On‐Demand Droplet Collection for Capturing Single Cells

Droplet‐based microfluidic techniques are extensively used in efficient manipulation and genome‐wide analysis of individual cells, probing the heterogeneity among populations of individuals. However, the extraction and isolation of single cells from individual droplets remains difficult due to the inevitable sample loss during processing. Herein, an automated system for accurate collection of defined numbers of droplets containing single cells is presented. Based on alternate sorting and dispensing in three branch channels, the droplet number can be precisely controlled down to single‐droplet resolution. While encapsulating single cells and reserving one branch as a waste channel, sorting can be seamlessly integrated to enable on‐demand collection of single cells. Combined with a lossless recovery strategy, this technique achieves capture and culture of individual cells with a harvest rate of over 95%. The on‐demand droplet collection technique has great potential to realize quantitative processing and analysis of single cells for elucidating the role of cell‐to‐cell variations.     

Standing Surface Acoustic Wave (SSAW)‐Based Fluorescence‐Activated Cell Sorter

Microfluidic fluorescence‐activated cell sorters (μFACS) have attracted considerable interest because of their ability to identify and separate cells in inexpensive and biosafe ways. Here a high‐performance μFACS is presented by integrating a standing surface acoustic wave (SSAW)‐based, 3D cell‐focusing unit, an in‐plane fluorescent detection unit, and an SSAW‐based cell‐deflection unit on a single chip. Without using sheath flow or precise flow rate control, the SSAW‐based cell‐focusing technique can focus cells into a single file at a designated position. The tight focusing of cells enables an in‐plane‐integrated optical detection system to accurately distinguish individual cells of interest. In the acoustic‐based cell‐deflection unit, a focused interdigital transducer design is utilized to deflect cells from the focused stream within a minimized area, resulting in a high‐throughput sorting ability. Each unit is experimentally characterized, respectively, and the integrated SSAW‐based FACS is used to sort mammalian cells (HeLa) at different throughputs. A sorting purity of greater than 90% is achieved at a throughput of 2500 events s^?1. The SSAW‐based FACS is efficient, fast, biosafe, biocompatible and has a small footprint, making it a competitive alternative to more expensive, bulkier traditional FACS.     

Lipoplex‐Mediated Single‐Cell Transfection via Droplet Microfluidics

While lipoplex (cationic lipid‐nucleic acid complex)‐mediated intracellular delivery is widely adopted in mammalian cell transfection, its transfection efficiency for suspension cells, e.g., lymphatic and hematopoietic cells, is reported at only ≈5% or even lower. Here, efficient and consistent lipoplex‐mediated transfection is demonstrated for hard‐to‐transfect suspension cells via a single‐cell, droplet‐microfluidics approach. In these microdroplets, monodisperse lipoplexes for effective gene delivery are generated via chaotic mixing induced by the serpentine microchannel and co‐confined with single cells. Moreover, the cell membrane permeability increases due to the shear stress exerted on the single cells when they pass through the droplet pinch‐off junction. The transfection efficiency, examined by the delivery of the pcDNA3‐EGFP plasmid, improves from ≈5% to ≈50% for all three tested suspension cell lines, i.e., K562, THP‐1, Jurkat, and with significantly reduced cell‐to‐cell variation, compared to the bulk method. Efficient targeted knockout of the TP53BP1 gene for K562 cells via the CRISPR (clustered regularly interspaced short palindromic repeats)–CAS9 (CRISPR‐associated nuclease 9) mechanism is also achieved using this platform. Lipoplex‐mediated single‐cell transfection via droplet microfluidics is expected to have broad applications in gene therapy and regenerative medicine by providing high transfection efficiency and low cell‐to‐cell variation for hard‐to‐transfect suspension cells.     

