An acoustofluidic device for the automated separation of platelet-reduced plasma from whole blood.

Separating plasma from whole blood is an important sample processing technique required for fundamental biomedical research, medical diagnostics, and therapeutic applications. Traditional protocols for plasma isolation require multiple centrifugation steps or multiunit microfluidic processing to sequentially remove large red blood cells (RBCs) and white blood cells (WBCs), followed by the removal of small platelets. Here, we present an acoustofluidic platform capable of efficiently removing RBCs, WBCs, and platelets from whole blood in a single step. By leveraging differences in the acoustic impedances of fluids, our device generates significantly greater forces on suspended particles than conventional microfluidic approaches, enabling the removal of both large blood cells and smaller platelets in a single unit. As a result, undiluted human whole blood can be processed by our device to remove both blood cells and platelets (>90%) at low voltages (25 Vpp). The ability to successfully remove blood cells and platelets from plasma without altering the properties of the proteins and antibodies present creates numerous potential applications for our platform in biomedical research, as well as plasma-based diagnostics and therapeutics. Furthermore, the microfluidic nature of our device offers advantages such as portability, cost efficiency, and the ability to process small-volume samples.

Microfluidic Assembly of Degradable, Stereocomplexed Hydrogel Microparticles.

Hydrogel microparticles (HMPs) have been investigated widely for their use in tissue engineering and drug delivery applications. However, translation of these highly tunable systems has been hindered by covalent cross-linking methods within microparticles. Stereocomplexation, a stereospecific form of physical cross-linking, provides a robust yet degradable alternative for creating translationally relevant HMPs. Herein, 4-arm polyethylene glycol (PEG) stars were used as macromolecular initiators from which oligomeric poly(l-lactic acid) (PLLA) was polymerized with a degree of polymerization (DPn) of 20 on each arm. Similarly, complementary propargyl-containing ABA cross-linkers with enantiomeric poly(d-lactic acid) (PDLA) segments (DPn = 20) on each arm. Droplets of these gel precursors were formed via a microfluidic organic-in-oil-in-water system where microparticles self-assembled via stereocomplexation and were stabilized after precipitation in deionized water. By varying the flow rate of the dispersed phase, well-defined microparticles with diameters of 33.7 ± 0.5, 62.4 ± 0.6, and 105.7 ± 0.8 µm were fabricated. Gelation due to stereocomplexation was confirmed via wide-angle X-ray scattering in which HMPs exhibited the signature diffraction pattern of stereocomplexed PLA at 2θ = 12.2, 21.2, 24.2°. Differential scanning calorimetry also confirmed stereocomplexation by the appearance of a crystallization exotherm (Tc = 37 °C) and a high-temperature endotherm (Tm = 159 °C) that does not appear in the homocrystallization of PLLA or the hydrogel precursors. Additionally, the propargyl handle present on the cross-linker allows for pre- or post-assembly thiol-yne "click" functionalization as demonstrated by the addition of thiol-containing fluorophores to the HMPs.

Capillary-based, multifunctional manipulation of particles and fluids via focused surface acoustic waves.

Surface acoustic wave (SAW)-enabled acoustofluidic technologies have recently atttracted increasing attention for applications in biology, chemistry, biophysics, and medicine. Most SAW acoustofluidic devices generate acoustic energy which is then transmitted into custom microfabricated polymer-based channels. There are limited studies on delivering this acoustic energy into convenient commercially-available glass tubes for manipulating particles and fluids. Herein, we have constructed a capillary-based SAW acoustofluidic device for multifunctional fluidic and particle manipulation. This device integrates a converging interdigitated transducer to generate focused SAWs on a piezoelectric chip, as well as a glass capillary that transports particles and fluids. To understand the actuation mechanisms underlying this device, we performed finite element simulations by considering piezoelectric, solid mechanic, and pressure acoustic physics. This experimental study shows that the capillary-based SAW acoustofluidic device can perform multiple functions including enriching particles, patterning particles, transporting particles and fluids, as well as generating droplets with controlled sizes. Given the usefulness of these functions, we expect that this acoustofluidic device can be useful in applications such as pharmaceutical manufacturing, biofabrication, and bioanalysis.

Acoustofluidic scanning fluorescence nanoscopy with a large field of view.

