(I-344) Electrokinetic Manipulation of Protein Aggregates

Introduction::
Isolation of extracellular vesicles, proteins, and nucleic acids from blood using dielectrophoresis (DEP) is a promising approach for liquid biopsy-based testing for diseases including cancer [1]. By applying an alternating current to an electrode array a DEP force is created that draws nanoparticles of a certain size range and material composition towards the high electric field gradient region around the electrode edge. This effectively recovers and concentrates diagnostically relevant biomarkers. However, when isolating biomarkers of interest from plasma, we have also observed the collection of serum albumin protein, contrary to classical predictions of low DEP forces for individual albumin molecules. Understanding the dielectrophoretic collection of proteins and their aggregates is crucial to enhance the collection of other particles with the most diagnostic value.



Materials and Methods::
Lyophilized bovine serum albumin (BSA) was dissolved in 0.5 X PBS to simulate plasma. Spontaneously forming aggregates of BSA were characterized using nanoparticle tracking analysis, fast protein liquid chromatography, and transmission electron microscopy. Aggregates of BSA were disrupted using the nonionic detergent Tween-20. Dielectrophoretic collection of naturally forming BSA aggregates and BSA treated with Tween-20 was performed using Biological Dynamics’ (San Diego, CA) electrokinetic chip-based Verita® platform with a 14 kHz alternating electric field at 10 Vpp for 10 minutes in 0.5X PBS (6.8 mS/cm). 100nm polystyrene beads were collected simultaneously to simulate biomarker collection. To identify a dielectrophoretic crossover frequency, the frequency was swept from direct current conditions to 2 MHz in varying buffer conductivities. The fluorescence intensity of collected or labeled material was quantified using a custom MATLAB program [2].



Results and Discussion::
We observed stable 236.8 nm diameter aggregates of BSA forming spontaneously when dissolved in 0.5X PBS. In agreement with classical DEP theory, the BSA aggregate particles experienced dielectrophoretic collection at much lower electric field gradient magnitudes than would be expected for a 7 nm diameter single protein of BSA. A crossover frequency for aggregates of BSA was observed at 70 Hz in a low conductance buffer (0.05X PBS, 0.68 mS/cm), above which negative DEP of BSA was observed. When the aggregates were treated with 5% Tween-20, the mean particle radius decreased significantly by nanoparticle tracking analysis, and these particles were not collected under the same DEP conditions as the larger aggregates. The presence of Tween-20 did not affect the simultaneous collection of 100nm polystyrene beads.



Conclusions::
DEP was found to be collecting aggregates of BSA rather than individual BSA molecules due to their larger radius. After disruption of the BSA aggregates using a nonionic detergent, the collection of BSA was significantly reduced and the variability of collection of desired biomarkers was decreased without significantly decreasing the collection efficiency. This study contributes to our understanding of protein electrokinetics and will increase control over the material isolated from plasma using dielectrophoresis to advance microfluidic diagnostic testing.



Acknowledgements::



This project was supported by the National Institutes of Health grant R37CA258787 to S.D.I.



This project was also supported by funding (Exploratory 2021-1445) from The Knight Cancer Institute’s Cancer Early Detection Advanced Research Center (CEDAR) at the Oregon Health and Science University.

Transmission electron microscopy was performed at the Multiscale Microscopy Core, a member of the OHSU University Shared Resource Cores.



References: :
1.
Ibsen, S.D., et al., Rapid Isolation and Detection of Exosomes and Associated Biomarkers from Plasma. 2017, American Chemical Society. p. 6641-6651.

2.
Gustafson, K.T., et al., Automated fluorescence quantification of extracellular vesicles collected from blood plasma using dielectrophoresis. Lab on a Chip, 2021. 21(7): p. 1318-1332.