Manipulating small model animals and biological nanoparticles via acoustofluidics

Abstract

As rapid developments in technology merge acoustics and microfluidics, acoustofluidic technology has been increasingly employed in biophysical and biomedical research to address various challenges, especially in the fields of tissue engineering, liquid biopsy, clinical diagnostics and therapeutics. Acoustofluidic technologies offer highly biocompatible, label-free and contact-less manipulation of objects based on differential effects including acoustic streaming and acoustic radiation force. However, acoustofluidic technologies have not been fully implemented into model animal studies to simplify the manipulation, lower the cost, and increase the throughput. In addition, despite the expansion of the scope of acoustic-based particle manipulation technologies from the micro to the nanoscale over the past decade, limitations continue to pose challenges in manipulating sub-100 nm particles using acoustic waves. As a result, developing an acoustic technique capable of manipulating sub-100 nm particles would strengthen the capabilities of acoustic manipulation and fulfill needs in areas such as biomedicine, biophysics, optics, electronics, and materials science.First, with the nematode Caenorhabditis elegans (C. elegans) employed as a model animal in the field of developmental biology, neuroscience, human diseases, aging and drug screening for more than 50 years, we sought to extend acoustofluidic technologies into C. elegans research to address the key drawbacks of current C. elegans analysis procedures. An acoustofluidic chip capable of rotating C. elegans in both static and continuous flow in a controllable, precise, high-throughput and stable manner was then developed. Rotational manipulation was achieved by exposing C. elegans to a surface acoustic wave (SAW) field that generated a vortex inside a microchannel. By controlling the propagation of the SAW, we achieved bidirectional and stepwise rotation of C. elegans. Using this chip, we have clearly imaged the dopaminergic neurons, as well as the vulval muscles and muscle fibers of the C. elegans in different orientations. These achievements are difficult to realize through conventional microscopy. After that, another tool for effectively isolating and categorizing large quantities of C. elegans based on different phenotypes was developed as an integrated acoustofluidic chip. This chip was capable of identifying worms of interest based on expression of a fluorescent protein in a continuous flow and then separating them accordingly in a high-throughput manner. For example, L3 worms can be processed at a throughput of around 70 worms/min with a sample purity over 99%, which remains over 90% when the throughput is increased to around 115 worms/min. In our acoustofluidic chip, the time period to complete the detection and sorting of one worm is only 50 ms, which outperforms nearly all existing microfluidics-based worm sorting devices and may be further reduced to achieve higher throughput. Moving forward with the experience we gained through manipulating C. elegans via acoustofluidics, we are aiming to solve a critical issue in current acoustofluidic technologies. Although acoustic fields have been increasingly used to pattern, focus, and separate micro- and nanometer-sized particles for biomedical applications, the acoustic-based separation of nanoscale bioparticles in sub-100 nm range remains a significant challenge. To address this problem, we present Acoustic Nanoscale Separation via Wave-pillar Excitation Resonance (ANSWER), allowing for the high-resolution, tunable separation of nanoscale bioparticles ranging from 50 nm to 1000 nm. We created virtual acoustic pillars that enable iterative deflection of particles for precision separation via an excitation resonance. Controlling the cut-off diameter is optimized by acoustic frequency, power, and microchannel dimensions in separating sub-100 nm particles. To demonstrate the potential of our ANSWER platform in biomedical applications, we have shown its ability to fractionate small extracellular vesicle (sEV) subpopulations. For the first time, sEV subpopulations can be rapidly separated (<10 minutes) directly from human plasma without sample preprocessing or complex nanofabrication. Due to its high separation purity (>96% small exosomes, >80% exomeres), ANSWER shows promise as a powerful tool that will enable more in-depth studies into the complexity, heterogeneity, and functionality of sEV subpopulations. To simplify the operation and keep the biological components in their native environment, separation without sheathflow was then discussed with the ANSWER platform. The same sheathless separation concept was then extended to the microscale for the isolation of plasma directly from human whole blood. The work in this dissertation presents a comprehensive investigation and exploration of both the mechanism of specialized acoustic field generation and modulation, as well as the application of highly controllable manipulation of model animals and nanoscale (< 100 nm) biological particles. We hope our work can benefit and enable new possibilities in the relevant research fields.