Reverse Engineering Human Pathophysiology with Organs-on-Chips.

While studies of cultured cells have led to new insights into biological control, greater understanding of human pathophysiology requires the development of experimental systems that permit analysis of intercellular communications and tissue-tissue interactions in a more relevant organ context. Human organs-on-chips offer a potentially powerful new approach to confront this long-standing problem.

Single-cell quantification of ribosome occupancy in early mouse development.

Translation regulation is critical for early mammalian embryonic development1. However, previous studies had been restricted to bulk measurements2, precluding precise determination of translation regulation including allele-specific analyses. Here, to address this challenge, we developed a novel microfluidic isotachophoresis (ITP) approach, named RIBOsome profiling via ITP (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key mechanism regulating genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high-coverage measurements enabled, to our knowledge, the first analysis of allele-specific ribosome engagement in early development. These led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts exhibiting allele-biased expression. By integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle-stage oocytes is the predominant determinant of protein abundance in the zygote. The Ribo-ITP approach will enable numerous applications by providing high-coverage and high-resolution ribosome occupancy measurements from ultra-low input samples including single cells.

Microfluidic chain reaction of structurally programmed capillary flow events.

Chain reactions, characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (for example, combustion engines), nuclear and biotechnological (for example, polymerase chain reaction) applications1-5. At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling dominoes and Rube Goldberg machines. On the other hand, the microfluidic lab-on-a-chip (also called a micro-total analysis system)6,7 was visualized as an integrated chip, akin to microelectronic integrated circuits, yet in practice remains dependent on cumbersome peripherals, connections and a computer for automation8-11. Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible12-19. Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are three-dimensionally printed, and powered by the free energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (1) the sequential release of 300 aliquots across chained, interconnected chips, (2) a protocol for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibodies detection in saliva and (3) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ and can form a frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.

Genetic and phenotypic profiling of single living circulating tumor cells from patients with microfluidics.

Accurate prediction of the efficacy of immunotherapy for cancer patients through the characterization of both genetic and phenotypic heterogeneity in individual patient cells holds great promise in informing targeted treatments, and ultimately in improving care pathways and clinical outcomes. Here, we describe the nanoplatform for interrogating living cell host-gene and (micro-)environment (NICHE) relationships, that integrates micro- and nanofluidics to enable highly efficient capture of circulating tumor cells (CTCs) from blood samples. The platform uses a unique nanopore-enhanced electrodelivery system that efficiently and rapidly integrates stable multichannel fluorescence probes into living CTCs for in situ quantification of target gene expression, while on-chip coculturing of CTCs with immune cells allows for the real-time correlative quantification of their phenotypic heterogeneities in response to immune checkpoint inhibitors (ICI). The NICHE microfluidic device provides a unique ability to perform both gene expression and phenotypic analysis on the same single cells in situ, allowing us to generate a predictive index for screening patients who could benefit from ICI. This index, which simultaneously integrates the heterogeneity of single cellular responses for both gene expression and phenotype, was validated by clinically tracing 80 non-small cell lung cancer patients, demonstrating significantly higher AUC (area under the curve) (0.906) than current clinical reference for immunotherapy prediction.

Massively multiplexed nucleic acid detection with Cas13.

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples1-3 while simultaneously testing for many pathogens4-6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents7 self-organize in a microwell array8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health9-11.

Ultrahigh-Throughput Enzyme Engineering and Discovery in In Vitro Compartments.

Novel and improved biocatalysts are increasingly sourced from libraries via experimental screening. The success of such campaigns is crucially dependent on the number of candidates tested. Water-in-oil emulsion droplets can replace the classical test tube, to provide in vitro compartments as an alternative screening format, containing genotype and phenotype and enabling a readout of function. The scale-down to micrometer droplet diameters and picoliter volumes brings about a >107-fold volume reduction compared to 96-well-plate screening. Droplets made in automated microfluidic devices can be integrated into modular workflows to set up multistep screening protocols involving various detection modes to sort >107 variants a day with kHz frequencies. The repertoire of assays available for droplet screening covers all seven enzyme commission (EC) number classes, setting the stage for widespread use of droplet microfluidics in everyday biochemical experiments. We review the practicalities of adapting droplet screening for enzyme discovery and for detailed kinetic characterization. These new ways of working will not just accelerate discovery experiments currently limited by screening capacity but profoundly change the paradigms we can probe. By interfacing the results of ultrahigh-throughput droplet screening with next-generation sequencing and deep learning, strategies for directed evolution can be implemented, examined, and evaluated.

