16 research outputs found
USING OXYGEN-CONSUMING THERMOSET PLASTICS TO GENERATE HYPOXIC CONDITIONS IN MICROFLUIDIC DEVICES FOR POTENTIAL CELL CULTURE APPLICATIONS
Development of thiol-ene-epoxy thermoset microdevices with automated fluid handling and nanolayer passivated microstructured impedimetric sensor for in vitro cell-based assays
Thiol-Ene Based Polymers as Versatile Materials for Microfluidic Devices for Life Sciences Applications
An all thiol-ene microchip for solid phase extraction featuring an in situ polymerized monolith and integrated 3D replica-molded emitter for direct electrospray mass spectrometry
Generation of transient and tunable oxygen gradients in microfluidic channels utilizing the oxygen scavenging properties of thiol-ene polymers
Chloroform compatible, thiol-ene based replica molded micro chemical devices as an alternative to glass microfluidic chips
A thiol-ene microfluidic device enabling continuous enzymatic digestion and electrophoretic separation as front-end to mass spectrometric peptide analysis
Thiol-ene microfluidic chip for fast on-chip sample clean-up, separation and ESI mass spectrometry of peptides and proteins
Thiol-ene microfluidic chip for performing hydrogen/deuterium exchange of proteins at sub-second timescales
Hydrogen/deuterium
exchange monitored by mass spectrometry (HDX-MS)
has become a routine approach for sensitive analysis of the dynamic
structure and interactions of proteins. However, transient conformational
changes and weak affinity interactions found in many biological systems
typically only perturb fast-exchanging amides in proteins. Detection
of HDX changes for such amides require shorter deuterium labeling
times (subsecond) than can be performed reproducibly by manual sample
handling. Here, we describe the development and validation of a microfluidic
chip capable of rapid on-chip protein labeling and reaction quenching.
The fastHDX thiol-ene microchip is fabricated entirely using thiol-ene
photochemistry. The chip has a three-channel design for introduction
of protein sample, deuterated buffer, and quench buffer. Thiol-ene
based monolith plugs (i.e., polymerized thiol-ene emulsions) situated
within microchannels are generated in situ using
a 3D-printed photolithography mask. We show that efficient on-chip
mixing can be achieved at channel junctions by spatially confined
in-channel monolith mixers. Using human hemoglobin (Hb), we demonstrate
the ability of the chip to perform highly reproducible HDX in the
0.14–1.1 s time frame. The HDX of Hb at 0.14–1.1 s,
resolved to peptide segments, correlates closely with structural features
of the crystal structure of the Hb tetramer, with helices exhibiting
no or minor HDX and loops undergoing pronounced HDX even at subsecond
time scales. On-chip HDX of Hb at time points ranging from 0.14–1.1
s demonstrates the ability to distinguish fast exchanging amides and
thus provides enhanced detection of transient structure and interactions
in dynamic or exposed regions of proteins in solution