Rapid and Accurate Antimicrobial Susceptibility Testing Using Label-Free Electrical Impedance-Based Microfluidic Platform.

Antimicrobial resistance has become a serious threat to the global public health. Accurate and rapid antimicrobial susceptibility testing (AST) allows evidence-based prescribing of antibiotics to improve patient care and clinical outcomes. Current culture-based AST assays are inherently limited by the doubling time of bacterial reproduction, which require at least 24 h to have a decisive result. Herein, a label-free electrical impedance-based microfluidic platform designed to expedite and streamline AST procedure for clinical practice is presented. Following a 30-min exposure of bacterial samples to antibiotics, the presented high-throughput, single-bacterium level impedance characterization platform enables a rapid 2-min AST assay. The platform facilitates accurate analysis of individual bacterial viability, as indicated by changes in electrical characteristics, thereby enabling the determination of antimicrobial resistance. Moreover, the potential clinical applicability of this platform is demonstrated by testing different E. coli strains against five antibiotics, yielding 100% categorical agreements compared to standard culture methods.

Molecular mechanism of different flower color formation of Cymbidium ensifolium.

Cymbidium ensifolium is one of the national orchids in China, which has high ornamental value with changeable flower colors. To understand the formation mechanism of different flower colors of C. ensifolium, this research conducted transcriptome and metabolome analyses on four different colored sepals of C. ensifolium. Metabolome analysis detected 204 flavonoid metabolites, including 17 polyphenols, 27 anthocyanins, 75 flavones, 34 flavonols, 25 flavonoids, 18 flavanones, and 8 isoflavones. Among them, purple-red and red sepals contain a lot of anthocyanins, including cyanidin, pelargonin, and paeoniflorin, while yellow-green and white sepals have less anthocyanins detected, and their metabolites are mainly flavonols, flavanones and flavonoids. Transcriptome sequencing analysis showed that the expression levels of the anthocyanin biosynthetic enzyme genes in red and purple-red sepals were significantly higher than those in white and yellow-green sepals of C. ensifolium. The experimental results showed that CeF3'H2, CeDFR, CeANS, CeF3H and CeUFGT1 may be the key genes involved in anthocyanin production in C. ensifolium sepals, and CeMYB104 has been proved to play an important role in the flower color formation of C. ensifolium. The results of transformation showed that the CeMYB104 is involved in the synthesis of anthocyanins and can form a purple-red color in the white perianth of Phalaenopsis. These findings provide a theoretical reference to understand the formation mechanism of flower color in C. ensifolium.

Uncovering early transcriptional regulation during adventitious root formation in Medicago sativa.

BACKGROUND: Alfalfa (Medicago sativa L.) as an important legume plant can quickly produce adventitious roots (ARs) to form new plants by cutting. But the regulatory mechanism of AR formation in alfalfa remains unclear. RESULTS: To better understand the rooting process of alfalfa cuttings, plant materials from four stages, including initial separation stage (C stage), induction stage (Y stage), AR primordium formation stage (P stage) and AR maturation stage (S stage) were collected and used for RNA-Seq. Meanwhile, three candidate genes (SAUR, VAN3 and EGLC) were selected to explore their roles in AR formation. The numbers of differentially expressed genes (DEGs) of Y-vs-C (9,724) and P-vs-Y groups (6,836) were larger than that of S-vs-P group (150), indicating highly active in the early AR formation during the complicated development process. Pathways related to cell wall and sugar metabolism, root development, cell cycle, stem cell, and protease were identified, indicating that these genes were involved in AR production. A large number of hormone-related genes associated with the formation of alfalfa ARs have also been identified, in which auxin, ABA and brassinosteroids are thought to play key regulatory roles. Comparing with TF database, it was found that AP2/ERF-ERF, bHLH, WRKY, NAC, MYB, C2H2, bZIP, GRAS played a major regulatory role in the production of ARs of alfalfa. Furthermore, three identified genes showed significant promotion effect on AR formation. CONCLUSIONS: Stimulation of stem basal cells in alfalfa by cutting induced AR production through the regulation of various hormones, transcription factors and kinases. This study provides new insights of AR formation in alfalfa and enriches gene resources in crop planting and cultivation.