Large-field nanoscale fluorescence imaging is invaluable for many applications, such as imaging subcellular structures, visualizing protein interactions, and high-resolution tissue imaging. Unfortunately, conventional fluorescence microscopy requires a trade-off between resolution and field of view due to the nature of the optics used to form the image. To overcome this barrier, we developed an acoustofluidic scanning fluorescence nanoscope that simultaneously achieves superior resolution, a large field of view, and strong fluorescent signals. The acoustofluidic scanning fluorescence nanoscope utilizes the superresolution capabilities of microspheres that are controlled by a programmable acoustofluidic device for rapid fluorescence enhancement and imaging. The acoustofluidic scanning fluorescence nanoscope resolves structures that cannot be resolved with conventional fluorescence microscopes with the same objective lens and enhances the fluorescent signal by a factor of ~5 without altering the field of view of the image. The improved resolution realized with enhanced fluorescent signals and the large field of view achieved via acoustofluidic scanning fluorescence nanoscopy provides a powerful tool for versatile nanoscale fluorescence imaging for researchers in the fields of medicine, biology, biophysics, and biomedical engineering.

Acoustothermal transfection for cell therapy.

Transfected stem cells and T cells are promising in personalized cell therapy and immunotherapy against various diseases. However, existing transfection techniques face a fundamental trade-off between transfection efficiency and cell viability; achieving both simultaneously remains a substantial challenge. This study presents an acoustothermal transfection method that leverages acoustic and thermal effects on cells to enhance the permeability of both the cell membrane and nuclear envelope to achieve safe, efficient, and high-throughput transfection of primary T cells and stem cells. With this method, two types of plasmids were simultaneously delivered into the nuclei of mesenchymal stem cells (MSCs) with efficiencies of 89.6 ± 1.2%. CXCR4-transfected MSCs could efficiently target cerebral ischemia sites in vivo and reduce the infarct volume in mice. Our acoustothermal transfection method addresses a key bottleneck in balancing the transfection efficiency and cell viability, which can become a powerful tool in the future for cellular and gene therapies.

Acoustic separation and concentration of exosomes for nucleotide detection: ASCENDx.

Efficient isolation and analysis of exosomal biomarkers hold transformative potential in biomedical applications. However, current methods are prone to contamination and require costly consumables, expensive equipment, and skilled personnel. Here, we introduce an innovative spaceship-like disc that allows Acoustic Separation and Concentration of Exosomes and Nucleotide Detection: ASCENDx. We created ASCENDx to use acoustically driven disc rotation on a spinning droplet to generate swift separation and concentration of exosomes from patient plasma samples. Integrated plasmonic nanostars on the ASCENDx disc enable label-free detection of enriched exosomes via surface-enhanced Raman scattering. Direct detection of circulating exosomal microRNA biomarkers from patient plasma samples by the ASCENDx platform facilitated a diagnostic assay for colorectal cancer with 95.8% sensitivity and 100% specificity. ASCENDx overcomes existing limitations in exosome-based molecular diagnostics and holds a powerful position for future biomedical research, precision medicine, and point-of-care medical diagnostics.

High-yield and rapid isolation of extracellular vesicles by flocculation via orbital acoustic trapping: FLOAT.

Extracellular vesicles (EVs) have been identified as promising biomarkers for the noninvasive diagnosis of various diseases. However, challenges in separating EVs from soluble proteins have resulted in variable EV recovery rates and low purities. Here, we report a high-yield ( > 90%) and rapid ( < 10 min) EV isolation method called FLocculation via Orbital Acoustic Trapping (FLOAT). The FLOAT approach utilizes an acoustofluidic droplet centrifuge to rotate and controllably heat liquid droplets. By adding a thermoresponsive polymer flocculant, nanoparticles as small as 20 nm can be rapidly and selectively concentrated at the center of the droplet. We demonstrate the ability of FLOAT to separate urinary EVs from the highly abundant Tamm-Horsfall protein, addressing a significant obstacle in the development of EV-based liquid biopsies. Due to its high-yield nature, FLOAT reduces biofluid starting volume requirements by a factor of 100 (from 20 mL to 200 µL), demonstrating its promising potential in point-of-care diagnostics.

Aerosol jet printing of surface acoustic wave microfluidic devices.

