Single-cell Droplet Research Articles

Artificial intelligence-enabled multipurpose smart detection in active-matrix electrowetting-on-dielectric digital microfluidics

An active-matrix electrowetting-on-dielectric (AM-EWOD) system integrates hundreds of thousands of active electrodes for sample droplet manipulation, which can enable simultaneous, automatic, and parallel on-chip biochemical reactions. A smart detection system is essential for ensuring a fully automatic workflow and online programming for the subsequent experimental steps. In this work, we demonstrated an artificial intelligence (AI)-enabled multipurpose smart detection method in an AM-EWOD system for different tasks. We employed the U-Net model to quantitatively evaluate the uniformity of the applied droplet-splitting methods. We used the YOLOv8 model to monitor the droplet-splitting process online. A 97.76% splitting success rate was observed with 18 different AM-EWOD chips. A 99.982% model precision rate and a 99.980% model recall rate were manually verified. We employed an improved YOLOv8 model to detect single-cell samples in nanolitre droplets. Compared with manual verification, the model achieved 99.260% and 99.193% precision and recall rates, respectively. In addition, single-cell droplet sorting and routing experiments were demonstrated. With an AI-based smart detection system, AM-EWOD has shown great potential for use as a ubiquitous platform for implementing true lab-on-a-chip applications.

Single Cell Droplet-Based Efficacy and Transcriptomic Analysis of a Novel Anti-KLRG1 Antibody for Elimination of Autoreactive T Cells.

Progress in developing improvements in the treatment of autoimmune disease has been gradual, due to challenges presented by the nature of these conditions. Namely, the need to suppress a patient's immune response while maintaining the essential activity of the immune system in controlling disease. Targeted treatments to eliminate the autoreactive immune cells driving disease symptoms present a promising new option for major improvements in treatment efficacy and side effect management. Monoclonal antibody therapies can be applied to target autoreactive immune cells if the cells possess unique surface marker expression patterns. Killer cell lectin like receptor G1 (KLRG1) expression on autoreactive T cells presents an optimal target for this type of cell depleting antibody therapy. In this study, we apply a variety of in vitro screening methods to determine the efficacy of a novel anti-KLRG1 antibody at mediating specific natural killer (NK) cell mediated antibody-dependent cellular cytotoxicity (ADCC). The methods include single-cell droplet microfluidic techniques, allowing timelapse imaging and sorting based on cellular interactions. Included in this study is the development of a novel method of sorting cells using a droplet-sorting platform and a fluorescent calcium dye to separate cells based on CD16 recognition of cell-bound antibody. We applied this novel sorting method to visualize transcriptomic variation between NK cells that are or are not activated by binding the anti-KLRG1 antibody using RNA sequencing. The data in this study reveals a reliable and target-specific cytotoxicity of the cell depleting anti-KLRG1 antibody, and supports our droplet-sorting calcium assay as a novel method of sorting cells based on receptor activation.

A Novel Method for Acquired Sex Chromosome Mosaicism.

The phenomenon of sex chromosome loss from hematopoietic cells is an emerging indicator of biological aging. While many methods to detect this loss have been developed, enhancing the field, these existing methods often suffer from being labor-intensive, expensive, and not sufficiently sensitive. To bridge this gap, a novel and more efficient technique is developed, named the SinChro assay. This method employs multiplexed single-cell droplet PCR, designed to detect cells with sex chromosome loss at single-cell resolution. Through the SinChro assay, the age-dependent increase in Y chromosome loss in male blood is successfully mapped. The age-dependent loss of the X chromosome in female blood is also identified, a finding that has been challenging with existing methods. The advent of the SinChro assay marks a significant breakthrough in the study of age-related sex mosaicism. Its utility extends beyond blood analysis, applicable to a variety of tissues, and it holds the potential to deepen the understanding of biological aging and related diseases.

