Mengxi Wu

Isolation of exosomes from whole blood by integrating acoustics and microfluidics

Significance We have developed a unique, integrated, on-chip technology that is capable of isolating exosomes or other types of extracellular vesicles, directly from undiluted whole-blood samples in an automated fashion. Automated exosome isolation enables biohazard containment, short processing time, reproducible results with little human intervention, and convenient integration with downstream exosome analysis units. Our method of integrating acoustics and microfluidics leads to the isolation of exosomes with high purity and yield. With its label-free, contact-free, and biocompatible nature, it offers the potential to preserve the structures, characteristics, and functions of isolated exosomes. This automated, point-of-care device can further help in advancing exosome-related biomedical research with potential applications in health monitoring, disease diagnostics, and therapeutics. Exosomes are nanoscale extracellular vesicles that play an important role in many biological processes, including intercellular communications, antigen presentation, and the transport of proteins, RNA, and other molecules. Recently there has been significant interest in exosome-related fundamental research, seeking new exosome-based biomarkers for health monitoring and disease diagnoses. Here, we report a separation method based on acoustofluidics (i.e., the integration of acoustics and microfluidics) to isolate exosomes directly from whole blood in a label-free and contact-free manner. This acoustofluidic platform consists of two modules: a microscale cell-removal module that first removes larger blood components, followed by extracellular vesicle subgroup separation in the exosome-isolation module. In the cell-removal module, we demonstrate the isolation of 110-nm particles from a mixture of micro- and nanosized particles with a yield greater than 99%. In the exosome-isolation module, we isolate exosomes from an extracellular vesicle mixture with a purity of 98.4%. Integrating the two acoustofluidic modules onto a single chip, we isolated exosomes from whole blood with a blood cell removal rate of over 99.999%. With its ability to perform rapid, biocompatible, label-free, contact-free, and continuous-flow exosome isolation, the integrated acoustofluidic device offers a unique approach to investigate the role of exosomes in the onset and progression of human diseases with potential applications in health monitoring, medical diagnosis, targeted drug delivery, and personalized medicine.

Enriching Nanoparticles via Acoustofluidics

Focusing and enriching submicrometer and nanometer scale objects is of great importance for many applications in biology, chemistry, engineering, and medicine. Here, we present an acoustofluidic chip that can generate single vortex acoustic streaming inside a glass capillary through using low-power acoustic waves (only 5 V is required). The single vortex acoustic streaming that is generated, in conjunction with the acoustic radiation force, is able to enrich submicrometer- and nanometer-sized particles in a small volume. Numerical simulations were used to elucidate the mechanism of the single vortex formation and were verified experimentally, demonstrating the focusing of silica and polystyrene particles ranging in diameter from 80 to 500 nm. Moreover, the acoustofluidic chip was used to conduct an immunoassay in which nanoparticles that captured fluorescently labeled biomarkers were concentrated to enhance the emitted signal. With its advantages in simplicity, functionality, and power consumption, the acoustofluidic chip we present here is promising for many point-of-care applications.

Acoustic Separation of Nanoparticles in Continuous Flow

The separation of nanoscale particles based on their differences in size is an essential technique to the nanoscience and nanotechnology community. Here, nanoparticles are successfully separated in a continuous flow by using tilted-angle standing surface acoustic waves. The acoustic field deflects nanoparticles based on volume, and the fractionation of nanoparticles is optimized by tuning the cutoff parameters. The continuous separation of nanoparticlesis demonstrated with a ≈90% recovery rate. The acoustic nanoparticle separation method is versatile, non-invasive, and simple.

