Single cells have unique biophysical signatures that can rapidly change during various disease states. For instance, cellular density is an inherent property differing between cell types. Characterizing changes in fundamental density properties down to the single-cell level can reveal sub-populations in pathological states. Here, we have developed a microfluidic, magnetic levitation-based assay (MagDense) that detects minute density differences of individual red blood cells (RBCs) down to 0.0001 g mL⁻¹ resolution. This assay fractionates RBCs based on their density profiles in a non-ionic paramagnetic medium flowing in a capillary microchannel placed between magnets with same poles facing each other. Based on precisely measured levitation height and density of individual RBCs at their specific equilibrium state, we demonstrated that MagDense can accurately analyze the density of sickle hemoglobin (HbS)-containing RBCs and normal hemoglobin (HbA)-containing RBCs. In addition, the precise density and cell size measurements at the single cell level showed three different sub-populations of RBCs in blood samples from individuals with homozygous sickle cell disease receiving blood transfusions; where less dense, HbA-containing RBCs levitated higher, while the denser, HbS-containing RBCs levitated lower. We compared the mean RBC densities of sickle cell disease subjects with healthy controls and found distinctly separated bands of RBC density for each group denoting the likely range of cell densities seen in the blood samples. The high resolution of our method enabled measurement of deviation from the mean RBC density. Moreover, we introduced a new term as a measure of density dispersion, “RBC levitational density width, RLDW”. Mean RBC density in sickle cell disease associated with hemoglobin from complete blood count (p = 0.032, linear regression) and RLDW associated with absolute reticulocyte count (ARC) and RBC distribution width (RDW) from complete blood count (p = 0.002 for ARC and p = 003 for RDW, linear regression). Our magnetic levitation-based assay enables rapid, accurate, density-based imaging, profiling and label-free monitoring of single RBCs. Our approach can be broadly applicable to investigate blood cell disorders and the effects of emerging pharmacological and curative therapies in patient outcomes.

This design demonstrates the isolation of extracellular vesicles (EVs) from human plasma, utilizing polymer beads as assay surfaces to capture EV subpopulations and magnetic levitation to sort beads at different outlets.

The biophysical properties of single cells are crucial for understanding cellular function and behavior in biology and medicine. However, precise manipulation of cells in 3-D microfluidic environments remains challenging, particularly for heterogeneous populations. Here, we present “Electro-LEV,” a unique platform integrating electromagnetic and magnetic levitation principles for dynamic 3-D control of cell position during separation. We demonstrated that small current adjustments in electromagnets significantly alter the levitation heights of diverse particles and cell types. By periodically modulating and tracking cell positions along the z-axis, Electro-LEV identified distinct levitation behaviors between single cells and cell clusters, with clusters responding more rapidly to magnetic field changes. Furthermore, we demonstrated that Electro-LEV significantly enhances the purity and efficiency of levitational sorting, achieving 10-fold enrichment of live cells from 50% starting viability samples and 18.8-fold enrichment from 10% starting viability samples. These results establish Electro-LEV as a powerful tool for investigating cellular heterogeneity, differentiating cell sizes and types, and improving cell sorting efficiency. Thus, Electro-LEV is broadly applicable, offering different possibilities for high-resolution cell analysis and label-free cell sorting in various biomedical fields, including but not limited to single-cell sequencing and drug screening.