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1. Cell lysis via acoustically oscillating sharp edges

   https://doi.org/10.1039/C9LC00498J  

   Wang, Zeyu ; Huang, Po-Hsun ; Chen, Chuyi ; Bachman, Hunter ; Zhao, Shuaiguo ; Yang, Shujie ; Huang, Tony J. ( December 2019 , Lab on a Chip)

   In this article, we demonstrate an acoustofluidic device for cell lysis using the acoustic streaming effects induced by acoustically oscillating sharp-edged structures. The acoustic streaming locally generates high shear forces that can mechanically rupture cell membranes. With the acoustic-streaming-derived shear forces, our acoustofluidic device can perform cell lysis in a continuous, reagent-free manner, with a lysis efficiency of more than 90% over a range of sample flow rates. We demonstrate that our acoustofluidic lysis device works well on both adherent and non-adherent cells. We also validate it using clinically relevant samples such as red blood cells infected with malarial parasites. Additionally, the unique capability of our acoustofluidic device was demonstrated by performing downstream protein analysis and gene profiling without additional washing steps post-lysis. Our device is simple to fabricate and operate while consuming a relatively low volume of samples. These advantages and other features including the reagent-free nature and controllable lysis efficiency make our platform valuable for many biological and biomedical applications, particularly for the development of point-of-care platforms. 

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2. Fluorescence-based sorting of Caenorhabditis elegans via acoustofluidics

   https://doi.org/10.1039/d0lc00051e  

   Zhang, Jinxin ; Hartman, Jessica H. ; Chen, Chuyi ; Yang, Shujie ; Li, Qi ; Tian, Zhenhua ; Huang, Po-Hsun ; Wang, Lin ; Meyer, Joel N. ; Huang, Tony Jun ( May 2020 , Lab on a Chip)

   Effectively isolating and categorizing large quantities of Caenorhabditis elegans ( C. elegans ) based on different phenotypes is important for most worm research, especially genetics. Here we present an integrated acoustofluidic chip capable of identifying worms of interest based on expression of a fluorescent protein in a continuous flow and then separate them accordingly in a high-throughput manner. Utilizing planar fiber optics as the detection unit, our acoustofluidic device requires no temporary immobilization of worms for interrogation/detection, thereby improving the throughput. Implementing surface acoustic waves (SAW) as the sorting unit, our device provides a contact-free method to move worms of interest to the desired outlet, thus ensuring the biocompatibility for our chip. Our device can sort worms of different developmental stages (L3 and L4 stage worms) at high throughput and accuracy. For example, L3 worms can be processed at a throughput of around 70 worms per min with a sample purity over 99%, which remains over 90% when the throughput is increased to around 115 worms per 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. 

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3. Surface acoustic waves enable rotational manipulation of Caenorhabditis elegans

   https://doi.org/10.1039/c8lc01012a  

   Zhang, Jinxin ; Yang, Shujie ; Chen, Chuyi ; Hartman, Jessica H. ; Huang, Po-Hsun ; Wang, Lin ; Tian, Zhenhua ; Zhang, Peiran ; Faulkenberry, David ; Meyer, Joel N. ; et al ( March 2019 , Lab on a Chip)

   Controllable, precise, and stable rotational manipulation of model organisms is valuable in many biomedical, bioengineering, and biophysics applications. We present an acoustofluidic chip capable of rotating Caenorhabditis elegans ( C. elegans ) in both static and continuous flow in a controllable manner. Rotational manipulation was achieved by exposing C. elegans to a surface acoustic wave (SAW) field that generated a vortex distribution inside a microchannel. By selectively activating interdigital transducers, we achieved bidirectional rotation of C. elegans , namely counterclockwise and clockwise, with on-demand switching of rotation direction in a single chip. In addition to continuous rotation, we also rotated C. elegans in a step-wise fashion with a step angle as small as 4° by pulsing the signal duration of SAW from a continuous signal to a pulsed signal down to 1.5 ms. Using this device, we have clearly imaged the dopaminergic neurons of C. elegans with pdat-1:GFP expression, as well as the vulval muscles and muscle fibers of the worm with myo-3::GFP fusion protein expression in different orientations and three dimensions. These achievements are difficult to realize through conventional ( i.e. , non-confocal) microscopy. The SAW manipulations did not detectably affect the health of the model organisms. With its precision, controllability, and simplicity in fabrication and operation, our acoustofluidic devices will be well-suited for model organism studies. 

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4. A Label-Free Electrical Impedance Spectroscopy for Detection of Clusters of Extracellular Vesicles Based on Their Unique Dielectric Properties

   https://doi.org/10.3390/bios12020104  

   Zhang, Yuqian ; Murakami, Kazutoshi ; Borra, Vishnupriya J. ; Ozen, Mehmet Ozgun ; Demirci, Utkan ; Nakamura, Takahisa ; Esfandiari, Leyla ( February 2022 , Biosensors)

   Extracellular vesicles (EVs) have gained considerable attention as vital circulating biomarkers since their structure and composition resemble the originating cells. The investigation of EVs’ biochemical and biophysical properties is of great importance to map them to their parental cells and to better understand their functionalities. In this study, a novel frequency-dependent impedance measurement system has been developed to characterize EVs based on their unique dielectric properties. The system is composed of an insulator-based dielectrophoretic (iDEP) device to entrap and immobilize a cluster of vesicles followed by utilizing electrical impedance spectroscopy (EIS) to measure their impedance at a wide frequency spectrum, aiming to analyze both their membrane and cytosolic charge-dependent contents. The EIS was initially utilized to detect nano-size vesicles with different biochemical compositions, including liposomes synthesized with different lipid compositions, as well as EVs and lipoproteins with similar biophysical properties but dissimilar biochemical properties. Moreover, EVs derived from the same parental cells but treated with different culture conditions were characterized to investigate the correlation of impedance changes with biochemical properties and functionality in terms of pro-inflammatory responses. The system also showed the ability to discriminate between EVs derived from different cellular origins as well as among size-sorted EVs harbored from the same cellular origin. This proof-of-concept approach is the first step towards utilizing EIS as a label-free, non-invasive, and rapid sensor for detection and characterization of pathogenic EVs and other nanovesicles in the future. 

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5. Metabolic tagging of extracellular vesicles and development of enhanced extracellular vesicle based cancer vaccines

   https://doi.org/10.1038/s41467-023-43914-8  

   Bhatta, Rimsha ; Han, Joonsu ; Liu, Yusheng ; Bo, Yang ; Lee, David ; Zhou, Jiadiao ; Wang, Yueji ; Nelson, Erik Russell ; Chen, Qian ; Zhang, Xiaojia Shelly ; et al ( December 2023 , Nature Communications)

   As key mediators of cellular communication, extracellular vesicles (EVs) have been actively explored for diagnostic and therapeutic applications. However, effective methods to functionalize EVs and modulate the interaction between EVs and recipient cells are still lacking. Here we report a facile and universal metabolic tagging technology that can install unique chemical tags (e.g., azido groups) onto EVs. The surface chemical tags enable conjugation of molecules via efficient click chemistry, for the tracking and targeted modulation of EVs. In the context of tumor EV vaccines, we show that the conjugation of toll-like receptor 9 agonists onto EVs enables timely activation of dendritic cells and generation of superior antitumor CD8⁺T cell response. These lead to 80% tumor-free survival against E.G7 lymphoma and 33% tumor-free survival against B16F10 melanoma. Our study yields a universal technology to generate chemically tagged EVs from parent cells, modulate EV-cell interactions, and develop potent EV vaccines.

    

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