Microfluidic Western Blotting of Low-Molecular-Mass Proteins

Abstract

We describe a microfluidic Western blot assay (μWestern) using a Tris tricine discontinuous buffer system suitable for analyses of a wide molecular mass range (6.5–116 kDa). The Tris tricine μWestern is completed in an enclosed, straight glass microfluidic channel housing a photopatterned polyacrylamide gel that incorporates a photoactive benzophenone methacrylamide monomer. Upon brief ultraviolet (UV) light exposure, the hydrogel toggles from molecular sieving for size-based separation to a covalent immobilization scaffold for in situ antibody probing. Electrophoresis controls all assay stages, affording purely electronic operation with no pumps or valves needed for fluid control. Electrophoretic introduction of antibody into and along the molecular sieving gel requires that the probe must traverse through (i) a discontinuous gel interface central to the transient isotachophoresis used to achieve high-performance separations and (ii) the full axial length of the separation gel. In-channel antibody probing of small molecular mass species is especially challenging, since the gel must effectively sieve small proteins while permitting effective probing with large-molecular-mass antibodies. To create a well-controlled gel interface, we introduce a fabrication method that relies on a hydrostatic pressure mismatch between the buffer and polymer precursor solution to eliminate the interfacial pore-size control issues that arise when a polymerizing polymer abuts a nonpolymerizing polymer solution. Combined with a new swept antibody probe plug delivery scheme, the Tris tricine μWestern blot enables 40% higher separation resolution as compared to a Tris glycine system, destacking of proteins down to 6.5 kDa, and a 100-fold better signal-to-noise ratio (SNR) for small pore gels, expanding the range of applicable biological targets.

Fabrication of an Open Microfluidic Device for Immunoblotting

“…11,38 . In situ photocapture is a critical step, as capture efficiency dictate the amount of protein available for further processing and analysis.…”

“…Our previous assay development of in-channel immunoprobing relied on electrophoresis to sweep antibody probe down the separation axis of the gel (i.e., 20 min of probe electromigration, followed by a 20 min antibody washout in the reverse direction to remove background). 11 …”

“…11,12 Microfluidic PAGE dissipates Joule heating efficiently owing to large surface area-to-volume ratios inherent to microchannels. This favorable thermal transport supports high electric field strength operation (e.g., 1000 V/cm in chip vs 10 V/cm in slab gel PAGE), thus resolving two proteins in a few seconds compared to hours on slab gel PAGE.…”

Recent advances in microscale western blotting

“…56,57,64–66,68 The general approach uses a bifunctional gel that serves as a sieving matrix for protein separation and immobilization scaffold, following brief UV exposure, capturing separated proteins for subsequent probing with antibodies. This method eliminates the transfer step and lends itself to scalable, microfabricated platforms.…”

“…64 A step in pore size from large to small at the interface transitioned the assay from transient isotachophoresis, used to stack the sample, to protein sizing in the PAGE separation. A discontinuous tris tricine buffer system was also used to improve separation efficiency and more effectively separate smaller proteins, which can remain stacked in other buffers.…”