Genome-Wide Identification and Analysis of bHLH Transcription Factors Related to Anthocyanin Biosynthesis in Cymbidium ensifolium.

The basic helix-loop-helix (bHLH) transcription factors are widely distributed across eukaryotic kingdoms and participate in various physiological processes. To date, the bHLH family has been identified and functionally analyzed in many plants. However, systematic identification of bHLH transcription factors has yet to be reported in orchids. Here, 94 bHLH transcription factors were identified from the Cymbidium ensifolium genome and divided into 18 subfamilies. Most CebHLHs contain numerous cis-acting elements associated with abiotic stress responses and phytohormone responses. A total of 19 pairs of duplicated genes were found in the CebHLHs, of which 13 pairs were segmentally duplicated genes and six pairs were tandemly duplicated genes. Expression pattern analysis based on transcriptome data revealed that 84 CebHLHs were differentially expressed in four different color sepals, especially CebHLH13 and CebHLH75 of the S7 subfamily. The expression profiles of CebHLH13 and CebHLH75 in sepals, which are considered potential genes regulating anthocyanin biosynthesis, were confirmed through the qRT-PCR technique. Furthermore, subcellular localization results showed that CebHLH13 and CebHLH75 were located in the nucleus. This research lays a foundation for further exploration of the mechanism of CebHLHs in flower color formation.

Automatic Microfluidic Cell Wash Platform for Purifying Cells in Suspension: Puriogen.

Cell wash is an essential cell sample preparation procedure to eliminate or minimize interfering substances for various subsequent cell analyses. The commonly used cell wash method is centrifugation which separates cells from other biomolecules in a solution with manual pipetting and has many drawbacks such as being labor-intensive and time-consuming with substantial cell loss and cell clumping. To overcome these issues, a centrifuge-free and automatic cell wash platform for cell purity generation, termed Puriogen, has been developed in this work. Compared with other developed products such as AcouWash, Puriogen can process samples with a high throughput of above 500 µL/min. Puriogen utilizes a uniquely designed inertial microfluidic device to complete the automatic cell wash procedure. One single-cell wash procedure with the Puriogen platform can remove more than 90% ambient proteins and nucleic acids from the cell sample. It can also remove most of the residual fluorescent dye after cell staining to significantly reduce the background signals for subsequent cell analysis. By removing the dead cell debris, it can increase the live cell percentage in the sample by 2-fold. Moreover, the percentage of single-cell population is also increased by 20% because of further disassociation of small-cell aggregates (e.g., doublets and triplets) into singlets. To freely adjust cell concentrations, the Puriogen platform can concentrate cells 5 times in a single flow-through process. The presented Puriogen cell wash solution has broad applications in cell preparation with its excellent simplicity in operation and wash efficiency, especially in single-cell sequencing.

Tunable and Dynamic Optofluidic Microlens Arrays Based on Droplets.

Microlens arrays (MLAs) are acquiring a key role in the micro-optical system, which have been widely applied in the fields of imaging processing, light extraction, biochemical sensing, and display technology. Compared with solid MLAs, liquid MLAs have received extensive attention due to their natural smooth interface and adjustability. However, manufacturing tunable liquid MLAs with ideal structures is still a key challenge for current technologies. In this paper, a novel and simple optofluidic method is demonstrated, enabling the tunable focusing and high-quality imaging of liquid MLAs. Tunable droplets are fabricated and self-assembled into arrays as the MLAs, which can be easily adjusted to focus, form images, and display different focal lengths. Tuning of MLAs' focusing properties (range from 550 to 5370 µm) is demonstrated by changing the refractive index (RI) of the droplets with a fixed size of 200 µm, which can be changed by adjusting the flow rates of the two branch streams. Also, the corresponding numerical apertures of the MLAs range from 0.026 to 0.26. Furthermore, the MLAs' functionality for microparticle imaging applications is also illustrated. Combining the MLAs with a 4× objective, microparticle imaging is magnified two times, and the resolution has also been improved on the original basis. Besides, both the size and RI of the MLAs in an optofluidic chip can be further adjusted to detect samples at different positions. These MLAs have the merits of high optical performance, a simple fabrication procedure, easy integration, and good tunability. Thus, it shows promising opportunities for many applications, such as adaptive imaging and sensing.