Phenome–Genome Profiling of Single Bacterial Cell by Raman‐Activated Gravity‐Driven Encapsulation and Sequencing

The small size and low DNA amount of bacterial cells have hindered establishing phenome–genome links in a precisely indexed, one‐cell‐per‐reaction manner. Here, Raman‐Activated Gravity‐driven single‐cell Encapsulation and Sequencing (RAGE‐Seq) is presented, where individual cells are phenotypically screened via single‐cell Raman spectra (SCRS) in an aquatic, vitality‐preserving environment, then the cell with targeted SCRS is precisely packaged in a picoliter microdroplet and readily exported in a precisely indexed, “one‐cell‐one‐tube” manner. Such integration of microdroplet encapsulation to Raman‐activated sorting ensures high‐coverage one‐cell genome sequencing or cultivation that is directly linked to metabolic phenotype. For clinical Escherichia coli isolates, genome assemblies derived from precisely one cell via RAGE‐Seq consistently reach >95% coverage. Moreover, directly from a urine sample of urogenital tract infection, metabolic‐activity‐based antimicrobial susceptibility phenotypes and genome sequence of 99.5% coverage are obtained simultaneously from precisely one cell. This single‐cell global mutation map corroborates resistance phenotype and genotype, and unveils epidemiological features with high specificity and sensitivity. The ability to profile and correlate bacterial metabolic phenome and high‐quality genome sequences at one‐cell resolution suggests broad application of RAGE‐Seq.     

Visual Quantitative Detection of Circulating Tumor Cells with Single‐Cell Sensitivity Using a Portable Microfluidic Device

Immunocytological technologies, molecular technologies, and functional assays are widely used for detecting circulating tumor cells (CTCs) after enrichment from patients' blood sample. Unfortunately, accessibility to these technologies is limited due to the need for sophisticated instrumentation and skilled operators. Portable microfluidic devices have become attractive tools for expanding the access and efficiency of detection beyond hospitals to sites near the patient. Herein, a volumetric bar chart chip (V‐Chip) is developed as a portable platform for CTC detection. The target CTCs are labeled with aptamer‐conjugated nanoparticles (ACNPs) and analyzed by V‐Chip through quantifying the byproduct (oxygen) of the catalytic reaction between ACNPs and hydrogen peroxide, which results in the movement of an ink bar to a concentration‐dependent distance for visual quantitative readout. Thus, the CTC number is decoded into visually quantifiable information and a linear correlation can be found between the distance moved by the ink and number of cells in the sample. This method is sensitive enough that a single cell can be detected. Furthermore, the clinical capabilities of this system are demonstrated for quantitative CTC detection in the presence of a high leukocyte background. This portable detection method shows great potential for quantification of rare cells with single‐cell sensitivity for various applications.     

A Microfluidic Strategy for Controllable Generation of Water‐in‐Water Droplets as Biocompatible Microcarriers

Droplet microfluidics has been widely applied in functional microparticles fabricating, tissue engineering, and drug screening due to its high throughput and great controllability. However, most of the current droplet microfluidics are dependent on water‐in‐oil (W/O) systems, which involve organic reagents, thus limiting their broader biological applications. In this work, a new microfluidic strategy is described for controllable and high‐throughput generation of monodispersed water‐in‐water (W/W) droplets. Solutions of polyethylene glycol and dextran are used as continuous and dispersed phases, respectively, without any organic reagents or surfactants. The size of W/W droplets can be precisely adjusted by changing the flow rate of dispersed and continuous phases and the valve switch cycle. In addition, uniform cell‐laden microgels are fabricated by introducing the alginate component and rat pancreatic islet (β‐TC6) cell suspension to the dispersed phase. The encapsulated islet cells retain high viability and the function of insulin secretion after cultivation for 7 days. The high‐throughput droplet microfluidic system with high biocompatibility is stable, controllable, and flexible, which can boost various chemical and biological applications, such as bio‐oriented microparticles synthesizing, microcarriers fabricating, tissue engineering, etc.     

