Deterministic Sorting of Submicrometer Particles and Extracellular Vesicles Using a Combined Electric and Acoustic Field.

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.

A low-cost and high-throughput benchtop cell sorter for isolating white blood cells from whole blood.

The ability to isolate and purify white blood cells (WBCs) from mixed ensembles such as blood would benefit autologous cell-based therapeutics as well as diagnosis of WBC disorders. Current WBCs isolation methods have the limitations of low purity or requiring complex and expensive equipment. In addition, due to the overlap in size distribution between lymphocytes (i.e., a sub-population of WBCs) and red blood cells (RBCs), it is challenging to achieve isolation of entire WBCs populations. In this work, we developed an inertial microfluidics-based cell sorter, which enables size-based, high-throughput isolation, and enrichment of WBCs from RBC-lysed whole blood. Using the developed inertial microfluidic chip, the sorting resolution is sharpened within 2 µm, which achieved separation between 3 and 5 µm diameter particles. Thus, with the present cell sorter, a full population of WBCs can be isolated from RBC-lysed blood samples with recovery ratio of 92%, and merely 5% difference in the composition percentage of the three subpopulations of granulocytes, monocytes, and lymphocytes compared to the original sample. Furthermore, our cell sorter is designed to enable broad application of size-based inertial cell sorting by supplying a series of microchips with different sorting cutoff size. This strategy allows us to further enrich the lymphocytes population by twofold using another microchip with a cutoff size between 10 and 15 µm. With simplicity and efficiency, our cell sorter provides a powerful platform for isolating and sorting of WBCs and also envisions broad potential sorting applications for other cell types.

Exosome Purification and Analysis Using a Facile Microfluidic Hydrodynamic Trapping Device.

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.

A deep learning approach for designed diffraction-based acoustic patterning in microchannels.

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.

Massively Multiplexed Submicron Particle Patterning in Acoustically Driven Oscillating Nanocavities.

Nanoacoustic fields are a promising method for particle actuation at the nanoscale, though THz frequencies are typically required to create nanoscale wavelengths. In this work, the generation of robust nanoscale force gradients is demonstrated using MHz driving frequencies via acoustic-structure interactions. A structured elastic layer at the interface between a microfluidic channel and a traveling surface acoustic wave (SAW) device results in submicron acoustic traps, each of which can trap individual submicron particles. The acoustically driven deformation of nanocavities gives rise to time-averaged acoustic fields which direct suspended particles toward, and trap them within, the nanocavities. The use of SAWs permits massively multiplexed particle manipulation with deterministic patterning at the single-particle level. In this work, 300 nm diameter particles are acoustically trapped in 500 nm diameter cavities using traveling SAWs with wavelengths in the range of 20-80 µm with one particle per cavity. On-demand generation of nanoscale acoustic force gradients has wide applications in nanoparticle manipulation, including bioparticle enrichment and enhanced catalytic reactions for industrial applications.

Submicron Particle Focusing and Exosome Sorting by Wavy Microchannel Structures within Viscoelastic Fluids.

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.

A MoS2-MWCNT based fluorometric nanosensor for exosome detection and quantification.

Circulating exosomes in body fluids are involved in many diseases and have important roles in pathophysiological processes. Specifically, they have emerged as a promising new class of biomarkers in cancer diagnosis and prognosis because of their high concentration and availability in a variety of biological fluids. The ability to quantitatively detect and characterize these nano-sized vesicles is crucial to make use of exosomes as a reliable biomarker for clinical applications. However, current methods are mostly technically challenging and time-consuming which prevents them from being adopted in clinical practice. In this work, we have developed a rapid sensitive platform for exosome detection and quantification by employing MoS2-multiwall carbon nanotubes as a fluorescence quenching material. This exosome biosensor shows a sensitive and selective biomarker detection. Using this MoS2-MWCNT based fluorometric nanosensor to analyze exosomes derived from MCF-7 breast cancer cells, we found that CD63 expression could be measured based on the retrieved fluorescence of the fluorophore with a good linear response range of 0-15% v/v. In addition, this nanosensing technique is able to quantify exosomes with different surface biomarker expressions and has revealed that exosomes secreted from MCF-7 breast cancer cells have a higher CD24 expression compared to CD63 and CD81.

A Microfluidic DNA Sensor Based on Three-Dimensional (3D) Hierarchical MoS2/Carbon Nanotube Nanocomposites.

In this work, we present a novel microfluidic biosensor for sensitive fluorescence detection of DNA based on 3D architectural MoS2/multi-walled carbon nanotube (MWCNT) nanocomposites. The proposed platform exhibits a high sensitivity, selectivity, and stability with a visible manner and operation simplicity. The excellent fluorescence quenching stability of a MoS2/MWCNT aqueous solution coupled with microfluidics will greatly simplify experimental steps and reduce time for large-scale DNA detection.

Thioglycolic Acid-Capped CdS Quantum Dots Conjugated to α-Amylase as a Fluorescence Probe for Determination of Starch at Low Concentration.

In the present research, water soluble thioglycolic acid-capped CdS quantum dots (QDs) were synthesized by chemical precipitation method. The characteristics of prepared quantum dots were determined using X-Ray Diffraction (XRD) and Transmission Electron Microscopy (TEM). The obtained results revealed that CdS QDs have 5.60 nm crystallite size, hexagonal wurtzite structure and spherical morphology with less than 10 nm diameter. The photoluminescence (PL) spectroscopy was performed in order to study the effect of the presence of starch solutions. Blue emission peaks were positioned at 488 nm and its intensity quenched by increasing the concentration of starch solutions. The result of PL quenches in range of studied concentrations (0-100 ppm) was best described by Michaelis-Menten model. The amount of Michaelis constant (Km) for immobilized α-amylase in this system was about 68.08 ppm which showed a great tendency of enzyme to hydrolyze the starch as substrate. Finally, the limit of detection (LOD) was found to be about 2.24 ppm.