Droplet-based microfluidic platform for detecting agonistic peptides that are self-secreted by yeast expressing a G-protein-coupled receptor

BackgroundSingle-cell droplet microfluidics is an important platform for high-throughput analyses and screening because it provides an independent and compartmentalized microenvironment for reaction or cultivation by coencapsulating individual cells with various molecules in monodisperse microdroplets. In combination with microbial biosensors, this technology becomes a potent tool for the screening of mutant strains. In this study, we demonstrated that a genetically engineered yeast strain that can fluorescently sense agonist ligands via the heterologous expression of a human G-protein-coupled receptor (GPCR) and concurrently secrete candidate peptides is highly compatible with single-cell droplet microfluidic technology for the high-throughput screening of new agonistically active peptides.ResultsThe water-in-oil microdroplets were generated using a flow-focusing microfluidic chip to encapsulate engineered yeast cells coexpressing a human GPCR [i.e., angiotensin II receptor type 1 (AGTR1)] and a secretory agonistic peptide [i.e., angiotensin II (Ang II)]. The single yeast cells cultured in the droplets were then observed under a microscope and analyzed using image processing incorporating machine learning techniques. The AGTR1-mediated signal transduction elicited by the self-secreted Ang II peptide was successfully detected via the expression of a fluorescent reporter in single-cell yeast droplet cultures. The system could also distinguish Ang II analog peptides with different agonistic activities. Notably, we further demonstrated that the microenvironment of the single-cell droplet culture enabled the detection of rarely existing positive (Ang II-secreting) yeast cells in the model mixed cell library, whereas the conventional batch-culture environment using a shake flask failed to do so. Thus, our approach provided compartmentalized microculture environments, which can prevent the diffusion, dilution, and cross-contamination of peptides secreted from individual single yeast cells for the easy identification of GPCR agonists.ConclusionsWe established a droplet-based microfluidic platform that integrated an engineered yeast biosensor strain that concurrently expressed GPCR and self-secreted the agonistic peptides. This offers individually isolated microenvironments that allow the culture of single yeast cells secreting these peptides and gaging their signaling activities, for the high-throughput screening of agonistic peptides. Our platform base on yeast GPCR biosensors and droplet microfluidics will be widely applicable to metabolic engineering, environmental engineering, and drug discovery.

Harnessing HetHydrogel: A Universal Platform to Dropletize Single-Cell Multiomics.

A universal platform is developed for dropletizing single cell plate-based multiomic assays, consisting of three main pillars: a miniaturized open Heterogeneous Hydrogel reactor (abbreviated HetHydrogel) for multi-step biochemistry, its tunable permeability that allows Tn5 tagmentation, and single cell droplet barcoding. Through optimizing the HetHydrogel manufacturing procedure, the chemical composition, and cell permeation conditions, simultaneous high-throughput mitochondrial DNA genotyping and chromatin profiling at the single-cell level are demonstrated using a mixed-species experiment. This platform offers a powerful way to investigate the genotype-phenotype relationships of various mtDNA mutations in biological processes. The HetHydrogel platform is believed to have the potential to democratize droplet technologies, upgrading a whole range of plate-based single cell assays to high throughput format.

Advancements in minimal residual disease detection: a practical approach using single-cell droplet PCR for comprehensive monitoring in hematological malignancy.

The identification of chromosomal abnormalities accompanied by copy number alterations is important for understanding tumor characteristics. Testing methodologies for copy number abnormality have limited sensitivity, resulting in their use only for the sample provided at the time of diagnosis or recurrence of malignancy, but not for the monitoring of minimal residual disease (MRD) during and after therapy. We developped the "DimShift" technology which enable to measure the copy number of target gene/chromosome in each cell, which is given by the single cell droplet PCR. Qualitative result of DimShift given by peripheral blood was perfectly concordant with that of bone marrow. These findings and performances are promising to be the new methodology for MRD detection in malignant diseases utilizing bone marrow as well as peripheral blood.

Multiplex fluorescence detection of single-cell droplet microfluidics and its application in quantifying protein expression levels.