Deterministic Sorting of Submicrometer Particles and Extracellular Vesicles Using a Combined Electric and Acoustic Field.

Sorting of extracellular vesicles has important applications in early stage diagnostics. Current exosome isolation techniques, however, suffer from being costly, having long processing times, and producing low purities. Recent work has shown that active sorting via acoustic and electric fields are useful techniques for microscale separation activities, where combining these has the potential to take advantage of multiple force mechanisms simultaneously. In this work, we demonstrate an approach using both electrical and acoustic forces to manipulate bioparticles and submicrometer particles for deterministic sorting, where we find that the concurrent application of dielectrophoretic (DEP) and acoustophoretic forces decreases the critical diameter at which particles can be separated. We subsequently utilize this approach to sort subpopulations of extracellular vesicles, specifically exosomes (<200 nm) and microvesicles (>300 nm). Using our combined acoustic/electric approach, we demonstrate exosome purification with more than 95% purity and 81% recovery, well above comparable approaches.

Label-Free Multivariate Biophysical Phenotyping-Activated Acoustic Sorting at the Single-Cell Level.

Biophysical markers of cells such as cellular electrical and mechanical properties have been proven as promising label-free biomarkers for studying, characterizing, and classifying different cell types and even their subpopulations. Further analysis or manipulation of specific cell types or subtypes requires accurate isolation of them from the original heterogeneous samples. However, there is currently a lack of cell sorting ability that could actively separate a large number of individual cells at the single-cell level based on their multivariate biophysical makers or phenotypes. In this work, we, for the first time, demonstrate label-free and high-throughput acoustic single-cell sorting activated by the characterization of multivariate biophysical phenotypes. Electrical phenotyping is implemented by single-cell electrical impedance characterization with two pairs of differential sensing electrodes, while mechanical phenotyping is performed by extracting the transit time for the single cell to pass through microconstriction from the recorded impedance signals. A real-time impedance signal processing and triggering algorithm has been developed to identify the target sample population and activate a pulsed highly focused surface acoustic wave for single-cell level sorting. We have demonstrated acoustic single-particle sorting solely based on electrical or mechanical phenotyping. Furthermore, we have applied the developed microfluidic system to sort live MCF-7 cells from a mixture of fixed and live MCF-7 population activated by a combined electrical and mechanical phenotyping at a high throughput >100 cells/s and purity ∼91.8%. This demonstrated ability to analyze and sort cells based on multivariate biophysical phenotyping provides a solution to the current challenges of cell purification that lack specific molecular biomarkers.

Single-Cell Stretching in Viscoelastic Fluids with Electronically Triggered Imaging for Cellular Mechanical Phenotyping.

Cellular mechanical phenotypes in connection to physiological and pathological states of cells have become a promising intrinsic biomarker for label-free cell analysis in various biological research and medical diagnostics. In this work, we present a microfluidic system capable of high-throughput cellular mechanical phenotyping based on a rapid single-cell hydrodynamic stretching in a continuous viscoelastic fluid flow. Randomly introduced single cells are first aligned into a single streamline in viscoelastic fluids before being guided to a flow splitting junction for consistent hydrodynamic stretching. The arrival of individual cells prior to the flow splitting junction can be detected by an electrical sensing unit, which produces a triggering signal to activate a high-speed camera for on-demand imaging of the cell motion and deformation through the flow splitting junction. Cellular mechanical phenotypes, including cell size and cell deformability, are extracted from the analysis of these captured single-cell images. We have evaluated the sensitivity of the developed microfluidic mechanical phenotyping system by measuring the synthesized hydrogel microbeads with known Young's modulus. With this microfluidic cellular mechanical phenotyping system, we have revealed the statistical difference in the deformability of microfilament disrupted, normal, and fixed NIH 3T3 fibroblast cells. Furthermore, with the implementation of a machine-learning-based classification of MCF-10A and MDA-MB-231 mixtures, our label-free cellular phenotyping system has achieved a comparable cell analysis accuracy (0.9:1, 5.03:1) with respect to the fluorescence-based flow cytometry results (0.97:1, 5.33:1). The presented microfluidic mechanical phenotyping technique will open new avenues for high-throughput and label-free single-cell analysis in diverse biomedical applications.