A Single‐Cell Assay for Time Lapse Studies of Exosome Secretion and Cell Behaviors

To understand the inhomogeneity of cells in biological systems, there is a growing demand on the capability of characterizing the properties of individual single cells. Since single‐cell studies require continuous monitoring of the cell behaviors, an effective single‐cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single‐cell technologies cannot provide proper environments to sustain cell growth and, proliferation of single cells and convenient, noninvasive tests of single‐cell behaviors from molecular markers. Here, a highly versatile single‐cell assay is presented that can accommodate different cellular types, enable easy and efficient single‐cell loading and culturing, and be suitable for the study of effects of in vitro environmental factors in combination with drug screening. One salient feature of the assay is the noninvasive collection and surveying of single‐cell secretions at different time points, producing unprecedented insight of single‐cell behaviors based on the biomarker signals from individual cells under given perturbations. Above all, the acquired information is quantitative, for example, measured by the number of exosomes each single‐cell secretes for a given time period. Therefore, our single‐cell assay provides a convenient, low‐cost, and enabling tool for quantitative, time lapsed studies of single‐cell properties.     

Label‐Free Optofluidic Nanobiosensor Enables Real‐Time Analysis of Single‐Cell Cytokine Secretion

Single‐cell analysis of cytokine secretion is essential to understand the heterogeneity of cellular functionalities and develop novel therapies for multiple diseases. Unraveling the dynamic secretion process at single‐cell resolution reveals the real‐time functional status of individual cells. Fluorescent and colorimetric‐based methodologies require tedious molecular labeling that brings inevitable interferences with cell integrity and compromises the temporal resolution. An innovative label‐free optofluidic nanoplasmonic biosensor is introduced for single‐cell analysis in real time. The nanobiosensor incorporates a novel design of a multifunctional microfluidic system with small volume microchamber and regulation channels for reliable monitoring of cytokine secretion from individual cells for hours. Different interleukin‐2 secretion profiles are detected and distinguished from single lymphoma cells. The sensor configuration combined with optical spectroscopic imaging further allows us to determine the spatial single‐cell secretion fingerprints in real time. This new biosensor system is anticipated to be a powerful tool to characterize single‐cell signaling for basic and clinical research.     

Solution‐Processing of High‐Purity Semiconducting Single‐Walled Carbon Nanotubes for Electronics Devices

High‐purity semiconducting single‐walled carbon nanotubes (s‐SWCNTs) are of paramount significance for the construction of next‐generation electronics. Until now, a number of elaborate sorting and purification techniques for s‐SWCNTs have been developed, among which solution‐based sorting methods show unique merits in the scale production, high purity, and large‐area film formation. Here, the recent progress in the solution processing of s‐SWCNTs and their application in electronic devices is systematically reviewed. First, the solution‐based sorting and purification of s‐SWCNTs are described, and particular attention is paid to the recent advance in the conjugated polymer‐based sorting strategy. Subsequently, the solution‐based deposition and morphology control of a s‐SWCNT thin film on a surface are introduced, which focus on the strategies for network formation and alignment of SWCNTs. Then, the recent advances in electronic devices based on s‐SWCNTs are reviewed with emphasis on nanoscale s‐SWCNTs' high‐performance integrated circuits and s‐SWCNT‐based thin‐film transistors (TFT) array and circuits. Lastly, the existing challenges and development trends for the s‐SWCNTs and electronic devices are briefly discussed. The aim is to provide some useful information and inspiration for the sorting and purification of s‐SWCNTs, as well as the construction of electronic devices with s‐SWCNTs.     

