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.

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.

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.

Acoustofluidics for simultaneous nanoparticle-based drug loading and exosome encapsulation.

Nanocarrier and exosome encapsulation has been found to significantly increase the efficacy of targeted drug delivery while also minimizing unwanted side effects. However, the development of exosome-encapsulated drug nanocarriers is limited by low drug loading efficiencies and/or complex, time-consuming drug loading processes. Herein, we have developed an acoustofluidic device that simultaneously performs both drug loading and exosome encapsulation. By synergistically leveraging the acoustic radiation force, acoustic microstreaming, and shear stresses in a rotating droplet, the concentration, and fusion of exosomes, drugs, and porous silica nanoparticles is achieved. The final product consists of drug-loaded silica nanocarriers that are encased within an exosomal membrane. The drug loading efficiency is significantly improved, with nearly 30% of the free drug (e.g., doxorubicin) molecules loaded into the nanocarriers. Furthermore, this acoustofluidic drug loading system circumvents the need for complex chemical modification, allowing drug loading and encapsulation to be completed within a matter of minutes. These exosome-encapsulated nanocarriers exhibit excellent efficiency in intracellular transport and are capable of significantly inhibiting tumor cell proliferation. By utilizing physical forces to rapidly generate hybrid nanocarriers, this acoustofluidic drug loading platform wields the potential to significantly impact innovation in both drug delivery research and applications.

Sonoporation: Past, Present, and Future.

A surge of research in intracellular delivery technologies is underway with the increased innovations in cell-based therapies and cell reprogramming. Particularly, physical cell membrane permeabilization techniques are highlighted as the leading technologies because of their unique features, including versatility, independence of cargo properties, and high-throughput delivery that is critical for providing the desired cell quantity for cell-based therapies. Amongst the physical permeabilization methods, sonoporation holds great promise and has been demonstrated for delivering a variety of functional cargos, such as biomolecular drugs, proteins, and plasmids, to various cells including cancer, immune, and stem cells. However, traditional bubble-based sonoporation methods usually require special contrast agents. Bubble-based sonoporation methods also have high chances of inducing irreversible damage to critical cell components, lowering the cell viability, and reducing the effectiveness of delivered cargos. To overcome these limitations, several novel non-bubble-based sonoporation mechanisms are under development. This review will cover both the bubble-based and non-bubble-based sonoporation mechanisms being employed for intracellular delivery, the technologies being investigated to overcome the limitations of traditional platforms, as well as perspectives on the future sonoporation mechanisms, technologies, and applications.

Harmonic acoustics for dynamic and selective particle manipulation.

Precise and selective manipulation of colloids and biological cells has long been motivated by applications in materials science, physics and the life sciences. Here we introduce our harmonic acoustics for a non-contact, dynamic, selective (HANDS) particle manipulation platform, which enables the reversible assembly of colloidal crystals or cells via the modulation of acoustic trapping positions with subwavelength resolution. We compose Fourier-synthesized harmonic waves to create soft acoustic lattices and colloidal crystals without using surface treatment or modifying their material properties. We have achieved active control of the lattice constant to dynamically modulate the interparticle distance in a high-throughput (>100 pairs), precise, selective and reversible manner. Furthermore, we apply this HANDS platform to quantify the intercellular adhesion forces among various cancer cell lines. Our biocompatible HANDS platform provides a highly versatile particle manipulation method that can handle soft matter and measure the interaction forces between living cells with high sensitivity.

Acoustofluidic Droplet Sorter Based on Single Phase Focused Transducers.

Droplet microfluidics has revolutionized the biomedical and drug development fields by allowing for independent microenvironments to conduct drug screening at the single cell level. However, current microfluidic sorting devices suffer from drawbacks such as high voltage requirements (e.g., >200 Vpp), low biocompatibility, and/or low throughput. In this article, a single-phase focused transducer (SPFT)-based acoustofluidic chip is introduced, which outperforms many microfluidic droplet sorting devices through high energy transmission efficiency, high accuracy, and high biocompatibility. The SPFT-based sorter can be driven with an input power lower than 20 Vpp and maintain a postsorting cell viability of 93.5%. The SPFT sorter can achieve a throughput over 1000 events per second and a sorting purity up to 99.2%. The SPFT sorter is utilized here for the screening of doxorubicin cytotoxicity on cancer and noncancer cells, proving its drug screening capability. Overall, the SPFT droplet sorting device shows great potential for fast, precise, and biocompatible drug screening.