Characterizing cell interactions at scale with made-to-order droplet ensembles (MODEs).

Cell-cell interactions are important to numerous biological systems, including tissue microenvironments, the immune system, and cancer. However, current methods for studying cell combinations and interactions are limited in scalability, allowing just hundreds to thousands of multicell assays per experiment; this limited throughput makes it difficult to characterize interactions at biologically relevant scales. Here, we describe a paradigm in cell interaction profiling that allows accurate grouping of cells and characterization of their interactions for tens to hundreds of thousands of combinations. Our approach leverages high-throughput droplet microfluidics to construct multicellular combinations in a deterministic process that allows inclusion of programmed reagent mixtures and beads. The combination droplets are compatible with common manipulation and measurement techniques, including imaging, barcode-based genomics, and sorting. We demonstrate the approach by using it to enrich for chimeric antigen receptor (CAR)-T cells that activate upon incubation with target cells, a bottleneck in the therapeutic T cell engineering pipeline. The speed and control of our approach should enable valuable cell interaction studies.

Biomolecular Interaction Analysis Quantification with a Low-Volume Microfluidic Chip and Particle Diffusometry.

Microfluidic techniques are widely applied in biomolecular analysis and disease diagnostic assays. While the volume of the sample that is directly used in such assays is often only femto-to microliters, the "dead volume" of solutions supplied in syringes and tubing can be much larger, even up to milliliters, increasing overall reagent use and making analysis significantly more expensive. To reduce the difficulty and cost, we designed a new chip using a low volume solution for analysis and applied it to obtain real-time data for protein-protein interaction measurements. The chip takes advantage of air/aqueous two-phase droplet flow, on-chip rapid mixing within milliseconds, and a droplet capture method, that ultimately requires only 2 µL of reagent solution. The interaction is analyzed by particle diffusometry, a nonintrusive and precise optical detection method to analyze the properties of microparticle diffusion in solution. Herein, we demonstrate on-chip characterization of human immunodeficiency virus p24 antibody-antigen protein binding kinetics imaged via fluorescence microscopy and analyzed by PD. The measured kon and koff are 1 × 106 M-1 s-1 and 3.3 × 10-4 s-1, respectively, and agree with independent measurement via biolayer interferometry and previously calculated p24-antibody binding kinetics. This new microfluidic chip and the protein-protein interaction analysis method can also be applied in other fields that require low-volume solutions to perform accurate measurement, analysis, and detection.

A Cascaded Phase-Transfer Microfluidic Chip with Magnetic Probe for High-Activity Sorting, Purification, Release, and Detection of Circulating Tumor Cells.

Microfluidic chips have emerged as a promising tool for sorting and enriching circulating tumor cells (CTCs) in blood, while the efficacy and purity of CTC sorting greatly depend on chip design. Herein, a novel cascaded phase-transfer microfluidic chip was developed for high-efficiency sorting, purification, release, and detection of MCF-7 cells (as a model CTC) in blood samples. MCF-7 cells were specifically captured by EpCAM aptamer-modified magnetic beads and then introduced into the designed cascaded phase-transfer microfluidic chip that consisted of three functional regions (sorting, purification, and release zone). In the sorting zone, the MCF-7 cells moved toward the inner wall of the channel and entered the purification zone for primary separation from white blood cells; in the purification zone, the MCF-7 cells were transferred to the phosphate-buffered saline flow under the interaction of Dean forces and central magnetic force, achieving high purification of MCF-7 cells from blood samples; in the release zone, MCF-7 cells were further transferred into the nuclease solution and fixed in groove by the strong magnetic force and hydrodynamic force, and the continuously flowing nuclease solution cleaved the aptamer on the trapped MCF-7 cells, causing gentle release of MCF-7 cells for subsequent inductively coupled plasma mass spectrometry (ICP-MS) detection or further cultivation. By measurement of the endogenous element Zn in the cells using ICP-MS for cell counting, an average cell recovery of 84% for MCF-7 cells was obtained in spiked blood samples. The developed method was applied in the analysis of real blood samples from healthy people and breast cancer patients, and CTCs were successfully detected in all tested patient samples (16/16). Additionally, the removal of the magnetic probes on the cell surface significantly improved cell viability up to 99.3%. Therefore, the developed cascaded phase-transfer microfluidic chip ICP-MS system possessed high integration for CTCs analysis with high cell viability, cell recovery, and purity, showing great advantages in early clinical cancer diagnosis.

