Digital Sort-Enabled Counting Allows Absolute Electrical Quantification of Target Nucleic Acid.

Quantitative nucleic acid amplification tests are of great importance for diagnostics, but current approaches require complex and costly optical setups that limit their nonlaboratory applications. Herein we describe the implementation of a microfluidics platform that can perform binary DNA-amplification-activated droplet sorting. The digital sort-enabled counting (DISCO) platform enables label-free absolute quantification of the nucleic acid. This is achieved by provoking a pH change in droplets through a loop-mediated isothermal amplification (LAMP) reaction, followed by using sorting by interfacial tension (SIFT) to direct positive and negative droplets to different outlets. With the use of on-chip electrodes at both outlets, we demonstrate that the digital electrical counting of target DNA and RNA can be realized. DISCO is a promising approach for realizing sensitive nucleic acid quantification in point-of-care settings.

Identification, sorting and profiling of functional killer cells via the capture of fluorescent target-cell lysate.

Assays for assessing cell-mediated cytotoxicity are largely target-cell-centric and cannot identify and isolate subpopulations of cytotoxic effector cells. Here we describe an assay compatible with flow cytometry for the accurate identification and sorting of functional killer-cell subpopulations in co-cultures. The assay, which we named PAINTKiller (for 'proximity affinity intracellular transfer identification of killer cells'), relies on the detection of an intracellular fluorescent protein 'painted' by a lysed cell on the surface of the lysing cytotoxic cell (specifically, on cell lysis the intracellular fluorescein derivative carboxyfluorescein succinimidyl ester is captured on the surface of the natural killer cell by an antibody for anti-fluorescein isothiocyanate linked to an antibody for the pan-leucocyte surface receptor CD45). The assay can be integrated with single-cell RNA sequencing for the analysis of molecular pathways associated with cell cytotoxicity and may be used to uncover correlates of functional immune responses.

Single-Cell Secretion Analysis via Microfluidic Cell Membrane Immunosorbent Assay for Immune Profiling.

Single-cell multiplexed phenotypic analysis expands the biomarkers for diagnosis, heralding a new era of precision medicine. Cell secretions are the primary measures of immune function, but single-cell screening remains challenging. Here, a novel cell membrane-based assay was developed using cholesterol-linked antibodies (CLAbs), integrating immunosorbent assays and droplet microfluidics to develop a flexible high-throughput single-cell secretion assay for multiplexed phenotyping. CLAb-grafted single cells were encapsulated in water-in-oil droplets to capture their own secretions. Subsequently, the cells were extracted from droplets for fluorescence labeling and screening. Multiple secretions and surface proteins were simultaneously measured from single cells by flow cytometry. To validate the approach, THP-1 cells, THP-1-derived M1 macrophages, and dendritic cells were assayed, indicating the differentiation efficiency of THP-1 cells under different chemical stimulations. Moreover, peripheral blood mononuclear cells from healthy donors under various stimuli showed varied active immune cell populations (6.62-47.14%). The peripheral blood mononuclear cells (PBMCs) of nasopharyngeal carcinoma patients were analyzed to identify a higher percentage of actively cytokine-secreted single cells in the basal state (2.82 ± 1.48%), compared with that in the health donors (0.70 ± 0.29%).

Rapid Information Retrieval from DNA Storage with Microfluidic Very Large-Scale Integration Platform.

Due to its high information density, DNA is very attractive as a data storage system. However, a major obstacle is the high cost and long turnaround time for retrieving DNA data with next-generation sequencing. Herein, the use of a microfluidic very large-scale integration (mVLSI) platform is described to perform highly parallel and rapid readout of data stored in DNA. Additionally, it is demonstrated that multi-state data encoded in DNA can be deciphered with on-chip melt-curve analysis, thereby further increasing the data content that can be analyzed. The pairing of mVLSI network architecture with exquisitely specific DNA recognition gives rise to a scalable platform for rapid DNA data reading.