Electrochemical micro-aptasensors for exosome detection based on hybridization chain reaction amplification.

Exosomes are cell-derived nanovesicles that have recently gained popularity as potential biomarkers in liquid biopsies due to the large amounts of molecular cargo they carry, such as nucleic acids and proteins. However, most existing exosome-based analytical sensing methods struggle to achieve high sensitivity and high selectivity simultaneously. In this work, we present an electrochemical micro-aptasensor for the highly sensitive detection of exosomes by integrating a micropatterned electrochemical aptasensor and a hybridization chain reaction (HCR) signal amplification method. Specifically, exosomes are enriched on CD63 aptamer-functionalized electrodes and then recognized by HCR products with avidin-horseradish peroxidase (HRP) attached using EpCAM aptamers as bridges. Subsequently, the current signal that is generated through the enzyme reaction between the HRP enzyme and 3,3',5,5'-tetramethylbenzidine (TMB)/H2O2 directly correlates to the amount of bound HRP on the HCR products and thus to the number of target exosomes. By introducing anti-EpCAM aptamers, micro-aptasensors can detect cancerous exosomes with high specificity. Due to the micropatterned electrodes and HCR dual-amplification strategy, the micro-aptasensors achieve a linear detection response for a wide range of exosome concentrations from 2.5×103 to 1×107 exosomes/mL, with a detection limit of 5×102 exosomes/mL. Moreover, our method successfully detects lung cancer exosomes in serum samples of early-stage and late-stage lung cancer patients, showcasing the great potential for early cancer diagnosis.

Acidic Submucosal Gland pH and Elevated Protein Concentration Produce Abnormal Cystic Fibrosis Mucus.

In response to respiratory insults, airway submucosal glands secrete copious mucus strands to increase mucociliary clearance and protect the lung. However, in cystic fibrosis, stimulating submucosal glands has the opposite effect, disrupting mucociliary transport. In cystic fibrosis (CF) pigs, loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channels produced submucosal gland mucus that was abnormally acidic with an increased protein concentration. To test whether these variables alter mucus, we produced a microfluidic model of submucosal glands using mucus vesicles from banana slugs. Acidic pH and increased protein concentration decreased mucus gel volume and increased mucus strand elasticity and tensile strength. However, once mucus strands were formed, changing pH or protein concentration largely failed to alter the biophysical properties. Likewise, raising pH or apical perfusion did not improve clearance of mucus strands from CF airways. These findings reveal mechanisms responsible for impaired mucociliary transport in CF and have important implications for potential treatments.

Acoustofluidic sonoporation for gene delivery to human hematopoietic stem and progenitor cells.

Advances in gene editing are leading to new medical interventions where patients' own cells are used for stem cell therapies and immunotherapies. One of the key limitations to translating these treatments to the clinic is the need for scalable technologies for engineering cells efficiently and safely. Toward this goal, microfluidic strategies to induce membrane pores and permeability have emerged as promising techniques to deliver biomolecular cargo into cells. As these technologies continue to mature, there is a need to achieve efficient, safe, nontoxic, fast, and economical processing of clinically relevant cell types. We demonstrate an acoustofluidic sonoporation method to deliver plasmids to immortalized and primary human cell types, based on pore formation and permeabilization of cell membranes with acoustic waves. This acoustofluidic-mediated approach achieves fast and efficient intracellular delivery of an enhanced green fluorescent protein-expressing plasmid to cells at a scalable throughput of 200,000 cells/min in a single channel. Analyses of intracellular delivery and nuclear membrane rupture revealed mechanisms underlying acoustofluidic delivery and successful gene expression. Our studies show that acoustofluidic technologies are promising platforms for gene delivery and a useful tool for investigating membrane repair.