Magnetic Microbead-Based Herringbone Chip for Sensitive Detection of Human Immunodeficiency Virus.

The microfluidic chip-based nucleic acid detection method significantly improves the sensitivity since it precisely controls the microfluidic flow in microchannels. Nonetheless, significant challenges still exist in improving the detection efficiency to meet the demand for rapid detection of trace substances. This work provides a novel magnetic herringbone (M-HB) structure in a microfluidic chip, and its advantage in rapid and sensitive detection is verified by taking complementary DNA (cDNA) sequences of human immunodeficiency virus (HIV) detection as an example. The M-HB structure is designed based on controlling the magnetic field distribution in the micrometer scale and is formed by accumulation of magnetic microbeads (MMBs). Hence, M-HB is similar to a nanopore microstructure, which has a higher contact area and probe density. All of the above is conducive to improving sensitivity in microfluidic chips. The M-HB chip is stable and easy to form, which can linearly detect cDNA sequences of HIV quantitatively ranging from 1 to 20 nM with a detection limit of 0.073 nM. Compared to the traditional herringbone structure, this structure is easier to form and release by controlling the magnetic field, which is flexible and helps in further study. Results show that this chip can sensitively detect the cDNA sequences of HIV in blood samples, demonstrating that it is a powerful platform to rapidly and sensitively detect multiple nucleic acid-related viruses of infectious diseases.

An Automated Single-Cell Droplet-Digital Microfluidic Platform for Monoclonal Antibody Discovery.

Monoclonal antibody (mAb) discovery plays a prominent role in diagnostic and therapeutic applications. Droplet microfluidics has become a standard technology for high-throughput screening of antibody-producing cells due to high droplet single-cell confinement frequency and rapid analysis and sorting of the cells of interest with their secreted mAbs. In this work, a new method is described for on-demand co-encapsulation of cells that eliminates the difficulties associated with washing in between consecutive steps inside the droplets and enables the washing and addition of fresh media. The new platform identifies hybridoma cells that are expressing antibodies of interest using antibody-characterization assays to find the best-performing or rare-cell antibody candidates.

Microfluidics-based EGFR mutation detection and its implication in the resource-limited clinical setting.

Management of lung cancer today obligates a mutational analysis of the epidermal growth factor receptor (EGFR) gene particularly when Tyrosine Kinase Inhibitor (TKI) therapy is being considered as part of prognostic stratification. This study evaluates the performance of automated microfluidics-based EGFR mutation detection and its significance in clinical diagnostic settings. Formalin-fixed, paraffin-embedded (FFPE) samples from NSCLC patients (n = 174) were included in a two-phase study. Phase I: Validation of the platform by comparing the results with conventional real-time PCR and next-generation sequencing (NGS) platform. Phase II: EGFR mutation detection on microfluidics-based platform as part of routine diagnostics workup. The microfluidics-based platform demonstrates 96.5% and 89.2% concordance with conventional real-time PCR and NGS, respectively. The system efficiently detects mutations across the EGFR gene with 88.23% sensitivity and 100% specificity. Out of 144 samples analysed in phase II, the platform generated valid results in 94% with mutation detected in 41% of samples. This microfluidics-based platform can detect as low as 5% mutant allele fractions from the FFPE samples. Therefore the microfluidics-based platform is a rapid, complete walkaway, with minimum tissue requirement (two sections of 5 µ thickness) and technical skill requirement. The method can detect clinically actionable EGFR mutations efficiently and can be considered a reliable diagnostic platform in resource-limited settings. From receiving samples to reporting the results this platform provides accurate data without much manual intervention. The study helped to devise an algorithm that emphasizes effective screening of the NSCLC cases for EGFR mutations with varying tumour content. Thus it helps in triaging the cases judiciously before proceeding with multigene testing.

Trends and challenges in microfluidic methods for protein manipulation-A review.