Mixing high-viscosity fluids via acoustically driven bubbles

We present an acoustofluidic micromixer which can perform rapid and homogeneous mixing of highly viscous fluids in the presence of an acoustic field. In this device, two high-viscosity polyethylene glycol (PEG) solutions were co-injected into a three-inlet PDMS microchannel with the center inlet containing a constant stream of nitrogen flow which forms bubbles in the device. When these bubbles were excited by an acoustic field generated via a piezoelectric transducer, the two solutions mixed homogenously due to the combination of acoustic streaming, droplet ejection, and bubble eruption effects. The mixing efficiency of this acoustofluidic device was evaluated using PEG-700 solutions which are ~106 times more viscous than deionized (DI) water. Our results indicate homogenous mixing of the PEG-700 solutions with a ~0.93 mixing index. The acoustofluidic micromixer is compact, inexpensive, easy to operate, and has the capacity to mix highly viscous fluids within 50 milliseconds.

Acoustofluidic coating of particles and cells

On-chip microparticle and cell coating technologies enable a myriad of applications in chemistry, engineering, and medicine. Current microfluidic coating technologies often rely on magnetic labeling and concurrent deflection of particles across laminar streams of chemicals. Herein, we introduce an acoustofluidic approach for microparticle and cell coating by implementing tilted-angle standing surface acoustic waves (taSSAWs) into microchannels with multiple inlets. The primary acoustic radiation force generated by the taSSAW field was exploited in order to migrate the particles across the microchannel through multiple laminar streams, which contained the buffer and coating chemicals. We demonstrate effective coating of polystyrene microparticles and HeLa cells without the need for magnetic labelling. We characterized the coated particles and HeLa cells with fluorescence microscopy and scanning electron microscopy. Our acoustofluidic-based particle and cell coating method is label-free, biocompatible, and simple. It can be useful in the on-chip manufacturing of many functional particles and cells.

Standing Surface Acoustic Wave (SSAW)‐Based Fluorescence‐Activated Cell Sorter

Microfluidic fluorescence-activated cell sorters (μFACS) have attracted considerable interest because of their ability to identify and separate cells in inexpensive and biosafe ways. Here a high-performance μFACS is presented by integrating a standing surface acoustic wave (SSAW)-based, 3D cell-focusing unit, an in-plane fluorescent detection unit, and an SSAW-based cell-deflection unit on a single chip. Without using sheath flow or precise flow rate control, the SSAW-based cell-focusing technique can focus cells into a single file at a designated position. The tight focusing of cells enables an in-plane-integrated optical detection system to accurately distinguish individual cells of interest. In the acoustic-based cell-deflection unit, a focused interdigital transducer design is utilized to deflect cells from the focused stream within a minimized area, resulting in a high-throughput sorting ability. Each unit is experimentally characterized, respectively, and the integrated SSAW-based FACS is used to sort mammalian cells (HeLa) at different throughputs. A sorting purity of greater than 90% is achieved at a throughput of 2500 events s-1 . The SSAW-based FACS is efficient, fast, biosafe, biocompatible and has a small footprint, making it a competitive alternative to more expensive, bulkier traditional FACS.

Clinical utility of non-EpCAM based circulating tumor cell assays

Methods enabling the isolation, detection, and characterization of circulating tumor cells (CTCs) in blood have clear potential to facilitate precision medicine approaches in patients with cancer, not only for prognostic purposes but also for prediction of the benefits of specific therapies in oncology. However, current CTC assays, which capture CTCs based on expression of epithelial cell adhesion molecule (EpCAM), fail to capture cells from de-differentiated tumors and carcinomas undergoing loss of the epithelial phenotype during the invasion/metastatic process. To address this limitation, many groups are developing non-EpCAM based CTC assays that incorporate nanotechnology to improve test sensitivity for rare but important cells that may otherwise go undetected, and therefore may improve upon clinical utility. In this review, we outline emerging non-EpCAM based CTC assays utilizing nanotechnology approaches for CTC capture or characterization, including dendrimers, magnetic nanoparticles, gold nanoparticles, negative selection chip or software-based on-slide methods, and nano-scale substrates. In addition, we address challenges that remain for the clinical translation of these platforms.