Exosomes, submicron membrane vesicles (30−200 nm) secreted by almost all cells, containing significant information such as proteins, microRNAs and DNAs, are closely associated with disease diagnostic and prognostic tests for liquid biopsy in clinical practice. However, their inherently small sizes lead to great challenges for isolating them from complex body fluids with high-throughput and high-purity. In this work, a reverse wavy channel structure using viscoelastic fluids with the addition of biocompatible polymer was presented for elasto-inertial focusing and sorting of submicron particles and exosomes. The microfluidic periodically reversed Dean secondary flow generated by repeated wavy channel structures could facilitate particle focusing compared with traditional straight channels. Four differently sized fluorescent submicron spheres (1 μm, 500 nm, 300 and 100 nm) were used to study the focusing behavior under various conditions. We have achieved simple, high-throughput, and label-free sorting of exosomes with purity higher than 92% and recovery higher than 81%. This developed elasto-inertial exosome sorting technique may provide a promising platform in various exosome-related biological research and pharmaceutical applications.

Exosomes are nanosized (30–150 nm) extracellular vesicles (EVs) secreted by various cell types. They are easily accessible in biological fluids and contain specific disease biomarkers, making them attractive for diagnosis and prognosis applications. Accurate biological characterization of exosomes is an important step toward clinical applications that require effective and precise isolation of subpopulations of exosomes. It is therefore of particular importance to develop an efficient and reliable exosome purification technique to isolate exosomes from the heterogeneous extracellular fluids. In this work, we intend to isolate and visualize exosomes by combining an affinity-based method and passive microfluidic particle trapping. Microbeads with a diameter of 20 μm are first functionalized with streptavidin and biotinylated antibodies and then used to immobilize and enrich exosomes on their surfaces using antigen–antibody affinity binding. We have developed a microfluidic device with trapping arrays to efficiently trap a large number of individual microbeads with enriched exosomes at the single-particle level, i.e., one single bead per trapping site, on the basis of a passive hydrodynamic trapping principle. The large-scale microfluidic single-bead trapping permits massively multiplexed fluorescence detection and quantification of the individual beads, which prevents the optical interfering of background noise as well as allowing one to acquire an average fluorescence density of a single bead for an accurate fluorescence-based exosome quantification. In addition, on-chip elusion and lysis of the protein and RNA content of captured exosomes enable further molecular analysis of exosomes, including Western blot and quantitative polymerase chain reaction. This microfluidic device provides a rapid and straightforward capturing and quantification method to analyze EVs for a variety of biological studies and applications.

Sorting of extracellular vesicles has important applications in early stage diagnostics. Current exosome isolation techniques, however, suffer from being costly, having long processing times, and producing low purities. Recent work has shown that active sorting via acoustic and electric fields are useful techniques for microscale separation activities, where combining these has the potential to take advantage of multiple force mechanisms simultaneously. In this work, we demonstrate an approach using both electrical and acoustic forces to manipulate bioparticles and submicrometer particles for deterministic sorting, where we find that the concurrent application of dielectrophoretic (DEP) and acoustophoretic forces decreases the critical diameter at which particles can be separated. We subsequently utilize this approach to sort subpopulations of extracellular vesicles, specifically exosomes (<200 nm) and microvesicles (>300 nm). Using our combined acoustic/electric approach, we demonstrate exosome purification with more than 95% purity and 81% recovery, well above comparable approaches.

Acoustic waves can be used to accurately position cells and particles and are appropriate for this activity owing to their biocompatibility and ability to generate microscale force gradients. Such fields, however, typically take the form of only periodic one or two-dimensional grids, limiting the scope of patterning activities that can be performed. Recent work has demonstrated that the interaction between microfluidic channel walls and travelling surface acoustic waves can generate spatially variable acoustic fields, opening the possibility that the channel geometry can be used to control the pressure field that develops. In this work we utilize this approach to create novel acoustic fields. Designing the channel that results in a desired acoustic field, however, is a non-trivial task. To rapidly generate designed acoustic fields from microchannel elements we utilize a deep learning approach based on a deep neural network (DNN) that is trained on images of pre-solved acoustic fields. We use then this trained DNN to create novel microchannel architectures for designed microparticle patterning. Microfluidic platforms are an important tool for precise micromanipulation, using force gradients on the length scale of cells and microparticles themselves. A major application space in microfluidics is the patterning and long-term retention of living cells; patterning is a powerful tool for the formation of cellular spheroids 1 , tissue engineering 2 , drug screening 1 and single-cell analysis 3 , cellular interactions 4 , and mechanobiology 5. The design of microfluidic systems for these activities, however, typically occurs on a first-principles basis and generally omits parametric optimization of the microchannel features. This has a large impact when hydrodynamic forces are involved, since the channel geometry can affect how, for example, lift and drag forces are distributed. In the case of hydrodynamic effects there has been some work using numerical simulations 6,7 to configure channels to optimize their mixing and dilution characteristics, showing the promise of modifying channel shapes for creating optimized microfluidic devices. Actively applied forces, rather than just hydrodynamic ones, however, are increasingly integrated into microfluidic devices for more refined and dynamic actuation. Acoustic fields are a particularly useful method for micromanipulation due to their biocompatibility, high force magnitudes arising from MPa order pressures and wavelengths that can match the length scale of individual cells. To date, acoustic fields have shown their capacity for versatile microscale actuation activities including patterning 8 , acoustic streaming 9-11 , droplet manipulation 12 , cell cultures 13,14 , mixing 15,16 and sorting 8 , with actuation down to the single cell level 12,17. In the case of patterning, these fields have typically been limited to creating simple lines 18 or grids 19 of cells and microparticles due to the limitations imposed by the transducers and channel geometries. For the most part these ...

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