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.     

Screening Therapeutic Agents Specific to Breast Cancer Stem Cells Using a Microfluidic Single‐Cell Clone‐Forming Inhibition Assay

Screens of cancer stem cells (CSCs)‐specific agents present significant challenges to conventional cell assays due to the difficulty in preparing CSCs ready for drug testing. To overcome this limitation, developed is a microfluidic single‐cell assay for screening breast cancer stem cell–specific agents. This assay takes advantage of the single‐cell clone‐forming capability of CSCs, which can be specifically inhibited by CSC‐targeting agents. The single‐cell assay is performed on a microfluidic chip with an array of 3840 cell‐capturing units; the single‐cell arrays are easily formed by flowing a cell suspension into the microchip. Achieved is a single cell‐capture rate of ≈60% thus allowing more than 2000 single cells to be analyzed in a single test. Over long‐term suspension culture, only a minority of cells survive and form tumorspheres. The clone‐formation rate of MCF‐7, MDA‐MB‐231, and T47D cells is 1.67%, 5.78%, and 5.24%, respectively. The clone‐forming inhibition assay is conducted by exposing the single‐cell arrays to a set of anticancer agents. The CSC‐targeting agents show complete inhibition of single‐cell clone formation while the nontargeting ones show incomplete inhibition effects. The resulting microfluidic single‐cell assay with the potential to screen CSC‐specific agents with high efficiency provides new tools for individualized tumor therapy.     

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.     

Dynamic Environmental Control in Microfluidic Single‐Cell Cultivations: From Concepts to Applications

Microfluidic single‐cell cultivation (MSCC) is an emerging field within fundamental as well as applied biology. During the last years, most MSCCs were performed at constant environmental conditions. Recently, MSCC at oscillating and dynamic environmental conditions has started to gain significant interest in the research community for the investigation of cellular behavior. Herein, an overview of this topic is given and microfluidic concepts that enable oscillating and dynamic control of environmental conditions with a focus on medium conditions are discussed, and their application in single‐cell research for the cultivation of both mammalian and microbial cell systems is demonstrated. Furthermore, perspectives for performing MSCC at complex dynamic environmental profiles of single parameters and multiparameters (e.g., pH and O₂) in amplitude and time are discussed. The technical progress in this field provides completely new experimental approaches and lays the foundation for systematic analysis of cellular metabolism at fluctuating environments.     

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.     

Microfluidic Single‐Cell Omics Analysis

The commonly existing cellular heterogeneity plays a critical role in biological processes such as embryonic development, cell differentiation, and disease progress. Single‐cell omics‐based heterogeneous studies have great significance for identifying different cell populations, discovering new cell types, revealing informative cell features, and uncovering significant interrelationships between cells. Recently, microfluidics has evolved to be a powerful technology for single‐cell omics analysis due to its merits of throughput, sensitivity, and accuracy. Herein, the recent advances of microfluidic single‐cell omics analysis, including different microfluidic platform designs, lysis strategies, and omics analysis techniques, are reviewed. Representative applications of microfluidic single‐cell omics analysis in complex biological studies are then summarized. Finally, a few perspectives on the future challenges and development trends of microfluidic‐assisted single‐cell omics analysis are discussed.     

A Microfabricated Sandwiching Assay for Nanoliter and High‐Throughput Biomarker Screening

Cells secrete substances that are essential to the understanding of numerous immunological phenomena and are extensively used in clinical diagnoses. Countless techniques for screening of biomarker secretion in living cells have generated valuable information on cell function and physiology, but low volume and real‐time analysis is a bottleneck for a range of approaches. Here, a simple, highly sensitive assay using a high‐throughput micropillar and microwell array chip (MIMIC) platform is presented for monitoring of biomarkers secreted by cancer cells. The sensing element is a micropillar array that uses the enzyme‐linked immunosorbent assay (ELISA) mechanism to detect captured biomolecules. When integrated with a microwell array where few cells are localized, interleukin 8 (IL‐8) secretion can be monitored with nanoliter volume using multiple micropillar arrays. The trend of cell secretions measured using MIMICs matches the results from conventional ELISA well while it requires orders of magnitude less cells and volumes. Moreover, the proposed MIMIC is examined to be used as a drug screening platform by delivering drugs using micropillar arrays in combination with a microfluidic system and then detecting biomolecules from cells as exposed to drugs.     