The addition of surface acoustic wave (SAW) technologies to microfluidics has greatly advanced lab-on-a-chip applications due to their unique and powerful attributes, including high-precision manipulation, versatility, integrability, biocompatibility, contactless nature, and rapid actuation. However, the development of SAW microfluidic devices is limited by complex and time-consuming micro/nanofabrication techniques and access to cleanroom facilities for multistep photolithography and vacuum-based processing. To simplify the fabrication of SAW microfluidic devices with customizable dimensions and functions, we utilized the additive manufacturing technique of aerosol jet printing. We successfully fabricated customized SAW microfluidic devices of varying materials, including silver nanowires, graphene, and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). To characterize and compare the acoustic actuation performance of these aerosol jet printed SAW microfluidic devices with their cleanroom-fabricated counterparts, the wave displacements and resonant frequencies of the different fabricated devices were directly measured through scanning laser Doppler vibrometry. Finally, to exhibit the capability of the aerosol jet printed devices for lab-on-a-chip applications, we successfully conducted acoustic streaming and particle concentration experiments. Overall, we demonstrated a novel solution-based, direct-write, single-step, cleanroom-free additive manufacturing technique to rapidly develop SAW microfluidic devices that shows viability for applications in the fields of biology, chemistry, engineering, and medicine.

Advances in Microsphere-based Super-resolution Imaging.

Techniques to resolve images beyond the diffraction limit of light with a large field of view (FOV) are necessary to foster progress in various fields such as cell and molecular biology, biophysics, and nanotechnology, where nanoscale resolution is crucial for understanding the intricate details of large-scale molecular interactions. Although several means of achieving super-resolutions exist, they are often hindered by factors such as high costs, significant complexity, lengthy processing times, and the classical tradeoff between image resolution and FOV. Microsphere-based super-resolution imaging has emerged as a promising approach to address these limitations. In this review, we delve into the theoretical underpinnings of microsphere-based imaging and the associated photonic nanojet. This is followed by a comprehensive exploration of various microsphere-based imaging techniques, encompassing static imaging, mechanical scanning, optical scanning, and acoustofluidic scanning methodologies. This review concludes with a forward-looking perspective on the potential applications and future scientific directions of this innovative technology.

Cellular immunity analysis by a modular acoustofluidic platform: CIAMAP.

The study of molecular mechanisms at the single-cell level holds immense potential for enhancing immunotherapy and understanding neuroinflammation and neurodegenerative diseases by identifying previously concealed pathways within a diverse range of paired cells. However, existing single-cell pairing platforms have limitations in low pairing efficiency, complex manual operation procedures, and single-use functionality. Here, we report a multiparametric cellular immunity analysis by a modular acoustofluidic platform: CIAMAP. This platform enables users to efficiently sort and collect effector-target (i.e., NK92-K562) cell pairs and monitor the real-time dynamics of immunological response formation. Furthermore, we conducted transcriptional and protein expression analyses to evaluate the pathways that mediate effector cytotoxicity toward target cells, as well as the synergistic effect of doxorubicin on the cellular immune response. Our CIAMAP can provide promising building blocks for high-throughput quantitative single-cell level coculture to understand intercellular communication while also empowering immunotherapy by precision analysis of immunological synapses.

Acoustofluidic Interfaces for the Mechanobiological Secretome of MSCs.

While mesenchymal stem cells (MSCs) have gained enormous attention due to their unique properties of self-renewal, colony formation, and differentiation potential, the MSC secretome has become attractive due to its roles in immunomodulation, anti-inflammatory activity, angiogenesis, and anti-apoptosis. However, the precise stimulation and efficient production of the MSC secretome for therapeutic applications are challenging problems to solve. Here, we report on Acoustofluidic Interfaces for the Mechanobiological Secretome of MSCs: AIMS. We create an acoustofluidic mechanobiological environment to form reproducible three-dimensional MSC aggregates, which produce the MSC secretome with high efficiency. We confirm the increased MSC secretome is due to improved cell-cell interactions using AIMS: the key mediator N-cadherin was up-regulated while functional blocking of N-cadherin resulted in no enhancement of the secretome. After being primed by IFN-γ, the secretome profile of the MSC aggregates contains more anti-inflammatory cytokines and can be used to inhibit the pro-inflammatory response of M1 phenotype macrophages, suppress T cell activation, and support B cell functions. As such, the MSC secretome can be modified for personalized secretome-based therapies. AIMS acts as a powerful tool for improving the MSC secretome and precisely tuning the secretory profile to develop new treatments in translational medicine.

Acoustic tweezers for high-throughput single-cell analysis.