The genome of Cymbidium sinense revealed the evolution of orchid traits.

The Orchidaceae is of economic and ecological importance and constitutes ˜10% of all seed plant species. Here, we report a genome physical map for Cymbidium sinense, a well-known species belonging to genus Cymbidium that has thousands of natural variation varieties of flower organs, flower and leaf colours and also referred as the King of Fragrance, which make it arose into a unique cultural symbol in China. The high-quality chromosome-scale genome assembly was 3.52 Gb in size, 29 638 protein-coding genes were predicted, and evidence for whole-genome duplication shared with other orchids was provided. Marked amplification of cytochrome- and photosystem-related genes was observed, which was consistent with the shade tolerance and dark green leaves of C. sinense. Extensive duplication of MADS-box genes, and the resulting subfunctional and expressional differentiation, was associated with regulation of species-specific flower traits, including wild-type and mutant-type floral patterning, seasonal flowering and ecological adaption. CsSEP4 was originally found to positively regulate gynostemium development. The CsSVP genes and their interaction proteins CsAP1 and CsSOC1 were significantly expanded and involved in the regulation of low-temperature-dependent flowering. Important genetic clues to the colourful leaf traits, purple-black flowers and volatile trait in C. sinense were also found. The results provide new insights into the molecular mechanisms of important phenotypic traits of Cymbidium and its evolution and serve as a powerful platform for future evolutionary studies and molecular breeding of orchids.

A low-cost and high-throughput benchtop cell sorter for isolating white blood cells from whole blood.

The ability to isolate and purify white blood cells (WBCs) from mixed ensembles such as blood would benefit autologous cell-based therapeutics as well as diagnosis of WBC disorders. Current WBCs isolation methods have the limitations of low purity or requiring complex and expensive equipment. In addition, due to the overlap in size distribution between lymphocytes (i.e., a sub-population of WBCs) and red blood cells (RBCs), it is challenging to achieve isolation of entire WBCs populations. In this work, we developed an inertial microfluidics-based cell sorter, which enables size-based, high-throughput isolation, and enrichment of WBCs from RBC-lysed whole blood. Using the developed inertial microfluidic chip, the sorting resolution is sharpened within 2 µm, which achieved separation between 3 and 5 µm diameter particles. Thus, with the present cell sorter, a full population of WBCs can be isolated from RBC-lysed blood samples with recovery ratio of 92%, and merely 5% difference in the composition percentage of the three subpopulations of granulocytes, monocytes, and lymphocytes compared to the original sample. Furthermore, our cell sorter is designed to enable broad application of size-based inertial cell sorting by supplying a series of microchips with different sorting cutoff size. This strategy allows us to further enrich the lymphocytes population by twofold using another microchip with a cutoff size between 10 and 15 µm. With simplicity and efficiency, our cell sorter provides a powerful platform for isolating and sorting of WBCs and also envisions broad potential sorting applications for other cell types.

Multi-frequency single cell electrical impedance measurement for label-free cell viability analysis.