DARESOME enables concurrent profiling of multiple DNA modifications with restriction enzymes in single cells and cell-free DNA.

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the most abundant DNA modifications that have important roles in gene regulation. Detailed studies of these different epigenetic marks aimed at understanding their combined effects and dynamic interconversion are, however, hampered by the inability of current methods to simultaneously measure both modifications, particularly in samples with limited quantities. We present DNA analysis by restriction enzyme for simultaneous detection of multiple epigenomic states (DARESOME), an assay based on modification-sensitive restriction digest and sequential tag ligation that can concurrently perform quantitative profiling of unmodified cytosine, 5mC, and 5hmC in CCGG sites genome-wide. DARESOME reveals the opposing roles of 5mC and 5hmC in gene expression regulation as well as their interconversion during aging in mouse brain. Implementation of DARESOME in single cells demonstrates pronounced 5hmC strand bias that reflects the semiconservative replication of DNA. Last, we showed that DARESOME enables integrative genomic, 5mC, and 5hmC profiling of cell-free DNA that uncovered multiomics cancer signatures in liquid biopsy.

Cell Granularity Reflects Immune Cell Function and Enables Selection of Lymphocytes with Superior Attributes for Immunotherapy.

In keeping with the rule of "form follows function", morphological aspects of a cell can reflect its role. Here, it is shown that the cellular granularity of a lymphocyte, represented by its intrinsic side scatter (SSC), is a potent indicator of its cell state and function. The granularity of a lymphocyte increases from naïve to terminal effector state. High-throughput cell-sorting yields a SSChigh population that can mediate immediate effector functions, and a highly prolific SSClow population that can give rise to the replenishment of the memory pool. CAR-T cells derived from the younger SSClow population possess desirable attributes for immunotherapy, manifested by increased naïve-like cells and stem cell memory (TSCM )-like cells together with a balanced CD4/CD8 ratio, as well as enhanced target-killing in vitro and in vivo. Altogether, lymphocyte segregation based on biophysical properties is an effective approach for label-free selection of cells that share collective functions and can have important applications for cell-based immunotherapies.

Melt-Encoded-Tags for Expanded Optical Readout in Digital PCR (METEOR-dPCR) Enables Highly Multiplexed Quantitative Gene Panel Profiling.

Digital PCR (dPCR) is an important tool for precise nucleic acid quantification in clinical setting, but the limited multiplexing capability restricts its applications for quantitative gene panel profiling. Here, this work describes melt-encoded-tags for expanded optical readout in digital PCR (METEOR-dPCR), a simple two-step assay that enables simultaneous quantification of a large panel of arbitrary genes in a dPCR platform. Target genes are quantitatively converted into DNA tags with unique melting temperatures through a ligation approach. These tags are then counted and distinguished by their melt-curve profiles on a dPCR platform. A multiplexing capacity of M^N, where M is the number of resolvable melting temperature and N is the number of fluorescence channel, can be achieved. This work validates METEOR-dPCR with simultaneous DNA copy number profiling of 60 targets using dPCR in cancer cells, and demonstrates its sensitivity for estimating tumor fraction in mixed tumor and normal DNA samples. The rapid, quantitative, and highly multiplexed METEOR-dPCR assay will have wide appeal for many clinical applications.

Rapid Single-Round Pool Testing of Infectious Disease Enabled by Multicolor Digital Melting PCR.