This study presented a platform of multiplex fluorescence detection of single-cell droplet microfluidics with demonstrative applications in quantifying protein expression levels. The platform of multiplex fluorescence detection mainly included optical paths adopted from conventional microscopy enabling the generation of three optical spots from three laser sources for multiple fluorescence excitation and capture of multiple fluorescence signals by four photomultiplier tubes. As to platform characterization, microscopic images of three optical spots were obtained where clear Gaussian distributions of intensities without skewness confirmed the functionality of the scanning lens, while the controllable distances among three optical spots validated the functionality of fiber collimators and the reflector lens. As to demonstration, this platform was used to quantify single-cell protein expression within droplets where four-type protein expression of α-tubulin, Ras, c-Myc, and β-tubulin of CAL 27 (Ncell = 1921) vs WSU-HN6 (Ncell = 1881) were quantitatively estimated, which were (2.85 ± 0.72) × 105 vs (4.83 ± 1.58) × 105, (3.69 ± 1.41) × 104 vs (5.07 ± 2.13) × 104, (5.90 ± 1.45) × 104 vs (9.57 ± 2.85) × 104, and (3.84 ± 1.28) × 105 vs (3.30 ± 1.10) × 105, respectively. Neural pattern recognition was utilized for the classification of cell types, achieving successful rates of 69.0% (α-tubulin), 75.4% (Ras), 89.1% (c-Myc), 65.8% (β-tubulin), and 99.1% in combination, validating the capability of this platform of multiplex fluorescence detection to quantify various types of single-cell proteins, which could provide comprehensive evaluations on cell status.

Multiplex single-cell droplet PCR with machine learning for detection of high-risk human papillomaviruses

High-risk human papillomavirus (HPV) testing can significantly decline the incidence and mortality of cervical cancer. Microfluidic technology provides an effective method for accurate detection of high-risk HPV by utilizing multiplex single-cell droplet polymerase chain reaction (PCR). However, current strategies are limited by low-integration microfluidic chip, complex reagent system, expensive detection equipment and time-consuming droplet identification. Here, we developed a novel multiplex droplet PCR method that directly detected high-risk HPV sequences in single cells. A multiplex microfluidic chip integrating four flow-focusing structures was designed for one-step and parallel droplet preparation. Using single-cell droplet PCR, multi-target sequences were detected simultaneously based on a monochromatic fluorescence signal. We applied machine learning to automatically identify the large populations of single-cell droplets with 97% accuracy. HPV16, 18 and 45 sequences were sensitively detected without cross-contamination in mixed CaSki and Hela cells. The approach enables rapid and reliable detection of multi-target sequences in single cells, making it powerful for investigating cellular heterogeneity related to cancer diagnosis and treatment.

High-throughput droplet-based analysis of influenza A virus genetic reassortment by single-virus RNA sequencing

The segmented RNA genome of influenza A viruses (IAVs) enables viral evolution through genetic reassortment after multiple IAVs coinfect the same cell, leading to viruses harboring combinations of eight genomic segments from distinct parental viruses. Existing data indicate that reassortant genotypes are not equiprobable; however, the low throughput of available virology techniques does not allow quantitative analysis. Here, we have developed a high-throughput single-cell droplet microfluidic system allowing encapsulation of IAV-infected cells, each cell being infected by a single progeny virion resulting from a coinfection process. Customized barcoded primers for targeted viral RNA sequencing enabled the analysis of 18,422 viral genotypes resulting from coinfection with two circulating human H1N1pdm09 and H3N2 IAVs. Results were highly reproducible, confirmed that genetic reassortment is far from random, and allowed accurate quantification of reassortants including rare events. In total, 159 out of the 254 possible reassortant genotypes were observed but with widely varied prevalence (from 0.038 to 8.45%). In cells where eight segments were detected, all 112 possible pairwise combinations of segments were observed. The inclusion of data from single cells where less than eight segments were detected allowed analysis of pairwise cosegregation between segments with very high confidence. Direct coupling analysis accurately predicted the fraction of pairwise segments and full genotypes. Overall, our results indicate that a large proportion of reassortant genotypes can emerge upon coinfection and be detected over a wide range of frequencies, highlighting the power of our tool for systematic and exhaustive monitoring of the reassortment potential of IAVs.