Acoustic tweezers provide an effective means for manipulating single cells and particles in a high-throughput, precise, selective and contact-free manner. The adoption of acoustic tweezers in next-generation cellular assays may advance our understanding of biological systems. Here we present a comprehensive set of instructions that guide users through device fabrication, instrumentation setup and data acquisition to study single cells with an experimental throughput that surpasses traditional methods, such as atomic force microscopy and micropipette aspiration, by several orders of magnitude. With acoustic tweezers, users can conduct versatile experiments that require the trapping, patterning, pairing and separation of single cells in a myriad of applications ranging across the biological and biomedical sciences. This procedure is widely generalizable and adaptable for investigations in materials and physical sciences, such as the spinning motion of colloids or the development of acoustic-based quantum simulations. Overall, the device fabrication requires ~12 h, the experimental setup of the acoustic tweezers requires 1-2 h and the cell manipulation experiment requires ~30 min to complete. Our protocol is suitable for use by interdisciplinary researchers in biology, medicine, engineering and physics.

Acoustofluidic scanning fluorescence nanoscopy with large field of view.

Nanoscale fluorescence imaging with a large-field view is invaluable for many applications such as imaging of subcellular structures, visualizing protein interaction, and high-resolution tissue imaging. Unfortunately, conventional fluorescence microscopy has to make a trade-off between resolution and field of view due to the nature of the optics used to form an image. To overcome this barrier, we have developed an acoustofluidic scanning fluorescence nanoscope that can simultaneously achieve superior resolution, a large field of view, and enhanced fluorescent signal. The acoustofluidic scanning fluorescence nanoscope utilizes the super-resolution capability of microspheres that are controlled by a programable acoustofluidic device for rapid fluorescent enhancement and imaging. The acoustofluidic scanning fluorescence nanoscope can resolve structures that cannot be achieved with a conventional fluorescent microscope with the same objective lens and enhances the fluorescent signal by a factor of ~5 without altering the field of view of the image. The improved resolution with enhanced fluorescent signal and large field of view via the acoustofluidic scanning fluorescence nanoscope provides a powerful tool for versatile nanoscale fluorescence imaging for researchers in the fields of medicine, biology, biophysics, and biomedical engineering.

Acousto-dielectric tweezers for size-insensitive manipulation and biophysical characterization of single cells.

The intrinsic biophysical properties of cells, such as mechanical, acoustic, and electrical properties, are valuable indicators of a cell's function and state. However, traditional single-cell biophysical characterization methods are hindered by limited measurable properties, time-consuming procedures, and complex system setups. This study presents acousto-dielectric tweezers that leverage the balance between controllable acoustophoretic and dielectrophoretic forces applied on cells through surface acoustic waves and alternating current electric fields, respectively. Particularly, the balanced acoustophoretic and dielectrophoretic forces can trap cells at equilibrium positions independent of the cell size to differentiate between various cell-intrinsic mechanical, acoustic, and electrical properties. Experimental results show our mechanism has the potential for applications in single-cell analysis, size-insensitive cell separation, and cell phenotyping, which are all primarily based on cells' intrinsic biophysical properties. Our results also show the measured equilibrium position of a cell can inversely determine multiple biophysical properties, including membrane capacitance, cytoplasm conductivity, and acoustic contrast factor. With these features, our acousto-dielectric tweezing mechanism is a valuable addition to the resources available for biophysical property-based biological and medical research.

A solution to the biophysical fractionation of extracellular vesicles: Acoustic Nanoscale Separation via Wave-pillar Excitation Resonance (ANSWER).

High-precision isolation of small extracellular vesicles (sEVs) from biofluids is essential toward developing next-generation liquid biopsies and regenerative therapies. However, current methods of sEV separation require specialized equipment and time-consuming protocols and have difficulties producing highly pure subpopulations of sEVs. Here, we present Acoustic Nanoscale Separation via Wave-pillar Excitation Resonance (ANSWER), which allows single-step, rapid (<10 min), high-purity (>96% small exosomes, >80% exomeres) fractionation of sEV subpopulations from biofluids without the need for any sample preprocessing. Particles are iteratively deflected in a size-selective manner via an excitation resonance. This previously unidentified phenomenon generates patterns of virtual, tunable, pillar-like acoustic field in a fluid using surface acoustic waves. Highly precise sEV fractionation without the need for sample preprocessing or complex nanofabrication methods has been demonstrated using ANSWER, showing potential as a powerful tool that will enable more in-depth studies into the complexity, heterogeneity, and functionality of sEV subpopulations.

Gold Nanopyramid Arrays for Non-Invasive Surface-Enhanced Raman Spectroscopy-Based Gastric Cancer Detection via sEVs.

