Bioanalytical Methods
Moving Molecules with Manic Precision
Biomolecules in biological samples are rarely present in concentrations, volumes and purity required by several downstream bioanalytical platforms such as sequencing machines and mass and optical spectrometers thus limiting sensitivity ranges. For instance, a cell contains roughly 10 pg of DNA, mRNA in the same range, and fM concentrations of proteins of interest. Biochemical surface assays-microarrays often have the same range of molecular concentration and density.
Demonstration of DNA separation and extraction using the Bidirectional Flow Filter (BFF). From Bacheva, V. et al., Anal. Chem. 94, 10584–10588 (2022).
One approach of quantifying these biomolecules is to amplify the signal before a readout using, for instance, enzymatic amplification. We believe a more promising approach for analyzing a low abundance biomolecules is to integrate low volume liquid handling with electrokinetic approaches to concentrate, isolate, and read signals with high sensitivity and specificity, while tuning the limits of detection based on the molecule of interest.
Schematic illustration of the PSiO2 biosensor setup and an ITP-based biosensing experiment. From Arshavsky-Graham, S. et al., ACS Sensors 2, 1767–1773 (2017).
Demonstration of bio-separation in a BFF. From Bacheva, V. et al., Angew. Chemie Int. Ed. 59, 12894–12899 (2020).
Bidirectional Flow Filter (BFF) Devices
Using AC-Field Effect Electro-Osmosis (AC-FEEO) and its ability to induce counter flows, we are able to separate biomolecules based on their diffusivity within a ‘reconfigurable microfluidic' chip. These devices have been characterized and used for separation of (1) labeled antibodies from unbound dyes, (2) oligonucleotides based on their length, and (3) microbeads based on their size.
Isotachophoretic Concentration of Analytes
Variable electrophoretic mobility of biomolecules in specific buffer systems in an electric field allows for focusing and concentration of these biomolecules in microfluidic channels. We use these devices for concentration and analysis of DNA and proteins, with 10⁵ and 10³ fold concentration enhancement respectively. Beyond being standalone devices, this forms a part of our tumor profiling workflows.
Photograph of a large-volume ITP device. Insets show a focused fluorescent dye. From van Kooten, X. F. et al., Sci. Rep. 7, 10467 (2017).