Centering Single Cells in Microgels via Delayed Crosslinking Supports Long‐Term 3D Culture by Preventing Cell Escape

Single‐cell‐laden microgels support physiological 3D culture conditions while enabling straightforward handling and high‐resolution readouts of individual cells. However, their widespread adoption for long‐term cultures is limited by cell escape. In this work, it is demonstrated that cell escape is predisposed to off‐center encapsulated cells. High‐speed microscopy reveals that cells are positioned at the microgel precursor droplets' oil/water interface within milliseconds after droplet formation. In conventional microencapsulation strategies, the droplets are typically gelled immediately after emulsification, which traps cells in this off‐center position. By delaying crosslinking, driving cells toward the centers of microgels is succeeded. The centering of cells in enzymatically crosslinked microgels prevents their escape during at least 28 d. It thereby uniquely enables the long‐term culture of individual cells within <5‐µm‐thick 3D uniform hydrogel coatings. Single cell analysis of mesenchymal stem cells in enzymatically crosslinked microgels reveals unprecedented high cell viability (>90%), maintained metabolic activity (>70%), and multilineage differentiation capacity (>60%) over a period of 28 d. The facile nature of this microfluidic cell‐centering method enables its straightforward integration into many microencapsulation strategies and significantly enhances control, reproducibility, and reliability of 3D single cell cultures.     

Tunable Confinement for Bridging Single‐Cell Manipulation and Single‐Molecule DNA Linearization

DNA linearization by nanoconfinement has offered a new avenue toward large‐scale genome mapping. The ability to smoothly interface the widely different length scales from cell manipulation to DNA linearization is critical to the development of single‐cell genomic mapping or sequencing technologies. Conventional nanochannel technologies for DNA analysis suffer from complex fabrication procedures, DNA stacking at the nanochannel entrance, and inefficient solution exchange. In this work, a dynamic and tunable confinement strategy is developed to manipulate and linearize genomic‐length DNA molecules from a single cell. By leveraging pneumatic microvalve control and elastomeric collapse, an array of nanochannels with confining dimension down to 20 nm and length up to sub‐millimeter is created and can be dynamically tuned in size. The curved edges of the microvalve form gradual transitions from microscale to nanoscale confinement, smoothly facilitating DNA entry into the nanochannels. A unified micro/nanofluidic device that integrates single‐cell trapping and lysis, DNA extraction, purification, labeling, and linearization is developed based on dynamically controllable nanochannels. Mbp‐long DNA molecules are extracted directly from a single cell and in situ linearized in the nanochannels. The device provides a facile and promising platform to achieve the ultimate goal of single‐cell, single‐genome analysis.     

Pick‐and‐Place Assembly of Single Microtubules

Intracellular transport is affected by the filament network in the densely packed cytoplasm. Biophysical studies focusing on intracellular transport based on microtubule–kinesin system frequently use in vitro motility assays, which are performed either on individual microtubules or on random (or simple) microtubule networks. Assembling intricate networks with high flexibility requires the manipulation of 25 nm diameter microtubules individually, which can be achieved through the use of pick‐and‐place assembly. Although widely used to assemble tiny objects, pick‐and‐place is not a common practice for the manipulation of biological materials. Using the high‐level handling capabilities of microelectromechanical systems (MEMS) technology, tweezers are designed and fabricated to pick and place single microtubule filaments. Repeated picking and placing cycles provide a multilayered and multidirectional microtubule network even for different surface topographies. On‐demand assembly of microtubules forms crossings at desired angles for biophysical studies as well as complex networks that can be used as nanotransport systems.     

Single‐Carbon‐Nanotube Manipulations and Devices Based on Macroscale Anthracene Flakes

Because of the outstanding mechanical and electrical properties of carbon nanotubes (CNTs), a CNT‐based torsion pendulum is demonstrated to show great potential in nano‐electromechanical systems. It is also expected to achieve various manipulations for further characterization and increase device sensitivity using ultrlong CNTs and macroscale moving parts. However, the reported top‐down method limits the CNT performance and device size by introducing inevitable contamination and destruction, which greatly hinders the development of single‐molecule devices. Here, a bottom‐up method is introduced to fabricate heterostructures of anthracene flakes (AFs) and suspended CNTs, providing a nondamaging CNT mechanical measurement before further applications, especially for the twisting behavior, and providing a controllable and clean transfer method to fabricate CNT‐based electrical devices under ambient conditions. Based on the unique geometry of CNT/AF heterostructures, various complex manipulations of single‐CNT devices are conducted to investigate CNT mechanical properties and prompt novel applications of similar structures in nanotechnology. The AF‐decorated CNTs show high Young's modulus (≈1 TPa) and tensile strength (≈100 GPa), and can be considered as the finest and strongest torsional springs. CNT‐based torsion balance enables to measure fN‐level forces and the torsional spring constant is two orders of magnitude lower than previously reported values.     