Pooled nucleic acid amplification test is a promising strategy to reduce cost and resources for screening large populations for infectious disease. However, the benefit of pooled testing is reversed when disease prevalence is high, because of the need to retest each sample to identify infected individual when a pool is positive. Split, Amplify, and Melt analysis of Pooled Assay (SAMPA) is presented, a multicolor digital melting PCR assay in nanoliter chambers that simultaneously identify infected individuals and quantify their viral loads in a single round of pooled testing. This is achieved by early sample tagging with unique barcodes and pooling, followed by single molecule barcode identification in a digital PCR platform using a highly multiplexed melt curve analysis strategy. The feasibility is demonstrated of SAMPA for quantitative unmixing and variant identification from pools of eight synthetic DNA and RNA samples corresponding to the N1 gene, as well as from heat-inactivated SARS-CoV-2 virus. Single round pooled testing of barcoded samples with SAMPA can be a valuable tool for rapid and scalable population testing of infectious disease.

Time-resolved assessment of single-cell protein secretion by sequencing.

Secreted proteins play critical roles in cellular communication. Methods enabling concurrent measurement of cellular protein secretion, phenotypes and transcriptomes are still unavailable. Here we describe time-resolved assessment of protein secretion from single cells by sequencing (TRAPS-seq). Released proteins are trapped onto the cell surface and probed by oligonucleotide-barcoded antibodies before being simultaneously sequenced with transcriptomes in single cells. We demonstrate that TRAPS-seq helps unravel the phenotypic and transcriptional determinants of the secretion of pleiotropic TH1 cytokines (IFNγ, IL-2 and TNF) in activated T cells. In addition, we show that TRAPS-seq can be used to track the secretion of multiple cytokines over time, uncovering unique molecular signatures that govern the dynamics of single-cell cytokine secretions. Our results revealed that early central memory T cells with CD45RA expression (TCMRA) are important in both the production and maintenance of polyfunctional cytokines. TRAPS-seq presents a unique tool for seamless integration of secretomics measurements with multi-omics profiling in single cells.

Heat selection enables highly scalable methylome profiling in cell-free DNA for noninvasive monitoring of cancer patients.

Genome-wide analysis of cell-free DNA methylation profile is a promising approach for sensitive and specific detection of many cancers. However, scaling such assays for clinical translation is impractical because of the high cost of whole-genome bisulfite sequencing. We show that the small fraction of GC-rich genome is highly enriched in CpG sites and disproportionately harbors most of the cancer-specific methylation signature. Here, we report on the simple and effective heat enrichment of CpG-rich regions for bisulfite sequencing (Heatrich-BS) platform that allows for focused methylation profiling in these highly informative regions. Our novel method and bioinformatics algorithm enable accurate tumor burden estimation and quantitative tracking of colorectal cancer patient's response to treatment at much reduced sequencing cost suitable for frequent monitoring. We also show tumor epigenetic subtyping using Heatrich-BS, which could enable patient stratification. Heatrich-BS holds great potential for highly scalable screening and monitoring of cancer using liquid biopsy.

Single-cell and bulk transcriptome sequencing identifies two epithelial tumor cell states and refines the consensus molecular classification of colorectal cancer.

The consensus molecular subtype (CMS) classification of colorectal cancer is based on bulk transcriptomics. The underlying epithelial cell diversity remains unclear. We analyzed 373,058 single-cell transcriptomes from 63 patients, focusing on 49,155 epithelial cells. We identified a pervasive genetic and transcriptomic dichotomy of malignant cells, based on distinct gene expression, DNA copy number and gene regulatory network. We recapitulated these subtypes in bulk transcriptomes from 3,614 patients. The two intrinsic subtypes, iCMS2 and iCMS3, refine CMS. iCMS3 comprises microsatellite unstable (MSI-H) cancers and one-third of microsatellite-stable (MSS) tumors. iCMS3 MSS cancers are transcriptomically more similar to MSI-H cancers than to other MSS cancers. CMS4 cancers had either iCMS2 or iCMS3 epithelium; the latter had the worst prognosis. We defined the intrinsic epithelial axis of colorectal cancer and propose a refined 'IMF' classification with five subtypes, combining intrinsic epithelial subtype (I), microsatellite instability status (M) and fibrosis (F).

Rapid microfluidic platform for screening and enrichment of cells secreting virus neutralizing antibodies.