Proteins are important molecules involved in an immensely large number of biological processes. Being capable of manipulating proteins is critical for developing reliable and affordable techniques to analyze and/or detect them. Such techniques would enable the production of therapeutic agents for the treatment of diseases or other biotechnological applications (e.g., bioreactors or biocatalysis). Microfluidic technology represents a potential solution to protein manipulation challenges because of the diverse phenomena that can be exploited to achieve micro- and nanoparticle manipulation. In this review, we discuss recent contributions made in the field of protein manipulation in microfluidic systems using different physicochemical principles and techniques, some of which are miniaturized versions of already established macro-scale techniques.

Modulating selective ionic enrichment and depletion zones in straight nanochannels via the interplay of surface charge modulation and electric field mediated fluid-thickening.

We report the possibilities of achieving highly controlled segregation of ion-enriched and ion-depleted regions in straight nanochannels. This is achieved via harnessing the interplay of an axial gradient of the induced transverse electric field on account of electrical double layer phenomenon and the localized thickening of the fluid because of intensified electric fields due to the large spatial gradients of the electrical potential in extreme confinements. By considering alternate surface patches of different charge densities over pre-designed axial spans, we illustrate how these effects can be exploited to realize selectively ion-enriched and ion-depleted zones. Physically, this is attributed to setting up of an axial concentration gradient that delves on the ionic advection due to the combined effect of an externally applied electric field and induced back-pressure gradient along the channel axis and electro-migration due to the combinatorial influences of the applied and the induced electrostatic fields. With an explicit handle on the pertinent parameters, our results offer insights on the possible means of imposing delicate controls on the solute-enrichment and depletion phenomena, a paradigm that remained unexplored thus far.

Analytical solutions for viscoelectric effects in electrokinetic nanochannels.

Understanding electrokinetic transport in nanochannels and nanopores is essential for emerging biological and electrochemical applications. The viscoelectric effect is an important mechanism implicated in the increase of local viscosity due to the polarization of a solvent under a strong electric field. However, most analyses of the viscoelectric effect have been limited to numerical analyses. In this work, we present a set of analytical solutions applicable to the physical description of viscoelectric effects in nanochannel electrokinetic systems. To achieve such closed-form solutions, we employ the Debye-Hückel approximation of small diffuse charge layer potentials compared to the thermal potential. We analyze critical parameters, including electroosmotic flow profiles, electroosmotic mobility, flow rate, and channel conductance. We compare and benchmark our analytical solutions with published predictions from numerical models. Importantly, we leverage these analytical solutions to identify essential thermophysical and nondimensional parameters that govern the behavior of these systems. We identify scaling parameters and relations among surface charge density, ionic strength, and nanochannel height.

Numerical and experimental investigation of the deviation of microparticles inside the microchannel using the vortices caused by the ICEK phenomenon.

One field of study in microfluidics is the control, trapping, and separation of microparticles suspended in fluid. Some of its applications are related to cell handling, virus detection, and so on. One of the new methods in this field is using ICEK phenomena and dielectrophoresis forces. In the present study, considering the ICEK phenomena, the microparticles inside the fluid are deviated in the desired ratio using a novel ICEK microchip. The deviation is such that after the microparticles reach the floating electrode, they are trapped in the ICEK flow vortex and deviated through a secondary channel that was placed crosswise and noncoplanar above the main channel. For simulation verification, an experimental test is done. The method used for making two noncoplanar channels and separating the particles in the desired ratio with a simple ICEK microchip is an innovation of the present study. Moreover, the adjustment of the percentage of separation of microparticles by adjusting the parameters of the applied voltage and fluid inlet velocity is one of the other innovations of the present experimental study. We observed that for input velocities of 150-1200 µm/s with applied voltages of 10-33 V, 100% of the particles can be directed toward the secondary-channel.

Microfluidic sperm sorting selects a subpopulation of high-quality sperm with a higher potential for fertilization.

STUDY QUESTION: Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods? SUMMARY ANSWER: The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential. WHAT IS KNOWN ALREADY: To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations. STUDY DESIGN, SIZE, DURATION: The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023-2024. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage. MAIN RESULTS AND THE ROLE OF CHANCE: In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P < 0.05). LIMITATION, REASONS FOR CAUTION: The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients. STUDY FUNDING/COMPETING INTEREST(S): The study is funded by the Iran University of Medical Sciences and no competing interest exists. TRIAL REGISTRATION NUMBER: N/A.