Ultrahigh Photocatalytic Rate at a Single‐Metal‐Atom‐Oxide

Metal oxides, as one of the mostly abundant and widely utilized materials, are extensively investigated and applied in environmental remediation and protection, and in energy conversion and storage. Most of these diverse applications are the result of a large diversity of the electronic states of metal oxides. Noticeably, however, many metal oxides present obstacles for applications in catalysis, mainly due to the lack of efficient active sites with desired electronic states. Here, the fabrication of single‐tungsten‐atom‐oxide (STAO) is demonstrated, in which the metal oxide's volume reaches its minimum as a unit cell. The catalytic mechanism in the STAO is determined by a new single‐site physics mechanism, named as quasi‐atom physics. The photogenerated electron transfer process is enabled by an electron in the spin‐up channel excited from the highest occupied molecular orbital to the lowest unoccupied molecular orbital +1 state, which can only occur in STAO with W⁵⁺. STAO results in a record‐high and stable sunlight photocatalytic degradation rate of 0.24 s^?1, which exceeds the rates of available photocatalysts by two orders of magnitude. The fabrication of STAO and its unique quasi‐atom photocatalytic mechanism lays new ground for achieving novel physical and chemical properties using single‐metal‐atom oxides (SMAO).     

A Droplet‐Based High‐Throughput SERS Platform on a Droplet‐Guiding‐Track‐Engraved Superhydrophobic Substrate

A novel droplet‐based surface‐enhanced Raman scattering (SERS) sensor for high‐throughput real‐time SERS monitoring is presented. The developed sensors are based on a droplet‐guiding‐track‐engraved superhydrophobic substrate covered with hierarchical SERS‐active Ag dendrites. The droplet‐guiding track with a droplet stopper is designed to manipulate the movement of a droplet on the superhydrophobic substrate. The superhydrophobic Ag dendritic substrates are fabricated through a galvanic displacement reaction and subsequent self‐assembled monolayer coating. The optimal galvanic reaction time to fabricate a SERS‐active Ag dendritic substrate for effective SERS detection is determined, with the optimized substrate exhibiting an enhancement factor of 6.3 × 10⁵. The height of the droplet stopper is optimized to control droplet motion, including moving and stopping. Based on the manipulation of individual droplets, the optimized droplet‐based real‐time SERS sensor shows high resistance to surface contaminants, and droplets containing rhodamine 6G, Nile blue A, and malachite green are successively controlled and detected without spectral interference. This noble droplet‐based SERS sensor reduces sample preparation time to a few seconds and increased detection rate to 0.5 µ L s^?1 through the simple operation mechanism of the sensor. Accordingly, our sensor enables high‐throughput real‐time molecular detection of various target analytes for real‐time chemical and biological monitoring.     

Single Upconversion Nanoparticle–Bacterium Cotrapping for Single‐Bacterium Labeling and Analysis

Detecting and analyzing pathogenic bacteria in an effective and reliable manner is crucial for the diagnosis of acute bacterial infection and initial antibiotic therapy. However, the precise labeling and analysis of bacteria at the single‐bacterium level are a technical challenge but very important to reveal important details about the heterogeneity of cells and responds to environment. This study demonstrates an optical strategy for single‐bacterium labeling and analysis by the cotrapping of single upconversion nanoparticles (UCNPs) and bacteria together. A single UCNP with an average size of ≈120 nm is first optically trapped. Both ends of a single bacterium are then trapped and labeled with single UCNPs emitting green light. The labeled bacterium can be flexibly moved to designated locations for further analysis. Signals from bacteria of different sizes are detected in real time for single‐bacterium analysis. This cotrapping method provides a new approach for single‐pathogenic‐bacterium labeling, detection, and real‐time analysis at the single‐particle and single‐bacterium level.     