Cell viability is a physiological status connected to cell membrane integrity and cytoplasmic topography, which is profoundly important for fundamental biological research and practical biomedical applications. A conventional method for assessing cell viability is through cell staining analysis. However, cell staining involves laborious and complicated processing procedures and is normally cytotoxic. Intrinsic cellular phenotypes thus provide new avenues for measuring cell viability in a stain-free and non-toxic manner. In this work, we present a label-free non-destructive impedance-based approach for cell viability assessment by simultaneously characterizing multiple electrical cellular phenotypes in a high-throughput manner (>1000 cells per min). A novel concept called the complex opacity spectrum is introduced for improving the discrimination of live and dead cells. The analysis of the complex opacity spectrum leads to the discovery of two frequency ranges that are optimized for characterizing membranous and cytoplasmic electrical phenotypes. The present impedance-based approach has successfully discriminated between living and dead cells in two different experimental scenarios, including mixed living and dead cells in both homogenous and heterogeneous cell samples. This impedance-based single cell phenotyping technique provides highly accurate and consistent cell viability analysis, which has been validated by commercial fluorescence-based flow cytometry (∼1% difference) using heterogeneous cell samples. This label-free high-throughput cell viability analysis strategy will have broad applications in the field of biology and medicine.

Genome-Wide Identification of the MYB Gene Family in Cymbidiumensifolium and Its Expression Analysis in Different Flower Colors.

MYB transcription factors of plants play important roles in flavonoid synthesis, aroma regulation, floral organ morphogenesis, and responses to biotic and abiotic stresses. Cymbidium ensifolium is a perennial herbaceous plant belonging to Orchidaceae, with special flower colors and high ornamental value. In this study, a total of 136 CeMYB transcription factors were identified from the genome of C. ensifolium, including 27 1R-MYBs, 102 R2R3-MYBs, 2 3R-MYBs, 2 4R-MYBs, and 3 atypical MYBs. Through phylogenetic analysis in combination with MYB in Arabidopsis thaliana, 20 clusters were obtained, indicating that these CeMYBs may have a variety of biological functions. The 136 CeMYBs were distributed on 18 chromosomes, and the conserved domain analysis showed that they harbored typical amino acid sequence repeats. The motif prediction revealed that multiple conserved elements were mostly located in the N-terminal of CeMYBs, suggesting their functions to be relatively conserved. CeMYBs harbored introns ranging from 0 to 13 and contained a large number of stress- and hormone-responsive cis-acting elements in the promoter regions. The subcellular localization prediction demonstrated that most of CeMYBs were positioned in the nucleus. The analysis of the CeMYBs expression based on transcriptome data showed that CeMYB52, and CeMYB104 of the S6 subfamily may be the key genes leading to flower color variation. The results lay a foundation for the study of MYB transcription factors of C. ensifolium and provide valuable information for further investigations of the potential function of MYB genes in the process of anthocyanin biosynthesis.

Submicron Particle Concentration and Patterning with Ultralow Frequency Acoustic Vibration.

Acoustofluidics have been widely used for particle and cell manipulations. Given the scaling of acoustic radiation forces and acoustic streaming flow velocities with increasing frequency, existing acoustofluidic manipulation of submicron particles require actuation at MHz and even GHz frequencies. In this work, we explore a novel acoustofluidic phenomenon, where an ultralow frequency (800 Hz) acoustic vibration is capable of concentrating and patterning submicron particles at two poles of each pillar in an array embedded in a microfluidic device. This unprecedented phenomenon is attributed to a collective effect of acoustic streaming induced drag force and non-Newtonian fluid induced elastic lift force, arising from symmetric acoustic microstreaming flows around each pillar uniformly across the entire pillar array. To our knowledge, this is the first demonstration that particles can be manipulated by an acoustic wave with a wavelength that is 6 orders of magnitude larger than the particle size. This ultralow frequency acoustofluidics will enable a simple and cost-effective solution to effective and uniform manipulation of submicron biological particles in large scales, which has the potential to be widely exploited in clinical and biomedical fields.

Exosome Purification and Analysis Using a Facile Microfluidic Hydrodynamic Trapping Device.