Multiphysics analytical and numerical studies of biomolecule preconcentration utilizing ion concentration polarization: a case study of convergent microchannels.

A convergent sector in microfluidic devices utilizing ion concentration polarization (ICP) can help increase the preconcentration rate and the concentration enhancement factor (CEF) of biomolecules. In this work, we present a detailed study of the nozzle-like-squeeze effect of a convergent channel on the preconcentration of biomolecules. By numerically solving coupled Nernst-Planck-Poisson-Navier-Stokes governing equations for the 2D channel model, we report the first study on the critical width of a convergent region in the channel to retain the advantage of the nozzle-like-squeeze effect in speeding up preconcentration and augmenting CEF. In addition, we investigated the impact of the location and the dimensions of a convergent sector on the mechanism of biomolecule preconcentration. The location of an ion-selective membrane was also determined to ensure that biomolecules are focused on the convergent region of the channel. Moreover, we introduce analytical studies to compare and verify simulation findings. Specifically, the formulas for the critical dimensions of a convergent channel, location of a preconcentrated biomolecule plug, and position of an ion-selective membrane are presented. Furthermore, important working parameters, including electric potentials and hydrostatic pressures, were examined to scrutinize their effect on convergent concentrators. These detailed analytical solutions and numerical simulation results are consistent with experimental observations, providing deep insights into the ICP phenomenon and the preconcentration mechanism of biomolecules in convergent microfluidic concentration devices.

Dynamic video recognition for cell-encapsulating microfluidic droplets.

Droplet microfluidics is a highly sensitive and high-throughput technology extensively utilized in biomedical applications, such as single-cell sequencing and cell screening. However, its performance is highly influenced by the droplet size and single-cell encapsulation rate (following random distribution), thereby creating an urgent need for quality control. Machine learning has the potential to revolutionize droplet microfluidics, but it requires tedious pixel-level annotation for network training. This paper investigates the application software of the weakly supervised cell-counting network (WSCApp) for video recognition of microdroplets. We demonstrated its real-time performance in video processing of microfluidic droplets and further identified the locations of droplets and encapsulated cells. We verified our methods on droplets encapsulating six types of cells/beads, which were collected from various microfluidic structures. Quantitative experimental results showed that our approach can not only accurately distinguish droplet encapsulations (micro-F1 score > 0.94), but also locate each cell without any supervised location information. Furthermore, fine-tuning transfer learning on the pre-trained model also significantly reduced (>80%) annotation. This software provides a user-friendly and assistive annotation platform for the quantitative assessment of cell-encapsulating microfluidic droplets.

Small extracellular vesicles' enrichment from biological fluids using an acoustic trap.

Small extracellular vesicles (sEVs), a form of extracellular vesicles, are lipid bilayered structures released by all cells. Large-scale studies on sEVs from clinical samples are necessary, but a major obstacle is the lack of rapid, reproducible, efficient, and low-cost methods to enrich sEVs. Acoustic microfluidics have the advantage of being label-free and biocompatible, which have been reported to successfully enrich sEVs. In this paper, we present a highly efficient acoustic microfluidic trap that can offer low and large volume compatible ways of enriching sEVs from biological fluids by flexible structure design. It uses the idea of pre-loading larger seed particles in the acoustic trap to enable sub-micron particle capturing. The microfluidic chip is actuated using a piezoelectric plate transducer attached to a silicon-glass bonding plate with circular cavities. Each cavity works as a resonant unit, excited at the frequency of both the half wave resonance in the main plane and inverted quarter wave resonance in the depth direction, which has the ability to strongly trap seed particles at the center, thereby improving the subsequent nanoparticle capture efficiency. Mean trapping efficiencies of 35.62% and 64.27% were obtained using 60 nm and 100 nm nanobeads, respectively. By the use of this technology, we have successfully enriched sEVs from cell culture conditioned media and blood plasma at a flow rate of 10 µL min-1. The isolated sEV subpopulations are characterized by NTA and TEM, and their protein cargo is determined by WB. This acoustic trapping chip provides a rapid and robust method to enrich sEVs from biofluids with high reproducibility and sufficient quantities. Therefore, it can serve as a new tool for biological and clinical research such as cancer diagnosis and drug delivery.