Dispersive phase microscopy incorporated with droplet-based microfluidics for biofactory-on-a-chip.

Biomolecular imaging of intracellular structures of a single cell and subsequent screening of the cells are of high demand in metabolic engineering to develop strains with the desired phenotype. However, the capability of current methods is limited to population-scale identification of cell phenotyping. To address this challenge, we propose to utilize dispersive phase microscopy incorporated with a droplet-based microfluidic system that combines droplet volume-on-demand generation, biomolecular imaging, and droplet-on-demand sorting, to achieve high-throughput screening of cells with an identified phenotype. Particularly, cells are encapsulated in homogeneous environments with microfluidic droplet formation, and the biomolecule-induced dispersive phase can be investigated to indicate the biomass of a specific metabolite in a single cell. The retrieved biomass information consequently guides the on-chip droplet sorting unit to screen cells with the desired phenotype. To demonstrate the proof of concept, we showcase the method by promoting the evolution of the Haematococcus lacustris strain toward a high production of natural antioxidant astaxanthin. The validation of the proposed system with on-chip single-cell imaging and droplet manipulation functionalities reveals the high-throughput single-cell phenotyping and selection potential that applies to many other biofactory scenarios, such as biofuel production, critical quality attribute control in cell therapy, etc.

ScRNA-seq for Microcephaly Research [I]: Single-Cell Droplet Encapsulation, mRNA Capture, and cDNA Synthesis.

Single-cell RNA sequencing (scRNA-seq) allows for the transcriptomic profiling of a sample tissue with single-cell resolution. The concept of scRNA-seq builds on traditional, "bulk" RNA-seq by recording and preserving the cellular origin of each transcript throughout library preparation. Here we describe an adaptation of the Drop-Seq method (Macosko et al. Cell 161, 1202-1214, 2015), in which nanoliter-scale droplets are used to physically separate dissociated cells, while a cell-specific DNA barcode is simultaneously introduced. Following barcoding, cDNAs can be mixed and pooled while retaining the identity of the cell of origin. The benefit of the Drop-Seq approach is high throughput from relatively small samples of tissue. The method described here is appropriate for processing an input of as few as 150,000 cells, with a final yield of as many as 5000 single-cell transcripts captured.

A Microfluidic Approach for Probing Heterogeneity in Cytotoxic T-Cells by Cell Pairing in Hydrogel Droplets.

Cytotoxic T-cells (CTLs) exhibit strong effector functions to leverage antigen-specific anti-tumoral and anti-viral immunity. When naïve CTLs are activated by antigen-presenting cells (APCs) they display various levels of functional heterogeneity. To investigate this, we developed a single-cell droplet microfluidics platform that allows for deciphering single CTL activation profiles by multi-parameter analysis. We identified and correlated functional heterogeneity based on secretion profiles of IFNγ, TNFα, IL-2, and CD69 and CD25 surface marker expression levels. Furthermore, we strengthened our approach by incorporating low-melting agarose to encapsulate pairs of single CTLs and artificial APCs in hydrogel droplets, thereby preserving spatial information over cell pairs. This approach provides a robust tool for high-throughput and single-cell analysis of CTLs compatible with flow cytometry for subsequent analysis and sorting. The ability to score CTL quality, combined with various potential downstream analyses, could pave the way for the selection of potent CTLs for cell-based therapeutic strategies.

