UV-Vis Spectra-Activated Droplet Sorting for Label-Free Chemical Identification and Collection of Droplets.

We introduce the UV-vis spectra-activated droplet sorter (UVADS) for high-throughput label-free chemical identification and enzyme screening. In contrast to previous absorbance-based droplet sorters that relied on single-wavelength absorbance in the visible range, our platform collects full UV-vis spectra from 200 to 1050 nm at up to 2100 spectra per second. Our custom-built open-source software application, "SpectraSorter," enables real-time data processing, analysis, visualization, and selection of droplets for sorting with any set of UV-vis spectral features.

Microfluidic platform enables tailored translocation and reaction cascades in nanoliter droplet networks.

In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and mimic reactions and processes at or across the membrane. In this context, we employ here a versatile microfluidic platform that enables precise positioning of nanoliter droplets with user-specified lipid compositions and in a defined pattern. Adjacent droplets make contact and form a droplet interface bilayer to simulate cellular membranes.

Droplet barcoding: tracking mobile micro-reactors for high-throughput biology.

Droplet microfluidics has become a powerful analytical platform in biological research for conducting high-throughput screening in millions of discrete micro-reactors per hour. While the method facilitates faster and cheaper testing than conventional microtiter plates, the mobile nature of droplets makes micro-reaction tracking a notable challenge. To address this, researchers are developing a variety of tracking methods, ranging from organizing droplets into an index or labeling droplets with a barcode.

Insulator Nanostructure Desorption Ionization Mass Spectrometry.

Surface-assisted laser desorption ionization (SALDI) is an approach for gas-phase ion generation for mass spectrometry using laser excitation on typically conductive or semiconductive nanostructures. Here, we introduce insulator nanostructure desorption ionization mass spectrometry (INDI-MS), a nanostructured polymer substrate for SALDI-MS analysis of small molecules and peptides. INDI-MS surfaces are produced through the self-assembly of a perfluoroalkyl silsesquioxane nanostructures in a single chemical vapor deposition silanization-step.

Ecosystem Fabrication (EcoFAB) Protocols for The Construction of Laboratory Ecosystems Designed to Study Plant-microbe Interactions.

Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions.

Profiling protein expression in circulating tumour cells using microfluidic western blotting.

Circulating tumour cells (CTCs) are rare tumour cells found in the circulatory system of certain cancer patients. The clinical and functional significance of CTCs is still under investigation. Protein profiling of CTCs would complement the recent advances in enumeration, transcriptomic and genomic characterization of these rare cells and help define their characteristics.

Single cell-resolution western blotting.

This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. Like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g.

Kinetic Rate Determination via Electrophoresis along a Varying Cross-Section Microchannel.

High throughput, efficient, and readily adoptable analytical tools for the validation and selection of reliable antibody reagents would impact the life sciences, clinical chemistry, and clinical medicine. To directly quantify antibody-antigen association and dissociation rate constants, kon and koff, in a single experiment, we introduce a microfluidic free-standing kinetic polyacrylamide gel electrophoresis (fsKPAGE) assay. Here, an antibody is immobilized in zones along the length of a single freestanding polyacrylamide gel lane of varying cross-sectional width.

Hydrogel Pore-Size Modulation for Enhanced Single-Cell Western Blotting.

Pore-gradient microgel arrays enable thousands of parallel high-resolution single-cell protein electrophoresis separations for targets accross a wide molecular mass (25-289 kDa), yet within 1 mm separation distances. Dual crosslinked hydrogels facilitate gel-pore expansion after electrophoresis for efficient and uniform immunoprobing. The photopatterned, light-activated, and acid-expandable hydrogel underpins single-cell protein analysis, here for oncoprotein-related signaling in human breast biopsy.

Microfluidics: reframing biological enquiry.

The underlying physical properties of microfluidic tools have led to new biological insights through the development of microsystems that can manipulate, mimic and measure biology at a resolution that has not been possible with macroscale tools. Microsystems readily handle sub-microlitre volumes, precisely route predictable laminar fluid flows and match both perturbations and measurements to the length scales and timescales of biological systems. The advent of fabrication techniques that do not require highly specialized engineering facilities is fuelling the broad dissemination of microfluidic systems and their adaptation to specific biological questions.

High-throughput electrophoretic mobility shift assays for quantitative analysis of molecular binding reactions.

We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mm-scale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand.

Photopatterned free-standing polyacrylamide gels for microfluidic protein electrophoresis.

Designed for compatibility with slab-gel polyacrylamide gel electrophoresis (PAGE) reagents and instruments, we detail development of free-standing polyacrylamide gel (fsPAG) microstructures supporting electrophoretic performance rivalling that of microfluidic platforms. For the protein electrophoresis study described here, fsPAGE lanes are comprised of a sample reservoir and contiguous separation gel. No enclosed microfluidic channels are employed.

Use of polyacrylamide gel moving boundary electrophoresis to enable low-power protein analysis in a compact microdevice.

In designing a protein electrophoresis platform composed of a single-inlet, single-outlet microchannel powered solely by voltage control (no pumps, values, injectors), we adapted the original protein electrophoresis format-moving boundary electrophoresis (MBE)-to a high-performance, compact microfluidic format. Key to the microfluidic adaptation is minimization of injection dispersion during sample injection. To reduce injection dispersion, we utilize a photopatterned free-solution-polyacrylamide gel (PAG) stacking interface at the head of the MBE microchannel.

Directed drop transport rectified from orthogonal vibrations via a flat wetting barrier ratchet.

We introduce the wetting barrier ratchet, a digital microfluidic technology for directed drop transport in an open air environment. Cyclic drop footprint oscillations initiated by orthogonal vibrations as low as 37 μm in amplitude at 82 Hz are rectified into fast (mm/s) and controlled transport along a fabricated ratchet design. The ratchet is made from a simple wettability pattern atop a microscopically flat surface consisting of periodic semi-circular hydrophilic features on a hydrophobic background.

Controlling liquid drops with texture ratchets.

Controlled vibration selectively propels multiple microliter-sized drops along microstructured tracks, leading to simple microfluidic systems that rectify oscillations of the three-phase contact line into asymmetric pinning forces that propel each drop in the direction of higher pinning.