Enhancing the Resolution of Micro Free Flow Electrophoresis through Spatially Controlled Sample Injection

Abstract

Free flow electrophoresis is a versatile technique for the continuous separation of mixtures with both preparative and analytical applications. Microscale versions of free flow electrophoresis are particularly attractive strategies because of their fast separation times, ability to work with small sample volumes, and large surface area to volume ratios facilitating rapid heat transfer, thus minimizing the detrimental effects of Joule heating even at high voltages. The resolution of microscale free flow electrophoresis, however, is limited by the broadening of the analyte beam in the microfluidic channel, an effect that becomes especially pronounced when the analyte is deflected significantly away from its original position. Here, we describe and demonstrate how restricting spatially the sample injection and collection to the regions where the gradients in the velocity distribution of the carrier medium are the smallest allows this broadening effect to be substantially suppressed and hence the resolution of microscale free flow electrophoresis devices to be increased. To demonstrate this concept, we fabricated microfluidic free flow electrophoresis devices with spatially restricted injection nozzles implemented through the use of multilayer soft-photolithography and further integrated quartz based observation areas for fluorescent detection and imaging. With these devices, we demonstrated a 5-fold reduction in the extent of beam broadening relative to conventional free flow electrophoresis approaches with nonrestricted sample introduction. The manifold enhancement in the achievable resolution of microscale free flow electrophoresis devices opens up the possibility of rapid separation and analysis of complex mixtures.

Direct digital sensing of protein biomarkers in solution

“…This feature is of utmost importance for effective and high-performance electrophoretic separation. [21][22][23] The α-synuclein fibrils were detected using T-SO508 aptamer 37 (5'-Alexa488-TTTTGCCTGTGGTGTTGGGGCGGGTGCG-3', HPLC purified; IDT). Prior to its use, the aptamer (100 µM stock in 1X TE buffer) was heated to 70°C and cooled to room temperature to facilitate correct folding.…”

“…To discriminate the free probe from its analyte-bound form, an electric potential is applied perpendicular to the flow direction using co-flowing electrolyte solutions that act as liquid electrodes and ensure the application of stable electric fields, as described earlier. 22,23 Simultaneously, laser-induced confocal fluorescence microscopy is used to detect individual molecules by scanning the confocal volume across the microfluidic chip.…”

Potential of Microfluidics and Lab-on-Chip Platforms to Improve Understanding of “prion-like” Protein Assembly and Behavior

“…In fact, a key aspect in evaluating the aggregation kinetics of biomolecular species is the ability to monitor fibrillar growth as a function of time (see above). The monitoring of this process can be developed in LOC [e.g., in hydrodynamic focusing systems (i.e., Fitzpatrick et al, 2013;Arosio et al, 2016)], or using electrophoretic approaches in these systems (i.e., Saar et al, 2018). However, it is well-known that the amyloid aggregation process is largely dependent on the interaction of the protein with several ions (i.e., Kim et al, 2018;Metrick et al, 2019), membranes (i.e., Terakawa et al, 2018;Alghrably et al, 2019), and other surfaces (e.g., water; Schladitz et al, 1999).…”

Application of a Micro Free-Flow Electrophoresis 3D Printed Lab-on-a-Chip for Micro-Nanoparticles Analysis

“…When operating at the microscale, thus acquiring several well-known advantages related to the scaling down of the dimensions, the microfluidic approach allows for the implementation of correspondingly scaled micro free-flow electrophoresis (µFFE) [24]. Except for the first µFFE devices, which were manufactured in silicon through a standard lithographic process [25], nowadays the majority of the µFFE devices are developed by exploiting various types of polymers through soft-lithographic or replica molding strategies [26][27][28]. So far, only one µFFE device, presented in literature, has been developed by an additive manufacturing process [29].…”