Gastric cancer (GC) is one of the most common and lethal types of cancer affecting over one million people, leading to 768,793 deaths globally in 2020 alone. The key for improving the survival rate lies in reliable screening and early diagnosis. Existing techniques including barium-meal gastric photofluorography and upper endoscopy can be costly and time-consuming and are thus impractical for population screening. We look instead for small extracellular vesicles (sEVs, currently also referred as exosomes) sized ⌀ 30-150 nm as a candidate. sEVs have attracted a significantly higher level of attention during the past decade or two because of their potentials in disease diagnoses and therapeutics. Here, we report that the composition information of the collective Raman-active bonds inside sEVs of human donors obtained by surface-enhanced Raman spectroscopy (SERS) holds the potential for non-invasive GC detection. SERS was triggered by the substrate of gold nanopyramid arrays we developed previously. A machine learning-based spectral feature analysis algorithm was developed for objectively distinguishing the cancer-derived sEVs from those of the non-cancer sub-population. sEVs from the tissue, blood, and saliva of GC patients and non-GC participants were collected (n = 15 each) and analyzed. The algorithm prediction accuracies were reportedly 90, 85, and 72%. "Leave-a-pair-of-samples out" validation was further performed to test the clinical potential. The area under the curve of each receiver operating characteristic curve was 0.96, 0.91, and 0.65 in tissue, blood, and saliva, respectively. In addition, by comparing the SERS fingerprints of individual vesicles, we provided a possible way of tracing the biogenesis pathways of patient-specific sEVs from tissue to blood to saliva. The methodology involved in this study is expected to be amenable for non-invasive detection of diseases other than GC.

Phase 2 of extracellular RNA communication consortium charts next-generation approaches for extracellular RNA research.

The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.

An acoustofluidic scanning nanoscope using enhanced image stacking and processing.

Nanoscale optical resolution with a large field of view is a critical feature for many research and industry areas, such as semiconductor fabrication, biomedical imaging, and nanoscale material identification. Several scanning microscopes have been developed to resolve the inverse relationship between the resolution and field of view; however, those scanning microscopes still rely upon fluorescence labeling and complex optical systems. To overcome these limitations, we developed a dual-camera acoustofluidic nanoscope with a seamless image merging algorithm (alpha-blending process). This design allows us to precisely image both the sample and the microspheres simultaneously and accurately track the particle path and location. Therefore, the number of images required to capture the entire field of view (200 × 200 µm) by using our acoustofluidic scanning nanoscope is reduced by 55-fold compared with previous designs. Moreover, the image quality is also greatly improved by applying an alpha-blending imaging technique, which is critical for accurately depicting and identifying nanoscale objects or processes. This dual-camera acoustofluidic nanoscope paves the way for enhanced nanoimaging with high resolution and a large field of view.

Intelligent nanoscope for rapid nanomaterial identification and classification.

Machine learning image recognition and classification of particles and materials is a rapidly expanding field. However, nanomaterial identification and classification are dependent on the image resolution, the image field of view, and the processing time. Optical microscopes are one of the most widely utilized technologies in laboratories across the world, due to their nondestructive abilities to identify and classify critical micro-sized objects and processes, but identifying and classifying critical nano-sized objects and processes with a conventional microscope are outside of its capabilities, due to the diffraction limit of the optics and small field of view. To overcome these challenges of nanomaterial identification and classification, we developed an intelligent nanoscope that combines machine learning and microsphere array-based imaging to: (1) surpass the diffraction limit of the microscope objective with microsphere imaging to provide high-resolution images; (2) provide large field-of-view imaging without the sacrifice of resolution by utilizing a microsphere array; and (3) rapidly classify nanomaterials using a deep convolution neural network. The intelligent nanoscope delivers more than 46 magnified images from a single image frame so that we collected more than 1000 images within 2 seconds. Moreover, the intelligent nanoscope achieves a 95% nanomaterial classification accuracy using 1000 images of training sets, which is 45% more accurate than without the microsphere array. The intelligent nanoscope also achieves a 92% bacteria classification accuracy using 50 000 images of training sets, which is 35% more accurate than without the microsphere array. This platform accomplished rapid, accurate detection and classification of nanomaterials with miniscule size differences. The capabilities of this device wield the potential to further detect and classify smaller biological nanomaterial, such as viruses or extracellular vesicles.

A sound approach to advancing healthcare systems: the future of biomedical acoustics.

Newly developed acoustic technologies are playing a transformational role in life science and biomedical applications ranging from the activation and inactivation of mechanosensitive ion channels for fundamental physiological processes to the development of contact-free, precise biofabrication protocols for tissue engineering and large-scale manufacturing of organoids. Here, we provide our perspective on the development of future acoustic technologies and their promise in addressing critical challenges in biomedicine.