Todd A. Duncombe

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. We describe how our understanding of molecular and cell biology is being and will continue to be advanced by precision microfluidic approaches and posit that microfluidic tools — in conjunction with advanced imaging, bioinformatics and molecular biology approaches — will transform biology into a precision science.

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.

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. Here we describe a microfluidic western blot for an eight-plex protein panel for individual CTCs derived from estrogen receptor-positive (ER+) breast cancer patients. The precision handling and analysis reveals a capacity to assay sparingly available patient-derived CTCs, a biophysical CTC phenotype more lysis-resistant than breast cancer cell lines, a capacity to report protein expression on a per CTC basis and two statistically distinct GAPDH subpopulations within the patient-derived CTCs. Targeted single-CTC proteomics with the capacity for archivable, multiplexed protein analysis offers a unique, complementary taxonomy for understanding CTC biology and ascertaining clinical impact.

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., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). The gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. To extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. Once the microdevice has been fabricated, the assay can be completed in 4–6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. The technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine.

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.

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. The fsPAG devices (120 μm tall) are directly photopatterned atop of and covalently attached to planar polymer or glass surfaces. Leveraging the fast <1 h design-prototype-test cycle - significantly faster than mold based fabrication techniques - we optimize the fsPAG architecture to minimize injection dispersion for rapid (<1 min) and short (1 mm) protein separations. The facile fabrication and prototyping of the fsPAGE provides researchers a powerful tool for developing custom analytical assays. We highlight the utility of assay customization by fabricating a polyacrylamide gel with a spatial pore-size distribution and demonstrate the resulting enhancement in separation performance over a uniform gel. Further, we up-scale from a unit separation to an array of 96 concurrent fsPAGE assays in 10 min run time driven by one electrode pair. The fsPAG array layout matches that of a 96-well plate to facilitate integration of the planar free standing gel array with multi-channel pipettes while remaining compatible with conventional slab-gel PAGE reagents, such as staining for label-free protein detection. Notably, the entire fsPAGE workflow from fabrication, to operation, and readout uses readily available materials and instruments - making this technique highly accessible.

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. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/min, notably more efficient than either conventional slab EMSAs (∼0.01 data/min) or even microchannel based microfluidic EMSAs (∼0.3 data/min). The free-standing polyacrylamide gel EMSAs yielded reliable quantification of molecular binding and associated mobility shifts for a riboswitch–ligand interaction, thus demonstrating a screening assay platform suitable for riboswitches and potentially a wide range of RNA and other macromolecular targets.

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. The nanoporous PAG molecular sieve physically induces a mobility shift that acts to enrich and sharpen protein fronts as proteins enter the microchannel. Various PAG configurations are characterized, with injection dispersion reduced by up to 85%. When employed for analysis of a model protein sample, microfluidic PAG MBE baseline-resolved species in 5 s and in a separation distance of less than 1 mm. PAG MBE thus demonstrates electrophoretic assays with minimal interfacing and sample handling, while maintaining separation performance. Owing to the short separation lengths needed in PAG MBE, we reduced the separation channel length to demonstrate an electrophoretic immunoassay powered with an off-the-shelf 9 V battery. The electrophoretic immunoassay consumed less than 3 μW of power and was completed in 30 s. To our knowledge, this is the lowest voltage and lowest power electrophoretic protein separation reported. Looking forward, we see the low-power PAG MBE as a basis for highly multiplexed protein separations (mobility shift screening assays) as well as for portable low-power diagnostic assays.

Droplet transport on flat chemically heterogeneous surfaces via periodic wetting barriers and vibration

We report on a Flat Surface Ratchet, capable of achieving droplet transport using curved hydrophilic rungs patterned onto a hydrophobic surface. These periodic asymmetric wetting barriers can be fabricated easily on a wide variety of surfaces. Transport of a 10 ¿l droplet is achieved by an electromagnetic speaker with vertical sinusoidal vibrational amplitudes as low as 37 ¿m at 82 Hz, making this technology implementable for a wide range of discrete microfluidic applications.

Integrating EWOD with Surface Ratchets for Active Droplet Transport and Sorting

Combining surface ratchets and electrowetting on dielectric (EWOD) produces novel microfluidic systems that achieve passive droplet transport by vibration along microscopically-rough surfaces and active droplet sorting by electric signals. The super-hydrophobic surface ratchet and EWOD plate sandwich a droplet; when vibrated the device adopts passive droplet transportation via the surface ratchet. The EWOD technology is utilized at particular junctions to produce several droplet specific control functions, including the combination of a new ratchet design with an EWOD plate to develop a switch (serving the same purpose as a switch in train tracks) that sorts 10 ¿l droplets at a junction.