Progress in Microfluidics‐Based Exosome Separation and Detection Technologies for Diagnostic Applications

Exosomes are secreted by most cell types and circulate in body fluids. Recent studies have revealed that exosomes play a significant role in intercellular communication and are closely associated with the pathogenesis of disease. Therefore, exosomes are considered promising biomarkers for disease diagnosis. However, exosomes are always mixed with other components of body fluids. Consequently, separation methods for exosomes that allow high‐purity and high‐throughput separation with a high recovery rate and detection techniques for exosomes that are rapid, highly sensitive, highly specific, and have a low detection limit are indispensable for diagnostic applications. For decades, many exosome separation and detection techniques have been developed to achieve the aforementioned goals. However, in most cases, these two techniques are performed separately, which increases operation complexity, time consumption, and cost. The emergence of microfluidics offers a promising way to integrate exosome separation and detection functions into a single chip. Herein, an overview of conventional and microfluidics‐based techniques for exosome separation and detection is presented. Moreover, the advantages and drawbacks of these techniques are compared.     

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.     

Stochastic Particle Barcoding for Single‐Cell Tracking and Multiparametric Analysis

This study presents stochastic particle barcoding (SPB), a method for tracking cell identity across bioanalytical platforms. In this approach, single cells or small collections of cells are co‐encapsulated within an enzymatically‐degradable hydrogel block along with a random collection of fluorescent beads, whose number, color, and position encode the identity of the cell, enabling samples to be transferred in bulk between single‐cell assay platforms without losing the identity of individual cells. The application of SPB is demonstrated for transferring cells from a subnanoliter protein secretion/phenotyping array platform into a microtiter plate, with re‐identification accuracies in the plate assay of 96±2%. Encapsulated cells are recovered by digesting the hydrogel, allowing subsequent genotyping and phenotyping of cell lysates. Finally, a model scaling is developed to illustrate how different parameters affect the accuracy of SPB and to motivate scaling of the method to thousands of unique blocks.     

Monoclonal Cell Line Generation and CRISPR/Cas9 Manipulation via Single‐Cell Electroporation

Stably transfected cell lines are widely used in drug discovery and biological research to produce recombinant proteins. Generation of these cell lines requires the isolation of multiple clones, using time‐consuming dilution methods, to evaluate the expression levels of the gene of interest. A new and efficient method is described for the generation of monoclonal cell lines, without the need for dilution cloning. In this new method, arrays of patterned cell colonies and single cell transfection are employed to deliver a plasmid coding for a reporter gene and conferring resistance to an antibiotic. Using a nanofountain probe electroporation system, probe positioning is achieved through a micromanipulator with sub‐micron resolution and resistance‐based feedback control. The array of patterned cell colonies allows for rapid selection of numerous stably transfected clonal cell lines located on the same culture well, conferring a significant advantage over slower and labor‐intensive traditional methods. In addition to plasmid integration, this methodology can be seamlessly combined with CRISPR/Cas9 gene editing, paving the way for advanced cell engineering.     

Inertial Microfluidic Cell Stretcher (iMCS): Fully Automated,High‐Throughput,and Near Real‐Time Cell Mechanotyping

Mechanical biomarkers associated with cytoskeletal structures have been reported as powerful label‐free cell state identifiers. In order to measure cell mechanical properties, traditional biophysical (e.g., atomic force microscopy, micropipette aspiration, optical stretchers) and microfluidic approaches were mainly employed; however, they critically suffer from low‐throughput, low‐sensitivity, and/or time‐consuming and labor‐intensive processes, not allowing techniques to be practically used for cell biology research applications. Here, a novel inertial microfluidic cell stretcher (iMCS) capable of characterizing large populations of single‐cell deformability near real‐time is presented. The platform inertially controls cell positions in microchannels and deforms cells upon collision at a T‐junction with large strain. The cell elongation motions are recorded, and thousands of cell deformability information is visualized near real‐time similar to traditional flow cytometry. With a full automation, the entire cell mechanotyping process runs without any human intervention, realizing a user friendly and robust operation. Through iMCS, distinct cell stiffness changes in breast cancer progression and epithelial mesenchymal transition are reported, and the use of the platform for rapid cancer drug discovery is shown as well. The platform returns large populations of single‐cell quantitative mechanical properties (e.g., shear modulus) on‐the‐fly with high statistical significances, enabling actual usages in clinical and biophysical studies.