As part of the body's immune response, antibodies (Abs) have the ability to neutralize pathogenic viruses to prevent infection. To screen for neutralizing Abs (nAbs) from the immune repertoire, multiple screening techniques have been developed. However, conventional methods have a trade-off between screening throughput and the ability to screen for nAbs via their functional efficacy. Although droplet microfluidic platforms have the ability to bridge this disparity, the majority of such reported platforms still rely on Ab-binding assays as a proxy for function, which results in irrelevant hits. Herein, we report the multi-module Droplet-based Platform for Effective Antibody RetrievaL (DROP-PEARL) platform, which can achieve high-throughput enrichment of Ab-secreting cells (ASCs) based on the neutralizing activity of secreted nAbs against the a target virus. In this study, in-droplet Chikungunya virus (CHIKV) infection of host cells and neutralization was demonstrated via sequential delivery of viruses and host cells via picoinjection. In addition, we demonstrate the ability of the sorting system to accurately discriminate and isolate uninfected droplets from a mixed population of droplets at a rate of 150 000 cells per hour. As a proof of concept, a single-cell neutralization assay was performed on two populations of cells (nAb-producing and non-Ab producing cells), and up to 2.75-fold enrichment of ASCs was demonstrated. Finally, we demonstrated that DROP-PEARL is able to achieve similar enrichment for low frequency (∼2%) functional nAb-producing cells in a background of excess cells secreting irrelevant antibodies, highlighting its potential prospect as a first round enrichment platform for functional ASCs. We envision that the DROP-PEARL platform could potentially be used to accelerate the discovery of nAbs against other pathogenic viral targets, and we believe it will be a useful in the ongoing fight against biological threats.

SAMBA: A Multicolor Digital Melting PCR Platform for Rapid Microbiome Profiling.

During the past decade, breakthroughs in sequencing technology have provided the basis for studies of the myriad ways in which microbial communities in and on the human body influence human health and disease. In almost every medical specialty, there is now a growing interest in accurate and quantitative profiling of the microbiota for use in diagnostic and therapeutic applications. However, the current next-generation sequencing approach for microbiome profiling is costly, requires laborious library preparation, and is challenging to scale up for routine diagnostics. Split, Amplify, and Melt analysis of BActeria-community (SAMBA), a novel multicolor digital melting polymerase chain reaction platform with unprecedented multiplexing capability is presented, and the capability to distinguish and quantify 16 bacteria species in mixtures is demonstrated. Subsequently, SAMBA is applied to measure the compositions of bacteria in the gut microbiome to identify microbial dysbiosis related to colorectal cancer. This rapid, low cost, and high-throughput approach will enable the implementation of microbiome diagnostics in clinical laboratories and routine medical practice.

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.

Microfluidic compartmentalization to identify gene biomarkers of infection.

Infectious diseases caused by pathogens, such as SARS-COV, H7N9, severe fever with thrombocytopenia syndrome virus, and human immunodeficiency virus, have fatal outcomes with common features of severe fever and subsequent bacterial invasion progressing to multiorgan failure. Gene biomarkers are promising to distinguish specific infections from others with similar presenting symptoms for the prescription of correct therapeutics, preventing pandemics. While routine laboratory methods based on polymerase chain reaction (PCR) to measure gene biomarkers have provided highly sensitive and specific viral detection techniques over the years, they are still hampered by their precision and resource intensity precluding their point-of-care use. Recently, there has been growing interest in employing microfluidic technologies to advance current methods for infectious disease determination via gene biomarker measurements. Here, based on the requirement of infection detection, we will review three microfluidic approaches to compartmentalize gene biomarkers: (1) microwell-based PCR platforms; (2) droplet-based PCR; and (3) point-of-care devices including centrifugal chip, SlipChip, and self-powered integrated microfluidic point-of-care low-cost enabling chip. By capturing target genes in microwells with a small sample volume (∼µl), sensitivity can be enhanced. Additionally, with the advance of significant sample volume minimization (∼pl) using droplet technology, gene quantification is possible. These improvements in cost, automation, usability, and portability have thereby allowed point-of-care applications to decentralize testing platforms from laboratory-based settings to field use against infections.

