Magnetic Levitation Patterns of Microfluidic-Generated Nanoparticle-Protein Complexes

Abstract

Magnetic levitation (MagLev) has recently emerged as a powerful method to develop diagnostic technologies based on the exploitation of the nanoparticle (NP)-protein corona. However, experimental procedures improving the robustness, reproducibility, and accuracy of this technology are largely unexplored. To contribute to filling this gap, here, we investigated the effect of total flow rate (TFR) and flow rate ratio (FRR) on the MagLev patterns of microfluidic-generated graphene oxide (GO)-protein complexes using bulk mixing of GO and human plasma (HP) as a reference. Levitating and precipitating fractions of GO-HP samples were characterized in terms of atomic force microscopy (AFM), bicinchoninic acid assay (BCA), and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE), and nanoliquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). We identified combinations of TFR and FRR (e.g., TFR = 35 μL/min and FRR (GO:HP) = 9:1 or TFR = 3.5 μL/min and FRR (GO:HP) = 19:1), leading to MagLev patterns dominated by levitating and precipitating fractions with bulk-like features. Since a typical MagLev experiment for disease detection is based on a sequence of optimization, exploration, and validation steps, this implies that the optimization (e.g., searching for optimal NP:HP ratios) and exploration (e.g., searching for MagLev signatures) steps can be performed using samples generated by bulk mixing. When these steps are completed, the validation step, which involves using human specimens that are often available in limited amounts, can be made by highly reproducible microfluidic mixing without any ex novo optimization process. The relevance of developing diagnostic technologies based on MagLev of coronated nanomaterials is also discussed.

Keywords: graphene oxide; magnetic levitation; microfluidics; protein corona.

Figure 1

Representative workflow of the study: (a) GO-HP incubation within a microfluidic cartridge at controlled flow rates. Collected samples were inserted in (b) a MagLev device, responsible for a (c) magnetic field and thus a dynamic balance between (d) the acting forces, i.e., magnetic (F_m) and gravitational (F_g) force. Images of the samples were (e) acquired and (f) processed frame by frame to compute (g) the corresponding intensity profiles of the investigated samples (black lines). The profile was fitted by using a multipeak Gaussian distribution (red line). More details about image processing and the determination of the experimental error can be found in Figures S6 and S7 in the Supplementary Materials.

Figure 2

Representative MagLev patterns for GO-HP samples obtained via (a) static incubation and microfluidic incubation at (b–d) different flow rates. (e) Corresponding MagLev profiles of the investigated samples.

Figure 3

Representative MagLev patterns for GO-HP samples obtained via microfluidic incubation (red) with a TFR of 3.5 μL/min at different flow ratios: 4:1 dashed pink line, 9:1 solid red line, and 19:1 dotted purple line. Static incubation references for each condition are reported as dashed grey line, solid grey line, and dotted grey line, respectively.

Figure 4

Atomic force microscopy (AFM) images for (a) levitating and (b) precipitating fractions of GO-HP samples collected after the MagLev measurements. (c) Z-profiles of the levitating (light green) and precipitating fractions (dark green). (d) Representative 1D SDS-PAGE image of the investigated samples (measurements were performed in triplicates) and (e) corresponding intensity profiles, which were normalized to the total lane intensity. The dominant protein contributions for the main peaks are reported in Table S2, as detected in nanoliquid chromatography–tandem mass spectrometry experiments.