Single-cell sorting using integrated pneumatic valve droplet microfluidic chip

Single-cell analysis and sorting have been increasingly prevalent in scientific study and industry in recent years. Droplet-based microfluidic systems are significant tools for single cell separation due to the advantages of high-throughput screening flux and relatively simple chip manufacturing. Herein, this work integrated single-cell droplet generation, incubation, and sorting functions into a single microfluidic chip using pneumatic microvalve for single-cell screening of hybridoma cells. The chip realized the generation, storage, incubation, and assay sorting of droplets in order by adjusting the switch of the microvalve. Numerical methods were used to simulate the effects of membrane size and pressure on valve deformation. Incubation results showed that fluorescent signals were generated in the droplets containing both hybridoma cells and microspheres under the control of pneumatic microvalves. In addition, the purity of target droplets was increased from 4.87% before sorting to 89.70% after sorting using fluorescence-activated droplet sorting platform, demonstrating the feasibility of the integrated chip with a pneumatic valve for single-cell screening. The integrated chip is proved to be operated efficiently with minimum cell loss and human labor through reducing the risk of droplet fusion and facilitating the development of automated devices. So, the present study provides an integrated design for microdroplet-based single-cell sorting with various applications, such as single-cell sequencing, antibody screening, and directed evolutionary studies.

Single-Cell RNA Sequencing in Yeast Using the 10× Genomics Chromium Device.

Single-cell RNA sequencing (scRNA-seq) is emerging as an essential technique for studying the physiology of individual cells in populations. Although well-established and optimized for mammalian cells, research of microorganisms has been faced with major technical challenges for using scRNA-seq, because of their rigid cell wall, smaller cell size and overall lower total RNA content per cell. Here, we describe an easy-to-implement adaptation of the protocolfor the yeast Saccharomyces cerevisiae using the 10× Genomics platform, originally optimized for mammalian cells. Introducing Zymolyase, a cell wall-digesting enzyme, to one of the initial steps of single-cell droplet formation allows efficient in-droplet lysis of yeast cells, without affecting the droplet emulsion and further sample processing. In addition, we also describe the downstream data analysis, which combines established scRNA-seq analysis protocols with specific adaptations for yeast, and R-scripts for further secondary analysis of the data.

Single-cell droplet microfluidics for biomedical applications.

Single-cell manipulation and analysis is critical to the study of many fundamental biological processes and uncovering cellular heterogeneity, and presents the potential for extremely valuable applications in biomedical fields, including neuroscience, regenerative therapy, early diagnosis, and drug screening. The use of microfluidic technologies in single-cell manipulation and analysis is one of the most promising approaches and enables the creation of innovative conditions that are impractical or impossible to achieve using conventional methods. Herein, an overview of the technological development of single-cell droplet microfluidics is presented. The significant advantages of microfluidic droplet technology, the dynamic parameters affecting droplet production, and the geometric structures of microfluidic devices are emphasized. Furthermore, the progress to date in passive and active droplet generation methods based on microfluidics and various microfluidic tools for the production of single-cell droplets and hydrogel microspheres are summarized. Their key features, achievements, and limitations associated with single-cell droplet and hydrogel formation are discussed. The recent popularized applications of single-cell droplet microfluidics in biomedicine involving small-molecule detection, protein analysis, and drug screening and genetic analysis of single cells are explored too. Finally, the challenges that must be overcome to enable future applications in single-cell droplet microfluidics are highlighted.

Recent Advancements in the Technologies Detecting Food Spoiling Agents.

To match the current life-style, there is a huge demand and market for the processed food whose manufacturing requires multiple steps. The mounting demand increases the pressure on the producers and the regulatory bodies to provide sensitive, facile, and cost-effective methods to safeguard consumers’ health. In the multistep process of food processing, there are several chances that the food-spoiling microbes or contaminants could enter the supply chain. In this contest, there is a dire necessity to comprehend, implement, and monitor the levels of contaminants by utilizing various available methods, such as single-cell droplet microfluidic system, DNA biosensor, nanobiosensor, smartphone-based biosensor, aptasensor, and DNA microarray-based methods. The current review focuses on the advancements in these methods for the detection of food-borne contaminants and pathogens.

Microfluidic technology for multiple single-cell capture.

Microfluidic devices are widely used in single-cell capture and for pairing single cells or groups of cells for cell-cell interaction analysis; these advances have improved drug screening and cell signal transduction analysis. The complex in vivo environment involves interactions between two cells and among multiple cells of the same or different phenotypes. This study reviewed the core principles and performance of several microfluidic multiple- and single-cell capture methods, namely, the microwell, valve, trap, and droplet methods. The advantages and disadvantages of the methods were compared, and suggestions regarding their application to multiple-cell capture were provided. The results may serve as a reference for research on microfluidic multiple single-cell coculture technology.