Massively parallel single-molecule telomere length measurement with digital real-time PCR.

Telomere length is a promising biomarker for age-associated diseases and cancer, but there are still substantial challenges to routine telomere analysis in clinics because of the lack of a simple and rapid yet scalable method for measurement. We developed the single telomere absolute-length rapid (STAR) assay, a novel high-throughput digital real-time PCR approach for rapidly measuring the absolute lengths and quantities of individual telomere molecules. We show that this technique provides the accuracy and sensitivity to uncover associations between telomere length distribution and telomere maintenance mechanisms in cancer cell lines and primary tumors. The results indicate that the STAR assay is a powerful tool to enable the use of telomere length distribution as a biomarker in disease and population-wide studies.

The Role of Single-Cell Technology in the Study and Control of Infectious Diseases.

The advent of single-cell research in the recent decade has allowed biological studies at an unprecedented resolution and scale. In particular, single-cell analysis techniques such as Next-Generation Sequencing (NGS) and Fluorescence-Activated Cell Sorting (FACS) have helped show substantial links between cellular heterogeneity and infectious disease progression. The extensive characterization of genomic and phenotypic biomarkers, in addition to host-pathogen interactions at the single-cell level, has resulted in the discovery of previously unknown infection mechanisms as well as potential treatment options. In this article, we review the various single-cell technologies and their applications in the ongoing fight against infectious diseases, as well as discuss the potential opportunities for future development.

Functional Stem Cell Sorting via Integrative Droplet Synchronization.

Stem cell regenerative medicine strategy requires selecting functional cells to trigger repair processes. Stem cell secretion measurement is important to evaluate cellular activities for functional cell sorting. At present, to determine single cell secretions, mixing chemical sensors and cells together in a chamber is a standard procedure. However, toxic chemical sensors, such as albumin assay kits, are used during this process, causing low viability (64%) and low functionality (30%). It is especially important for stem cell profiling, as the toxicity of chemical sensors such as albumin permanently changes stem cell phenotypes, leading to unwanted analysis outcomes. Moreover, because of the sensor toxicity, the challenge of culturing sorted cells remain. In this study, an integrative synchronized droplet screen system was developed to separate a large droplet with cell encapsulation into two daughter droplets: one droplet containing cell secretions and the other droplet containing a single cell. These two daughter droplets moved along the channels at the same speed in synchronization. By injecting toxic chemical sensors into one daughter droplet, the single-cell secretions were determined without affecting the cells in the corresponding droplet. Based on the daughter droplet synchronization, the cells without mixing toxicity sensors were sorted for cell culturing. For example, to identify hepatocytes, the albumin secretion of undifferentiated HepaRG stem cells was measured in daughter droplets by injecting a toxic albumin assay kit for functional stem cell sorting. With synchronized sorting, functional hepatocytes were collected without exposure to toxic chemical sensors, showing high viability (78%) and active functionality (89%).

DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells.

Genome-wide profiling of copy number alterations and DNA methylation in single cells could enable detailed investigation into the genomic and epigenomic heterogeneity of complex cell populations. However, current methods to do this require complex sample processing and cleanup steps, lack consistency, or are biased in their genomic representation. Here, we describe a novel single-tube enzymatic method, DNA Analysis by Restriction Enzyme (DARE), to perform deterministic whole genome amplification while preserving DNA methylation information. This method was evaluated on low amounts of DNA and single cells, and provides accurate copy number aberration calling and representative DNA methylation measurement across the whole genome. Single-cell DARE is an attractive and scalable approach for concurrent genomic and epigenomic characterization of cells in a heterogeneous population.