Microfluidic‐Nanofiber Hybrid Array for Screening of Cellular Microenvironments

Cellular microenvironments are generally sophisticated, but crucial for regulating the functions of human pluripotent stem cells (hPSCs). Despite tremendous effort in this field, the correlation between the environmental factors—especially the extracellular matrix and soluble cell factors—and the desired cellular functions remains largely unknown because of the lack of appropriate tools to recapitulate in vivo conditions and/or simultaneously evaluate the interplay of different environment factors. Here, a combinatorial platform is developed with integrated microfluidic channels and nanofibers, associated with a method of high‐content single‐cell analysis, to study the effects of environmental factors on stem cell phenotype. Particular attention is paid to the dependence of hPSC short‐term self‐renewal on the density and composition of extracellular matrices and initial cell seeding densities. Thus, this combinatorial approach provides insights into the underlying chemical and physical mechanisms that govern stem cell fate decisions.     

Site‐Specific SERS Assay for Survivin Protein Dimer: From Ensemble Experiments to Correlative Single‐Particle Imaging

An assay for Survivin, a small dimeric protein which functions as modulator of apoptosis and cell division and serves as a promising diagnostic biomarker for different types of cancer, is presented. The assay is based on switching on surface‐enhanced Raman scattering (SERS) upon incubation of the Survivin protein dimer with Raman reporter‐labeled gold nanoparticles (AuNP). Site‐specificity is achieved by complexation of nickel‐chelated N‐nitrilo‐triacetic acid (Ni‐NTA) anchors on the particle surface by multiple histidines (His₆‐tag) attached to each C‐terminus of the centrosymmetric protein dimer. Correlative single‐particle analysis using light sheet laser microscopy enables the simultaneous observation of both elastic and inelastic light scattering from the same sample volume. Thereby, the SERS‐inactive AuNP‐protein monomers can be directly discriminated from the SERS‐active AuNP‐protein dimers/oligomers. This information, i.e. the percentage of SERS‐active AuNP in colloidal suspension, is not accessible from conventional SERS experiments due to ensemble averaging. The presented correlative single‐particle approach paves the way for quantitative site‐specific SERS assays in which site‐specific protein recognition by small chemical and in particular supramolecular ligands can be tested.     

Receptor‐Mediated Cellular Uptake of Folate‐Conjugated Fluorescent Nanodiamonds: A Combined Ensemble and Single‐Particle Study

Fluorescent nanodiamonds (FNDs) are nontoxic and photostable nanomaterials, ideal for long‐term in vivo imaging applications. This paper reports that FNDs with a size of ≈140 nm can be covalently conjugated with folic acid (FA) for receptor‐mediated targeting of cancer cells at the single‐particle level. The conjugation is made by using biocompatible polymers, such as polyethylene glycol, as crosslinked buffer layers. Ensemble‐averaged measurements with flow cytometry indicate that more than 50% of the FA‐conjugated FND particles can be internalized by the cells (such as HeLa cells) through receptor‐mediated endocytosis, as confirmed by competitive inhibition assays. Confocal fluorescence microscopy reveals that these FND particles accumulate in the perinuclear region. The absolute number of FNDs internalized by HeLa cells after 3 h of incubation at a particle concentration of 10 µg mL^?1 is in the range of 100 particles per cell. The receptor‐mediated uptake process is further elucidated by single‐particle tracking of 35‐nm FNDs in three dimensions and real time during the endocytosis.