Exosomes are nanosized (30-150 nm) extracellular vesicles (EVs) secreted by various cell types. They are easily accessible in biological fluids and contain specific disease biomarkers, making them attractive for diagnosis and prognosis applications. Accurate biological characterization of exosomes is an important step toward clinical applications that require effective and precise isolation of subpopulations of exosomes. It is therefore of particular importance to develop an efficient and reliable exosome purification technique to isolate exosomes from the heterogeneous extracellular fluids. In this work, we intend to isolate and visualize exosomes by combining an affinity-based method and passive microfluidic particle trapping. Microbeads with a diameter of 20 µm are first functionalized with streptavidin and biotinylated antibodies and then used to immobilize and enrich exosomes on their surfaces using antigen-antibody affinity binding. We have developed a microfluidic device with trapping arrays to efficiently trap a large number of individual microbeads with enriched exosomes at the single-particle level, i.e., one single bead per trapping site, on the basis of a passive hydrodynamic trapping principle. The large-scale microfluidic single-bead trapping permits massively multiplexed fluorescence detection and quantification of the individual beads, which prevents the optical interfering of background noise as well as allowing one to acquire an average fluorescence density of a single bead for an accurate fluorescence-based exosome quantification. In addition, on-chip elusion and lysis of the protein and RNA content of captured exosomes enable further molecular analysis of exosomes, including Western blot and quantitative polymerase chain reaction. This microfluidic device provides a rapid and straightforward capturing and quantification method to analyze EVs for a variety of biological studies and applications.

Enhanced Molecular Diagnosis of Bloodstream Candida Infection with Size-Based Inertial Sorting at Submicron Resolution.

Inertial microfluidics has been proven to be a powerful tool for high-throughput, size-based cell sorting in diverse biomedical applications. In the case of Candida-related sepsis, Candida species and major blood cells (i.e., red blood cells and white blood cells) have a size distribution of 3-5 and 6-30 µm, respectively. To effectively retrieve a majority of Candida species and remove most of the interfering blood cells for accurate molecular analysis, inertial sorting of micron-sized biological particles with submicron size difference is highly desired, but far unexplored till now. In this work, we present a new channel design for an inertial microfluidic sorting device by embedding microsquares to construct periodic contractions along a series of repeating curved units. This unique channel design allows us to enhance inertial lift force at the microsquare zone and produce localized secondary Dean flow drag force in addition to global Dean flow drag force. This inertial sorting device has successfully separated 5.5 µm particles from 6.0 µm particles with a recovery ratio higher than 80% and a purity higher than 92%, demonstrating a size-based inertial sorting at submicron resolution (i.e., 0.5 µm). We further applied this inertial sorting device to purify Candida species from whole blood sample for enhanced molecular diagnosis of bloodstream Candida infection and especially compared it with the commonly used lysis-centrifugation-based purification method (STEM method) by recovering two species of Candida (Cornus glabrata and Candida albicans) from Candida-spiked blood samples. Through quantitative polymerase chain reaction (qPCR) analysis, we found that our inertial sorting approach has nearly 3-fold improvement on the pathogen recovery than the STEM method at pathogen abundances of 103 cfu/mL and 102 cfu/mL. The present inertial sorting at submicron resolution provides a simple, rapid, and efficient pathogen purification method for significantly improved molecular diagnosis of bloodstream Candida infection.

Massively Multiplexed Submicron Particle Patterning in Acoustically Driven Oscillating Nanocavities.

Nanoacoustic fields are a promising method for particle actuation at the nanoscale, though THz frequencies are typically required to create nanoscale wavelengths. In this work, the generation of robust nanoscale force gradients is demonstrated using MHz driving frequencies via acoustic-structure interactions. A structured elastic layer at the interface between a microfluidic channel and a traveling surface acoustic wave (SAW) device results in submicron acoustic traps, each of which can trap individual submicron particles. The acoustically driven deformation of nanocavities gives rise to time-averaged acoustic fields which direct suspended particles toward, and trap them within, the nanocavities. The use of SAWs permits massively multiplexed particle manipulation with deterministic patterning at the single-particle level. In this work, 300 nm diameter particles are acoustically trapped in 500 nm diameter cavities using traveling SAWs with wavelengths in the range of 20-80 µm with one particle per cavity. On-demand generation of nanoscale acoustic force gradients has wide applications in nanoparticle manipulation, including bioparticle enrichment and enhanced catalytic reactions for industrial applications.