High-resolution characterization of gene function using single-cell CRISPR tiling screen

Identification of novel functional domains and characterization of detailed regulatory mechanisms in cancer-driving genes is critical for advanced cancer therapy. To date, CRISPR gene editing has primarily been applied to defining the role of individual genes. Recently, high-density mutagenesis via CRISPR tiling of gene-coding exons has been demonstrated to identify functional regions in genes. Furthermore, breakthroughs in combining CRISPR library screens with single-cell droplet RNA sequencing (sc-RNAseq) platforms have revealed the capacity to monitor gene expression changes upon genetic perturbations at single-cell resolution. Here, we present “sc-Tiling,” which integrates a CRISPR gene-tiling screen with single-cell transcriptomic and protein structural analyses. Distinct from other reported single-cell CRISPR screens focused on observing gene function and gene-to-gene/enhancer-to-gene regulation, sc-Tiling enables the capacity to identify regulatory mechanisms within a gene-coding region that dictate gene activity and therapeutic response.

Single-cell lipidomics with high structural specificity by mass spectrometry

Single-cell analysis is critical to revealing cell-to-cell heterogeneity that would otherwise be lost in ensemble analysis. Detailed lipidome characterization for single cells is still far from mature, especially when considering the highly complex structural diversity of lipids and the limited sample amounts available from a single cell. We report the development of a general strategy enabling single-cell lipidomic analysis with high structural specificity. Cell fixation is applied to retain lipids in the cell during batch treatments prior to single-cell analysis. In addition to tandem mass spectrometry analysis revealing the class and fatty acyl-chain for lipids, batch photochemical derivatization and single-cell droplet treatment are performed to identify the C=C locations and sn-positions of lipids, respectively. Electro-migration combined with droplet-assisted electrospray ionization enables single-cell mass spectrometry analysis with easy operation but high efficiency in sample usage. Four subtypes of human breast cancer cells are correctly classified through quantitative analysis of lipid C=C location or sn-position isomers in ~160 cells. Most importantly, the single-cell deep lipidomics strategy successfully discriminates gefitinib-resistant cells from a population of wild-type human lung cancer cells (HCC827), highlighting its unique capability to promote precision medicine.

Multiplexed Single-Cell Leukocyte Enzymatic Secretion Profiling from Whole Blood Reveals Patient-Specific Immune Signature.

Enzymatic secretion of immune cells (leukocytes) plays a dominant role in host immune responses to a myriad of biological triggers, including infections, cancers, and cardiovascular diseases. Current tools to probe these leukocytes inadequately profile these vital biomarkers; the need for sample preprocessing steps of cell lysis, labeling, washing, and pipetting inevitably triggers the cells, changes its basal state, and dilutes the individual cell secretion in bulk assays. Using a fully integrated system for multiplexed profiling of native immune single-cell enzyme secretion from 50 μL of undiluted blood, we eliminate sample handling. With a total analysis time of 60 min, the integrated platform performs six tasks of leukocyte extraction, cell washing, fluorescent enzyme substrate mixing, single-cell droplet making, droplet incubation, and real-time readout for leukocyte secretion profiling of neutrophil elastase, granzyme B, and metalloproteinase. We calibrated the device, optimized the protocols, and tested the leukocyte secretion of acute heart failure (AHF) patients at admission and predischarge. This paper highlights the presence of single-cell enzymatic immune phenotypes independent of CD marker labeling, which could potentially elucidate the innate immune response states. We found that patients recovering from AHF showed a corresponding reduction in immune-cell enzymatic secretion levels and donor-specific enzymatic signatures were observed, which suggests patient-to-patient heterogeneous immune response. This platform presents opportunities to elucidate the complexities of the immune response from a single drop of blood and bridge the current technological, biological, and medical gap in understanding immune response and biological triggers.