Combining Affinity Selection and Specific Ion Mobility for Microchip Protein Sensing

Abstract

The sensitive detection of proteins is a key objective in many areas of biomolecular science, ranging from biophysics to diagnostics. However, sensing in complex biological fluids is hindered by nonspecific interactions with off-target species. Here, we describe and demonstrate an assay that utilizes both the chemical and physical properties of the target species to achieve high selectivity in a manner not possible by chemical complementarity alone, in complex media. We achieve this objective through a combinatorial strategy, by simultaneously exploiting free-flow electrophoresis for target selection, on the basis of electrophoretic mobility, and conventional affinity-based selection. In addition, we demonstrate amplification of the resultant signal by a catalytic DNA nanocircuit. This approach brings together the inherent solution-phase advantages of microfluidic sample handling with isothermal, enzyme-free signal amplification. With this method, no surface immobilization or washing steps are required, and our assay is well suited to monoepitopic targets, presenting advantages over conventional ELISA techniques.

Aptamers against Immunoglobulins: Design, Selection and Bioanalytical Applications

“…Wide range of microfluidic devices were used including gel-shift detection platform incorporating competitive inhibition strategy [170], DNA mobility shift assay [171] and frontal analysis [172]. Since the aptamer-IgE complex is difficult to measure with conventional methods-due the slow migration of the complex-free flow electrophoresis (FFE) was applied with fast measurements and accurate results [173,174], i.e., in FFE, the sample is progressed by pressure driven flow and the electrical field applied perpendicularly to the direction of the flow only deviates the various components based on their mass/charge ratio from their linear trajectory. Perhaps, the best analytical performance has been achieved with a homogeneous mobility shift assay employing a complex amplification mechanism.…”

Direct digital sensing of protein biomarkers in solution

“…Fluid waste is guided out of the device by tubing inserted into device outlets. Importantly, the free-flow electrophoresis functionality of the device is attained by a design that makes use of liquid electrolyte electrodes, 21,22 where an electric potential is applied outside and downstream of the microfluidic separation chamber in separate electrolyte channels, and thereby allowed to propagate back to the separation area through the use of a co-flowing, highly conductive electrolyte solution (i.e., generated electrolysis products to be flushed out of the chip and Joule heating to be reduced, permitting strong electric fields to be applied in a stable manner. This feature is of utmost importance for effective and high-performance electrophoretic separation.…”

“…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.…”

“…Moreover, by incorporating microchip electrophoresis in addition to affinity selection, DigitISA provides an additional criterion for signal generation to afford highly specific protein sensing; this enhances the selectivity in protein sensing. 21 Finally, the surface-free nature of the assay not only reduces false-positive signaling by non-specific surface adsorption, it also allows the assay to be operated in a single step. Given that DigitISA uses only a single affinity reagent per target, the complexity of assay design is reduced because validated, noncross-reactive affinity probe pairs and multi-epitopic targets are not required.…”

Current status and issues of protein solution biophysics—Session 1SDP

“…Precise thermodynamic measurements for drug development are now possible for very weak but specific interactions of biomolecules (Nagatoishi et al 2018). Mr. William E. Arter (Department of Chemistry, University of Cambridge) introduced biophysical analyses by microchip electrophoresis, focusing on alpha-synuclein oligomers (Arter et al 2018).…”