Submicron Particle Focusing and Exosome Sorting by Wavy Microchannel Structures within Viscoelastic Fluids.

Exosomes, submicron membrane vesicles (30-200 nm) secreted by almost all cells, containing significant information such as proteins, microRNAs and DNAs, are closely associated with disease diagnostic and prognostic tests for liquid biopsy in clinical practice. However, their inherently small sizes lead to great challenges for isolating them from complex body fluids with high-throughput and high-purity. In this work, a reverse wavy channel structure using viscoelastic fluids with the addition of biocompatible polymer was presented for elasto-inertial focusing and sorting of submicron particles and exosomes. The microfluidic periodically reversed Dean secondary flow generated by repeated wavy channel structures could facilitate particle focusing compared with traditional straight channels. Four differently sized fluorescent submicron spheres (1 µm, 500 nm, 300 and 100 nm) were used to study the focusing behavior under various conditions. We have achieved simple, high-throughput, and label-free sorting of exosomes with purity higher than 92% and recovery higher than 81%. This developed elasto-inertial exosome sorting technique may provide a promising platform in various exosome-related biological research and pharmaceutical applications.

Detachable Acoustophoretic System for Fluorescence-Activated Sorting at the Single-Droplet Level.

Droplet-based single-cell sequencing has emerged as a very powerful tool to study the cellular heterogeneity in diseased tissues for a variety of biological problems. However, the current droplet generation with a single particle and cell encapsulation is a random process and suffers from a low yield that is unable to fulfill the high-throughput analysis requirement. In this work, we present a new fluorescence-activated droplet sorting (FADS) system that can isolate single-cell droplets at high accuracy and high yield using a highly focused surface acoustic wave (HFSAW) with a beam width around 50 µm. The acoustic wave is locally coupled into the microfluidic channel for droplet sorting through a micropillar waveguide structure between the channel and the interdigitated transducer (IDT). This detachable acoustic sorting system allows the disposal of the microfluidic channel after a single use to avoid cross-contamination and keeps the expensive IDT device reusable. We have achieved rapid and accurate isolation of single-cell droplets with purity higher than 90% at ∼1 kHz sorting rate with three different encapsulation contents. In addition, with the uniformly produced droplet size at ∼40 µm, the present acoustic FADS system enables effective sorting of small particles down to submicrometer size, which is challenging for existing fluorescence-activated cell sorting systems.

Sheathless Acoustic Fluorescence Activated Cell Sorting (aFACS) with High Cell Viability.

In this work, we demonstrate a sheathless acoustic fluorescence activated cell sorting (aFACS) system by combining elasto-inertial cell focusing and highly focused traveling surface acoustic wave (FTSAW) to sort cells with high recovery rate, purity, and cell viability. The microfluidic sorting device utilizes elasto-inertial particle focusing to align cells in a single file for improving sorting accuracy and efficiency without sample dilution. Our sorting device can effectively focus 1 µm particles which represents the general minimum size for a majority of cell sorting applications. Upon the fluorescence interrogation at the single cell level, individual cells are deflected to the target outlet by a ∼50 µm wide highly focused acoustic field. We have applied our aFACS to sort three different cell lines (i.e., MCF-7, MDA-231, and human-induced pluripotent stem-cell-derived cardiomyocytes; hiPSC-CMs) at ∼kHz with a sorting purity and recovery rate both of about 90%. A further comparison demonstrates that the cell viability drops by 35-45% using a commercial FACS machine, while the cell viability only drops by 3-4% using our aFACS system. The developed aFACS system provides a benchtop solution for rapid, highly accurate single cell level sorting with high cell viability, in particular for sensitive cell types.