A disposable acoustofluidic chip for nano/microparticle separation using unidirectional acoustic transducers.

Separation of nano/microparticles based on surface acoustic waves (SAWs) has shown great promise for biological, chemical, and medical applications ranging from sample purification to cancer diagnosis. However, the permanent bonding of a microchannel onto relatively expensive piezoelectric substrates and excitation transducers renders the SAW separation devices non-disposable. This limitation not only requires cumbersome cleaning and increased labor and material costs, but also leads to cross-contamination, preventing their implementation in many biological, chemical, and medical applications. Here, we demonstrate a high-performance, disposable acoustofluidic platform for nano/microparticle separation. Leveraging unidirectional interdigital transducers (IDTs), a hybrid channel design with hard/soft materials, and tilted-angle standing SAWs (taSSAWs), our disposable acoustofluidic devices achieve acoustic radiation forces comparable to those generated by existing permanently bonded, non-disposable devices. Our disposable devices can separate not only microparticles but also nanoparticles. Moreover, they can differentiate bacteria from human red blood cells (RBCs) with a purity of up to 96%. Altogether, we developed a unidirectional IDT-based, disposable acoustofluidic platform for micro/nanoparticle separation that can achieve high separation efficiency, versatility, and biocompatibility.

Low-frequency flexural wave based microparticle manipulation.

Manipulation of microparticles and bio-samples is a critical task in many research and clinical settings. Recently, acoustic based methods have garnered significant attention due to their relatively simple designs, and biocompatible and precise manipulation of small objects. Herein, we introduce a flexural wave based acoustofluidic manipulation platform that utilizes low-frequency (4-6 kHz) commercial buzzers to achieve dynamic particle concentration and translation in an open fluid well. The device has two primary modes of functionality, wherein particles can be concentrated in pressure nodes that are present on the bottom surface of the device, or particles can be trapped and manipulated in streaming vortices within the fluid domain; both of these functions result from flexural mode vibrations that travel from the transducers throughout the device. Throughout our research, we numerically and experimentally explored the wave patterns generated within the device, investigated the particle concentration phenomenon, and utilized a phase difference between the two transducers to achieve precision movement of fluid vortices and the entrapped particle clusters. With its simple, low-cost nature and open fluidic chamber design, this platform can be useful in many biological, biochemical, and biomedical applications, such as tumor spheroid generation and culture, as well as the manipulation of embryos.

Acoustofluidic Salivary Exosome Isolation: A Liquid Biopsy Compatible Approach for Human Papillomavirus-Associated Oropharyngeal Cancer Detection.

Previous efforts to evaluate the detection of human papilloma viral (HPV) DNA in whole saliva as a diagnostic measure for HPV-associated oropharyngeal cancer (HPV-OPC) have not shown sufficient clinical performance. We hypothesize that salivary exosomes are packaged with HPV-associated biomarkers, and efficient enrichment of salivary exosomes through isolation can enhance diagnostic and prognostic performance for HPV-OPC. In this study, an acoustofluidic (the fusion of acoustics and microfluidics) platform was developed to perform size-based isolation of salivary exosomes. These data showed that this platform is capable of consistently isolating exosomes from saliva samples, regardless of viscosity variation and collection method. Compared with the current gold standard, differential centrifugation, droplet digital RT-PCR analysis showed that the average yield of salivary exosomal small RNA from the acoustofluidic platform is 15 times higher. With this high-yield exosome isolation platform, we show that HPV16 DNA could be detected in isolated exosomes from the saliva of HPV-associated OPC patients at 80% concordance with tissues/biopsies positive for HPV16. Overall, these data demonstrated that the acoustofluidic platform can achieve high-purity and high-yield salivary exosome isolation for downstream salivary exosome-based liquid biopsy applications. Additionally, HPV16 DNA sequences in HPV-OPC patients are packaged in salivary exosomes and their isolation will enhance the detection of HPV16 DNA.

