifications. Therefore, we envision the K-channel as a versatile, easy to use microfluidic component enabling diverse, in-droplet (bio)chemical manipulations.

Encapsulation of Fluidic Tubing and Microelectrodes in Microfluidic Devices: Integrating Off-Chip Process and Coupling Conventional Capillary Electrophoresis with Electrochemical Detection.

In this paper, an approach to fabricate epoxy or polystyrene microdevices with encapsulated tubing and electrodes is described. Key features of this approach include a fixed alignment between the fluidic tubing and electrodes, the ability to polish the device when desired, and the low dead volume nature of the fluidic interconnects. It is shown that a variety of tubing can be encapsulated with this approach, including fused silica capillary, polyetheretherketone (PEEK), and perfluoroalkoxy (PFA), with the resulting tubing/microchip interface not leading to significant band broadening or plug dilution. The applicability of the devices with embedded tubing is demonstrated by integrating several off-chip analytical methods to the microchip. This includes droplet transfer, droplet desegmentation, and microchip-based flow injection analysis. Off-chip generated droplets can be transferred to the microchip with minimal coalescence, while flow injection studies showed improved peak shape and sensitivity when compared to the use of fluidic interconnects with an appreciable dead volume. Importantly, it is shown that this low dead volume approach can be extended to also enable the integration of conventional capillary electrophoresis (CE) with electrochemical detection. This is accomplished by embedding fused silica capillary along with palladium (for grounding the electrophoresis voltage) and platinum (for detection) electrodes. With this approach, up to 128,000 theoretical plates for dopamine was possible. In all cases, the tubing and electrodes are housed in a rigid base; this results in extremely robust devices that will be of interest to researchers wanting to develop microchips for use by non-experts.