Acoustofluidic methods in cell analysis.

Cellular analysis is a central concept for both biology and medicine. Over the past two decades, acoustofluidic technologies, which marry acoustic waves with microfluidics, have significantly contributed to the development of innovative approaches for cellular analysis. Acoustofluidic technologies enable precise manipulations of cells and the fluids that confine them, and these capabilities have been utilized in many cell analysis applications. In this review article, we examine various applications where acoustofluidic methods have been implemented, including cell imaging, cell mechanotyping, circulating tumor cell phenotyping, sample preparation in clinics, and investigation of cell-cell interactions and cell-environment responses. We also provide our perspectives on the technological advantages, limitations, and potential future directions for this innovative field of methods.

Applications of Acoustofluidics in Bioanalytical Chemistry.

Acoustics has a broad spectrum of applications, ranging from noise cancelation to ultrasonic imaging. In the past decade, there has been increasing interest in developing acoustic-based methods for biological and biomedical applications. This Perspective summarizes the recent progress in applying acoustofluidic methods (i.e., the fusion of acoustics and microfluidics) to bioanalytical chemistry. We describe the concepts of acoustofluidics and how it can be tailored to different types of bioanalytical applications, including sample concentration, fluorescence-activated cell sorting, label-free cell/particle separation, and fluid manipulation. Examples of each application are given, and the benefits and limitations of these methods are discussed. Finally, our perspectives on the directions that developing solutions should take to address the bottlenecks in the acoustofluidic applications in bioanalytical chemistry are presented.

Microfluidic approaches for cell-based molecular diagnosis.

The search for next-generation biomarkers has enabled cell-based diagnostics in a number of disciplines ranging from oncology to pharmacogenetics. However, cell-based diagnostics are still far from clinical reality due to the complex assays and associated protocols which typically require cell isolation, lysis, DNA extraction, amplification, and detection steps. Leveraging recent advances in microfluidics, many biochemical assays have been translated onto microfluidic platforms. We have compared and summarized recent advances in modular approaches toward the realization of fully-integrated, cell-based molecular diagnostics for clinical and point-of-care applications.

High-throughput cell focusing and separation via acoustofluidic tweezers.

Separation of particles and cells is an important function in many biological and biomedical protocols. Although a variety of microfluidic-based techniques have been developed so far, there is clearly still a demand for a precise, fast, and biocompatible method for separation of microparticles and cells. By combining acoustics and hydrodynamics, we have developed a method which we integrated into three-dimensional acoustofluidic tweezers (3D-AFT) to rapidly and efficiently separate microparticles and cells into multiple high-purity fractions. Compared with other acoustophoresis methods, this 3D-AFT method significantly increases the throughput by an order of magnitude, is label-free and gently handles the sorted cells. We demonstrate not only the separation of 10, 12, and 15 micron particles at a throughput up to 500 µl min-1 using this 3D-AFT method, but also the separation of erythrocytes, leukocytes, and cancer cells. This 3D-AFT method is able to meet various separation demands thus offering a viable alternative with potential for clinical applications.

Circulating Tumor Cell Phenotyping via High-Throughput Acoustic Separation.

The study of circulating tumor cells (CTCs) offers pathways to develop new diagnostic and prognostic biomarkers that benefit cancer treatments. In order to fully exploit and interpret the information provided by CTCs, the development of a platform is reported that integrates acoustics and microfluidics to isolate rare CTCs from peripheral blood in high throughput while preserving their structural, biological, and functional integrity. Cancer cells are first isolated from leukocytes with a throughput of 7.5 mL h-1 , achieving a recovery rate of at least 86% while maintaining the cells' ability to proliferate. High-throughput acoustic separation enables statistical analysis of isolated CTCs from prostate cancer patients to be performed to determine their size distribution and phenotypic heterogeneity for a range of biomarkers, including the visualization of CTCs with a loss of expression for the prostate specific membrane antigen. The method also enables the isolation of even rarer, but clinically important, CTC clusters.